key: cord- - sltubqk authors: horiuchi, sho; saito, yuichi; matsui, atsuka; takahashi, nobumasa; ikeya, tomohiko; hoshi, eishin; shimizu, yoshihiko; yasuda, masanori title: a novel loop-mediated isothermal amplification method for efficient and robust detection of egfr mutations date: - - journal: int j oncol doi: . /ijo. . sha: doc_id: cord_uid: sltubqk the activation of somatic mutations conferring sensitivity to epidermal growth factor receptor (egfr) tyrosine kinase inhibitors has been widely used in the development of advanced or metastatic primary lung cancer therapy. therefore, identification of egfr mutations is essential. in the present study, a loop-mediated isothermal amplification (lamp) method was used to identify egfr mutations, and its efficiency was compared with the therascreen quantitative pcr assay. using lamp and therascreen to analyze surgically resected tissue samples from patients with pulmonary adenocarcinoma, egfr mutations were observed in / tumor samples (lamp) and / tumor samples (therascreen). notably, the lamp assay identified one tumor as wild-type, which had previously been identified as a deletion mutation in exon via the therascreen assay (case x). however, the direct sequencing to confirm the egfr status of the case x adhered to the results of the lamp assay. further experiments using case x dna identified this exon deletion mutation using both methods. in addition, a novel deletion mutation in exon of the egfr was identified. overall, the present study shows that the lamp method may serve as a valuable alternative for the identification oncogene mutations. lung cancer has the highest morbidity and mortality of all malignancies in the usa in and ( , ) . non-small cell lung carcinoma (nsclc) is the most common form of lung cancer, accounting for - % of cases and is primarily treated using systemic chemotherapy ( ) . however, nsclc treatment has evolved due to the development of therapy targeting the activation of mutations in the epidermal growth factor receptor (egfr) ( ) . egfr activation is caused by genetic mutations, conferring susceptibility to egfr tyrosine kinase inhibitor (tki) treatment, and was first reported in ( ). egfr mutations of pulmonary adenocarcinoma are present in - % of the caucasian population and - % of the east asian population ( ) ( ) ( ) . clinical trials have shown that patients with pulmonary adenocarcinoma with an egfr mutation exhibit clinical responses to orally administered egfr inhibitors ( , , ) . in , lin et al ( ) reported that / ( . %) patients with egfr-mutant metastatic lung adenocarcinoma had a survival time of -years ( ) . therefore, detection of egfr mutations is an important step in the treatment-decision pathway for patients with pulmonary adenocarcinoma. previously, direct dna sequencing was the standard method for detecting genetic mutations ( ) ; however, at present, several alternative methods for mutation testing have been developed ( , ) . for example, the therascreen egfr pcr kit ® (qiagen, inc.) is a commercial quantitative (q)pcr kit and has been widely adopted for clinical practice; however, this method is time-consuming and possesses certain procedural complexities, for example, requiring several temperature changes during dna amplification ( ) . next-generation sequencing has improved the efficiency of oncogene testing by high-throughput sequencing, which can detect dozen of mutations at the same time, but the high-cost of this technique limits its clinical usage ( , ) . therefore, detecting oncogenic mutations using a simple, easy and highly reproducible method remains a challenge. loop-mediated isothermal amplification (lamp) is a new pcr based method with high levels of specificity and a novel loop-mediated isothermal amplification method for efficient and robust detection of egfr mutations amplification efficiency and utilizes six primers ( ) . this method is performed under isothermal conditions, thereby enabling rapid amplification. due to the high specificity and rapid detection quality of lamp, this method has been widely used in the fields of bacteriology ( ) and virology ( , ) . however, to the best of our knowledge, there are very few studies reporting the value of lamp in determining egfr mutations. therefore, the present study aimed to detect egfr mutations in surgically resected tumor tissues from patients with pulmonary adenocarcinoma using this method, as detection of egfr mutations is one a key examination for patients with pulmonary adenocarcinoma ( , , , ) . in addition, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of lamp was evaluated and compared with the therascreen egfr pcr kit ® . tumor tissue samples. the tumor tissues were surgically resected from consecutive patients diagnosed with pulmonary adenocarcinoma by the expert pathologist at the saitama cardiovascular and respiratory center (kumagaya, japan) between january and october . all pathological diagnosis was determined on the basis of the who classification version ( ) by an expert pathologist, who normally makes a diagnosis using he-stained slides using light microscope (nikon co., eclipse ni-u) from a low magnification to a high magnification. the inclusion criteria were as follows: i) surgically resected tissue of primary lung cancer; ii) pulmonary adenocarcinoma; iii) enough volume materials for molecular testing; and iv) informed written consent from patients. conversely, cases with no informed consent or less volume sample were excluded. clinical characteristics of the patients are presented in table i . the mean patient age was . years and included males and females. all samples were fixed with % buffer formalin at room temperature ( - h) to create formalin-fixed, paraffin-embedded (ffpe) tumor blocks at department of pathology in saitama cardiovascular and respiratory center. hematoxylin-eosin staining was performed by the standard method using tissue-tek prisma (sakura finetek japan co., ltd.) according to the manufacturer's protocol. prior to dna extraction, the tumor content of each sample was assessed using light microscopy at x and x magnification, to ensure efficient pcr amplification. after sections were deparaffinized with xylene and hydrated through a graded series of ethanol ( , , and % ethanol), dna from the tissue blocks was extracted using the qiaamptm dna ffpe tissue kit ® (qiagen, inc.) and analyzed using a qiacube robot ® (qiagen, inc.) according to the manufacturer's protocols ( ) . therascreen qpcr mutation analysis. the presence of egfr mutations was determined using a therascreen egfr pcr kit ® (qiagen, inc.) according to the manufacturer's protocols ( ) . primer design. a primer set for lamp amplification of the partial sequence of the egfr gene (ng_ ) was designed using primer explorer (primerexplorer.jp/e/). fig. presents the sequences of the primers used with forward and backward outer primers (f and b ), forward and backward inner primers (fip and bip) and forward and back-loop primers (lf and lb). the primers were synthesized and purified by eurofin genomics. block oligo and fluorophore-labelled probes were synthesized and purified by japan bio services co., ltd., or gene design, inc. (table ii) . lamp egfr mutation analysis. the lamp assay detected egfr wild types and egfr mutations (table ii) . among egfr mutations, exon l r point mutations ( . %), exon deletions ( . %), simultaneous exon g x point mutation/exon s i point mutations ( . %) and exon ss i point mutation alone ( . %) were identified (table ii) . concentration for egfr mutation detection was . % of dna sample in therascreen assay (table si) . on the other hand, . % was the minimum concentration in lamp assay, since lamp assay demonstrated one success of the detection per tests at the level of . , . and . % concentrations and all positive per tests at > . % concentration (table si) . direct sequencing of case x. it was observed that a single case deviated in the identification of the mutation in exon between therascreen pcr and the lamp assay. to confirm this result, direct sequencing of the target site of exon was performed. direct sequencing results demonstrated no mutation in exon in case x, which was concordant with the results of the lamp assay. to reconfirm the status of egfr mutation in case x, four additional ffpe tissue blocks in case x were used to extract further dna samples. the hematoxylin-eosin (he) images of these ffpe blocks are presented in fig. . following removal of normal lung tissues, dna samples were extracted as aforementioned, and investigated using therascreen egfr pcr and a lamp assay. in all the four samples, the deletion mutation in exon was identified using both therascreen pcr and lamp assays. furthermore, direct sequencing revealed a novel exon egfr deletion mutation in samples a and b; ng_ . : g. _ delinsgca represented the deletion of nucleotides g. to g. (attaagagaagcaacatc, data not shown), which were replaced by a gca nucleotide triplet, changing ggaattaagagaagcaacatctcc to ggagcatcc (data not shown), resulting in shortening substation in the protein (p.leu _ser delinshis). egfr mutations in pulmonary adenocarcinoma are associated with sensitivity to tki therapy. hence the identification of egfr mutations has become a standard analysis in the treatment pathway of patients with pulmonary adenocarcinoma. although there are a number of methods available, there is no standardized approach to satisfy the practical clinical requirements of simplicity, rapid and cheap. several pcr based methods have previously been used as a routine test for the detection of egfr status in united states, european union, japan and china, including the scorpion amplification refractory mutation system (arms) ® ( ) , such as therascreen pcr assay. this method was approved by the fda as a standard approach for egfr gene analysis in lung cancer (fda. gov/medical-devices/recently-approved-devices/therascreenr-fg fr-rgq-pcr-kit-p ); however, although stable and reliable, this approach has procedural complexities, including complex settings and controls for the temperature at several times using a ( ) and cycleave pcr™ ( ) , have been developed in japan and are commercially used in centralized laboratories. the sensitivity of the pna-lna pcr clamp is > % with % specificity ( ) and the accuracy of cycleave pcr is . % ( ) , so it was the same for our results as it was those. lamp is a new pcr method and is considered to be a robust approach for gene analysis as it does not require sophisticated or expensive equipment, such as a thermal cycler necessary for pcr ( ) . therefore, the lamp method may have potential to decrease the costs of gene analysis. previous studies have demonstrated the value of lamp in field of bacteriology and virology ( , , , ) ; however, few studies have reported the value of lamp in oncology. ikeda et al ( ) used lamp assays to detect egfr mutations in nsclc, demonstrating the value of lamp, but only the l r mutation was studied. therefore, the present study aimed to investigate other egfr mutations, including those in exon , exon and other minor mutations using lamp assays. the sensitivity, specificity, ppv, npv and accuracy values of lamp for egfr mutations compared with the therascreen assay method were , , , . and . %, respectively, demonstrating the potential of lamp as an efficient alternative approach t oncogene mutation analyses. to evaluate the sensitivity of these two assays, a genetic analysis was performed to investigate the minimum concentration of egfr mutation detection in both methods. the minimum concentration for lamp was . % and there were no detection under . % in the therascreen assay. therefore, the minimum concentration for the lamp assay is ~ times higher than . % in the therascreen assay. with almost equal efficiency, detection of the exon mutation in case x using the therascreen method suggested that lamp, as well as direct sequence methods, could identify false negatives. however, additional experiments investigating case x tumor tissues demonstrated the presence of deletion mutations in exon . the direct sequence method has reported low sensitivity ( , ) , whereas the therascreen method was generally recognized as a promising method ( , ) . however, the identified mutation should be simplified, and an improved, faster and cheaper method of identification should be developed for its clinical application. the additional case x experiments also identified a novel egfr mutation using direct sequencing which was not identified using therascreen or lamp; however, this mutation may have been detected as an exon deletion by the primers of the similarly targeted mutation. however, the details of the primer of lamp method could not be disclosed due to the policies of eiken co., ltd., and further information concerning the primers of therascreen egfr pcr kit could not be obtained due to the patent. furthermore, the present study noted that this novel mutation was not included in the cosmic database and that no previous studies had reported this mutation. therefore, the present study is the first report this egfr mutation, to the best of our knowledge. in conclusion, the lamp method may be a valuable alternative for the identification of oncogenic mutations in lung cancer. currently, the study group is developing a new method of detecting oncogenes using liquid biopsies (data not shown). in addition, a novel mutation, ng_ . :g. _ delinsgca, was identified exon of egfr. however, this needs further validation before clinical use. the present study was funded by eiken chemical co., ltd. the datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. cancer statics introduction to the world health organization classification of tumors of the lung, pleura, thymus, and heart five-year survival in egfr-mutant metastatic lung adenocarcinoma treated with egfr-tkis activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib ret fusion gene: translation to personalized lung cancer therapy chipping away at the lung cancer genome genotyping and genomic profiling of non-small-cell lung cancer: implications for current and future therapies egfr mutations in lung cancer: correlation with clinical response to gefitinib therapy activity of epidermal growth factor receptor-tyrosine kinase inhibitors in patients with non-small cell lung cancer harboring rare epidermal growth factor receptor mutations first-line erlotinib versus gemcitabine/cisplatin in patients with advanced egfr mutation-positive non-small-cell lung cancer: analyses from the phase iii, randomized, open-label, ensure study afatinib versus cisplatin-based chemotherapy for egfr mutation-positive lung adenocarcinoma (lux-lung and lux-lung ): analysis of overall survival data from two randomised, phase trials current and future molecular testing in nsclc, what can we expect from new sequencing technologies? cost of cancer diagnosis using next-generation sequencing targeted gene panels in routine practice: a nationwide french study loop-mediated isothermal amplification of dna sensitive and rapid detection of mycoplasma pneumoniae by loop-mediated isothermal amplification rapid detection of the severe acute respiratory syndrome (sars) coronavirus by a loop-mediated isothermal amplification assay rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific loop-mediated isothermal amplification method the iaslc lung cancer staging project: proposals for revision of the tnm stage groupings in the forthcoming (eighth) edition of the tnm classification for lung cancer a commercial real-time pcr kit provides greater sensitivity than direct sequencing to detect kras mutations: a morphology-based approach in colorectal carcinoma efficiency of the therascreen® rgq pcr kit for the detection of egfr mutations in non-small cell lung carcinomas detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer sensitive detection of dna polymorphisms by the serial invasive signal amplification reaction genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid pcr clamp a rapid, sensitive assay to detect egfr mutation in small biopsy specimens from lung cancer reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers accuracy of the cobas egfr mutation assay in non-small-cell lung cancer compared with three laboratory-developed tests mycoplasma pneumoniae from the respiratory tract and beyond an updated loop-mediated isothermal amplification method for rapid diagnosis of h n avian influenza viruses detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification non-isotopic silver-stained sscp is more sensitive than automated direct sequencing for the detection of pten mutations in a mixture of dna extracted from normal and tumor cells epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast taqman pcr assay the authors would like to thank mr satoru michiyuki, employee of eiken chemical co., ltd., otawara, japan, for his valuable comments and suggestions concerning the lamp sh analyzed the data and wrote the initial draft of the manuscript. ys designed the study, analyzed and interpreted the data and assisted in preparing the manuscript. am performed all experiments and analyzed all results in the study. ys made pathological diagnosis in all cases and provided all tissue samples. nt, ti and eh contributed to data collection and interpretation. my performed some of the experiments, and critically reviewed the manuscript, and organized research group in this study. besides am, all authors approved the final version of the manuscript and they agree to be accountable for all aspects of the work. the present study was approved by the institutional review board of the saitama cardiovascular and respiratory center (approval no. ). written informed consent was provided by all patients. not applicable. am is an employee of eiken chemical co., ltd, and was the only author who conducted all the experiments. eiken chemical co., ltd, provided the research grant for the present study, and provided the lamp assay which is not currently commercially available. eiken chemical co., ltd., had no control over the interpretation, writing, or publication of the present study. key: cord- -wmmbkmrg authors: wang, de-guo; brewster, jeffrey d.; paul, moushumi; tomasula, peggy m. title: two methods for increased specificity and sensitivity in loop-mediated isothermal amplification date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: wmmbkmrg the technique of loop-mediated isothermal amplification (lamp) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional pcr methods. the high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. in this study, a set of lamp primers were designed targeting the prfa gene sequence of listeria monocytogenes, and dimethyl sulfoxide (dmso) as well as touchdown lamp were employed to increase the sensitivity and specificity of the lamp reactions. the results indicate that the detection limit of this novel lamp assay with the newly designed primers and additives was fg per reaction, which is ten-fold more sensitive than a commercial isothermal amplification kit and hundred-fold more sensitive than previously reported lamp assays. this highly sensitive lamp assay has been shown to detect strains of listeria monocytogenes, and does not detect other listeria species (including listeria innocua and listeria invanovii), providing some advantages in specificity over commercial isothermal amplification kits and previously reported lamp assay. loop-mediated isothermal amplification, developed and reported by notomi et al., in [ ] , can specifically, sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing independent sequences of a target gene under isothermal conditions. moreover, nagamine et al., has advanced the method by putting forward loop primers that accelerate the lamp reaction [ ] . therefore, the lamp assay theoretically has the advantage of specificity, selectivity, and rapidity over polymerase chain reaction (pcr) [ , ] , nucleic acid sequence based amplification (nasba) [ , ] , strand displacement amplification (sda) [ ] , rolling circle amplification (rca) [ ] , helicase dependent amplification (hda) [ ] , and cross-priming amplification assay (cpa) [ , ] . for practical application of lamp as well as reduction of the rate of false positive results in lamp reactions, most researches currently focus on development of closed-tube detection to reduce aerosol pollution and cross pollution, which include the use of turbidity [ ] , sybr green i [ ] , picogreen [ ] , gelred tm [ , ] , lateral flow dipstick [ ] , hydroxynaphthol blue dye [ ] , malachite green [ ] , microfluidic chips and gmr sensors as well as calcein used by eiken chemical co., ltd [ , ] . however, there is still no report studying non-specific amplification and cause of false positive results in lamp reactions at present. the objective of this paper is to study the cause and limit the rate of false positive results in lamp reactions targeting l. monocytogenes as well as to increase the specificity and sensitivity of these lamp reactions using dmso and touchdown lamp. although there were only two primers, non-specific amplification occurred in the isothermal amplification of four primer combinations out of seven combinations, and it was more obvious in the reaction of three primer combinations (hlya-fip + hlya-lf, hlya-fip + hlya-lb, hlya-fip + hlya-b ), as table shown. analysis on the three combinations indicated that they had the common situation that - bases at ' end of both primers had two complementary sequences on a same primer, and such situation had been avoided when lamp primers for prfa of l. monocytogenes were designed and screened in this study. moreover, it was proved by the experiment that non-specific amplification caused by primer dimers was one reason for false positive results of lamp. the lamp reaction mixtures containing varying concentrations of dmso were heated at °c for min ( s per cycle), as indicated in figure . when % dmso was added, the detection time for pg l. monocytogenes genomic dna was less than min; however, one of the two negative controls amplified as well. the tm value and melt curve were obviously different from those of the positive controls, and were therefore as attributed to non-specific amplification, which may be caused by partial complementation among primers of lamp. the detection time with % dmso was slightly longer than with . %. overall, the results showed that the lower concentration of dmso does not inhibit non-specific amplification while the higher concentration of dmso may inhibit the activity of bst . warmstart dna polymerase, and therefore, . % dmso was determined to be the optimal among these three concentrations. . % dmso was added to lamp reaction mixtures and the reactions were carried out at varying temperatures for min, as shown in table . with a reaction temperature of °c, only one of two positive controls ( pg l. monocytogenes genomic dna) was detected. the threshold time obtained using a reaction temperature of °c was shorter than that obtained using °c reaction temperature and slightly shorter than that obtained using the other temperatures. therefore, °c was chosen as the most suitable reaction temperature. the optimized lamp reaction conditions were used with the conventional lamp methodology with a serial dilution of l. monocytogenes dna template was and these mixtures were heated at °c for min. as shown in table , the detection limit of the optimized reaction mixture using the conventional lamp technique was found to be fg l. monocytogenes dna template. the optimized lamp mixture was used with the touchdown methodology to detect a serial dilution of l. monocytogenes dna template. after the mixtures were preheated at °c for min and bst . warmstart dna polymerase (new england biolabs, beverly, ma, usa) were added, they were heated at °c for min, at °c for min, at °c for min and then at °c for min, and, as indicated in table , the sensitivity of touchdown lamp was found to be fg of l. monocytogenes dna. therefore comparing these identical reaction mixtures in the conventional lamp assay and the touchdown lamp assay shows that the touchdown lamp method increases the overall sensitivity of lamp assay. the detection limit of the original reported lamp method by tang, et al (tang method) tested using fg l. monocytogenes dna template, as well. only one of positives controls amplified using these conditions. moreover, one of four negative controls showed non-specific amplification, as reported in table . sensitivity of both the isothermal master mix using our own designed lamp primers as well as the loopamp ® listeria monocytogenes detection kit to detect l. monocytogenes was tested. the results indicate that the detection limit of both commercial lamp kits is fg l. monocytogenes dna template per reaction, as shown in table . therefore, the sensitivity of the newly developed lamp assay presented here was ten-fold higher than that obtained using the commercial isothermal amplification kits and hundred-fold higher than the originally reported tang lamp assay. this newly developed lamp assay was tested with listeria monocytogenes strains ( stereotypes) and as shown in table , all were successfully detected. the assay was also tested with five other listeria species. in the initial experiment, listeria invanovii atcc was falsely detected and one of three reactions amplified, while the other species had negative results. the experiment with l. invanovii atcc was repeated four times and all four repeated reactions were negative. therefore, the initial false positive result may have been caused by slight aerosol pollution of dna templates. [ ] ; however, the optimized lamp assay required an extended amplification time of min, and even with the lengthy reaction time, two strains (listeria monocytogenes j - (stereotype: / b) and listeria monocytogenes atcc (stereotype: b) were not detected, as shown in table . extending the amplification time further to h, led to non-specific amplification of negative controls. the two commercial lamp kits were able to distinguish listeria monocytogenes from other listeria species. there were, however, two negative controls that exhibited non-specific amplification, and, sometimes, l. monocytogenes j - (stereotype: a) was not detected, as shown in table . therefore, the newly developed lamp assay presented here can detect stereotypes of listeria monocytogenes selectively while not detecting other listeria species (including listeria innocua and listeria invanovii), and had some advantages over commercial isothermal amplification kit and the original tang lamp assay in specificity. the lamp primers targeting specific gene hlya of listeria monocytogenes reported by tang, et al., are used for studying non-specific amplification of lamp [ ] , as shown in table . targeting the specific gene prfa (genbank locus: ay . ) of l. monocytogenes, a set of lamp primers were designed and selected with primerexplorer and oligo according to the reported methodology [ ] , and are listed in table . the isothermal amplification was performed in a total μl reaction mixture containing . [ , ] . %, . % and % dmso were added into different reaction tubes. lamp was carried out at °c for min and a melt curve was obtained using a stepone tm system. lamp was performed as above in a μl reaction mixture containing pg l. monocytogenes dna template as well as the optimized concentration dmso at , , , and °c for min, and a melt curve was obtained using a stepone tm system. the optimized lamp mixture was combined with serial dilutions of dna template of listeria monocytogenes ranging from to fg, and the reaction mixtures were heated at selected temperature °c for min in stepone tm system, and the detection limit of conventional lamp was determined. in the case of touchdown lamp, the reaction mixture was preheated at °c for min. after min, bst . warmstart dna polymerase (large fragment) was added and the reaction mixture was heated at temperatures °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min and then at selected temperature for min, and the sensitivity of touchdown lamp was determined and compared with that of conventional lamp. for comparison, the detection limit of the reported method by tang, et al., (tang lamp assay) was determined by carrying out lamp reactions according to the conditions specified in their publication [ ] . isothermal master mix and loopamp ® listeria monocytogenes detection kits were purchased from optigene limited (west sussex, uk) and eiken chemical co., ltd (tochigi, japan), respectively, and lamp was carried out according to the manufacturers' instructions using a set of serially diluted dna template of l. monocytogenes ranging from to fg. eleven strains of l. monocytogenes (different stereotype) and other listeria species (including l. innocua and l. invanovii) were used for the specificity study (table ) . listeria strains were cultured overnight at °c in difco tm buffered listeria enrichment broth base (becton, dickinson and company, san jose, ca, usa) and the others in luria-bertani (lb) broth. dna from these pure cultures was extracted according to the manufacturer's handbook of dneasy ® blood and tissue kit (qiagen ltd, north manchester, uk), and these dna templates was used for determining the specificity of the optimized touchdown lamp assay, the tang lamp assay and the lamp assay utilizing the commercial isothermal amplification kit. the amount of dna template used is pg per reaction. it is difficult to avoid primer dimers and non-specific amplification when couple numerous sets of primers are used in lamp assays. this is especially true when the concentrations of primers, mg + , dntps and dna polymerase in reaction mixtures are as high as those used in real-time pcr. the concentrations of these factors must be strictly controlled to avoid non-specific amplification in real-time pcr [ ] as well as lamp reactions. there are instances in which standard pcr amplification reaction conditions do not produce acceptable results. addition of dmso and use of touchdown temperature conditions have been used improve pcr results. we investigate these approaches for the first time for optimization of lamp reactions. unfortunately, with the information presently available it is not possible to predict which enhancing agent is best for any particular target. but dmso has been frequently used in this capacity [ ] . the results presented here using dmso in lamp reaction mixtures indicate that, dmso can increase amplification with lamp at low concentration and can inhibit activity of bst . warmstart dna polymerase. we had tried to enhance the reaction of lamp with betaine, tetramethylammonium chloride, tetramethylene sulfoxide, and formamide, but their effect was not as good as that of dmso, because of the limitation of length, no more tautology here. while dmso may not necessarily be the best enhancing agent [ , ] , i.e., the manufacturer of the commercial isothermal amplification kit used in this experiment may have found some favorable enhancing agent, but their reagents are proprietary, and dmso served to decrease non-specific amplification in the specific experiments presented here. touchdown pcr offered a simple and rapid means to optimize pcrs, increasing specificity, sensitivity and yield, without the need for lengthy reaction times and/or the redesigning of primers [ , ] . touchdown lamp, compared to conventional lamp methods, results in increased sensitivity and yield of lamp. this improvement may be due to the high temperature inhibiting the formation of primer dimers and promoting the correct combination of primers and template. the biggest advantage of lamp is that the reaction can be performed isothermally, and people argue all the time that all we need is a simple water bath for the rapid detection, so the advantage may be compromised by the developed touchdown lamp assay, we made such efforts here just to reveal or verify the main cause for false positive results of lamp and inspire people to modify and improve lamp technology, my colleagues and i have also been looking for a more suitable method, which can not only keep the advantage but also improve the sensitivity and specificity of lamp. in summary, non-specific amplification was a limiting factor in the applicability of the lamp methodology. a few different options to eliminate this issue have been reported here to successfully selectively and sensitively detect l. monocytogenes. designing ideal primers, additives such as dmso, and method modifications such as touchdown lamp may be favorable alternatives for increased specificity and sensitivity in lamp in other applications as well. loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia primer-directed enzymatic amplification of dna with a thermostable dna polymerase nucleic acid sequence-based amplification self-sustained sequence replication ( sr): an isothermal transcription-based amplification system alternative to pcr strand displacement amplification-an isothermal, in vitro dna amplification technique mutation detection and single-molecule counting using isothermal rolling-circle amplification helicase-dependent isothermal dna amplification cross-priming amplification for rapid detection of mycobacterium tuberculosis in sputum specimens the development and evaluation of cross-priming amplification for the detection of avian reovirus optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of macrobrachium rosenbergii noda virus and extra small virus in macrobrachium rosenbergii rapid identification of virus-carrying mosquitoes using reverse transcription-loop-mediated isothermal amplification development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus loop-mediated isothermal amplification for the detection of goose circovirus a loop-mediated isothermal amplification (lamp) method for the identification of species within the echinococcus granulosus complex the development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of vibrio parahaemolyticus visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for leishmania infection a novel hbv genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and gmr sensors a new surveillance and response tool: risk map of infected oncomelania hupensis detected by loop-mediated isothermal amplification (lamp) from pooled samples rapid and sensitive detection of listeria monocytogenes by loop-mediated isothermal amplification simple and rapid method for detecting foodborne shigella by a loop-mediated isothermal amplification kinetic pcr analysis: real-time monitoring of dna amplification reactions betaine and dmso: enhancing agents for pcr a specificity enhancer for polymerase chain reaction. nucl. acid res improvement of pcr amplified dna sequencing with the aid of detergents touchdown pcr for increased specificity and sensitivity in pcr amplification touchdown" pcr to circumvent spurious priming during gene amplification this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to acknowledge the china scholarship council. this work was supported by natural science foundation of china ( ), nsfc-henan talent the authors declare no conflict of interest. key: cord- -lrkscs authors: kurosaki, yohei; martins, danyelly bruneska gondim; kimura, mayuko; catena, andriu dos santos; borba, maria amélia carlos souto maior; mattos, sandra da silva; abe, haruka; yoshikawa, rokusuke; de lima filho, josé luiz; yasuda, jiro title: development and evaluation of a rapid molecular diagnostic test for zika virus infection by reverse transcription loop-mediated isothermal amplification date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: lrkscs the recent outbreak of zika virus (zikv) disease caused an enormous number of infections in central and south america, and the unusual increase in the number of infants born with microcephaly associated with zikv infection aroused global concern. here, we developed a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay using a portable device for the detection of zikv. the assay specifically detected zikv strains of both asian and african genotypes without cross-reactivity with other arboviruses, including dengue and chikungunya viruses. the assay detected viral rna at . tcid( )/ml in virus-spiked serum or urine samples within min, although it was slightly less sensitive than reference real time rt-pcr assay. we then evaluated the utility of this assay as a molecular diagnostic test using plasma or serum samples and urine samples collected from suspected cases of arbovirus infection in the states of paraíba and pernambuco, brazil in . the results of this assay were consistent with those of the reference rt-pcr test. this portable rt-lamp assay was highly specific for zikv, and enable rapid diagnosis of the virus infection. our results provide new insights into zikv molecular diagnostics and may improve preparedness for future outbreaks. zikv is a positive-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. zikv shares its vector, the aedes mosquito, with other flaviviruses, including denv, yellow fever virus (yfv), and chikv . zikv has been isolated from humans in east and west africa and in southeast asia and polynesian countries where the host mosquitoes, a. aegypti and a. albopictus, are found , . based on phylogenetic analyses, these isolates can be categorised into two genotypes, african and asian. epidemiological studies have revealed that the recent outbreak of zikv in brazil occurred via the introduction of a virus from french polynesia, where an outbreak of the disease occurred in . all of the viruses isolated in brazil and other countries on the american continent belong to the asian genotype . in patients with zikv infection, the virus can be detected in several sample types, including blood, urine, saliva, and other body fluids [ ] [ ] [ ] [ ] [ ] . the viral load in blood reaches a peak at to days after the onset of illness, but decreases rapidly thereafter. therefore, it is difficult to detect zikv in blood samples from patients after the acute phase of infection, even with sensitive molecular diagnostic methods, such as reverse transcription-polymerase chain reaction (rt-pcr) , , . the virus can be detected in urine samples for longer durations (> - days after the onset of symptoms) than in those for blood samples . currently, blood and urine samples are typically used for the molecular diagnosis of zikv. zikv infection is diagnosed in the laboratory by nucleic acid amplification tests (naats) to detect viral rna [ ] [ ] [ ] [ ] [ ] or by elisa to detect igm or igg antibodies , . the naats such as rt-pcr and other technologies (e.g. recombinase polymerase amplification) are highly accurate, and rt-pcr is considered the gold standard to confirm zikv infection , . rt-pcr, however, requires a step for viral rna extraction prior to the assay and the use of expensive equipment, such as thermal cycler, to conduct the test. moreover, there is a risk of reduced sample quality due to rna degradation during transportation to the laboratory. for elisa, serological cross-reaction between zikv and other circulating flaviviruses like denv makes accurate diagnosis with serology difficult , . therefore, novel diagnostic technologies that can be conducted at the point-of-care or in regional laboratories are greatly needed to control zikv infections. reverse transcription loop-mediated isothermal amplification (rt-lamp) is a rapid, sensitive rna detection method performed under isothermal conditions using four or six unique oligonucleotide primers , . since lamp reactions can be performed with simple inexpensive equipment, rt-lamp assays can be conducted in the field and by under-funded laboratories . we previously developed a rt-lamp assay using a portable isothermal amplification and detection device for ebola virus in response to the recent outbreak of ebola virus disease in west africa, and the assay has been deployed for field surveillance in guinea , . here, we developed a rt-lamp assay for the detection of zikv with a portable battery-powered device. then, we evaluated the utility of this assay for molecular diagnosis using clinical specimens collected from the recent zikv outbreak in brazil. sensitivity. we designed zikv genotype-specific lamp primers that targeted conserved sequences in the e protein-coding region (table ) . each genotype-specific primer recognised the same genomic position. to detect all known zikv strains, we used a mixture of primers specific for each genotype in a single reaction. first, we examined the sensitivity of the assay using serial -fold dilutions of in vitro synthesised standard rnas from strain uganda, which was isolated from a rhesus macaque in uganda, and strain prvabc , which was isolated at the centres for disease control and prevention (cdc) from a patient who travelled to puerto rico in . ten copies of the rna standards were detected from both strains in quadruplicate reactions (fig. a) . the times to obtain positive results (tp) for rna standards ranging from to copies were mostly less than min, and within this range, tp was correlated with the number of rna copies ( fig. b and c) . single copies of the standard rnas from the uganda and prvabc strains were detected with % and % positivity, respectively, and tp values were dispersed. these results suggested that the rt-lamp assay could be used as a rapid, sensitive diagnostic test for zikv, the tp value (i.e., less than min) can be used as an indicator of the number of rna copies in each reaction. we evaluated the specificity of each primer in silico using zikv strain sequences available in genbank as of november . twenty-seven sequences of african genotype isolates collected in - and sequences of asian genotype isolates collected in southeast asia and polynesia in - as well as from the current outbreak in the americas were used for this analysis. the lamp primers consist of nucleotides in total length and recognise eight separate sites on zikv genome (fig. a) . we determined the proportion of sequences that had identical nucleotides at each position for either the asian or african genotype primers (fig. b) . african genotype zikv sequences had identical residues at out of positions ( . %) in the african or asian genotype-specific primers. at positions in the primer recognition sites, more than % of african genotype zikv sequences had mismatched nucleotides. at six positions, scattered in the f , f , lf, and b sites in the asian and african genotype primers, more than % of the african genotype zikv sequences had nucleotide differences (fig. b, upper panel) . asian genotype sequences showed greater identity than african genotype sequences to the lamp primers. asian genotype sequences had identical residues at out of positions ( . %) in the asian or african genotype primers. for one residue at the ′ terminus of the f site of the fip primers, . % of the asian genotype sequences had nucleotide differences (fig. b, lower panel) . to assess the primer specificity for zikv strains, we synthesised rnas with the partial genome sequences of two african genotype zikv strains, -dak and ard , and two asian genotype zikv strains, p - and cpc- , which had more mismatched nucleotides against the primer sequences compared with the average for all strains. these rna sequences were also detected using the rt-lamp assay, in addition to the sequences in the uganda and prvabc strains (table ) . furthermore, no cross-reactions with other tested arboviruses, including denv, yfv, west nile virus (wnv), chikv, and rift valley fever virus (rvfv), and plasmodium falciparum were observed. these results suggested that the rt-lamp assay developed here was highly specific for detecting zikv strains of both african and asian genotypes. and/or urine is used, since viral rna can be detected in these clinical specimens during the acute phase of infection. the feasibility of using the rt-lamp assay for clinical specimens was evaluated using zikv-spiked human serum and urine samples. we prepared human serum and urine spiked with four-fold serially diluted zikv strain uganda, and obtained samples with titres of . - . tcid /ml. the sensitivity of the rt-lamp assay was compared to that of the real time rt-pcr (rrt-pcr) assay developed by the cdc . using the rt-lamp assay, we detected viral rna in both serum and urine samples at a titre of . tcid /ml in quadruplicate reactions. the ct values in the rrt-pcr were . ± . and . ± . for serum and urine samples, respectively, which corresponded to . and . genome equivalents (geq) per reaction, respectively (table ). using the rrt-pcr assay, we detected viruses in both serum and urine samples at a titre of . tcid /ml, which corresponded to . and . geq per reaction, respectively; however, the rt-lamp assay failed for these samples, suggesting that the rt-lamp assay was less sensitive than the cdc rrt-pcr assay for zikv detection. together with the results table ). the rt-lamp assay did not show any false-positive results, even for six confirmed denv samples (data not shown). the ct values of these eight zikv-positive samples were . - . , and the viral loads were estimated to be . × - . × geq/ml using the viral rna standards (table ) . these viral titres were higher than those reported in previous studies. to examine whether the assay can detect viral rna in samples with lower titres, we randomly selected two zikv-positive samples confirmed in this study, mrl and mrl , and conducted a dilution test (table ). while the rt-lamp failed to detect samples with the ct value > , however, it detected viral rna at the ct < , consistent with our earlier results obtained using the virus-spiked serum and urine samples (table ). these results show that the rrt-lamp assay had sufficient specificity for the detection of zikv as a molecular diagnostic test. the assay can be used to detect an amount of viral rna equivalent to that yielding ct values of - in the reference rrt-pcr test. we developed a rapid molecular detection assay for zikv in response to the recent outbreak in south america. lamp assays and modified diagnostic methods for zikv have been reported; however, these molecular techniques have never been evaluated for clinical use [ ] [ ] [ ] [ ] . this is the first evaluation of the clinical usage of a lamp assay for molecular diagnostic testing in the recent outbreak of zikv infections. since zikv shares a vector with denv and chikv, these viral diseases can occur simultaneously, and northeast brazil is an endemic area for dengue and chikungunya . numerous severe mosquito-borne diseases, including arbovirus infections as well as malaria, share clinical symptoms during the acute phase. however, zikv infection is generally associated with mild symptoms. a major concern with respect to molecular diagnostic testing for zikv is the potential for cross-reactivity with other flaviviruses, especially dnev, which have close antigenic relation with zikv , , , . in contrast, our assay showed no cross-reactions with other arboviruses or p. falciparum, and did not show false-positive results when applied to zikv-negative samples. these results indicated that the rt-lamp assay is specific for the detection of zikv and is a reliable molecular diagnostic test. another potential limitation of molecular diagnostic testing is that zikv-infected samples often have low titres after the acute or early phase of infection due to rapid clearance by the host immune system. this makes it difficult to identify zikv cases, even using rt-pcr-based tests. the limit of detection for this assay was copies table . detection of zikv by rt-lamp and rrt-pcr using diluted zikv-confirmed samples. . for both genotypes. the assay was slightly less sensitive than the cdc rrt-pcr test, which was commonly used to confirm zikv infection during the recent outbreak. zikv-infected clinical samples often show high ct values (> ) , . however, the zikv-positive samples detected in this evaluation showed ct values of less than . (more than . × geq/ml), which was a higher titre than that reported in other studies. to confirm its clinical utility, this assay should be tested using samples with lower titres or borderline zikv infections. it has been reported that viral rna can be detected for longer periods in urine than in blood , . therefore, we considered urine to be one of the best sample types for detecting zikv infections. recently, paz-bailey et al. reported contradictory results for the persistence of viral rna in blood samples of zikv patients; rna can be detected or weeks after the onset of illness . in some cases, viral rna can also be detected at higher titres in saliva than in blood, but persists for shorter periods , . it is necessary to determine the sample types suitable for the rt-lamp assay and to establish a standardised rna extraction protocol adjusted to each clinical specimen type in order to improve the sensitivity of this assay. owing to the sequence diversity among zikv isolates, we designed lamp primers specific for each genotype and used a mixture of these primers to detect all known isolates of both african and asian genotypes. as shown in fig. , we conducted an in silico evaluation of each primer using available zikv sequences. african genotype strains supposedly have a longer history of circulation in african mosquitos and humans than that of asian genotype strains , and african genotype sequences showed a lower identity at some positions in the lamp primers. the lamp primers designed here showed high identities at most positions against the sequences of strains involved in the recent outbreak on the american continent, as well as its ancestral southeast asian and polynesian isolates. during the outbreak of zikv in americas, confirmed or probable zikv-infected cases has been continuously reported in southeast asia . our assay will be useful for virus detection and may contribute to preparedness for future outbreaks in these zikv endemic countries as well as in asia and africa. however, the evolution of zikv sequences must be constantly monitored to guarantee primer specificity. using samples obtained from subjects with suspected arbovirus infection, we did not find any zikv-positive samples in paraíba in march or july by rrt-pcr or our rt-lamp test. these samples were collected from patients within or weeks after the onset of arbovirus infection-like symptoms as part of an education and follow-up campaign for cardiovascular diseases. many samples might have been collected after the acute or early stage of infection. in addition, when this campaign was conducted, the prevalence of zikv infection may have been low, since most cases were reported from november to march , which is closely linked to the ecology of the vector aedes mosquito. the main advantages of this assay are its speed (positive results can be obtained within min) and the use of a battery-operated portable device. since the device has a user-friendly interface, training is not necessary to conduct the assay and interpret the results. recently, freeze-dried reagents for lamp assays have been made available, making cold-chain-free lamp assays a possibility. our assay is suitable for use in field surveillance or remote areas where it is difficult to implement laboratory diagnostic tests. the assay should be evaluated in a prospective study to confirm its utility for molecular diagnostic testing, especially under limited resources and by field laboratories in zikv endemic countries. in this paper, we successfully developed a rt-lamp assay for the detection of zikv by designing asian and african genotype-specific primers. the assay showed results consistent with those of the reference rrt-pcr assay in diagnostic tests with suspected cases of zikv infection. our results provide a potential new molecular diagnostic test for zikv and may serve as a basis for the development of alternative rapid diagnostic techniques to prepare for potential outbreaks. and were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % penicillin/streptomycin and % foetal bovine serum (fbs). zikv strain uganda was kindly provided by dr. shigeru tajima (national institute of infectious diseases; niid). the virus was propagated in vero cells grown in dmem supplemented with % fbs. two days after infection, culture supernatants were harvested, clarified by low-speed centrifugation, and then stored as virus stock at − °c until use. the infectious titre of the virus stock was determined by the % tissue culture infective dose (tcid ) using vero cells; titres are expressed as tcid / ml. viral rna was extracted from μl of infected culture supernatant using the qiaamp viral rna mini kit according to the manufacturer's protocol. the rna was eluted in μl of elution buffer and stored at − °c until use. viral rna from zikv strain prabc was kindly provided by dr. shigeru tajima (niid). viral rnas from other arboviruses, including denv serotype - , yfv, wnv, chikv, and rvfv, as well as genomic dna from p.falciparum strain d were kindly provided by dr. kouichi morita and dr. osamu kaneko (institute of tropical medicine, nagasaki university). preparation of rna standards. rna standards, consisting of partial genome sequences of zikv strains uganda and prvab c , were amplified by rt-pcr using for ward ( ′-ggagtcaggatggtacttgtacc- ′) and reverse ( ′-aaaattggatattcaggaacc- ′) primers with the primescriptii high fidelity one step rt-pcr kit (takara bio, shiga, japan). the reactions were performed using the takara pcr thermal cycler dice with the following program: °c for min, °c for min, followed by cycles of °c for s, °c for s, and °c for s. amplified pcr fragments were cloned into the pcr . vector using the topo-ta-cloning kit (invitrogen, carlsbad, ca, usa). the plasmids were digested with bamhi, purified from the agarose gel slice using a column purification kit (qiagen, hilden, germany), and used as templates for rna synthesis. the partial genomic rnas of each zikv strain were synthesised in vitro using t rna polymerase (promega, madison, wi, usa) and purified using the rneasy mini kit (qiagen). the rna concentration was determined by measuring the optical density at nm (od ) with scientific reports | : | doi: . /s - - - a nanodrop (thermo fisher scientific, waltham, ma, usa), and the rnas were diluted in depc-treated water to achieve the desired concentrations. primer design. lamp primers for zikv detection were designed based on the coding sequences for the e protein. the zikv sequences available in genbank were aligned using clustalx to identify conserved regions. a consensus sequence for a region in the e gene was used to design lamp primers using lamp designer (optigene; http://www.optigene.co.uk/lamp-designer/). primers specific for asian genotype viruses were designed first, and then african genotype-specific primers were designed by adapting each position to the african genotype consensus sequence. the rt-lamp assay required a set of six primers, two outer primers (f and b ), a forward inner primer (fip), a reverse inner primer (bip), a forward loop primer (lf), and a reverse loop primer (lb). the fip consisted of the f c sequence, which was complementary to the f and f sequences. the bip consisted of the b c sequence, which was complementary to the b and b sequences . the lb primer was designed to detect both asian and african genotype sequences. the sequences and locations of the oligonucleotide primers are shown in table . rt-lamp was performed with isothermal master mix reagent (optigene, west sussex, uk) using the genelyzer fiii real-time fluorescence detection platform (toshiba medical systems, otawara, japan). the reaction mixture (total volume, µl) contained µl of isothermal master mix; µl of warmstart rtx reverse transcriptase ( u; new england biolabs, ipswich, ma, usa); µl of the lamp primer mix consisting of pmol f and b , pmol fip and bip, pmol lf and lb; and µl of rna sample (template). the assay was carried out using a mixture of primers specific for the asian and african genotypes. all primers were cartridge-purified oligonucleotides purchased from hokkaido system science (sapporo, japan). the reaction was performed at °c for min, followed by a dissociation analysis at °c- °c. depc-treated distilled water and rna synthesised from uganda or prvabc were used for the negative and positive controls, respectively. nonspecific amplification was excluded by comparing the melting temperature to that of the positive control . real time rt-pcr. real time rt-pcr for zikv was performed using the quantitect probe rt-pcr kit (qiagen) as reported previously . the reaction mixture (total volume, µl) contained . µl of × quantitect probe rt-pcr master mix, . µl of quantitect rt mix, pmol each of primers and c, and pmol fam-labelled probe for zikv. then, aliquots of the rna samples ( µl) were added to the -µl reaction mixtures. each reaction was performed using the real-time pcr system (applied biosystems, tokyo, japan) with a thermal cycle profile consisting of °c for min, °c for min, followed by cycles of °c for s and °c for min. cut-off values were set at ct . . to quantify viral rna, a standard curve, generated with -fold serial dilutions of synthesised standard rna from uganda or prvabc , was used. table except zikv was quantified by droplet digital pcr (ddpcr). the complementary dna (cdna) of each arbovirus rna was synthesised from an extracted rna stock using the superscript iii first-strand synthesis system (invitrogen) with forward primer for rvfv and reverse primers for denv, wnv, yfv, and chikv, respectively (supplementary table ). the primers used for ddpcr were designed using primer (supplementary table ). all -μl ddpcr mixtures contained × evagreen ddpcr supermix (bio-rad, hercules, ca, usa), . μm forward and reverse primers, and μl of cdna. each oil compartment of the droplet generator dg cartridge (bio-rad) was filled with μl of droplet generation oil for evagreen (bio-rad), and approximately , droplets were generated in each well by the qx droplet generator (bio-rad). the reactions were performed in a -μl droplet emulsion using a geneamp pcr system (applied biosystems) under the following thermal cycling conditions: °c for min, followed by cycles of °c for s and °c for min, with a final step at °c for min. controls without the template were used to monitor for signals from contamination or primer-dimer formation. the cycled droplets were read individually using the qx droplet reader (bio-rad) and analysed with quantasoft droplet reader software (bio-rad). clinical specimens. peripheral blood and urine samples were obtained from patients between and year old with suspected arbovirus infection, who presented with fever, rash, and/or arthralgia symptoms. venous whole blood samples were collected in one vacuette ® z serum separator clot activator and two vacuette ® edta tubes (greiner bio-one, kremsmünster, austria). to one edta tube, rnalater (thermo fisher scientific) was added at half the volume of the collected blood samples to prevent rna degradation during transport. in total, plasma/serum and urine samples from patients with suspected arbovirus infection, including paired samples from cases, were used in this study. the separated plasma or serum samples and urine samples were stored at − °c until use. rnas were extracted from sera and urine using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. rna samples were eluted with µl of elution buffer and stored at − °c until use. ethical declaration. this study was approved by the ccs-ufpe ethical committee (caae: . . . ) and all patients gave informed consent. whole blood and urine samples were collected as part of an education and follow-up campaign for arboviruses and cardiovascular diseases conducted by lika in the states of paraíba and pernambuco, brazil in february-july . all experiments were 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based on loop-mediated isothermal amplification and ac susceptometry instrument-free point-of-care molecular detection of zika virus simple and highly sensitive molecular diagnosis of zika virus by lateral flow assays investigation into an outbreak of dengue-like illness in pernambuco, brazil, revealed a cocirculation of zika, chikungunya, and dengue virus type zika virus infections imported to italy: clinical, immunological and virological findings, and public health implications zika virus in asia pan american health organization and world health organization. zika -epidemiological update the authors would like to thank sayaka okada, shota koyano and olamide k. oloniniyi for technical assistance with the experiments at nagasaki university, renato p. melo neto and carlos henrique m. castelletti for bioinformatics support at lika, and all members of the staff for their hospitality during the visit when the main results of this paper were obtained. supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -dtvzwin authors: jeong, joojin; cho, sang-yun; lee, wang-hyu; lee, kui-jae; ju, ho-jong title: development of a rapid detection method for potato virus x by reverse transcription loop-mediated isothermal amplification date: - - journal: plant pathol j doi: . /ppj.oa. . . sha: doc_id: cord_uid: dtvzwin the primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. reverse transcription loop-mediated isothermal amplification (rt-lamp) has been used to detect viral rna molecules because of its simplicity and high sensitivity for a number of viruses. rt-lamp for the detection of potato virus x (pvx) was developed and compared with conventional reverse transcription polymerase chain reaction (rt-pcr) to demonstrate its advantages over rt-pcr. rt-lamp reactions were conducted with or without a set of loop primers since one out of six primers showed pvx specificity. based on real-time monitoring, rt-lamp detected pvx around min, compared to min for rt-pcr. by adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. rt-lamp was conducted using simple inexpensive instruments and a regular incubator to evaluate whether rna could be amplified at a constant temperature instead of using an expensive thermal cycler. this study shows the potential of rt-lamp for the diagnosis of viral diseases and pvx epidemiology because of its simplicity and rapidness compared to rt-pcr. lamp will be useful not only for the detection of infected plants but also quarantine. the reactions are easily monitored by detecting the turbidity caused by the production of a large amount of target dna (webster et al., ) . the reaction time for rt-lamp is less than min and this time can be further reduced by adding two more loop primers (ju, ; nagamine et al., ) . in addition, if a fluorescent dye like sybr green is present in the reaction, pcr products in the reaction tubes can be seen with the naked eye under a uv lamp (cardoso et al., ) . viruses infecting potatoes including plrv and pvy have been detected by rt-lamp (ju, ; nie, ) . the rt-lamp method has not been reported for the diagnosis of pvx. the purpose of this study was to develop a system to diagnose pvx by rt-lamp based on its unique nucleotide sequences encoding a coat protein. the specificity, sensitivity, and rapidity of rt-lamp were also assessed to optimize the detection of pvx. preparation of pvx-infected plant material. a pspvxp binary vector, containing pvx full-length cdna, was kindly provided by dr. kim from seoul national university, seoul korea (park and kim, ). an agro-infiltration method previously described by english et al. ( ) has been applied for pvx infection to nicotiana benthamiana. after two or three weeks, the treated-leaves were collected from healthy and diseased plants. total rna extraction. total rna was extracted from the leaves of pvx-infected or non-infected n. benthamiana using the easy-blue rna extraction kit (intron, republic of korea) as directed by the manufacturer's instructions. primer design. primer sets were designed from the coat protein (cp) gene of pvx (fig. ) . three primer sets were designed using primer explorer v (eiken chemical co. ltd., japan), and the other three primer sets were designed using lamp designer (premier biosoft, usa). all primers were purified by hplc. lamp. the rt-lamp reaction was conducted by incubating a total reaction mixture of μl at o c for min. a homemade mixture (table s ) and two commercial mixtures (loopamp rna amplification kit (eiken chemical co. ltd., japan) and isothermal master mix (imm kit) (optigene ltd., england)) were used. in order to optimize the rt-lamp mixtures, different concentrations of dntps and primers were tested as follows: dntps from . to . mm, fip and bip (inner primers) from to pmole, and f and b (outer primers) from to pmole. real-time monitoring. rt-lamp amplification was spectrophotometrically monitored by recording the optical density at nm with a real-time turbidimeter (eiken co. ltd., tokyo, japan). visualization under uv. rt-lamp amplification products were visualized by adding μl of fluorescent detection reagent (eiken co. ltd., tokyo, japan) to the reaction mixture before incubation. the products were examined under natural light and under uv light. using the maxime tm rt-pcr premix kit (in-tron biotechnology, korea), an one-step rt-pcr reaction was conducted. the mixture was incubated at o c for min and denatured at o c for min, followed by cycles of o c for sec, . o c for sec and o c for selection of specific primer sets over pvx. in order to examine the specificity of six primer sets, three replicates of the experiments were conducted. when rt-lamp assays were conducted using the rt-lamp mixture, primer sets a, c, and f showed ladder-like bands on the agarose gel only for the pvx rna containing samples (fig. s ). by repeating the experiments with primer sets a, c, and f, we confirmed true positive reactions for primer sets a and f. to evaluate the sensitivity of the two primer sets, a dilution series of pvx rna was used for rt-lamp. the detection limits of primer sets a and f were observed at . ng and . µg, respectively ( fig. a and b ). in addition, the detection limit of rt-pcr was . ng (fig. c ). in comparison, rt-lamp conducted with primer set a ( fig. a) showed higher sensitivity (about times) than conventional rt-pcr ( fig c) . finally, primer set a was selected and used for rt-lamp pcr. the rt-lamp products of rna from leaves infected by pvx showed ladder-like bands on the agarose gel, but not rom rt-lamp products of rnas from healthy plant leaves and plrv-infected plant leaves, indicating that primer set a is specific for pvx (fig. s ). to determine the optimal rt-lamp reaction, various parameters including ampli- fication temperature and the concentration of dntps and outer (f and b ) and inner (fip and bip) primers were tested. rt-lamp reactions were carried out using gradient pcr setting the temperature range from o c to o c. fig. a shows that all temperatures were capable of producing rt-lamp amplicons. a similar amount of product was also generated at temperatures ranging from to . o c. however, temperatures under o c or above o c showed decreased yield. fig. b shows that the concentration of dntps affected the amount of rt-lamp products. a minimum concentration of . mm dntps was needed to acquire a positive rt-lamp result. the greatest amount of product was for both . and . mm suggesting that the optimum concentration of dntps was either . or . mm. the concentrations of primers also affected the amount of rt-lamp products, but the effect of primer concentration depended on the types of primers (fig. c) . a greater amount of amplification products were generated from reactions at both and pmole (fig. c - ) of inner primers compared to pmole of inner primer. using the same inner primers (fip and bip), a similar amount of rt-lamp products was obtained regardless of the concentrations of outer primers (f and b ); thus, showing that the concentrations of fip and bip were more effective than those of f and b . effect of loop primers. rt-lamp reactions were performed using primer set a with or without loop primers to examine the effect of loop primers on shortening the reaction time according to the turbidity of reaction mixtures with or without loop primers. the results showed that the time required for the initiation of rt-lamp amplification was or min with or without loop primers, respec-tively (fig. ) . evidently, the use of loop primers accelerated the amplification of pvx, reducing the reaction time to almost half. the rt-lamp amplification products in the reaction tubes were visualized using fluorescent dyes under ambient light or uv light, which also allowed for determination of the detection limit of pvx rna. rt-lamp products showing positive results had a light green color under ambient light, whereas the negative control was light yellow-orange (fig. ) . under uv light, positive tubes emitted bright fluorescent light, but negative samples showed dim or no fluorescent light (lane and ). in addition, rt-lamp amplification products could be visualized when µg to . ng of pvx rna was used as the template, whereas no products were visualized when . ng of pvx rna was used (lane ), indicating that . ng of pvx rna is the detection limit in this study. plant diseases have been increasing due to globalization of trade, increased human mobility, global warming, pathogen's evolution, and improper crop management (anderson et al., ; garrett et al., ) . therefore, it is essential to diagnose disease agents for proper disease control strategies. specifically, accurate, rapid, and efficient diagnostic tools are crucial to control viral diseases because agrochemicals to cure plant viral diseases have not been commercialized. many techniques have been developed and used for the diagnosis of potato viral diseases including pvx (el-araby et al., ; khan et al., ) . elisa has been accepted as the most common and reliable detection method for viruses including pvx because it has long history of use and it is also rapid and inexpensive (clark and adams, ; makkouk and kumari, ) . in general, elisa methods have drawbacks such as long reaction time, less specificity caused by cross reactivity, fewer numbers of antibodies available, and less sensitivity (el-araby et al., ). since molecular-based assays are known to have higher sensitivity, rt-pcr may reduce shortcomings caused by less sensitivity. rt-pcr-based assays have also been shown to remedy additional defects of elisa (agindotan et al., ; el-araby et al., ). however, one of the limitations of rt-pcr is that the assay needs sophisticated and expensive instruments because it depends on thermal cycling for denaturation of double stranded dna into single stranded dna and enzymatic replication of the dna (saiki et al., ) . lamp pcr is relatively new and does not require thermal cycling because it is an assay based on isothermal amplification (notomi et al., ) . rt-lamp pcr, one of the variants of lamp pcr, has been used for the diagnosis of plant rna viruses (ju, ; nie, ) . the specificity of lamp is generally high because the rt-lamp pcr assay uses four primers that perceive six regions on the target gene. however, primer design seems to be an important limiting factor for the general use of rt-lamp pcr in pvx diagnosis, as previous studies reported that more nonspecific reactions among primers are often found in lamp pcr compared to conventional pcr (boubourakas et al., ; notomi et al., ; wei et al., ) . in order to determine the rt-lamp primer sets that could be used for detection of pvx, six sets of primers were designed. only one primer set (set a) was selected based on its specificity ( fig. s and s ) and sensitivity (fig. ) for highly successful detection of pvx. although the sensitivity for detecting classical swine fever virus (yin et al., ) is slightly lower for rt-pcr compared to rt-lamp, most rt-lamp assays are more sensitive than regular rt-pcr or nested pcr. the rt-lamp pcr for pvx that was developed in this study showed , -fold greater sensitivity than conventional rt-pcr assays, similar to most previous reports (fig. ) (ju, ; kuan et al., ; parida et al., ; venkatesan et al., ) . the optimum temperature for different rt-lamp assays might be different because of different primer sets, for example . and o c for peach latent mosaic viroid and to o c for squash leaf curl virus (boubourakas et al., ; kuan et al., ) . in fig. a , the optimum temperature of the rt-lamp to detect pvx seemed to range from to . o c. as shown in many studies, increasing or decreasing the reaction temperature beyond this tempera-ture range results in decreased yield (fukuda et al., ; wei et al., ) . this might be because of inactivation of enzymes or reaction instability caused by too high or low temperatures. different target genes may require different concentrations of dntps when those were amplified by lampbased methods. this study showed that amplification products were obtained using a concentration of dntps ranging from . to . mm, but no reaction products at . mm. the concentration of dntps required to detect roundup ready soybeans by lamp ranges from . to . mm (wang et al., ) . in general, most rt-pcr amplification takes a couple of hours, including min of an additional reverse transcription step. however, rt-lamp amplification takes less than min, even with the reverse transcription step (fukuda et al., ) . many studies have revealed a reaction time of less than min for rt-lamp (kuan et al., ; soliman and el-matbouli, ) . in addition, virus can be detected within min by real-time rt-lamp when two loop primers are applied (fukuta et al., ; ju, ; parida et al., ) . this study showed similar results in that the rt-lamp assay included two loop primers and took only min for detection of pvx. in conclusion, the rt-lamp assay developed to diagnose pvx in this study is rapid, cost-effective, specific, and sensitive for the detection of pvx. moreover, this assay can be extended many other applications for diagnostic purposes. diagnosis and control of cereal viruses in the middle east a sensitive and reliable rt-nested pcr assay for detection of citrus tristeza virus from naturally infected citrus plants simultaneous detection of potato viruses, plrv, pva, pvx and pvy from dormant potato tubers by taqman real-time rt-pcr emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnological drivers sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses biological, serological and molecular diagnosis of three major potato viruses in egypt requirement of sense transcription for homology-dependent virus resistance and trans-inactivation development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum climate change effects on plant disease: genomes and ecosystems immunocapture reverse transcription-polymerase chain reaction combined with nested pcr greatly increases the detection of prunus necrotic ring spot virus in the peach enhanced detection of prune dwarf virus in peach leaves by immunocapture-reverse transcription-polymerase chain reaction with nested polymerase chain reaction (ic-rt-pcr nested pcr) simple and rapid detection of potato leafroll virus (plrv) by reverse transcription loop-mediated isothermal amplification (rt-lamp) further studies on resistance -breaking strains of potato virus x detection of important plant viruses in in vitro regenerated potato plants by double antibody sandwich method of elisa rapid detection of squash leaf curl virus by loop-mediated isothermal amplification molecular diagnosis of plant viruses estimation of vector propensity for lettuce mosaic virus based on viral detection in single aphids a single tube, quantitative real-time rt-pcr assay that detects four potato viruses simultaneously accelerated reaction by loop-mediated isothermal amplification using loop primers reverse transcription loop-mediated isothermal amplification of dna for detection of potato virus y loop-mediated isothermal amplification of dna new device and method for capture, reverse transcription, and nested pcr in a single closed-tube rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay a new generation of innovative gene amplification technique perspectives in clinical diagnosis of infectious diseases agroinfiltration-based potato virus x replicons to dissect the requirements of viral infection primerdirected enzymatic amplification of dna with a thermostable dna polymerase reverse transcription loo-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of product development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples development of a rapid detection method for pvx by rt potato viruses in china detection of roundup ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick detection of mix-infected potato viruses with multiplex rt-pcr diagnosis of plant viral pathogens one-step detection of bean pod mottle virus in soybean seed by the reverse-transcription loop-mediated isothermal amplification this paper was supported by rural development administration (rda) fund pj , republic korea. key: cord- - nh gc authors: tian, fei; liu, chao; deng, jinqi; han, ziwei; zhang, lu; chen, qinghua; sun, jiashu title: a fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing date: - - journal: sci china chem doi: . /s - - - sha: doc_id: cord_uid: nh gc the outbreak of virus-induced infectious diseases poses a global public-health challenge. nucleic acid amplification testing (naat) enables early detection of pandemic viruses and plays a vital role in preventing onward transmission. however, the requirement of skilled operators, expensive instrumentation, and biosafety laboratories has hindered the use of naat for screening and diagnosis of suspected patients. here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing. the release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification (rt-lamp) were integrated into the reaction units of a microfluidic disc. the whole processing steps such as injection of reagents, fluid actuation by rotation, heating and temperature control, and detection of fluorescence signals were carried out automatically by a customized instrument. we validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus (sars-cov- ) armored rna particles. the estimated limit of detection for armored rna particles is copies per reaction, the throughput is reactions per disc, and the assay sample-to-answer time is approximately min. this enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol, and can be readily adapted for virus detection outside the diagnostic laboratory. electronic supplementary material: supplementary material is available for this article at . /s - - - and is accessible for authorized users. infectious diseases caused by pathogens are one of the leading causes of death in the world. according to the statistics provided by the world health organization (who), the number of deaths attributable to pathogen infections such as hepatitis, tuberculosis and malaria is . million worldwide in [ ] . the emergence and spread of pathogens such as middle east respiratory syndrome (mers) coronavirus, ebola virus, h n virus, zika virus, and severe acute respiratory syndrome coronavirus (sars-cov- ) severely affect global public health. early detection of these pathogens is of great importance in providing timely treatment and preventing onward transmission [ ] [ ] [ ] . especially in a virusinduced public health emergency, there is an urgent need to develop accurate, rapid, and on-site assays for screening suspected patients and diagnosing viral infection [ ] . nucleic acid amplification testing (naat) is an effective tool for clinical pathogen detection, owing to its capability of specific amplification and sensitive identification of target sequences [ ] [ ] [ ] [ ] [ ] . compared with polymerase chain reaction in detecting pathogens [ ] , isothermal nucleic acid amplification obviates the need for thermocycling [ ] [ ] [ ] , making naat more rapid and convenient [ ] [ ] [ ] . however, the general requirements for repetitive manual operations such as nucleic acid extraction and amplification, expensive instrumentation, and high-level biosafety laboratories have hindered the use of naat for screening and diagnosis of infectious diseases outside the clinical diagnostic laboratory [ ] . microfluidic technology capable of manipulating small volumes of fluid and integrating a variety of reactions holds great promise for viral nucleic acid testing [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, microfluidics combined with a commercially available coffee mug or portable heating device allowed for sensitive and rapid detection of zika virus by integrating rna extraction, enrichment, and amplification in a microfluidic device [ ] [ ] [ ] . in another work, a multifunctional microfluidic device pre-loaded with wax-sealed reagents was designed for extraction and amplification of viral nucleic acids in a point-of-care format [ ] . our group presented a microcapillary-based assay for sample-to-answer detection of hiv virus [ , ] . to enable nucleic acid testing without the need of electricity, we developed a microfluidic disc combined with a hand-powered centrifugal device for sample-to-answer diagnostics of pathogens [ ] . despite the great potential of microfluidic naat, there is still a lack of fully automated and enclosed microfluidic systems with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing [ , ] . in this work, we develop an automated centrifugal microfluidic system (figure ) consisting of a microfluidic disc and a customized instrument for rapid sample-to-answer detection of sars-cov- armored rna particles with high sensitivity and specificity. the microfluidic disc contains identical and independent reaction units, each of which integrates the on-chip release of nucleic acids and reverse transcription loop-mediated isothermal amplification (rt-lamp). after injection of oropharyngeal swab samples into the microfluidic disc, the whole processing steps including sample treatment, rt-lamp, and fluorescence signal detection were carried out automatically. this enclosed and automated microfluidic system could rapidly detect viral nucleic acid without resorting to skilled operators and diagnostic laboratories, promoting diagnosis efficiency of infectious diseases. the virus lysis/nucleic acid release kit was purchased from sansure biotech (china). the nucleic release reagent comprised of tris-hcl, triton x- , tween- , and octylphenoxy-poly(ethoxyethanol), which can stabilize ph of reaction solution to a lamp-compatible level, protect single-stranded rna after virus lysis, and preserve the enzyme activity. three rt-lamp primer sets targeted to n, e, and orf ab (o) genes of sars-cov- and two types of plasmids containing the n genes of sars-cov or sars-cov- were synthesized by sangon biotech (china). bst dna polymerase, avian myeloblastosis virus (amv) reverse transcriptase, calcein kit, and × reaction buffer for rt-lamp (composed of tris-hcl, ph . , mm; kcl, mm; mgso , mm; (nh ) so , mm; tween , . %; betaine, . m; dntps, . mm for each type), positive control rna (pc rna), and primer mix. rna (pm rna) were purchased from eiken china (china). tris buffer containing m tris-hcl (ph . ) was purchased from solarbio (china). dnase-free water was purchased from sangon biotech (china). the mineral oil was purchased from sigma-aldrich (usa). the armored rna particles are a complex of a singlestranded rna and ms bacteriophage coat protein. three types of sars-cov- armored rna particles ( copies/ ml) containing ms bacteriophage coat protein and nucleotide rna sequence (the part sequence of n gene, the whole sequence of e gene, or the part sequence of o gene, table s , supporting information online) were constructed by xiamen zeesan biotech (china). briefly, the recombinant plasmids encoding a rna sequence and the phage coat protein were transformed in escherichia coli and then expressed by adding the expression inducer. after cell lysis, the armored rna particles were separated and quantified by reverse transcription-polymerase chain reaction (rt-pcr). table s ). the melting temperature (t m ), gibbs free energy (Δg), and g/c rate of the primer sets are summarized in table s . they were further analyzed with rnafold web server (http://rna.tbi.univie.ac.at/cgi-bin/ rnawebsuite/rnafold.cgi) to ensure the absence of hairpins and self-complementarity. the performance of the designed primer sets for rt-lamp detection of target sequences was tested in tube. sars-cov- armored rna particles loaded with rna sequence of n, e, or o gene were suspended in μl tris buffer at a concentration of copies/μl and mixed with μl lysis buffer. for the sensitivity test, sars-cov- armored rna particles loaded with rna sequence of n gene were prepared at different concentrations of - copies/μl in tris buffer and subjected to rt-lamp following the above procedures using n primer set. after the completion of rt-lamp, the fluorescence signal arising from calcein was detected under the excitation at nm by a hand-held uv-flashlight and the colorimetric signal was directly detected under bright field. the centrifugal microfluidic disc consisting of identical reaction units is designed for multiplex viral nucleic acid testing. the disc is assembled from three layers: channel layer i with embedded reaction units, channel layer ii with sample and reagent injection holes, and the sealing layer from bottom to top. channel layer i is a mm thick poly (methyl methacrylate) (pmma) layer with identical reaction units. each unit contains chambers: chamber i for loading reagents, chamber ii for loading sample, and chamber iii for nucleic acid release and rt-lamp reaction the disc center to the edge. all the chambers have a uniform depth of mm. the diameter of chamber i and chamber ii is mm. the length and width of chamber iii are . and mm. the connection channels between chambers are mm wide and . mm deep. channel layer ii is a mm thick pmma layer with sample injection holes ( mm in diameter) and reagent injection holes ( mm in diameter). all samples generated signals appearing as green after amplification. tris buffer was used as nc. the primers and pc-rna in the commercial kit were used as pc. scale bar, cm (color online). the sealing layer is a poly(dimethylsiloxane) (pdms) layer for preventing the aerosol contamination during the entire testing procedures. all the pmma layers were fabricated using a computer numerical control (cnc) machine. the channel layer and sealing layer were bonded at °c and n force conditions using vacuum hot press bonding machine (wh- a, wenhao, china). the automated instrument for sample-to-answer detection of viral nucleic acids is composed of multiple functional modules for automated reagent injection, centrifugationbased flow actuation, temperature control, and fluorescence imaging. the overall size of the customized instrument is mm (depth)× mm (width)× mm (height). the injection module is equipped with syringe pumps (lead fluid, china) for loading different reagents and reservoirs for washing buffer ( ml in volume, dow corning, usa). the precision of automated reagent injection is ensured by a linear module (dizi, china) and a linear module controller (vince, china). in the flow actuation module, the high-speed rotation of the microfluidic disc is precisely controlled by a servo motor (fuji electric, japan) and a servo motor controller (fuji, japan). the temperature control module consists of a proportional-integral-derivative (pid) temperature controller (yexian, china), thermo electric coolers (shenzhen tecooler, china), and water coolers (beijing jizhi, china). the fluorescence imaging module is equipped with a light-emitting diode (led) light source ( w power, mshot, china), a fluorescence filter (e x / e m : / nm, chroma, usa), an objective lens ( . numerical aperture, . mm working distance, olympus, japan), an electric autofocus system ( mm focusing range, hiwin, china), and a cmos camera ( % quantum efficiency, tucsen, china). the fluorescence signal recorded by the imaging module was automatically processed by a build-in program. these functional modules are controlled by an industrial personal computer (zhanmei, china) for instrument automation. the instrument is also integrated with a -inch display (weichensi, china) to provide a touchscreen user interface (labview, , usa) for monitoring the workflow of sample-to-answer nucleic acid testing and a real-time view of fluorescence signal at the reaction unit. for the sample-to-answer detection of viral nucleic acids, μl sample fluid was injected into the chamber ii through the sample injection hole and sealed by silica gel. meanwhile, the lysis buffer, rt-lamp reagents (composed of n primer set, reaction buffer, enzyme mix, and calcein fluorescence indicator), and mineral oil were preloaded into different syringes controlled by the reagent injection module. the sample-contained microfluidic disc was then mounted in the instrument, followed by an automated workflow for rt-lamp detection of viral nucleic acids: ( ) controlled the syringe to puncture through the pdms sealing layer and injected μl lysis buffer into chamber i; ( ) rotated the microfluidic disc at r/min for s to centrifuge both the sample and lysis buffer into chamber iii and incubated at room temperature for min to release the nucleic acids; ( ) injected μl rt-lamp reagent into chamber i; ( ) rotated the microfluidic disc at r/min for s to mix the lysed sample with the rt-lamp reagent in chamber iii; ( ) injected μl mineral oil into chamber i and rotated the microfluidic disc at r/min for s. this procedure was performed by twice to completely fill chamber iii with mineral oil for tight sealing; ( ) heated the microfluidic disc at °c for min to carry out rt-lamp; ( ) captured the fluorescence signal of chamber iii and automatically calculated the fluorescence intensity. for the specificity test, two types of plasmids containing n genes of sars-cov or sars-cov- ( copies/μl) were suspended in dnase-free water to serve as the samples. dnase-free water was used as the nc. for the sensitivity test, sars-cov- armored rna particles loading with n gene were suspended in tris buffer at different concentrations ( . , , , , and copies/μl) to serve as the samples. tris buffer was used as nc. to explore feasibility in virus detection at clinical setting, oropharyngeal swab samples from healthy volunteer and spiked with sars-cov- armored rna particles containing n gene ( . and copies/μl) were used as samples. tris buffer was used as nc. theoretically, a given sample that contains at least one copy of the target rna will be detected as positive by lamp. in our case, the probability p to successfully detect target rna from μl sample containing copies on average was estimated to be % using the poisson distribution where λ= is the average number of target rna per μl sample and k= represents the absence of target rna within the sample. to investigate the impact-induced mixing between sample and reagent solutions within the reaction unit, the computational fluid dynamic (cfd) simulation was performed using a finite-volume solver (fluent . , ansys inc., usa). the motion of liquid and air phases within the reaction unit was obtained by using the volume of fluid (vof) model, in which continuity and momentum equations depending on the volume fraction of each phase were solved: where α i , ρ i , and u i are the volume fraction, density, and velocity vector of i-th phase, respectively, p is the pressure, η is the dynamic viscosity of fluid, g is the vector of the centrifugal acceleration, and f is the surface tension force per unit volume. to solve the equations, least square cell based gradient was used for spatial discretization, quick scheme was used for momentum discretization, and presto was used for pressure discretization. time step was set to be μs. the fully automated centrifugal microfluidic system for sample-to-answer detection of viral nucleic acids is comprised of a customized instrument, an integrated microfluidic disc, and a reagent set for the release of nucleic acids and rt-lamp detection (figure , figure s , supporting information online). the instrument consists of multiple functional modules for automated reagent injection, centrifugationbased flow actuation, temperature control, fluorescence imaging, and data processing, allowing for automatically streamlining the entire procedures of viral nucleic acid testing. the microfluidic disc with independent reaction units was loaded with virus samples and then mounted onto the automated instrument. virus lysis buffer, rt-lamp reagents, and mineral oil were sequentially injected into the microfluidic disc for on-chip release of viral nucleic acids, amplification of target rna, and sealing of reaction unit. after each injection, the microfluidic disc was under program-controlled high-speed rotation for actuating and mixing different fluids. the on-chip release of viral nucleic acids was performed at room temperature for min, and rt-lamp reaction was at °c up to min using pid temperature controller. fluorescence signal indicating the presence of target rna was recorded by the imaging module and processed by the built-in program. this automated centrifugal microfluidic system enables the sample-toanswer detection of viral nucleic acids in min with minimal operation. we designed three primer sets targeted to n, e and o genes of sars-cov- ( figure , table s ) and tested their performance for rt-lamp detection of target sequences in tube. the primers and pc rna in the commercial kit were used as pc. tris buffer was used as nc. /μl sars-cov- armored rna particles loaded with rna sequences of n, e or o gene were first treated with the lysis buffer to release nucleic acids at room temperature for min, and then subjected to rt-lamp at °c for min. as shown in figure , all samples generated signals appearing as green after amplification, indicating that the n, e and o primer sets can be used for rt-lamp in tube. as sars-cov- shows a higher transcript level of n gene than that of o gene, the detection of n gene may provide better diagnostic performance [ ] . to assess the sensitivity of rt-lamp for detecting n gene of sars-cov- , serial dilution test was performed using armored rna particles with n gene. after lysis treatment and rt-lamp, the bright green fluorescence for armored rna particles from to copies/μl was observed in tubes using the n primer set, indicating the high sensitivity of rt-lamp ( figure ). the centrifugal microfluidic disc has identical reaction units enabling the multiplex analysis of viral nucleic acids with sample-in-answer-out capability (figure (a) ). each reaction unit consists of chambers, including a chamber for loading reagents (chamber i), a chamber for loading samples (chamber ii), and a reaction chamber (chamber iii) from the disc center to the edge. after loading the samples by pipette into chamber ii, the microfluidic disc was tightly sealed by the silica gel (figure (b) ). virus lysis buffer, rt-lamp reagents, and mineral oil were sequentially injected into chamber i. the release of viral nucleic acids and amplification of target rna were performed in chamber iii. the fluorescence signal indicating the presence of target rna can be directly observed in chamber iii as the entire microfluidic disc was made from transparent materials (pmma and pdms). the sealing of reaction unit was tested by heating the microfluidic chip at °c for min (to mimic lamp amplification) ( figure s ). no leakage or liquid evaporation was observed as rhodamine b liquid segment inside the reaction unit remained unchanged. the complete seal of reaction unit ensured that the amplicons will not become aerosol to contaminate the next lamp. to enable the rapid, on-demand mixing of samples with reagents in the chamber iii, two adjacent chambers were bridged by a microchannel with μm in width (w) and μm in depth (h). under stagnant conditions, a convex liquid front was formed at the junction of the microchannel and the chamber due to the sudden expansion configuration, resulting in a pressure barrier (Δp) directing toward the liquid phase to stop the flow [ ] : c c m where γ= . × − n/m is the liquid-air surface tension, θ c = °is the contact angle, β= °is the wedge angle of the sudden expansion, ρ= kg/m is the fluid density, r m is the average distance between liquid front, and Δr is the length of the liquid body. given an r m of mm and a Δr of . mm, the minimum rotation rate required to overcome the pressure barrier was estimated to be r/min. therefore, the setting rotation rate of r/min was high enough to actuate the fluid into chamber iii ( figure s ). cfd simulation revealed an impact-induced mixing between sample and reagent solutions at r/min, which significantly reduced the characteristic diffusion length to~ . mm within ms ( figure ). the minimal diffusion time for complete mixing was estimated to be s using the following equation: where l= . mm is the characteristic diffusion length and d= − m /s is the diffusion coefficient estimated for bst enzyme ( . kda). for sample-in-answer-out detection of viral nucleic acids, an automated, stand-alone instrument was designed (figure (a)). the instrument is composed of ( ) syringe pumps equipped with injection needles for loading reagents; ( ) a motor for rotating the microfluidic disc at high speed; ( ) a temperature controlling system for carrying out rt-lamp reaction; ( ) an imaging system for real-time quantifying the fluorescence signal; and ( ) an industrial personal computer for controlling the modules to streamline the entire procedures. the operator only needs to pipette the samples into the microfluidic disc and mount it into the instrument for rapid step-by-step illustration of viral nucleic acid release, amplification, and detection within the integrated microfluidic disc (color online). detection of viral nucleic acids, without the requirement of skilled personnel and clinical diagnostic laboratory. to characterize the performance of customized instrument, its accuracy for reagent injection was first tested (figure (b)). we set the injection volumes to be μl (for nucleic acid release reagents) and μl (for lamp reagents), and measured the actual volumes after automated injection. a standard deviation of < % was obtained, showing the good accuracy and reproducibility of the injection system. we next assessed the rotation stability of instrument ( figure (c) ). by setting the rotation rate at r/min for s, we observed a sudden increase in measured rotation rate from to r/min, followed by a plateau at r/min for s. in addition, a temperature of °c (for rt-lamp reaction) could be maintained more than min using the temperature controlling system (figure (d) ). the imaging system for collecting signals showed an intra-unit variation of < % and an inter-unit variation of < % (figure (e)). to demonstrate the automated detection of viral nucleic acids by the centrifugal microfluidic system, we loaded different concentrations of sars-cov- armored rna particles with n gene ( . , , , , copies/μl) into five reaction units of the microfluidic disc, and used the instrument to carry out on-chip release of viral nuclei acids, rt-lamp, and realtime fluorescence signal detection. tris buffer was used as nc. the amplification curve of armored rna particles at copies/μl showed a sharp increase at~ min and reached a maximum fluorescent intensity at min after amplification (figure (a) ). all samples with different rna concentrations from . to copies/μl could be amplified and detected within min. the limit of detection of this automated centrifugal microfluidic system was copies per reaction for detecting sars-cov- armored rna particles. the entire assay sample-to-answer time was less than min. to test the specificity of the microfluidic assay, plasmids containing n gene of sars-covor sars-cov- were used. based on the analysis using the basic local alignment search tool (blast), the amplification sequences of n gene of sars-cov and sars-cov- had a similarity of % ( figure s ). as shown in figure (b), sars-cov- plasmids ( copies/μl) could be detected by the automated microfluidic system, whereas sars-cov plasmids ( copies/μl) and negative control (nc, water) could not be amplified (figure (b) ). this is attributed to the use of lamp primers that specifically recognize six regions of target sequences, allowing for high specificity detection of sars-cov- using the automated microfluidic system. we next demonstrated the practical applications of automated microfluidic system for viral detection using oropharyngeal swab samples from healthy volunteer and spiked with sars-cov- armored rna particles ( . , copies/μl) (figure (a) ). no fluorescence signals were detected for the swab sample without armored rna particles as well as for nc (tris buffer) after microfluidic sample treatment and rt-lamp (figure (b) ). in contrast, the swab sample spiked with even . copies/μl armored rna particles showed a positive signal. these results collectively demonstrate that the automated microfluidic system with good performance can be adapted to detect viral nucleic acids in a sample-to-answer manner. in this work, we developed a fully automated centrifugal microfluidic system for sample-to-answer detection of viral nucleic acids in min with minimal operation. the microfluidic system was comprised of a customized instrument, an integrated microfluidic disc, and a reagent set for the release of nucleic acids and rt-lamp detection. the limit of detection for sars-cov- armored rna particles was copies per reaction, and the assay did not cross-react with sars-cov. the oropharyngeal swab samples spiked with sars-cov- armored rna particles down to . copies/μl could be detected. to make our system more compatible to naat outside the diagnostic laboratory, technical improvements are required to further reduce the instrument size, including minimizing the diameter of the microfluidic disc by using higher rotation speed, simplifying flow control modulus by preloading lyophilized reagents in the microfluidic device, and optimizing the arrangement of optical path and using photomultiplier tube instead of cmos camera for fluorescence detection. we envisioned that this microfluidic system free of aerosol contamination may facilitate viral nucleic acid detection outside the diagnostic laboratory, promoting diagnosis efficiency of infectious diseases and preventing onward transmission. and the strategic priority research program of chinese academy of sciences (xdb ) monitoring health for the sdgs acknowledgements this work was supported by the national natural science foundation of china ( , ), chinese academy of the authors declare no conflict of interest. the supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/ . the supporting materials are published as submitted, without typesetting or editing. the responsibility for scientific accuracy and content remains entirely with the authors. key: cord- -gmjnbnx authors: yang, limin; li, jing; bi, yuhai; xu, lei; liu, wenjun title: development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of duck hepatitis a virus type date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: gmjnbnx we developed and evaluated a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for detecting duck hepatitis a virus type (dhav- ). the amplification could be finished in h under isothermal conditions at °c by employing a set of four primers targeting the c gene of dhav- . the rt-lamp assay showed higher sensitivity than the rt-pcr with a detection limit of . eld( ) . ml(− ) of dhav- . the rt-lamp assay was highly specific; no cross-reactivity was observed from the samples of other related viruses, bacteria, allantoic fluid of normal chicken embryos, or the livers of uninfected ducks. thirty clinical samples were subjected to detection by rt-lamp, rt-pcr, and virus isolation, which obtained completely consistent, positive results. as a simple, rapid, and accurate detection method, this rt-lamp assay has important potential applications in the clinical diagnosis of dhav- . duck hepatitis virus type (dhv- ), a member of family picornaviridae and genus avihepatovirus, is a kind of single-stranded rna virus that causes an acute, highly lethal disease in young ducklings called duck hepatitis. duck hepatitis leads to severe economic losses for duck raising farms. duck hepatitis virus includes three serotypes dhv- , dhv- , and dhv- . dhv- is distributed widely, while dhv- and dhv- have only been reported in the uk and the usa, respectively [ ] [ ] [ ] [ ] [ ] [ ] [ ] . duck hepatitis caused by dhv- can lead to mortality up to % in young ducklings during the first week of life, thus accurate and efficient diagnosis is extremely useful to control the initial disease outbreak [ ] . recently, dhv- was renamed to dhav, and dhav has three genotypes (dhav- , ,and ) [ ] [ ] [ ] [ ] . dhav- is distributed widely and prevalent in china, dhav- have been only isolated in taiwan until now [ , ] , while dhav- was first isolated in south korea [ ] , but now it is also epidemic in mainland of china. the traditional detection methods, including virus isolation and neutralization tests, are generally reliable for the diagnosis of dhv- [ ] , but these methods have shortcomings, such as labor intensive, time consuming, and have insufficient sensitivity which cannot detect extremely low viral loads. to address this, a virus antigen-based elisa was first established in [ ] , then a recombinant vp protein-based elisa was developed, which showed agreement with the neutralization test [ ] . nucleic acid-based assays such as rt-pcr, real-time rt-pcr, and real-time quantitative pcr were developed and showed high specificity and sensitivity [ , , [ ] [ ] [ ] . however, these assays need specialized and expensive equipment such as a thermal cycler or real-time pcr system, thus they are of limited application in rural areas. loop-mediated isothermal amplification (lamp) assay was developed in [ ] , which is a novel nucleic acid amplification method that occurs under isothermal conditions. this method employs a dna polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target dna, which can be amplified with high specificity. lamp continues with the accumulation of copies of target in less than an hour [ ] . as a simple and efficient diagnostic technique, lamp has been used in the detection of various rna or dna viruses, such as avian leukosis virus [ , ] , barley yellow dwarf virus [ ] , swine transmissible gastroenteritis coronavirus [ ] , avian influenza virus [ ] , and foot-and-mouth disease virus [ ] . here, we report a one-step, single-tube rt-lamp assay for the rapid detection dhav- , and its specificity and sensitivity were assessed. this method has potential applications in the early diagnosis and forecasting of dhav- . the dhav- strain (dhav-sd, stored at the china general microbiological culture collection center, cgmcc no. ) was propagated in the allantoic cavities of -day old spf chicken embryos. the embryos that died - h post inoculation were collected. allantoic fluid was centrifuged ( , g at °c for min) and the suspension was stored at - °c until it was used for rna extraction [ ] . duck enteritis virus (dev), muscovy parvovirus (mpv), avian influenza virus (aiv, h n ), riemerella anatipestifer (ra), salmonella enteritidis, and escherichia coli (o ), which were maintained in our laboratory, were propagated and the nucleic acids were extracted [ , [ ] [ ] [ ] [ ] . total rna was extracted from allantoic fluid and liver samples using trizol reagent (invitrogen, carlsbad, usa) according to the manufacturer's instructions. dhav- total rna concentration was measured spectrophotometrically at a and a . this rna was stored at - °c before use. the primers for the rt-lamp amplification of dhav- were designed based on the conserved region in the c gene (genbank accession no. jx ). primers f , b , fip, and bip were designed by means of the primer software primer explorer v (http://primerexplorer.jp/elamp . . /index.html; eiken chemical co., japan). the primer sequences are shown in table . the rt-lamp reaction was carried out in a total ll reaction volume containing thermopol reaction buffer, u of bst dna polymerase, u amv reverse transcriptase (new england biolabs, ma, usa), mm dntp mix (newpep, beijing, china), . m betaine, mm mgso , . lm of each of the f and b primers, . lm of each of the bip and fip primers, and . ll of the target rna. the mixture was incubated at °c for h followed by min at °c. after the reaction, the amplified dna products were detected by electrophoresis on a . % agarose gel (biowest agarose, spain) followed by ethidium bromide staining under ultraviolet light [ ] . in order to compare the sensitivity of the rt-lamp assay with other conventional assays, an rt-pcr assay was developed using two pairs of primers (for and rev; f and b ) according to the early report with some changes ( table ) [ ] . the rt-pcr was carried out in a ll total reaction volume using the one-step rt-pcr kit (newpep, beijing, china) with . lm of each of the upstream and downstream primers and ll of target rna, according to sensitivity comparison of rt-lamp to rt-pcr to detect the limit of the rt-lamp and rt-pcr assay, dhav- total rnas were extracted from the serially -fold diluted allantoic fluid, ranging from to - % egg lethal dose (eld ) per ll. this single dilution series was used as a template for the two assays. the products were detected by agarose gel electrophoresis as described above ( . % agarose, tae) [ ] . to assess the specificity of rt-lamp, including potential cross-reactions with dhav- , dev, mpv, aiv, r. anatipestifer (ra), s. enteritidis, and e. coli (o ) were examined. total rna from the allantoic fluid of normal chicken embryos and livers of uninfected ducks were also assayed. to evaluate the reliability of the rt-lamp assay, clinical liver samples were collected from dhav-suspected ducks in different provinces of china, including shandong, hebei, sichun, and beijing. rna was extracted from these samples and detected by both the rt-lamp and rt-pcr. the products were detected by agarose gel electrophoresis ( . % agarose, tae). the virus isolation method was also applied to the clinical liver samples using the method previously described [ ] . in order to obtain more specificity and detect multiple strains of dhav- , the rt-lamp primers were designed based on a highly conserved region of the c gene of the dhav- strain. the one-step, single-tube, rt-lamp assay was optimized with the selected primer set by varying the ratio of the concentrations of mgso and dntp, the reaction temperature, and time. to compare the sensitivity of the rt-lamp assay with the conventional rt-pcr, the two assays were used to detect the same rnas which were extracted from -fold serial dilutions (from to - eld per ll) of allantoic fluid. dhav- total rna concentrations were also measured spectrophotometrically at a and a . therefore, the corresponding rna concentration range is from pg to - pg per assay. the results are shown in fig. . the detection limit of the rt-lamp assay was . eld per ll, equivalent to - pg dhav- total rna per reaction, which was -fold higher than the rt-pcr assay. in addition, the rt-pcr assay using two pairs of primers have the same sensitivity. the cross-reactivity of the dhav- rt-lamp assay was evaluated with rna from dev, mpv, aiv, ra, s. enteritidis, e. coli (o ), allantoic fluid of normal chicken embryos, and liver of uninfected duck. all these reactions were negative (fig. ) . to evaluate the feasibility of rt-lamp of detecting dhav- in clinical specimens, clinical specimens collected over the past years were assayed by rt-lamp and rt-pcr. in parallel, virus isolation was also performed. the results showed that of the samples tested contained dhav- by virus isolation, the same clinical specimens were also positive by both rt-lamp and rt-pcr (fig. ) . the results of rt-lamp, rt-pcr, and virus isolation were % correlated. several nucleic acid amplification techniques have been developed for the specific and sensitive detection of dhv- , including rt-pcr and real-time pcr. however, these assays require considerable operator skills, expensive equipment, and - h for amplification; thus, the application of these assays is limited in the field. compared to traditional pcr technology, lamp has more advantages. first, lamp is more specific since it requires or primers to identify or specific domains [ , ] , while pcr uses only two primers. second, lamp is more sensitive, for the amplification of lamp is more efficient than pcr. third, lamp does not require expensive and complex equipment, instead it can be performed using a water bath or heat block for incubation under isothermal conditions. finally, lamp is time saving, the assay can be accomplished within h, whereas the pcr technology typically requires - h [ ] . in addition, the lamp amplification products can be observed by the naked eye directly, as sometimes a white precipitate of magnesium pyrophosphate form during the reaction [ ] . after a comparison of different dhav- subgroup genomes, the conserved domain c of the genome was selected as the domain for lamp primer design and was used for screening a group of primers with good amplification efficiency. the d gene has also been used to design primers for detection of dhv- in the early reports, which encodes an rna-dependent rna polymerase [ , , ] . given that many viruses have rna polymerase gene, we prefer to choose c gene as a detecting marker. a one-step rt-lamp assay with high specificity and sensitivity was developed for rapid diagnosis of dhav- , which has no cross-reaction with dev, mpv, aiv, r. anatipestifer (ra), s. enteritidis, and e. coli (o ), suggesting that this technique has high specificity to distinguish among some common avian viruses and bacteria at the nucleic acid level. the rt-lamp has a detection limit of . eld per ll, equivalent to - pg dhav- total rna per reaction, which was times more sensitive than the conventional rt-pcr, which suggested that this method is useful for the detection of low levels of dhav- and is also useful for confirming the early stages of dhav- infection when viral titers are relatively low. the rt-lamp method was also used to detect dhav- in clinical samples. the results from the rt-lamp assay were consistent with the rt-pcr and viral isolation methods, further confirming the reliability of the rt-lamp assay. considering that dhav- rt-lamp has many advantages, such as being highly sensitive, simple, specific, less time consuming, and not requiring expensive equipment, it is therefore more suitable for use as a dhav- diagnostic tool in the field or rural areas than other nucleic acid-based assays. in summary, the dhav- rt-lamp assay we developed could be a potential diagnostic method for use in the surveillance, control, and molecular epidemiological screening of dhav- for using in developing countries. disease of poultry th edn acknowledgments financial support was provided by the special fund for the agro-scientific research in the public interest ( ) and the nature science foundation of china (nsfc ). key: cord- -ti rpt q authors: zhao, kai; hu, ruili; ni, jianping; liang, jieling; he, xizhong; du, yanan; xu, yan; zhao, binan; zhang, qi; li, chunhua title: establishment of a porcine parvovirus (ppv) lamp visual rapid detection method date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: ti rpt q porcine parvovirus (ppv) is one of the major causes of reproductive pig disease. due to its serious nature, wide spread and consequent great damage to the swine industry, an effective, rapid and convenient method for its detection is needed. a loop-mediated isothermal amplification (lamp) assay was established to detect ppv infection. two pairs of primers were specifically designed to recognize the six different sequences of open reading frame (orf ) gene. the optimized lamp program was as follows: min at °c followed by min at °c.the amplified products were analyzed both by visual inspection after staining with sybr green i dye and by conventional agarose gel electrophoresis. both methods showed the same sensitivity. the limit of detection (lod) for ppv by lamp was copies, which is -fold lower than conventional pcr. our lamp assay did not cross-react with other viruses. we used the established lamp system to test field samples and detected positives. the lamp detection method for ppv represents a visual, sensitive and rapid assay which can detect the virus in the field, offering an attractive alternative for the ppv detection methods currently in use. porcine parvovirus (ppv), a virus belonging to the parvoviridae family, causes maternal reproductive failure of swine known as porcine reproductive system disease which is a serious problem in the pig breeding industry. the characteristics of ppv infection in infected sows (especially primiparous sows) are stillbirth, fetal malformation and mummification, but the infection also can cause neonatal death and piglet disease including diarrhea and dermatitis (yin and liu, ) . all kinds of pigs can be infected by ppv, such as domestic pigs, wild boar, newborn piglets, finishing j o u r n a l p r e -p r o o f pigs and spf pigs. however, the pregnant sow itself and some infected pigs do not have evident clinical symptoms (kennedy et al., ; ellis et al., ) . ppv has caused huge losses to the pig industry. therefore, an effective method is necessary to detect the ppv infection. currently, conventional pcr is used to detect and identify the virus (caprioli et al., ; huang et al., ; jiang et al., ) . but its amplification efficiency is affected by many disturbing inhibitors (wilson, ; abu and rådström, ) . enzyme linked immunosorbent assay (elisa) is also a common way to detect ppv (jenkins, ) . however, infected swine are difficult to diagnose by this method because they are prone to false-positive results during the analytical process (westenbrink et al., ) .though the common pcr, elisa and real-time pcr methods (zheng et al., ; pérez et al., ; ) are also suitable for the qualitative and quantitative analysis of ppv, it requires highly skilled laboratory technicians. therefore, an alternative quick, accurate and simple method is still needed to detect ppv. some years ago, a novel nucleic acids amplification technique was introduced which was called loop-mediated isothermal amplification (lamp) (notomi et al., ) . the method is simple and extremely specific to the target sequence, since the four primers can identify the six target sequences and amplify it (mori et al., ; zhang et al., ) . compared with other detection methods, the lamp method has many advantages, in particular specificity, sensitivity and rapidity. the lamp products have a typical ladder-like pattern and can be detected by adding sybr green i dye (zhang et al., ; iwamoto et al., ) . the lamp amplification solution can be visually turned to green in the presence of a dye sybr green i, while the lamp solution remains orange in the absence of amplification (iwamoto et al., ) . the lamp method has become a useful assay for the fast detection of food borne pathogenic microorganisms and infectious diseases (he et al., ) . other examples are the detection of heat-labile i and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by lamp (yano et al., ) . in this study, a detection method based on the lamp technology is described which is suitable for the clinical detection of porcine parvovirus. the diagnostic kit was j o u r n a l p r e -p r o o f developed, tested and applied. the present study offers the necessary technological basis for the prevention and control of porcine parvovirus infection. the viral strains used for the lamp assays were obtained from the institute of animal husbandry and veterinary science, shanghai academy of agricultural sciences. porcine parvoviruse (ppv), classical swine fever virus (csfv), porcine circovirus type (pcv ), porcine pseudorabies virus (prv) and porcine reproductive and respiratory syndrome virus (prrsv) were included. the geographical origin, and year of isolation of these viruses were summarized in table . pig sera were gathered from a slaughterhouse in shanghai (china) and used as clinical samples for the detection of ppv by lamp. dna was extracted from ppv, pcv and prv by the blood viral dna/rna kit (biomiga inc, san diego, ca). the dna from ppv obtained in the previous step was used as template to optimize the test reaction temperature. rna from prrsv and csfv was extracted following the same method as the dna. the process from rna to cdna was achieved by reverse transcription (takara corp., japan). these cdna templates were used for the next specific experiment. two pairs of primers were designed by primer explorer based on the ppv vp gene (capsid protein ) gene of ppv genome (https://www.ncbi.nlm.nih.gov/ nuccore kf . ). they were called fip, bip, f and b and the information of them were shown in table . the f and b primers were used in the pcr reaction and the target sequence was bp. pcr assays were performed in μl reaction volumes containing . μl ×buffer (takara), . mm dntps, . μm each of f and b , . u taq dna polymerase j o u r n a l p r e -p r o o f (takara biotechnology co., ltd, dalian, china) and μl template dna. the program consisted of an initial denaturation at °c for min, followed by cycles at °c for s, °c for s, and °c for s, and a final extension at °c for min in an applied biosystem thermal cycler (applied biosystem., us). the pcr products were sequenced and analyzed. meanwhile, the bp pcr products were purified using the qiaquick pcr purification kit (qiagen, germany),following the manufacturer's instructions. then the fragments were cloned into peasy®-t cloning vector and transformed into trans -t competent cell using peasy®-t cloning kit (transgen biotech co., ltd., beijing, china). the positive plasmid were obtained according to blue/white selection and identified by colony pcr and sequencing. the resulting positive plasmid containing vp gene fragment of ppv was extracted for further experiments. lamp reactions were performed in volumes of μl, which contained . μl ×buffer (-mg + )(takara), . mm mg + , . mm dntps, . μm each of fip and bip, . μm each of f and b , m betaine (sigma), and . u bst dna polymerase (vazyme biotech co.,ltd). after adding μl template dna, the mixture was incubated for min at °c and cooled on ice for min, after which the bst polymerase was added. the lamp reaction was performed in a conventional heating block. to optimize the reaction temperature, lamp was carried at ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃ . ℃ and ℃ for min and terminated at ℃ for min, respectively. the reaction system was the same as method . mentioned above, and the template was plasmid containing ppv vp gene fragment with the concentration copies per μl. two reactions with and without inner set of primers were carried out at . ℃ as control. j o u r n a l p r e -p r o o f to verify the specificity of ppv detection by lamp, the dna (ppv, pcv , prv) and cdna samples (prrsv, csfv) were amplified as sample templates by the lamp reaction at . ℃ for min and terminated at ℃ for min, respectively. as a positive control we used the ppv vp plasmid; as a no template control (ntc) water was used. all reactions were repeated in duplicates. ten-fold serial dilutions of the ppv plasmid were made to obtain a gradient of to copy per μl. these plasmids of different concentration were prepared to define the limit of detection (lod) of ppv dna by lamp and pcr assays. the amplification products were checked on agarose gel electrophoresis. in addition, the lamp products were also checked by adding a dye sybr green i. lamp and pcr amplified products were detected by % (w/v) agarose gel electrophoresis and observed by staining with goldview (sbs genetech co., ltd., shanghai). the lamp products were also directly observed depending on their color by mixing each sample with μl : -diluted sybr green i (thermo fisher scientific, usa). the positive sample will turn green while the negative sample will still remain orange. whole blood samples were collected as random from "duroc ×landrace ×yorkshire" pigs with the weight of - kg and - days old. these blood samples were placed at ℃ for hours, then the sera were separated by centrifugation at r/min for minutes. serum samples were tested using the lamp and pcr method for the presence of ppv to determine if their source were infected with ppv. we also compared the lamp results of straightly using the serum samples with the dna extraction by the blood viral dna/rna kit. samples of serum were used as template in lamp reactions, meanwhile the genomic dna of these serum samples were extracted as template to perform this experiment. all reactions were carried out at . ℃ for min and terminated at ℃ for min, respectively. the optimum temperature for the lamp reaction was determined to be ± °c ( fig. ). at this temperature, the typical ladder-like pattern was the brightest and clearest, corresponding to the highest amount of product. the comparison between reactions with and without inner set primers confirmed the feasibility of this lamp protocol. in this assay, only the genomic dna samples of ppv strains and ppv plasmid (positive control) used as template gave the typical ladder-like bands and a green color, while the dna/cdna samples obtained for other virus species as well as the ntc had no bands and showed an orange color (fig. a and b) . the results indicated the primers could only amplify ppv nucleic acid. consistently, the length of the amplified product was bp as predicted. its sequence was confirmed to be % identical to the corresponding sequence in the ppv vp gene. the lower lod of ppv by lamp was found to be copies based on the results of the agarose gel electrophoresis analysis (fig. a) and visual observation (fig. b ). in contrast, the lod for the pcr was copies (fig. ) . these results indicate that the lamp method is about times more sensitive than the conventional pcr assay and this sensitivity is in line with the daily testing requirements. a total of serum samples were tested using the lamp and pcr methods to determine whether their sources were infected by ppv. among them, clinical samples were found to be positive by lamp and samples were positive by pcr (table ). in summary, serum samples were detected positive and samples were negative by both lamp and pcr. the coincidence rate of these two methods was . % ( / ) for clinical samples detection. the comparison between serum template and genomic dna template showed that dna extraction is not necessary and can be omitted. using the serum samples straightly as template doesn't affect the lamp results ( figure ). in this study, we developed a method for the visual and rapid detection of ppv using an optimized lamp technique. lamp has a number of advantages when compared to pcr, particularly its high sensitivity, easy manipulation in addition to its visual and time saving detection. we investigated the optimized ppv lamp method and observed its high specificity for ppv, showing no amplification products for any of the other viruses tested. importantly, two pairs of primers were used to identify the target gene by lamp (nagamine et al., ) , while only one pair of primers was used in conventional pcr. our lamp method has a high specificity because it targets the conserved region of ppv orf gene in the design of the two pairs of primers. the lamp reaction can be carried out under isothermal conditions in a relatively short time, without specific equipment like a pcr thermo cycler. the lamp reaction took only min total time and did not need either intricate pretreatment or an expensive apparatus. the reaction was more quickly than the enzyme linked immunosorbent assay and the real-time pcr. hence, the lamp method we developed has the advantages of easy manipulation and easy popularization. the lamp detection limit for ppv based on visual observation by addition of j o u r n a l p r e -p r o o f sybr green i and by gel electrophoresis analysis was copies. the sensitivity of detection by lamp was times higher than by conventional pcr. this indicated that the visual observation method could be used to analyze the lamp product and could reliably replace the conventional agarose gel electrophoresis . the sensitivity of the lamp method is in line with reports about other virus species, such as swine transmissible gastroenteritis coronavirus, h avian influenza virus and yellow head virus imai et al., ; mekata et al., ) . in the analysis of clinical samples, dna extraction and purification steps were not needed. serum samples can be used straightly as templates for the lamp reaction. the sensitivity of the lamp method was less affected by the composition of the clinical samples than observed with pcr. this feature not only can decrease the time and cost of the lamp reaction, but also can simplify many troublesome programs. for the evaluation of clinical samples, we tested randomly sera by lamp and pcr, and verified this feature. compared to conventional pcr, the detection rate of ppv by elisa was . % (jekins, ) . furthermore, elisa is known to easily cause false-positive results (westenbrink et al., ) . moreover, the elisa method is troublesome and time-consuming in contrast of the lamp method. although the lod by real-time pcr is times lower than by conventional pcr, the detection rate of real-time pcr for ppv is only . % ~ % (zheng et al., ; ). lamp method can make it applicable to laboratories, small-scale hospitals, private clinics and pig industry. for a reliable lamp test, some precautions should be adopted to prevent the occurrence of false positive results. for example, separate work areas and aerosol-resistant pipette tips should be used. meanwhile, the used pipette tips and reaction vessels should also be collected in airtight containers. moreover, it is advisable to divide reagents into aliquots in order to avoid contaminations. notably, negative control samples should be firstly finished as soon as possible when total reaction system is finished to aliquot. a lamp method for ppv has been developed successfully by others (chen et al., j o u r n a l p r e -p r o o f ; liu et al., ; qu et al., ) . chen et al chose to amplify the vp gene of ppv by using a set of four primers at ℃ for min. in this study, we selected four different primers to amplify the vp gene and used the sybr green i dye for the detection of ppv. although both lamp methods were established using specific primers based on the highly conserved ppv ns protein gene (qu et al., ) , the primers we designed are in different region of ns from them. we had aligned many ppv genome sequences and chose the most conserved region for primer design. furthermore, we combined with the dye sybr green i and realized the visual detection for ppv lamp instead of by the conventional gel electrophoresis analysis or fluorescent detection . in addition, dna extraction was omitted in order to save time. in the present study, porcine serum as sample can be used straightly in the lamp assay without dna extraction, as the lamp reaction is well tolerant against biological substances. therefore, in the lamp assay the dna extraction step can be ignored (kaneko et al., ) . thus, the lamp method can be used to detect ppv in the field without the need of a pcr thermocycle instrument, electrophoresis apparatus or turbidimeter. hence, our lamp protocol provides an attractive new method for the detection of ppv. the results of this study illustrate that lamp detection offers a convenient visual approach to detect ppv rapidly, sensitively, specifically and simply. above all, the lamp method was improved from the point of high reaction efficiency and accurateness. for ppv detection, it supplements and extends the former approach; the method was developed into a diagnostic kit that is well received and applied in the field. the authors declare no conflict of interest. all relevant data are within the paper and its supporting information. and ℃, respectively. the other reactions with and without inner set of primers were carried out at . ℃ as control. plasmids containing ppv vp gene fragment with the concentration copies per μl were as template. all reactions were incubated at above temperature for min and terminated at ℃ for min. the amplification products were detected by % (w/v) agarose gel electrophoresis staining with goldview. all reactions were carried out at . ℃ for min and terminated at ℃ for min. a. results on % (w/v) agarose gel electrophoresis staining with goldview. b. visual results by adding sybr green i. m: dl dna marker; n: no template control; pcr was carried out at to copies of plasmids of ppv as template, respectively. the amplification products were detected by % (w/v) agarose gel electrophoresis staining with goldview. (collins et al., ) j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f capacity of nine thermostable dna polymerases to mediate dna amplification in the presence of pcr-inhibiting samples pcr detection of porcine circovirus type (pcv ) dna in blood, tonsillar and faecal swabs from experimentally infected pigs rapid detection of porcine parvovirus dna by sensitive loop-mediated isothermal amplification a taqman-based real-time polymerase chain reaction for the detection of porcine parvovirus detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome porcine circovirus type (pcv ): genetic variation and newly emerging genotypes in china rapid and sensitive detection of lily symptomless virus by reverse transcription loop-mediated isothermal amplification multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. j o u r n a l p r e -p r o o f intracellulare in sputum samples an enzyme-linked immunosorbent assay for detection of porcine parvovirus in fetal tissues simultaneous detection of porcine circovirus type , classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction tolerance of loop-mediated isothermal amplification to a culture medium and biological substances reproduction of lesions of post weaning multisystem wasting syndrome by infection of conventional pigs with porcine circovirus type alone or in combination with porcine parvovirus development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus establishment of porcine parvovirus detection by loop-mediated isothermal amplification assay detection of yellow head virus in shrimp by loop-mediated isothermal amplification (lamp) detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna isolation and identification of porcine parvovirus s- strain (in chinese) isolation and identification of porcine parvovirus s- strain (in chinese) a multiple sybr green i-based real-time pcr system forthe simultaneous dete ction of porcinecircovirus type , porcine parvovirus, pseudorabies virus and to rque teno sus virus and inpigs rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (lamp) method some multiplication properties of the lapinized chinese strain (c strain) of classical swine fever virus in primary bovine testicular cells (in chinese) an enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus inhibition and facilitation of nucleic acid amplification rapid and sensitive detection of heat-labile i and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by loop-mediated isothermal amplification the pseudorabies vaccination research. i: pseudorabies attenuated vaccine research (in chinese) p r e -p r o o f rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines isolation and identification of nj strain of porcine parvovirus(in chinese) simultaneous detection of porcine parvovirus and porcine circovirus type by duplex real-time pcr and amplicon melting curve analysis using sybr green we are grateful to professor peter j.m. rottier for editing the manuscript. key: cord- -cpjlzutk authors: ablordey, anthony; amissah, diana ackon; aboagye, isaac frimpong; hatano, ben; yamazaki, toshio; sata, tetsutaro; ishikawa, koichi; katano, harutaka title: detection of mycobacterium ulcerans by the loop mediated isothermal amplification method date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: cpjlzutk background: buruli ulcer (bu) caused by mycobacterium ulcerans (m. ulcerans) has emerged as an important public health problem in several rural communities in sub-saharan africa. early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where m. ulcerans is most prevalent. methodology: we compared conventional and pocket warmer loop mediated isothermal amplification (lamp) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of m. ulcerans in clinical specimens. the effect of purified and crude dna preparations on the detection rate of the lamp assays were also investigated and compared with that of is pcr, a reference assay for the detection of m. ulcerans. thirty clinical specimens from suspected bu cases were examined by lamp and is pcr. principal findings: the lower detection limit of both lamp methods at °c was copies of is and copies of is for the conventional lamp at °c. when purified dna extracts were used, both the conventional lamp and is pcr concordantly detected positive cases, while the pocket warmer lamp detected cases. nine of samples were positive by both the lamp assays as well as is pcr when crude extracts of clinical specimens were used. conclusion/significance: the lamp method can be used as a simple and rapid test for the detection of m. ulcerans in clinical specimens. however, obtaining purified dna, as well as generating isothermal conditions, remains a major challenge for the use of the lamp method under field conditions. with further improvement in dna extraction and amplification conditions, the pwlamp could be used as a point of care diagnostic test for bu buruli ulcer (bu) caused by mycobacterium ulcerans (m. ulcerans) is a necrotizing skin disease endemic mostly in rural wetland of tropical countries of africa, america, asia and australia. the disease also occurs in non-tropical areas of australia, china and japan. globally, bu has been reported in over countries [ ] [ ] [ ] . the burden of bu is however most severe in sub saharan africa where the true incidence of the disease is difficult to determine as a result of poor surveillance measures and case confirmation [ ] . available data however reveals an increase in bu incidence over the last several years in the west african countries of ivory coast, ghana and benin. in these countries bu has replaced leprosy as the second most prevalent mycobacterial disease [ ] , [ ] [ ] [ ] . bu begins as painless nodule, papule, plaque or edema that evolves into characteristic ulcers with undermined edges. if untreated, extensive ulceration (that can cover % of the body), scarring and contractures may cause serious functional disabilities in patients [ ] [ ] [ ] . unfortunately most patients seek treatment late and present with large ulcers [ ] [ ] [ ] . previously treatment of such lesions involved surgical removal of all the affected tissue and part of the surrounding tissues, eventually followed by skin grafting [ ] [ ] [ ] [ ] . in antimycobacterial treatment alone (if necessary in combination with surgery) was introduced and has since been considered as the treatment of choice for bu [ ] , [ ] [ ] [ ] [ ] . laboratory confirmation of clinically suspected bu cases has therefore become crucial for the clinical management of bu [ ] . four laboratory tests are recommended for the diagnosis of bu. these include microscopic examination, culture, is pcr and histopathological analysis. microscopic examination detects %- % of clinically suspected bu cases and is currently the only rapid and affordable test available for bu diagnosis in many endemic areas. the detection rate of culture is between %- % and takes an average of - weeks to yield positive results. culture therefore cannot be used for rapid laboratory confirmation of bu. histopathological analysis is reported to detect % additional cases than other confirmatory tests, however this technique is restricted to external reference laboratories and are unavailable in peripheral health centres or district or regional hospitals. is pcr has close to % sensitivity and is considered the method of choice for laboratory confirmation of bu [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the who recommends that at least % of cases must be confirmed by is pcr before commencement of antibiotic therapy [ ] [ ] [ ] [ ] . however technical difficulties (eg, cold chain requirement, stable power supply and qualified laboratory staff) limit the use of this diagnostic test in bu endemic areas. a dry reagent pcr consisting of lyophilized pcr mix which is reconstituted with water for testing dna was developed to simplify bu diagnosis by pcr [ ] but this method also requires the use of a thermocycler, electrophoresis and gel imaging equipment and therefore similarly makes the use of this diagnostic test for bu diagnosis in endemic areas unlikely. the loop mediated isothermal amplification (lamp) is a novel nucleic acid amplification method for molecular detection and identification [ ] . the principle of lamp is autocycling strand displacement dna synthesis in the presence of bst dna polymerase with high strand displacement activity under isothermal conditions between - uc within minutes [ ] . the assay is highly specific due to the recognition of target dna by to independent sequences and the amplification efficiency of lamp is equivalent to that of pcr based methods ( [ ] , [ ] , [ ] ). the lamp reaction enables easy identification of positive tests due to the accumulation of high amounts of amplification products in the reaction tubes. further improvement in visual identification can be realized through the addition of intercalating dyes such as sybr green or hydroxynapthtol blue to reaction tubes [ ] . this therefore precludes the need for post amplification analysis and hence reduces cost and labour. lamp has also been shown to be less affected by a number of inhibitors of conventional pcr [ ] . additionally the closed tube format of this assay reduces problem of carry over contamination which is likely in less controlled environments [ ] . with all of these characteristics lamp of dna has emerged as a powerful tool to facilitate point of care diagnostic test [ ] . in order to develop a field applicable technique that offers high detection sensitivity and specificity for the diagnosis of bu, we explored the use of the pocket warmer lamp (pwlamp) technique, a dna amplification method using isothermal conditions ( uc) provided by a disposable pocket warmer [ ] . ethical approval for analysing patients' specimens was obtained from the ethical review board of the noguchi memorial institute for medical research. specimens used were anonymously taken from an already existing collection of patients' specimens processed for diagnosis of bu from agogo presbyterian hospital in ghana. thirty clinical specimens consisting of swabs and fine needle aspirates taken respectively from ulcers and pre-ulcerative lesions of suspected bu patients were used in this study. the fine needle aspirate specimens were kept in ml phosphate buffered saline (pbs) and swabs were stored dry in sterile tubes. each swab was transferred into a tube containing ml milli-q purified water (millipore corporation, billerica, ma) and gently vortexed for sec and then removed. portions ml of the sample suspensions were transferred to separate new sterile eppendorf tubes containing ml of lysis buffer ( . m guhcl, mm tris ph . , % triton x- , mm edta, tween- %), ml proteinase-k and ml glass beads. the mixtures were incubated horizontally in a shaker ( rpm) at uc overnight. to capture the dna, ml of diatomaceous earth solution ( g diatomaceous earth obtained from sigma aldrich chemi gmbh in ml of h o containing ml of % (wt/vol) hcl) was added to the suspensions and incubated at uc with shaking ( rpm) for min. the mixtures were centrifuged at , rpm for sec and the resulting pellets were washed twice with ml of % ethanol ( - uc) followed by ml of acetone. the pellets were dried at uc for min and resuspended in ml milli q purified water and centrifuged at , rpm for sec. the purified dna was used as templates for both is pcr and lamp assays to detect m. ulcerans. to investigate the performance of the lamp assay on crude dna preparations, we obtained types of dna extracts for each clinical specimen. one crude extract consisted of ml suspensions of the specimen boiled for min followed by centrifugation at , rpm for min (boiled extract). the other crude extract used was a ml suspension of the unboiled specimen. ten m. ulcerans strains grown on lj slants were harvested and dna was extracted as previously described [ ] . serial dilutions of purified m. ulcerans dna containing , , , , , and copies of is element per ml were prepared. the number of copies of the insertion sequence element was determined based on the genome size of , kb and presence of an average number of copies of is . this was used to determine the detection limit of the lamp assays. in order to develop a simple and rapid test that can be used to diagnose buruli ulcer under field conditions, we modified the conventional lamp assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified dna as the diagnostic specimen. thirty clinical specimens from suspected buruli ulcer patients were investigated by the modified lamp (or pocket warmer lamp) and the conventional lamp, as well as is pcr, a reference method for the detection of mycobacterium ulcerans. there was no significant difference in the detection rate ( - %) in all of the methods when purified samples were used for the tests. on the other hand the use of crude specimen preparation resulted in a drop in detection rate ( - %) . this study demonstrates that the lamp test can be used for rapid detection of m. ulcerans when purified dna preparations are used. with further improvements in the sample reaction, as well as in specimen purification, the pocket warmer lamp may provide a simple and rapid diagnostic test for buruli ulcer. lamp for detection of mycobacterium ulcerans www.plosntds.org pcr for is pcr targeting is was performed as described previously [ ] . the first and second round pcrs used primers pgp : -agggcagcgcggtgatacgg- and pgp : -cagtg-gattggtgccgatcgag- and pgp : -ggcgcagat-caacttcgcggt- and pgp : -ctgcgtggtgcttt-acgcgc- , respectively. for the first round, the ml reaction volume contained ml dna, pmol/ml of each primer (pgp and pgp ), ml of pcr buffer (containing . mm magnesium chloride), . ml qsolution, . mm deoxynucleotide triphosphates (dntps) and . u hotstar taq polymerase (qiagen). for the second run, ml of the first run product was added to ml reaction volume containing, pmol/ml of each primer (pgp and pgp ), . pocket warmer lamp (pwlamp) was performed using a loopamp dna amplification kit (eiken chemical) described previously [ ] . each ml reaction mixture contained . mm each of fip and bip, . mm each of f and b , . mm each of lf and lb, reaction mixture ( . ml), ml of bst dna polymerase, ml of fluorescence detection reagent (eiken chemical), . ml distilled water and ml sample. reaction tubes were incubated at uc for min in the heat block (geneamp , applied biosystems, foster city, ca) while with the pwlamp, the tubes were sandwiched in a twofold pocket warmer (hokaron haru-type, lotte health products, tokyo, japan) surrounded by a paper towel, and put in a styrofoam box for min ( min reaction incubation). the reaction was terminated at uc for min and the results were read by eye in ambient light and also using uv illumination. a chi-squared test was performed to reveal the statistical difference using spss (version . ; spss inc., chicago, il) software. lamp reaction requires a constant temperature of about u- uc for min for amplification of dna [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in a previous study a pocket warmer reached uc in min and stayed around uc for more than min in a styrofoam box [ ] . the pocket warmers (of a pack of hand warmers) tested in this study achieved a temperature of uc after min and maintained this temperature for about min. the pocket warmer thus provided a suitable temperature ( uc) and time range ( min) for amplification. both pwlamp and the conventional lamp assays were able to detect to the limit of copies of the target sequence after min of amplification. this limit improved to copies when the conventional lamp was carried out at uc (the pocket warmer was not able to attain this temperature and was therefore not investigated). the sensitivity and specificity of the lamp assays for the detection of m. ulcerans is shown in tables and . under ambient illumination, positive specimens in the lamp assay produced greenish colouration (figure ). when purified dna extracts were used, ( swabs, fine needle aspirates) ( %) of clinical specimens were positive by is pcr as well as by the conventional lamp. none of the pcr positive specimens were negative by conventional lamp. however samples of purified dna extracts were positive with the pwlamp, but the . % sensitivity ( / ) of the pwlamp compared to the results by is pcr was not statistically significant (p = . , chi-square test). all negative specimens in is pcr were negative in both lamp assays, indicating specificities of both lamp assays to the reference method were %. twelve unboiled ( swabs and fine needle aspirates) and boiled ( swabs and fine needle aspirates) extracts were positive by all detection assays with sensitivities of . % (unboiled, / ) and . % (boiled, / ) compared to results using purified dna extracts respectively for both lamp and is pcr assays. the positivity of swabs was found to be in the range of % to % compared to % to % for fine needle aspirates. when the positivities in crude dna specimens were compared with those in purified dna, the differences were statistically significant by chi-square test (unboiled vs purified dna, p = . , and boiled vs purified dna, p = . ). none of the is pcr negatives was positive in the lamp assays irrespective of the dna extracts type used. these data suggest that sensitivity of lamp and pcr assays for the detection of m. ulcerans in clinical specimens is enhanced when purified dna extracts are used. bu is a neglected tropical disease that mostly affects the poor in resource limited communities in sub-saharan africa [ ] [ ] [ ] . is pcr, the method of choice for confirmation of bu diagnosis cannot be operational in bu endemic areas [ ] , [ ] , [ ] [ ] . the development of rapid and reliable point of care diagnostic assays is of high priority to bu management and prevention. this study explored the potential use of the lamp method for field diagnosis of bu. some important limitations to the use of this assay in the field include specimen purification and difficulty in maintaining isothermal condition for the reaction. a study suggested that omission of dna extraction or the use of crude dna extracts have no effect on the lamp test [ ] . hatano et al used a disposable pocket warmer to provide isothermal condition for lamp reaction [ ] . based on this knowledge, we applied the pwlamp to crude and purified dna extracts in order to determine whether this method will be suitable for use as a point of care diagnostic test for bu. although the pocket warmers used in this study reached uc after hr (instead of min in previous studies [ ] ), both devices achieved the requisite temperature and holding time for executing lamp reaction, a major advantage in the use of amplification based assay for the detection of an infectious agent under field condition. the pwlamp did not cross-react with other mycobacteria ( figure ) . moreover, the experiment on clinical specimens demonstrated that the pwlamp had a % specificity in clinical specimens of bu. the pwlamp was found to have comparable sensitivity as the conventional lamp at uc as both assays were able to detect copies of is element (equivalent of . genomes of m. ulcerans). the detection limit of the conventional lamp at uc improved to copies of is and this level of sensitivity may probably be achieved with a pocket warmer that can attain a temperature of uc and maintain a holding time of min. when applied to purified dna extracts of clinical specimens, the pwlamp, conventional lamp and is pcr yielded concordant results (tables and ) . however, of the samples that were positive by is pcr/ conventional lamp were negative by the pw lamp. none of the is pcr negative samples were positive in both types of lamp assays. on the other hand we observed a drop in detection rate from - % to - % when crude extracts of clinical specimens were used (tables and ) . this indicates that the use of crude dna extracts as template may not be appropriate for the detection of m. ulcerans by the lamp method. this observation contradicts a previous study that suggested omission of dna extraction had no effect on sensitivity of the lamp assay [ ] . for the crude preparations however, it is noteworthy that the detection rate of the lamp assay was significantly higher for the unboiled extracts than for the boiled extracts. explanations for these results were not explored. the observation that the lamp assay was not inhibited especially for the unboiled specimens is quite consistent with previous work that have shown lamp to be tolerant to culture medium and to certain biological substances including phosphate buffered saline, serum, plasma, urine and vitreous [ ] . in conclusion, the study demonstrates that the lamp assay yields comparable results as is pcr when it is performed at u- uc for min on purified dna extracts and further supports the use of the pocket warmer as a device for providing isothermal amplification condition for the lamp assay. this therefore is a potential boost to the application of pwlamp in resource poor settings. challenges of obtaining pure dna extracts of clinical specimen as well as the use of a pocket warmer capable of maintaining uc for hr, however needs to be addressed in order to improve the performance of the pwlamp assay. further development and testing in larger numbers of specimens is therefore necessary to access the potential use of pwlamp as a simple and rapid point of care diagnostic test for bu. buruli ulcer. mycobacterium ulcerans infection distribution of mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in ghana mycobacterium ulcerans infection: an overview of reported cases globally mycobacterium ulcerans infection: control, diagnosis, and treatment buruli ulcer in ghana: results of a national case search buruli ulcer: management of mycobacterium ulcerans disease: a manual for health care providers mycolactone: a polyketide toxin from mycobacterium ulcerans required for virulence mycobacterium ulcerans disease: role of age and gender in incidence and morbidity mycobacterium ulcerans infection (buruli ulcer): first reported patients in togo socio-economic implications of buruli ulcer in ghana: a three-year review buruli ulcer. mycobacerium ulcerans infection. who/cds/cpe/gbui mycobacterium ulcerans infection and buruli ulcer disease: emergence of a public health dilemma provisional guidance on the role of specific antibiotics in the management of mycobacterium ulcerans disease (buruli ulcer). world health organization efficacy of the combination rifampin-streptomycin in preventing growth of mycobacterium ulcerans in early lesions of buruli ulcer in humans promising clinical efficacy of streptomycin-rifampin combination for treatment of buruli ulcer (mycobacterium ulcerans disease) mycobacterium ulcerans infection (buruli or bairnsdale ulcer): challenges in developing management strategies laboratory confirmation of buruli ulcer disease in togo consensus recommendations for the diagnosis, treatment and control of mycobacterium ulcerans infection (bairnsdale or buruli ulcer buruli ulcer. diagnosis of mycobacterium ulcerans disease: a manual for health providers. world health organization (who) development of a pcr assay for rapid diagnosis of mycobacterium ulcerans infection identification and characterization of is and is : two distinct repeated sequences for detection of mycobacterium ulcerans by pcr buruli ulcer: progress report buruli ulcer: advances in understanding mycobacterium ulcerans infection comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of buruli ulcer disease laboratory diagnosis of buruli ulcer disease dry-reagentbased pcr as a novel tool for laboratory confirmation of clinically diagnosed mycobacterium ulcerans-associated disease in areas in the tropics where m. ulcerans is endemic loop-mediated isothermal amplification of dna loop-mediated isothermal amplificationmethod for rapid detection of the toxic dinoflagellate alexandrium, which causes algal blooms and poisoning of shellfish accelerated reaction by loop-mediated isothermal amplification using loop primers rapid detection of the severe acute respiratory syndrome (sars) coronavirus by a loop-mediated isothermal amplification assay loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium and m. intracellulare in sputum samples tolerance of loop-mediated isothermal amplification to a culture medium and biological substances poon loop-mediated isothermal amplification for influenza a (h n ) virus. emerging infectious diseases n www lamp using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism a comparison of dna extraction procedures for the detection of mycobacterium ulcerans, the causative agent of buruli ulcer, in clinical and environmental specimens key: cord- -nnx nwf authors: ren, xiaofeng; li, pengchong title: development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: nnx nwf in this study, a reverse transcription loop-mediated isothermal amplification (rt-lamp) was developed for detection of porcine epidemic diarrhea virus (pedv). six primers were designed to amplify the nucleocapsid (n) gene of pedv. the optimization, sensitivity, and specificity of the rt-lamp were investigated. the results showed that the optimal reaction condition for rt-lamp amplifying pedv n gene was achieved at °c for min. the rt-lamp assay was more sensitive than gel-based rt-pcr and enzyme-linked immunosorbent assay. it was capable of detecting pedv from clinical samples and differentiating pedv from porcine transmissible gastroenteritis virus, porcine rotavirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, and avian infectious bronchitis virus. porcine epidemic diarrhea (ped) is an infectious enteric disease characterized by acute enteritis and diarrhea in pigs, and the infection is more severe in neonates [ ] . at present, ped has been a major concern in the swine industry, particularly, in the asia and europe, resulting in large economic losses [ ] [ ] [ ] . the causative agent of ped is porcine epidemic diarrhea virus (pedv), an enveloped and single-stranded rna virus that belongs to the family coronaviridae [ ] . coronavirus comprises three major viral structural proteins: spike (s, - kda), membrane (m, - kda), and nucleocapsid (n, - kda) proteins [ , ] . the s protein is a major viral antigen, binds to a cellular receptor for virus attachment to enter target cells and mediates viral attachment to target cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the m protein is a trans-membrane protein [ ] and it is involved in the assembly process of viral nucleocapsid and membrane [ , ] . the n protein of coronaviruses is a phosphorylated protein that interacts with virus genomic rna, forming a helical ribonucleoprotein [ ] . therefore, it plays important roles in viral genome transcription, core formation and virus assembly [ ] . viral n protein is also conserved and can be used as a diagnostic target for detecting viral infection. loop-mediated isothermal amplification (lamp) is a recently developed dna amplification method [ ] . lamp uses four to six primers that recognize six to eight regions of target dna, in conjunction with the enzyme bst polymerase, which has strand-displacement activity. the synchronization dna synthesis by these primers maintains the specificity of the method. interestingly, the amplification step can be performed under isothermal conditions, resulting in the synthesis of a large amount of dna. lamp proceeds when the forward inner primer (fip) anneals to the complementary region in the target dna and initiates the first-strand synthesis. next, the outer forward primer (f ) hybridists and displaces the first strand, forming a loop structure at one end. the resulting single-stranded dna serves as a template for backward inner primer (bip)-initiated dna synthesis and subsequent outer backward (b )-primed strand-displacement dna synthesis. the formed dumbbell-shaped dna stem loop structure serves as a template for subsequent hybridization between one inner primer and the loop, initiating the displacement dna synthesis. the lamp method may form the original stem loop and a new stem loop that is twice as long as the original one. the final products are stem loop dnas with several inverted repeats of the target dna and cauliflower-like structures bearing multiple loops [ ] . at present, the lamp approach has been applied for detecting of infectious pathogens. examples include h n avian influenza virus [ ] , hepatitis b virus [ ] , foot-andmouth disease virus [ ] , etc. in this study, the authors developed a reverse transcription (rt)-lamp using primers directed toward the n gene of pedv. the convenience, sensitivity, and specificity of the established rt-lamp indicate its advantages and utility in detecting pedv. pedv isolate hljby, porcine transmissible gastroenteritis virus (tgev), porcine pseudorabies virus (prv), porcine rotavirus (prv), porcine reproductive and respiratory syndrome virus (prrsv) and avian infectious bronchitis virus (ibv) are propagated in susceptible cells. based on the n gene sequence of pedv (genbank accession number: gu ), a total of six primers targeting the n gene were designed using the primer explorer version (http://primerexplorer.jp/lamp . . /index.html). they include an outer pair (f , b ), an inner pair (fip, bip), and a loop pair (f-loop, b-loop). a pair of primers (named ped and ped ) was used for rt-pcr amplifying the n gene. information regarding the primer names and sequences is shown in table . pedv propagation and rna extraction pedv was propagated in african green monkey kidney (vero) cells according to reference with modification [ ] . in brief, vero cells were cultured in dulbecco's modified eagle medium (dmem) supplementary with % newborn bovine serum (excell bio, china) in six-well plates at °c to allow the formation of cell monolayer. the cells were washed with pbs and infected with pedv ( ll/ well) at an multiplicity of infection (moi) of at °c for h in the presence of edta-free trypsin at a final concentration of lg/ml. dmem containing edta-free trypsin ( lg/ml) was then added into the wells ( . ml/ well) and the culture was maintained at °c for - h. the titer of pedv was . tcid /ml. the total rnas were extracted from the culture supernatants of pedv, tgev, prv, prrsv, and ibv using the rna extraction kit (keygen biotech, china) and the genomic dna of prv was extracted from virus-infected vero cell culture using the dna extraction kit (omega, norcross, usa) according to the manufacturer's instructions. the extracted rna was subjected to reverse transcription (rt) to synthesize the cdna using reverse primer ped and a cdna synthesis kit (haigene, china) according to the manufacturer's instructions. the reaction mixture contained rna template ( lg sterile water was used as a negative control template. the amplified dna products from the rt-lamp were analyzed by separating ll of rt-lamp reaction mixture in ethidium bromide-stained % agarose gel electrophoresis, where the positive reaction mixtures showed a characteristic ladder of multiple bands. the relative quantification of the dna was performed using the gel documentation system (uvitec, cambridge, uk) and determined with gel analyzer software (copyright by dr. istvan lazar) according to the manufacturer's instructions. the reaction result was also observed directly without staining because of the white precipitate of magnesium pyrophosphate or the green color produced by the intercalating dye picogreen Ò (invitrogen, wisconsin, usa) in positive reactions. after the amplification was completed, ll of coloring agent ( loading buffer:gene finder = : ) was added to each test tube and mixed. the test tubes were then examined visually. to determine the optimal reaction temperature, the rt-lamp reaction mixtures were incubated at , , , or °c for min. the optimal reaction time was determined by performing the rt-lamp at the optimal temperature for , , , , or min. finally, the reaction was terminated by heat inactivation at °c for min. the amplified dna products from the rt-lamp assays were visualized by agarose gel electrophoresis as above. the concentration of pedv rna was determined using an ultra-violet photometer (type , shanghai spectrum instrument company) according to the manufacturer's instructions. then the tenfold serial dilutions of the rna ( lg/ml) were used as template for rt-lamp and a conventional rt-pcr. the rt-lamp was performed as above. the rt-pcr was performed using a rt-pcr kit (haigene, china). elisa was performed to compare its sensitivity with rt-lamp. in brief, purified pedv particles ( . lg/ll) were serially diluted in carbonate-bicarbonate buffer ( mm na co , mm nahco , ph . ) and the viruses were coated into elisa plates ( ll/well) at °c overnight. the next day, the plates were blocked with % non-fat dry milk in pbs- . % tween (pbst) at °c for h. subsequently, the wells were incubated with serially diluted anti-pedv polyclonal antibody ( : dilution) or control serum from a non-immunized rabbit at °c for h, after triple wash with pbst. the plates were incubated with horseradish peroxidase-conjugated goat anti-rabbit igg (boster, china : dilution in pbst) at °c for h. the wells were incubated with o-phenylenediamine dihydrochloride (opd) substrate for min after complete washing with pbst. the od value was examined using an elisa reader. the od value of anti-pedv serum positive well (p)/the od value of control serum well (n) [ was regarded as positive. twenty clinical feces of piglets (approx. weeks) with diarrhea symptom were collected from a pig farm in heilongjiang province in . the samples were prepared as a % (w/v) suspension in pbs (ph . ) and centrifuged at g at °c for min. the supernatant was subjected to rna extraction with above-mentioned rna extraction kit. the resulting rna was used as a template for rt-pcr and rt-lamp according to above-mentioned protocols. at the same time, the equal supernatant was used as coating antigen in elisa as above. the detection limit of the three methods was compared. to analyze the specificity of the rt-lamp, pedv, tgev, prv, prv, prrsv, and ibv were used as templates and subjected to rt-lamp as above. using pedv rna and six primers targeting the pedv n gene, an rt-lamp was done at °c in a water bath for h. the resulting amplified dna products showed a characteristic ladder of multiple bands, indicating that the final products were the mixtures of stem loop dnas with various stem lengths (fig. a) . in contrast, the negative control did not show the characteristic bands. the results of virus genes ( ) : - the rt-lamp reaction were also determined directly by visual inspection. if the reaction product is positive, the gene finder dye inserts into the double-stranded dna after the reaction and the product becomes green; otherwise, the dye does not insert into the double-stranded dna and the reaction sample remains blue (fig. b) . the effect of reaction temperature and incubation time on the rt-lamp was investigated. as shown in fig. a , the dna products of the rt-lamp at different temperatures showed multiple of characteristic ladder bands; however, the intensity of dnas determined by gel analyzer software from the reactions at °c was stronger than that at other reaction temperatures, which was judged as the optimal temperature for rt-lamp amplifying pedv n gene. the rt-lamp was then performed at °c for different time points. the results indicated that the dna products showed the highest intensity when the reaction was performed for min (fig. b) . therefore, the optimal reaction condition of the current rt-lamp for pedv was °c for min. the sensitivity of the rt-lamp assay was first compared with the conventional rt-pcr amplifying the tenfold serial dilutions of rna templates of pedv. the detection limit of rt-pcr was . - lg/ml which equal to a virus titer of . tcid /ml, while, the rt-lamp had a detection limit of . - ( . tcid /ml) which was much higher than that of rt-pcr (fig. ) . after applying the same concentration of pedv particles in rt-lamp and elisa, the minimal required virus template amount for the both assays was analyzed. the results showed that the detection limit of rt-lamp was - lg. in contrast, elisa had a detection limit of - lg (fig. ) . table ). the rt-lamp had a similar sensitivity with elisa and was somewhat sensitive than rt-pcr in detection of clinical samples. to analyze the utility of the rt-lamp, several related porcine viruses (i.e., tgev, prv, and prv) and an avian coronavirus, ibv were used as templates and included in the rt-lamp. the result indicated that no positive dna products of the rt-lamp assay were observed among these control viruses. when the pedv was used as template, the positive bands were amplified as expected (fig. ) . the result demonstrated that the rt-lamp assay is specific and can be applied in discriminating elisa for distinguishing pedv from other viruses. there are ped epidemics in china, although inactivated vaccines are used in some regions in china. establishment of rapid, sensitive, and cost-effective diagnostic assays for detecting pedv is highly desirable. virus isolation has been a popular detection method; nevertheless, the virological diagnosis is somewhat difficult for detecting pedv, since it was not possible until to propagate porcine epidemic diarrhea virus in cell culture [ ] . even now, the viral titer of pedv in cell culture is still low. other diagnostic methods for detecting pedv include immunohistochemistry, in situ hybridization, dot-blot hybridization, rt-pcr, and real-time rt-pcr [ ] [ ] [ ] [ ] [ ] . these methods may require either high-precision instruments or complicated procedures. therefore, they are unsuitable for detecting pedv in fields and in less well-equipped laboratories. the rt-lamp method established in this study is a valuable alternative for detection of pedv, since the novel dna amplification technology owns numerous advantages such as simplicity, rapidity, and inexpensiveness. the isothermal conditions required for lamp can be provided with a conventional water bath or heat block. therefore, the current method can be applied less in well-equipped laboratories and fields for rapid detection of pedv. in general, the lamp can be carried out under isothermal conditions ( - °c). in this study, the authors optimized the reaction conditions of the rt-lamp by performing the test at different temperatures and time points. subsequently, its sensitivity was compared with that of rt-pcr. the results showed that the rt-lamp specific for pedv was approx. , times sensitive than the rt-pcr. nevertheless, it is necessary to screen other optimal primers to further compare the sensitivity between rt-lamp and rt-pcr in the future. moreover, the sensitivity between the rt-lamp and conventional elisa was compared using the inactivated pedv as template. the former is more sensitive than the latter. two reports have pointed out that the detection limit of rt-pcr for pedv was . tcid /ml [ , ] . detection limit of a commercially available elisa kit (jinma, shanghai) used in china was . ng/ml, which was the same as the detection limit of the rt-lamp developed in this study. this result further confirmed the sensitivity of the rt-lamp for amplifying the n gene of pedv. nonetheless, when the authors used these methods to detect pedv from clinical samples, the sensitivity of rt-lamp was somewhat higher than rt-pcr and had a similar sensitivity with elisa. more experiments are needed to clarify this point in the future; however, the rt-lamp still has advantages including simplicity, rapidity, and convenience. to analyze the specificity of the rt-lamp for pedv, several related or unrelated viruses were used as control templates. for example, pedv and tgev belong to the group i coronaviruses which are closely related [ ] . ibv and prrsv belong to the group iii coronavirus and arterivirus, respectively; however, both viruses belong to the order nidovirales [ , ] . the structural similarity between the n proteins of ibv and prrsv suggests that members of the coronaviridae and arteriviridae families share a mechanism of filamentous nucleocapsid formation, with suitable alterations necessary to interact specifically with their respective genomes [ , ] . prv and prv are members of the families herpesviridae and reoviridae, respectively. these viruses such as pedv, tgev, prv, prv, or prrsv may cause co-infection in pigs. the results showed that the rt-lamp is successful only if the pedv served as template, indicating that the established method is specific and applicable for differentiation diagnosis. to the knowledge, this is the first report regarding the establishment and optimization of a rt-lamp for pedv n gene. the assay may be useful for the clinical diagnosis of pedv infection. proceedings of the international pig veterinary society congress veterinary virology the coronaviridae acknowledgments the authors acknowledge funding supported by program for new century excellent talents in heilongjiang provincial university ( -ncet- ). key: cord- - sjh mw authors: rödel, jürgen; egerer, renate; suleyman, aynur; sommer-schmid, beatrice; baier, michael; henke, andreas; edel, birgit; löffler, bettina title: use of the variplex(tm) sars-cov- rt-lamp as a rapid molecular assay to complement rt-pcr for covid- diagnosis date: - - journal: j clin virol doi: . /j.jcv. . sha: doc_id: cord_uid: sjh mw background: molecular assays based on reverse transcription-loop-mediated isothermal amplification (rt-lamp) may be useful for rapid diagnosis of the severe acute respiratory syndrome coronavirus- (sars-cov- ) because of the easy performance and the option to bypass rna extraction. objectives: this study was designed to evaluate the clinical performance of the ce-labeled variplextm real time sars-cov- rt-lamp assay in comparison to commercial rt-pcrs. study design: rna extracted from pharyngeal swabs was tested by variplex™ rt-lamp and corman’s lightmix™ e gene rt-pcr as reference. samples of respiratory secretions from coronavirus infection disease (covid- ) and negative control patients were analyzed by variplex™ without rna extraction and tested in parallel with the allplex™ and viasure bd max rt-pcrs. results: using isolated rna variplex™ rt-lamp showed a sensitivity of % compared to lightmix e gene rt-pcr but contrary to the latter it produced no false-positive results. for the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ rt-lamp conducted on unprocessed samples and allplex™ and viasure rt-pcrs (cohen’s κ ranging from . - . ). using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: . % for variplex™, . % for allplex™ and . % for viasure. however, when results of rt-pcr and rt-lamp were combined diagnostic sensitivity was increased to - %. conclusion: the variplex rt-lamp may serve as a rapid test to be combined with a rt-pcr assay to increase the diagnostic accuracy in patients with suspected covid- infection. the severe acute respiratory syndrome coronavirus- (sars-cov- ) pandemic has already caused an enormous burden on healthcare systems worldwide [ high analytical sensitivity several studies reported on false-negative as well as fluctuating results in patients whose clinical diagnosis using chest ct was in accordance with covid- [ , ] . problems with clinical sensitivity of nucleic acid amplification tests can be due to analytical errors of rna isolation procedures and choose of inadequate primers. other challenges in diagnostics are associated with the significantly increased requests for testing, resulting in time delays to generate diagnostic reports [ , ] . moreover, mass testing has rapidly caused serious shortages in the supply of rna purification kits in many countries [ , ] . for a rapid diagnosis of sars-cov- cost-effective methods with low hands-on time that circumvent limitations of rt-pcr may be helpful tools for a routine diagnostic workflow [ , ] . rt-loop-mediated isothermal amplification (lamp) may offer the possibility to be established as an alternative diagnostic technique [ ] [ ] [ ] . the combination of rt with bst polymerase possessing a dna strand displacement activity allows amplification of target genes at a constant temperature in less than one hour. rna purification can be bypassed j o u r n a l p r e -p r o o f depending on the sample type and different transport media because of the robustness of the polymerase. there are several studies that demonstrated satisfying sensitivity and specificity of rt-lamp for sars-cov- detection but little is known about its performance of testing clinical samples directly without rna extraction [ ] [ ] [ ] [ ] . in this study we evaluated the newly introduced ce-labeled variplex sars-cov- lamp assay and compared the clinical performance with commercial rt-pcr tests. testing was performed using pharyngeal washes and samples from respiratory secretions, including sputum, endotracheal secretions, and bronchoalveolar lavage. pharyngeal specimens were collected using eswab™ transport systems (copan, brescia, italy). total viral rna was extracted from l of the sample medium using the qiasymphony dsp virus/pathogen mini kit (qiagen, hilden, germany. extraction was performed on the automated qiasymphony sp instrument (qiagen). purified rna was eluted in l ave buffer and divided into two parts for testing. to rule out cross-reactivity with human coronaviruses e and oc external quality assessment samples (instand e.v., düsseldorf, germany) were processed in a similar manner. reference rt-pcr was performed using the lightmix modular sars-cov e-gene primers (tib molbiol, berlin, germany) and the lightcycler multiplex rna virus master (roche, penzberg, germany) [ ] . rt-pcr was run on a lightcycler (roche, penzberg, germany). the variplex sars-cov- is a qualitative molecular assay using a mix of oligonucleotide primers targeting a -bp sequence of the membrane protein (m) gene. for a single test l of rt master mix and l of eluted rna were pipetted into two wells of a genie test strip (amplex diagnostics). l of the primer mixes for sars-cov- or the inhibition control were added to one each well. tests were run at o c for min using a genie ii mk a device (amplex diagnostics). amplification was measured by real-time fluorescence detection using a dna intercalating dye. data interpretation and calculations were automatically performed by the integrated eazyreport tm software (amplex diagnostics). for the viasure assay l of the sample was used for rna extraction. viasure rehydration buffer and gene reaction tubes containing a ready-to-use master mix were loaded onto bd max exk tna- reagent strips. nucleic acid extraction and real time rt-pcr were performed on the automated bd max system (bd). to assess the analytical sensitivity of the assays the sars-cov- isolate jena/ / propagated and titrated on vero- cells was used. -fold serial dilutions of a virus stock of tcid /ml in a pharyngeal wash were mixed with copan sl solution and processed for the different assays as described above. the qualitative performance of the assays was assessed by calculating the specificity, sensitivity, negative and positive prospective values, and accuracy. for reference a sample was defined as true-positive when at least two different tests gave a positive result. concordance of two diagnostic tests was examined by cohen's  coefficient analysis. pearson coefficient analysis. first, we analyzed a panel of pharyngeal swabs sent to the laboratory for routine sars-cov-aliquots were tested. samples with divergent results between lightmix rt-pcr and rt-lamp were verified by viasure and allplex assays in order to identify false-positively tested specimen. out of rna eluates that were lightmix e-positive could not be confirmed by a second test and were defined as false-positive. their median ct value was . (iqr . - . ). in contrast, no false-positive results were observed using the variplex rt-lamp. however, the sensitivity of rt-lamp was only % ( to verify the sensitivity of rt-lamp extracted rna from a log-dilution series of a virus stock was tested. the variplex assay achieved a reliable detection at tcid /ml, corresponding to . tcid /reaction. in comparison lightmix rt-pcr showed % detection down to . tcid /ml. this concentration was positive by rt-lamp in % of the samples (table ). next, we investigated a panel of clinical samples that were tested by rt-lamp without rna extraction. only samples from respiratory secretions and pharyngeal washes were used because in preliminary experiments we observed inhibitory effects by transport media of swabs on rt. a total of specimens collected from patients were included. from patients or more samples were obtained during the course of the disease. as controls we examined samples from patients that were repeatedly tested negative by lightmix screening rt-pcr. respiratory secretions from covid- patients were often highly viscous and tough. to homogenize the specimens they were mixed with copan sl solution. this procedure was applied to all samples to standardize the methodology. homogenized samples diluted in lptv buffer were directly pipetted into the master mix for rt-lamp. for comparative analysis two aliquots were subjected to rna isolation and rt-pcr using the j o u r n a l p r e -p r o o f allplex and viasure bd max assays. all tests did not produce false-positives results in the group of control patients. from the samples of covid- patients heterogeneous results were obtained. as expected a high agreement of results was found for the three different targets of the allplex assay (table ) . the results obtained with the variplex rt-lamp only showed a moderate agreement to both the allplex and viasure rt-pcr results (table ) . to calculate how the moderate cohen's  concordance coefficients were related to different sensitivities of the assays we defined a sample as true-positive when at least two target genes of the virus were detected. when only one target gave a positive signal the sample was tested by the lightmix rt-pcr to verify the result. using this approach, all assay had sensitivities < % (table ). the allplex rdrp assay offered the highest sensitivity of %, followed by e and n gene tests from the same kit. combining the three targets of allplex did not result in a higher positive rate of the samples. the sensitivity of the viasure assay was only . % and that of the variplex rt-lamp was in between, at . % (table ). however, when results of the variplex rt-lamp were combined with those of the viasure s or allplex rdrp rt-pcr diagnostic sensitivity was increased to and %, respectively (table ) table shows the course of testing an icu patient over days, illustrating the fluctuating results by different assays. in comparison the different sensitivities of the assays were also examined using simulated samples. for these experiments we started at tcid /ml because of the dilution of the samples in lptv buffer for direct rt-lamp testing. as shown in table the allplex rt-pcrs reached higher sensitivities than the other assays. the lower sensitivity of the variplex rt-lamp was probably caused by the relatively high dilution of the sample in lptv buffer because the limit of detection of . tcid /reaction was satisfying in comparison to the allplex rt-pcr. timely and accurate laboratory diagnosis of patients with the suspicion of sars-cov- infection is important for optimizing patient treatment and preventing transmission to other persons [ ]. rt-pcr is the standard method to detect an acute infection and is also used to identify asymptomatic carriers [ , ] . however, several studies have reported false-negative results in initial testing of symptomatic patients as well as during the course of the disease in no small measure that can have an impact on isolation or discharge of patients [ , ]. it has been suggested that a single rt-pcr assay should not be the only laboratory diagnostic marker [ , ] . the data of this study demonstrate that the variplex lamp sars-cov- assay may be suitable as an additional tool to close gaps in covid- diagnosis. by using extracted rna the variplex rt-lamp assay showed a lower sensitivity, compared to our screening e gene rt-pcr, but performance was acceptable when only e gene ct values < were considered. this cut-off has been chosen because on one hand it has been proposed that patients diagnosed with high ct values are rather non-infectious and on the other hand we could identify several false-positive rt-pcr tests that were associated with a high ct value [ ] . a major advantage of rt-lamp is that it allows a simple testing of specimens when unprocessed samples are used, bypassing the bottleneck of rna extraction [ , ] . against j o u r n a l p r e -p r o o f the background of irregularities regarding the delivery of rna isolation kits by many manufacturers rt-lamp would be highly attractive as an alternative easy-to-use technology [ , ] . for direct testing we focused on samples from respiratory secretions and pharyngeal washes instead of swabs because several transport media can inhibit or reduce rt activity, as reported in recent studies [ , ] . another reason was that the supply of swabs with fluid transport media was running into a critical shortage in a phase of significantly increased demand for testing. . by using rt-lamp to test unprocessed samples this problem is avoided but rt activity may be inhibited by carbohydrates and salts depending on the sample composition [ ] . in this context suitable specimen types have to be carefully evaluated. saliva which has been described to contain high virus copy numbers may also represent a potential specimen type for direct rt-lamp testing [ , ] . in conclusion this study shows that the variplex sars-cov- rt-lamp assay may serve as an easy-to perform rapid molecular test to be combined with rt-pcr in order to ensure an efficient workflow of timely and accurate diagnosis even at times of high work load and increased testing requests. the major limitation of this work was the relatively small sample size due to low numbers of covid- patients in our hospital. future studies are needed to examine the utility of rt-lamp under routine conditions with high sample throughput. the authors declare no conflicts of interest. the authors declare no conflicts of interest. [ sars-cov- detection by direct rrt-pcr without rna extraction rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification development of a reverse transcription-loopmediated isothermal amplification as a rapid early-detection method for novel easycov: lamp-based rapid detection of sars-cov- in saliva rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay detection of novel coronavirus ( -ncov) by real-time rt-pcr the allplex -ncov (seegene) assay: which performances are for sars-cov- infection diagnosis? eur comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of sars-cov- viral rna load as determined by cell culture as a management tool for discharge of sars-cov- patients from infectious disease wards rapid and extraction-free deetction of sars-cov- from saliva with colorimetric lamp rapid implementation and validation of a cold-chain free sars-cov- diagnostic testing workflow to support surge capacity we thank katrin schützler, sylvia stoll, beate haschke, and christina gödicke for excellent technical assistance. the study was supported by internal funding. key: cord- -fpoztmcl authors: almasi, mohammad amin; almasi, galavizh title: loop mediated isothermal amplification (lamp) for embryo sex determination in pregnant women at eight weeks of pregnancy date: journal: j reprod infertil doi: nan sha: doc_id: cord_uid: fpoztmcl background: in human, sry (sex-determining region of the y chromosome) is the major gene for the testis-determining factor which is found in normal xy males and in the rare xx males, and it is absent in normal xx females and many xy females. there are several methods which can indicate a male genotype by the presence of the amplified product of sry gene. the aim of this study was to identify the sry gene for embryo sex determination in human during pregnancy using loop mediated isothermal amplification (lamp) method. methods: a total of blood samples from pregnant women at eight weeks of pregnancy were collected, and plasma dna was extracted. lamp assay was performed using dna obtained for detection of sry gene. furthermore, colorimetric lamp assay for rapid and easy detection of sry gene was developed. results: lamp results revealed that the positive reaction was highly specific only with samples containing xy chromosomes, while no amplification was found in samples containing xx chromosomes. a total of blood samples from pregnant women were seven male embryos ( . %) and eight female embryos ( . %). all used visual components in the colorimetric assay could successfully make a clear distinction between positive and negative ones. conclusion: the lamp assay developed in this study is a valuable tool capable of monitoring the purity and detection of sry gene for sex determination. introduction olecular mechanisms of sex determination involve a growing network of genes, a large number, which are transcription factors. the transcription factors so far are identified as having a major role in sex determination ( ) . in humans, the gene for the testis-determining factor resides on the short arm of the y chromosome. individuals who are born with the short arm but not the long arm of the y chromosome are male, while individuals born with the long arm of the y chromosome but not the short arm are female ( ) . by analyzing the dna of rare xx men and xy women, the position of the testis-determining gene has been narrowed down to a , -base-pair region of the y chromosome located near the tip of the short arm ( ) . in this region, a male-specific dna se-quence was found that could encode a peptide of amino acids. this peptide is probably a transcription factor, since it contains a dna-binding domain called the hmg (high-mobility group) box ( , ) . this domain is found in several transcription factors and non-histone chromatin proteins, and it induces bending in the region of dna to which it binds ( ) . this gene is called sry (sex-determining region of the y chromosome), and there is extensive evidence that it is indeed the gene that encodes the human testis-determining factor ( ) ( ) ( ) ( ) . methods for sex determination using pcr for amplification of sry gene have also been reported ( , ) . these methods can indicate a male genotype by the presence of the amplified product of jri sry gene. nonetheless, the need for trained staff, operating space, equipment, and reagents has impeded its usefulness ( ) . hence, there has been an increasing demand for simple and cost-effective molecular tests. loop mediated isothermal amplification (lamp), a novel nucleic acid amplification method that relies on an auto-cycling strand displacement dna synthesis, is performed by bst dna polymerase ( ) ( ) ( ) ( ) . four or six primers that identify six or eight distinct regions are used in this method. it is operated under a constant temperature ( to °c), and it eliminates the need for specialized thermal cycler equipment ( ) ( ) ( ) ( ) ( ) ( ) . an important advantage of lamp is its ability to amplify specific sequences of dna under isothermal conditions ( , ) . detection of amplification product can be determined via turbidity caused by an increasing quantity of the magnesium pyrophosphate precipitate in solution ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . lamp positive amplicons have been confirmed by adding a number of fluorescent dsdna intercalating dye including ethidium bromide and sybr ® premix ex taq tm ii after the reaction is completed or metal indicators such as magnesium sulphate (mgso ), calcium chloride (cacl ), sybr green i, propidium iodide, genefinder tm , calcein, hydroxynaphthol blue (hnb), phenol red and gel-red tm prior to the reaction, allowing observation with the naked eye ( ) ( ) ( ) ( ) . the purpose of this study was to establish a rapid, sensitive, specific, and easy method to detect sry gene using a visualized detection system. sample collection and plasma dna extraction: a total of blood samples from pregnant women at eight weeks of pregnancy were collected. another set of blood samples from non-pregnant women and men were also obtained using them as plasma negative and positive controls for the study, respectively. fresh blood samples were drawn into vacutainer edta tubes (bd cat. ) immediately and were centrifuged at rpm for min in a refrigerated centrifuge; their plasma fractions were collected and stored at - °c for further analysis. plasma dna was extracted using qiaamp circulating nucleic acid kit (qiagen, cat. ) by following manufacturer's protocol. briefly, ml of plasma was mixed with μl proteinase k, . ml on acl buffer by pulsevortexing for min in a ml conical tube. then the mixture was incubated in a °c water bath for min. then, . ml acb buffer was added to the sample lysate and mixed by pulse-vortexing for min, incubated for min on ice and applied to the qiaamp mini column. following the multiple wash and centrifugation steps, columns were incubated for min at °c and dna was eluted with μl of ave buffer and stored at - °c for further analyses. the extraction quality of nucleic acid was confirmed by . % agarose gel electrophoresis. the nucleic acid was stained with ethidium bromide. the purity of dna was estimated by measuring the absorbance values at nm and nm using uv-vis spectrophotometer (shimadzu uv- , japan). by means of the primer explorer v. software (specific for lamp), specific primers were designed based on sry genes (genbank accession jq . ) (table ). figure a shows the schematic position of lamp primers within sry gene. a basic set of four primers was needed, including fip (forward inner primer), bip (backward inner primer), f (forward outer primer), and b (backward outer primer) to identify six distinct regions on the target dna. to accelerate the lamp reaction, two additional primers, i.e., lf (loop forward primer) and lb (loop backward primer) were also used. fip consisted of the complementary sequence of f , a t-t-t-t linker, and f . bip consisted of b c, a t-t-t-t linker, and the complementary sequence of b c. primers f and b were located outside f and b regions, while loop primers lf and lb were located between f and f and between b and b , respectively ( ) ( ) ( ) ( ) . finally, dna must be purified from cellular material in such a manner to prevent degradation. the absorbance ratios (a /a ) of dna samples were found to be in range of . - . , indicating the purity of dna ( figure b ). the initial optimization of the assay was conducted using a set of four basic primers, and optimal concentrations were determined. the reaction mixture was incubated at temperatures ranging from to °c. the optimal temperature was found to be °c for min. values and concentrations of other components of the reaction were optimized. after detection of lamp by gel electrophoresis, the smeared bands of amplified product indicated the positive reaction (figure a) . our results revealed the positive reaction was highly specific only with fresh blood samples containing xy chromosomes, while no amplification was found in blood samples containing xx chromosomes. our new lamp protocol could successfully identify positive samples and positive control. lamp amplicons were electrophoresed and a large number of fragments (a ladder like pattern) were eventually visualized. as shown in figure a typical ladder pattern of the amplified lamp products with agarose gel electrophoresis was only observed for the positive samples and positive control. the lamp assay showed no detectable amplification of other samples, including the negative control and negative samples. a total of blood samples from pregnant women were seven male embryos ( . %) and eight female embryos ( . %). the authenticity of the results by the follow up was performed after delivery and finally the diagnosis of gender in pregnant women was determined by plasma lamp method. colorimetric assay: lamp amplicons could be detected with the naked eye by adding different visual dyes followed by color changing in the solutions. in this regard, all used visual components could successfully make a clear distinction between positive and negative ones ( figure b ). it is noticeable that positive results by mgso , gene-finder tm , sybr green i, hnb, phenol red, gel-red tm , ethidium bromide and sybr ® premix ex taq tm ii were turbidity, green, green, sky blue, red, orange, yellow and green, respectively. the lamp method, generally, had the following advantages over the other methods such as easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost and being user friendly. in reality, except for section one which takes equal time (see "material and methods" section), lamp overall requires just min to accomplish (as the least demanding detection method) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this, in turn, would simplify the detection procedure and result in saving significant time needed for separating the amplified products on the gel and analyzing the data which are commonly used in other pcr-based methods, including nested pcr, reverse transcription pcr, multiplex pcr and immunocapture pcr ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . such visual methods not only involve some expensive instruments but also during a period of time, exposure to the uv light (because it is harmful to the eyes, even watching for a short period would irritate the eyes and cause symptoms similar to conjunctivitis) as well as ethidium bromide could induce a number of serious negative effects on researchers who use these methods ( ) . more surprisingly, in lamp amplified products can be easily visualized by means of different in-tube color indicators with no essential requirement of additional staining systems; thus, toxic staining materials would be significantly avoided ( ) . simple, cost effective and user friendly equipped labs with some molecular instruments as well as trained personnel are prerequisites to perform pcr assays ( ) . on the contrary, lamp can be easily accomplished just in a water bath or temperature block with no need of thermocycler and gel electrophoresis as the same results were recorded by other researchers ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . likewise, exclusive of the primer designing process which is somehow complicated and sensitive, other phases are simply applicable. on the other hand, the presence of lamp positive amplicons proved to be confirmed by adding a number of fluorescent or metal dyes to the reaction tubes, allowing observation with the naked eye ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in the current study, therefore, lamp amplified products were confirmed by adding all aforementioned visual systems (see "methods" section), either prior to or after the reaction along with forming diverse color patterns depending upon the chemical characteristics of the applied chemical substances as dye. according to our results, despite the precise detection of positive lamp products using all dyes, some were significantly superior when the time of stability, cost and the safety were taken into consideration ( table ) . for instance, regardless of its reasonable color change stability (≥ weeks), when employing ethidium bromide, a uv transilluminator must be available; both of them are toxic, resulting in deleterious consequences on human health and the environment ( ) ( ) ( ) ( ) ( ) ( ) . more interestingly, in some cases, just a little fluorescent emission could be observed in negative tubes, presumably arising from the presence of primers and/or dna templates, leading to an increase in false-positive results ( ) ( ) ( ) ( ) ( ) ( ) . alternative to such problems, the first metal dye called magnesium sulphate was utilized, but just an ephemeral color change (i.e. turbidity; no more than a few seconds) was observed as the same result was reported ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . even though short stability of the color change probably cannot be a significant problem as long as just a few numbers of suspicious samples are used, during assessment of a great quantity of infected samples, most of the time, inaccurate results will be accordingly achieved ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the method, nevertheless, could be thereby attributed as a reliable visual observation approach since on the one hand, it is exploited prior to the reaction and on the other hand, there is no need for toxic devices. anyway, to provide a situation to increase time stability, the second fluorescent dye sybr ® premix ex taq tm ii was employed, leading to a clear green color pattern. notably, regardless of observing a significant growth on time stability (about - days), the method not only requires uv transilluminator but also its stability is more negatively sensitive (in daylight). indeed, since the dye must be added after the amplification as it requires opening of the tubes, the occurrence of cross-contamination risk will be accordingly enhanced ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to avoid such contaminations, using separate rooms can be a solution for lamp setup and analysis. more recently, it has also been pointed out that the visualized lamp assay demands a high concentration of sybr green i, which will hamper the lamp reaction if it is added before lamp reaction, so the close-tube lamp detection based on sybr green i might be difficult ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to abbreviate the contamination hazard and also increase color stability, as a result, two additional metal indicators (i.e. hnb and genefinder tm ) known as close-tube lamp detection were lastly used. interestingly, among several different visual dyes to accurately detect lamp products, both hnb and genefinder tm could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk as the best alternatives ( , , ) . two additional metal indicators (phenol red and gelred tm ) were added before lamp reaction but uv transilluminator was required. meanwhile, the visual inspection of lamp products by means of hnb and genefinder tm metal dyes was seen as advantageous as there was no need for electrophoresis and subsequent staining with carcinogenic ethidium bromide ( ) . lastly, the color brightness and stability of both hnb and genefinder tm in the solutions with positive/negative reactions remained constant after weeks of exposure to ambient light, whereas turbidity caused by precipitation of mgso or sybr ® premix ex taq tm ii was stable for only - s and - days, respectively, which were in agreement with the results reported by previous researchers ( , , ) . however, the results obtained here represent those achievable in the research laboratory setting, and it is uncertain whether these results also apply to actual specimens compared with other lamp assays (table ) . in conclusion, sex determination using detection of sry gene by lamp method in fresh human blood shows the smear and/or ladder band only in male blood samples, but not in female samples. the lamp assay developed in this study is a valuable tool capable of helping in monitoring the purity and identification of sry gene for sex determination. lamp method, all in all, had the following advantages over the other mentioned procedures: (i) no requirement for expensive and sophisticated tools in amplification and detection; (ii) no post-amplification treatment of the amplicons; (iii) a flexible and easy detection approach, by detecting with naked eyes using diverse visual dyes; (iv) high sensitivity; (v) high efficiency; (vi) safety; and (vii) being user friendly. this robust assay is quick, sensitive and specific enough to be applied for gene detection. forensic study of sex determination using pcr on teeth samples novel mutations affecting sry dna-binding activity: the hmg box n h associated with , xy pure gonadal digenesis and the familial non-hmg box r i associated with variable phenotypes identification and molecular modelling of a novel familial mutation in the sry gene implicated in the pure gonadal dysgenesis a novel frame shift mutation in the hmg box of the sry gene in a patient with complete , xy pure gonadal dysgenesis. diagn mol pathol swyer syndrome sry and human sex determination: the basic tail of the hmg box functions as a kinetic clamp to augment dna bending structure-function relationships in human testis-determining factor sry: an aromatic buttress underlies the specific dna-bending surface of a high mobility group (hmg) box xx man with sry gene translocation: cytogenetic characteristics, clinical features and management two new novel point mutations localized upstream and downstream of the hmg box region of the sry gene in three indian ,xy females with sex reversal and gonadal tumour formation mammalian sex--origin and evolution of the y chromosome and sry reliability of dna-based sex tests is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis? loop-mediated isothermal amplification of dna rapid sex chromosomal chimerism analysis in heterosexual twin female calves by loop-mediated isothermal amplification codeposition of dntps detection for rapid lamp-based sexing of bovine embryos a novel loop-mediated isothermal amplification approach for sex identification of columbidae birds development of loop mediated isothermal amplification (lamp) of sry gene in almasi ma and almasi g jri human blood samples for sex determination. rangsit loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples loop-mediated isothermal amplification (lamp): a rapid, accurate, and costeffective diagnostic method for infectious diseases fast and reliable detection of plum pox virus in woody host plants using the blue lamp protocol visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye colorimetric detection of loopmediated isothermal amplification reaction by using hydroxyl naphthol blue applications of loop-mediated isothermal dna amplification detection of coat protein gene of the potato leafroll virus by reverse transcription loop-mediated isothermal amplification development and evaluation of a loopmediated isothermal amplification assay for detection of erwinia amylovora based on chromosomal dna development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza a h n virus loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products development of a loop-mediated isothermal amplification for rapid detection of orf virus visual detection of potato leafroll virus by one-step reverse transcription loop-mediated isothermal amplification of dna with hydroxynaphthol blue dye novels reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus comparison and evaluation of three diagnostic methods for detection of beet curly top virus in sugar beet using different visualizing systems assessment of performance ability of three diagnostic methods for detection of potato leafroll virus (plrv) using different visualizing systems immunocapture loop mediated isothermal amplification for rapid detection of tomato yellow leaf curl virus (tylcv) without dna extraction development and application of loop-mediatred isothermal amplification assay for rapid detection of fusarium oxysporum f. sp. lycopersici a novel and rapid loop-mediated isothermal amplification assay for the specific detection of verticillium dahlia detection of japanese yam mosaic virus by rt-lamp visual detection of potato leafroll virus by one-step reverse transcription loop-mediated isothermal amplification of dna with the genefinder tm dye development of colorimetric loopmediated isothermal amplification assay for rapid detection of the tomato yellow leaf curl virus comparison and evaluation of two diagnostic methods for de-jri tection of npt ii and gus genes in nicotiana tabacum establishment and application of a reverse transcription loop-mediated isothermal amplification assay for detection of grapevine fanleaf virus this study was funded by young researchers and elites club, north tehran branch, islamic azad university, tehran, iran. the authors have no conflict of interest. this study was supported by islamic azad university, north tehran branch. key: cord- - k pqbc authors: lee, j. y.; kim, j. h.; rho, j. y. title: development of rapid and specific detection for the human aichivirus a using the loop-mediated isothermal amplification from water samples date: - - journal: indian j microbiol doi: . /s - - - sha: doc_id: cord_uid: k pqbc human aichivirus a (aiv-a) is classified as a kobuvirus, group iv positive sense single strand rna viruses. the first outbreak of aiv-a was reported from aichi prefecture, japan in . aiv-a exists not only among clinical patients, such as diarrhea, but also in a variety of water environments, as its occurrence is reported across a wide geographical range, from developing to advanced countries. for diagnose of aiv-a from water samples, mostly polymerase chain reaction (pcr) system have been developed. however, loop-mediated isothermal amplification (lamp) assay has not been applied. in this study, developed a lamp method to achieve a rapid, specific and highly sensitive detection of aiv-a. the method developed in this study is aimed specifically at aiv-a. through a specific and non-specific selection and sensitivity test process for the five prepared lamp primer sets, one primer set and optimum reaction temperature were selected. a newly developed method was more rapid (approximately – h), specific and equivalent detection of aiv-a than with the conventional pcrs. in addition, confirm system of positive lamp reaction was developed by using the restriction enzyme aci i and hae iii. for evaluation and verification of developing lamp assay, a was applied to twenty cdna from groundwater samples. this study proved rapid and specific diagnosis of aiv-a from water samples, and it is also demanded to be applicable to other environmental, clinical and food samples. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. evaluation and verification of developing lamp assay, a was applied to twenty cdna from groundwater samples. this study proved rapid and specific diagnosis of aiv-a from water samples, and it is also demanded to be applicable to other environmental, clinical and food samples. keywords aiv-a Á human aichivirus a Á lamp Á loopmediated isothermal amplification Á water sample aichivirus a (aiv-a) is classified as kobuvirus, picornaviridae and group iv (?) ssrna virus [ ] . aiv-a was first reported in in a stool specimen of non-bacterial gastroenteritis in the aichi prefecture, japan [ ] . aiv-a has been believed to be a local, endemic disease until the virus was detected among travelers to japan and southeast asia [ ] . however, it is now reported worldwide in asia, europe, north america, and south america [ ] [ ] [ ] [ ] . although the aiv-a can be detected in patients with diarrhea, detection frequency is very low at - . % [ ] . in addition, mixed infection with other waterborne viruses can occur (i.e. norovirus), and aiv-a characterized as being present in a wide range of geographical locations, from developing countries to advanced countries [ , ] . it has also been reported that the aiv-a may exist in an aquatic environment under natural conditions [ ] . according to a survey of water sources, effluents, rivers, and streams, the aiv-a gene was detected at a high rate in japan, in the united states, and in europe [ ] [ ] [ ] . a method for detecting aiv-a specific genes by using reverse transcription nested polymerase chain reaction (rt-pcr) assay, have been reported [ ] [ ] [ ] . it was also developed on the basis of rt-nested pcr for detection of aiv-a from national project in - , korea [ ] . since the conventional pcr method entails a long time after dna electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. extraction [ ] , it required extensive laboratory effort and labor costs. thus, the loop-mediated isothermal amplification (lamp) has been proposed as one of the methods for the rapid monitoring and mass treatment of specimens [ ] . a number of pcr-based aiv-a detecting system has been developed, however, lamp assay has not been applied. in this study, developed a rapid, specific and highly sensitive detection of aiv-a by using a lamp assay. in the design of a lamp primers to detect aiv-a, the following sequences were collected from the national center for biotechnology information (ncbi); ( ) target viruses (aiv-a strains), ( ) taxonomically similar viruses (aiv-b and c), and ( ) eighteen enteric waterborne viruses. five lamp primer sets were designed by using primerexplorer that targets the p a-b region of human aiv-a (table s ) . it was genetically synthesized to human aiv-a (nc_ ; location - nucleotide), and nucleic acid or plasmid of sixteen reference viruses (rotavirus a, the aiv-a was subjected to a specific reaction for five lamp primer sets. the lamp reaction mixture amounted to a total of ll, containing ll of the template (aiv-a plasmid), ll of the lamp primers [f and b ( pmol)], ll of fip and bip ( pmol), . ll of bstpolymerase ( unit/ll), ll of dntp ( mm), ll of buffer, and . ll of nucleic acid free water. regarding the lamp condition, it reacted for min at °c before reacting for h under four different conditions [ - °c ( °c interval)], thereby completing the reaction of all five lamp primer sets. among the five lamp primer sets, a non-specific reaction occurred in the c and e sets. the aiv-a specific reaction was found in the lamp primers a and b sets, while no reaction was observed under the four conditions in set d. in particular, lamp primer set a exhibited specific reactions under all four conditions. among the varying temperatures in which a reaction occurred in sets a and b, °c was selected (fig. s ) . a check of the non-specific reaction of sixteen reference viruses in the two lamp primer sets with aiv-a specific reaction showed no response in the references of both primer sets (fig. s ) . furthermore, the aiv-a gene was diluted tenfold to evaluate the relative detection sensitivity of the two lamp primer sets. the detection sensitivity of set a was about times higher than that of set b (fig. s a) . for the comparison of lamp and conventional pcr, conventional pcr primer set was selected according to recently reported with high sensitivity [ ] . a comparison of the detection sensitivity of the human aiv-a with conventional pcr sets showed that the highest detection sensitivity was at - , which was about fg, and was equivalent to that of lamp primer set a (fig. s b) . the specific response to lamp primer set a was reverified, and pcr amplification was performed on the lamp outer primer set (f and b ) with its composition shown below: a total of ll was added with ll of the template (aiv-a plasmid) ll each of f and b ( pmol), . ll of hs taq polymerase ( . units/ll, geneall, korea), ll of dntp ( mm), . ll of buffer, and . ll of nucleic acid free water. the conditions were min at °c, and min at °c after repeating cycles ( °c for s, °c for s, and °c for s). the pcr product ( bp) reacted using two restriction enzyme, aci i (c/cgc) and hae iii (gg/ cc) ( units) with °c for h. restriction enzyme treatment resulted with gel electrophoresis, pcr products were digested with ? bp (aci i) and ? bp (hae iii) ( fig. s and s ) . the cdnas of a total of twenty samples from groundwater, were collected to verify aiv-a detection. the collected cdnas reacted by using the most sensitive method among the lamp primer set a developed in this study, and the conventional pcr method [ ] . the lamp results showed that aiv-a was positive in of samples ( %), but conventional pcr results showed of samples ( %). among the aiv-a positive samples of conventional pcr and lamp assay, samples were same positive results, additionally samples were positive results at lamp assay (sample number , , , and ) . one sample showed positive results at conventional pcr but result of lamp assay was negative result (sample number ). however, conventional pcr showed that nonspecific reaction from sample tests (sample number , and ) . for verification of lamp reaction, pcr amplification with positive samples was performed on the lamp outer primer set. after amplification, samples showed that bp positive reaction from lamp outer primer set. outer primer positive samples were cut due to the restriction enzyme, hae iii ( units) from pcr products, with °c, h. digested from pcr products were confirmed with ? bp (fig. ) . in this study, developed a lamp assay that could rapid, specific, and sensitive detection of aiv-a from water samples. there have been cases of sybr or hydroxynaphthol blue application for more rapid monitoring of the lamp assay, which may reduce the time and cost of electrophoresis [ ] . comparison between lamp assay and conventional pcr, lamp assay have advantages in high sensitivity, high specificity and reaction time. lamp assay can be shortened the total duration time about - h as compared to conventional pcr methods, or by about h when compared to the rt-nested pcr used in the environment. also, despite of same sensitivity results from conventional pcr and lamp assay (fig. s a) , lamp assay can detection lower concentration virus in environmental samples, almost % were highly specific and sensitive compare to conventional pcr methods (fig. ) . since the primers used in the lamp reaction anneal specifically to the six locations, only a specific pathogen can be detected more accurately than through the conventional pcr. furthermore, since lamp amplification can be re-verified with the restriction enzyme hae iii. this study is expected to be useful as a diagnose aiv-a from water environment, and also applicable to other environmental, clinical and food samples. virus taxonomy at the xith international congress of virology aichi virus strains in children with gastroenteritis isolation of cytopathic small round virus (aichi virus) from pakistani children and japanese travelers from southeast asia isolation of cytopathic small round viruses with bs-c- cells from patients with gastroenteritis epidemiology of human parechovirus, aichi virus and salivirus in fecal samples from hospitalized children with gastroenteritis in hong kong aichi virus shedding in high concentrations in patients with acute diarrhea molecular characterization of the first aichi viruses isolated in europe and in south america detection and genomic characterization of aichi viruses in stool samples from children in monastir, tunisia etiological role of viruses in outbreaks of acute gastroenteritis in the netherlands from through aichi virus, norovirus, astrovirus, enterovirus, and rotavirus involved in clinical cases from a french oysterrelated gastroenteritis outbreak isolation and molecular characterization of aichi viruses from fecal specimens collected in japan, bangladesh, thailand, and vietnam molecular detection and nucleotide sequence analysis of a new aichi virus closely related to canine kobuvirus in sewage samples aichi virus in sewage and surface water, the netherlands molecular detection and characterization of aichi viruses in sewage-polluted waters of venezuela prevalance and genetic diversity of aichi viruses in wastewater and river water in japan aichi virus : enviromental occurrence and behavior. pathogens development and verification of genetically diagnostic method for the detection of non-regulated viruses from water environment (i) loop-mediated isothermal amplification assay to rapidly detect wheat streak mosaic virus in quarantined plants development of rapid and highly sensitive detection of bean common mosaic necrosis virus in leguminous crops using loop-mediated isothermal amplification assay publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -krim zt authors: wang, deguo title: one-pot detection of covid- with real-time reverse-transcription loop-mediated isothermal amplification (rt-lamp) assay and visual rt-lamp assay date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: krim zt background rapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by covid- . objective this study was to establish one-pot real-time reverse-transcription loop-mediated isothermal amplification (rt-lamp) assay and one-pot visual rt-lamp assay for the detection of covid- . methods six specific lamp primers targeting the n gene of covid- were designed, the rt-lamp reaction system was optimized with plasmid puc containing n gene sequence, the detection limit was determined with a serial dilution of the plasmid puc containing n gene sequence, and the one-pot real-time rt-lamp assay and one-pot visual rt-lamp assay for the detection of covid- were established. results our results showed that the one-pot rt-lamp assays can detect covid- with a limit of ≥ copies per μl− of puc containing n gene sequence. conclusion this study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of covid- . the outbreak of covid- , firstly reported from chinese wuhan in december , has spread to many other countries in the last few weeks , and has caused almost infections and more than people died. the virus spreads so quickly that it has attracted the globe attention, and rapid diagnosis is one of the effective ways to prevent and control the diseases. chest ct and rt-pcr has been used for the clinical diagnosis of the coronavirus pneumonia , , however, chest ct needs ct equipment and put the professional operator at risk of infection, and rt-pcr has high professional and technical requirements for operators. loop-mediated isothermal amplification (lamp) can amplify nucleic acids under isothermal conditions , and it had the advantages over real-time pcr assays in specificity sensitivity, cost effectiveness and rapidity , , . the objective of this study was to develop the one-pot real-time rt-lamp assay and the one-pot visual rt-lamp assay for diagnosis of the pneumonia caused by covid- , which would be suitable for use at less developed areas. the rt-lamp primers targeting the n gene of covid- (genbank accession no. mn . ) were designed using primerexplorer v (http://primerexplorer.jp/e/) and oligo (molecular biology insights, inc. colorado springs, co, usa) software packages. the primer sequences are gcc aaa agg ctt cta cgc a (f ), ttt ggc ctt gtt gtt gtt gg (b ), tcc cct act gct gcc tgg agt ttt cgg cag tca agc ctc ttc (fip), tcc tgc tag aat ggc tgg caa ttt ttt ttg ctc tca agc tgg ttc a (bip) , cga cta cgt gat gag gaa cga (lf) and gcg gtg atg ctg ctc t (lb), table , and the length of the targeted sequence was bp. the n gene (genbank accession no. mn . ) of covid was chemically synthesized and cloned into puc plasmid (herein referred to as puc -n dna) by general biosystems (anhui) co., ltd, the puc -n dna was used as the template for optimization of the rt-lamp system, as well as for determination of sensitivity. the real-time rt-lamp assay with above designed rt-lamp primers was performed in a -μl reaction mixture containing . mm each of forward inner primer (fip) and backward inner primer (bip), . mm each of forward outer primer (f ) and backward outer primer (b ), . mm of forward loop primer (lf) and backward loop primer (lb), . mm dntps, × bst dna polymerase buffer (zhengzhou shenxiang industrial co., ltd, china), × evagreen, × rox, pg puc -n dna, and u bst dna/rna polymerase . (new england biolabs, inc., ma, usa) , . the reaction mixtures were heated at °c , °c , °c and °c for min ( s per cycle), individually. the amplification plot and melt curve were obtained using a stepone tm system (applied biosystems, foster city, ca, usa). because the rna extraction step was omitted, the effect of inhibitors in blood samples on the amplification efficiency of the rt-lamp assays was to be determined. µl, . µl, µl and . µl pork blood samples (purchased from xuchang market, china) free of covid- were dissolved in µl -ncov-fast-sample nucleic acid releasing agent, respectively, which were added to above µl real-time rt-lamp reaction system and heated at the optimized temperature for min ( s per cycle) in stepone tm system (applied biosystems, foster city, ca, usa). the analytic sensitivities of the newly developed real-time rt-lamp assay and visual rt-lamp assay were determined with puc -n dna ranging from - . fg, and the reaction mixtures were heated at the optimal temperature for min in a steponetm system (applied biosystems, foster city, ca, usa) or in a water bath, when water bath was used, the reaction tube was sunk into water. the stability and the repeatability of the real-time lamp system were tested with one-month interval. the test was carried out at the optimized temperature determined as described above with four positive controls ( pg puc -n dna) and four negative controls (ddh o) in a stepone tm system (applied biosystems, foster city, ca, usa). for the rt-lamp reaction, as shown in figure , all positive controls (with puc -n dna as template) had amplification, and the results of all negative controls (dna template substituted by ddh o) were negative at °c, °c, °c and °c , however, there was no significant difference in the amplification efficiency, and °c was temporarily was selected for the subsequent experiments. the effect of inhibitors in blood samples on the amplification efficiency had been determined, as figure indicated, when the real-time rt-lamp reaction system was added with µl blood samples dissolved in µl -ncov-fast-sample nucleic acid releasing agent (hunan shengxiang biology technology co., ltd, china), there was no amplification; when . µl blood samples and µl -ncov-fast-sample nucleic acid releasing agent added, there was slight amplification; when µl or . µl blood samples dissolved in µl -ncov-fast-sample nucleic acid releasing agent added, there was acceptable effect on the real-time rt-lamp reaction. therefore, the blood sample volume of µl was selected for µl one-pot real-time or visual rt-lamp reaction. the detection limits of the one-pot real-time rt-lamp assay and the one-pot visual rt-lamp assay were determined using puc -n dna ranging from - . fg at °c for min in a steponetm system (applied biosystems, foster city, ca, usa) or in a water bath. the detection limits of both the one-pot real-time rt-lamp assay and the one-pot visual rt-lamp assay were found to be ≥ . fg puc -n dna (figure ) , which was equivalent to ≥ copies. the stability and the repeatability of the real-time lamp system were tested with one-month interval. as shown in figure , the reactions of four positive controls were positive with the same amplification plot and melt curve, while the reactions of all negative controls were negative with the same melt curve. this demonstrated that the newly established real-time lamp assay was robust and repeatable. the one-pot real-time rt-lamp assay and one-pot visual rt-lamp assay for detection of covid- were established in the study, and the detection limit was ≥ copies. although the specificity of the established rt-lamp assays had been not determined, they were still considered to be highly specific for following reason. upon alignment in dna data bank of japan, the target sequence of established rt-lamp assays had % identities ( / ) with that of covid- strains, and had % identities ( / ) with that of congeneric sars coronavirus strains, among which there were bp on the rt-lamp primers, it was reported that bp primer-template mismatches extend the detection time from min to min , therefore, the established rt-lamp assays were theoretically highly specific. the bst dna/rna polymerase . (new england biolabs, inc., ma, usa) had been used in the study. the enzyme has faborable perforance of both amplification and reverse transcription activity, so it can use either dna or rna as template , , no reverse transcriptase is needed, and the estalished assays can be directly used for detection of the rna virus. the nucleic acid extraction had been omitted in the established one-pot real-time or visual rt-lamp assays, which had greatly reduced the infection risk of the operators. furthermore, it was recommended the visual rt-lamp assay over the real-time rt-lamp assay, one of four negative controls in above sensitivity determination of the real-time rt-lamp assay had amplification, while all four negative controls of the visual rt-lamp assay had no amplification, because the aerosol that may leak was washed by water in water bath, the false positive rate caused by aerosol of the real-time rt-lamp assay was higher than that of the visual rt-lamp assay which sunk the reaction tube in water. this study had established the one-pot real-time rt-lamp assay and one-pot visual rt-lamp assay for the detection of covid- , and provided the rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of covid- . no external funding the covid- epidemic chest imaging appearance of covid- infection detection of novel coronavirus ( -ncov) by real-time rt-pcr loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers development and evaluation of a loop-mediated isothermal amplification (lamp) method for detecting listeria monocytogenes in raw milk rapid detection of listeria monocytogenes in raw milk with loop-mediated isothermal amplification and chemosensor evaluation and improvement of lamp assays for detection of escherichia coli serogroups o , o , o , o , o , o , and o . afri health sci development of a real-time loop-mediated isothermal amplification (lamp) assay and visual lamp assay for detection of african swine fever virus (asfv) effect of internal primer-template mismatches on loop-mediated isothermal amplification a novel thermostable polymerase for rna and dna loop-mediated isothermal amplification (lamp) innate reverse transcriptase activity of dna polymerase for isothermal rna direct detection the author declares no competing interests. key: cord- -zlah u s authors: günther, sonja; felten, sandra; wess, gerhard; hartmann, katrin; weber, karin title: detection of feline coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zlah u s feline infectious peritonitis (fip) is a fatal disease in cats worldwide. the aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect feline coronavirus (fcov) in body cavity effusions of cats with and without fip, in order to minimize the time from sampling to obtaining results. rna was extracted from body cavity effusion samples of cats, including samples from cats with a definitive diagnosis of fip, and samples of control cats with similar clinical signs but other confirmed diseases. two reaction mixtures (isothermal mastermix, optigene ltd.and pcrun™ molecular detection mix, biogal) were tested using the same primers, which were designed to bind to a conserved region of the fcov membrane protein gene. both assays were conducted under isothermal conditions ( °c– °c). using the isothermal mastermix of optigene ltd., amplification times ranged from and min with a sensitivity of . % and a specificity of . % for the reported sample group. using the pcrun™ molecular detection mix of biogal, amplification times ranged from to min with a sensitivity of . % and a specificity of . %. although the rt-lamp assay is less sensitive than real time reverse transcription pcr (rt-pcr), it can be performed without the need of expensive equipment and with less hands-on time. further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of fip. feline coronavirus (fcov), a member of the genus alphacoronavirus of the subfamily coronavirinae, family coronaviridae within the order nidovirales (de groot et al., ) , belongs to a group of enveloped, positive-sense rna viruses that cause diseases in several species, such as severe acute respiratory syndrome (sars) in humans or transmissible gastroenteritis (tge) in pigs. despite the high prevalence of fcov infections in the cat population worldwide, only - % of fcov-infected cats develop the fatal disease feline infectious peritonitis (fip) (addie and jarrett, ) . this change of virulence of a harmless fcov biotype that usually causes no clinical signs into the pathogenic variant is thought to be caused by mutations in the fcov spike protein gene (chang et al., ; vennema et al., ) . these mutations cause a change in tropism from enterocytes to macrophages, giving fcov the ability to infect and effectively replicate within cells of the macrophage lineage and cause a lethal systemic disease with multi-organ involvement (pedersen, ) . the median survival time of cats with effusive fip is only a few days (ritz et al., ) , and the diagnosis of fip commonly leads to euthanasia, since to date, no treatment has been proven to be effective. cats with fip show nonspecific clinical signs such as fever, weight loss and anorexia, often accompanied by body cavity effusions and/or ocular and neurological signs. a definitive diagnosis of fip ante mortem remains challenging, especially when no body cavity effusions can be detected (hartmann et al., ) . presently, the gold standard for the diagnosis of fip is considered to be immunostaining of fcov antigen in macrophages within tissue lesions, a technique that requires invasive tissue collection (kipar and meli, ) . in cats with fip, fcov can be detected by rt-pcr in cell-free body cavity effusions in more than % of the cases, while serum or blood samples often are negative (doenges et al., ) . for both immunostaining and rt-pcr, samples have to be sent to specialized laboratories, resulting in the delay of diagnostic results. this leads to further unnecessary testing for other diseases, to withholding necessary therapy of other treatable diseases, or to delayed euthanasia in cats suffering from severe signs of fip. therefore, a fast and simple point of care test would be very beneficial in the diagnostic process. loop-mediated isothermal amplification (lamp) is a simple, rapid, and cost-effective nucleic acid amplification method (notomi et al., ) and is already used for the detection of coronaviruses in humans and several animal species (hong et al., ; nemoto et al., ) . a set of four to six primers is used, that form products with self-hybridizing loop structures. by using a dna polymerase with strand displacement activity, no melting or annealing steps are required, and amplification products of different lengths are formed at a constant temperature of - °c (nagamine et al., ) . since lamp reactions only require a simple heat block with constant temperature, and dna amplification can be detected by fluorescence or color change, the method can be applied for point-of-care diagnostics (surabattula et al., ) . the aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription lamp (rt-lamp) to detect fcov in body cavity effusions of cats with and without fip, and to minimize the time from sampling to obtaining results. this study included cats that were presented to the clinic of small animal internal medicine, lmu munich, germany. all cats included had body cavity effusions. in every cat presenting with body cavity effusions, fip is a potential differential diagnosis. an earlier study showed that fip is responsible for about % of effusions, while most of the remaining cases were caused by malignomas, cardiac insufficiency or purulent serositis (hirschberger et al., ) . the fip group (n = ) included cats with a definitive diagnosis of fip by one or more methods: all effusions of cats with fip tested positive for fcov by rt-pcr by a commercial laboratory, and in / samples putative disease-causing mutations could be detected. the rt-pcr detection method has been described previously (felten et al., ) . in / cats fip diagnosis was achieved by post-mortem examination, including full body necropsy with histopathological examination. diagnosis of fip was confirmed when typical histologic lesions where detected (surfacebound multi-systemic pyogranulomatous and fibrinonecrotic disease with venulitis with or without high-protein exudate). in / cats with full body necropsy immunohistological staining for fcov-antibody was done on tissue sections and returned a positive result. immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, and all samples returned a positive result. a summary of the cases in the fip group can be found in the supplementary table . cats were included in the control group (n = ) if they were definitively diagnosed with a disease other than fip that explained the effusion. cats of the control group suffered from neoplasia (n = ), decompensated cardiac diseases (n = ), inflammatory diseases (n = ), such as bacterial peritonitis and pleurisy, or other diseases (n = ). one cat had chronic thoracic chylous effusion of unknown origin and secondary fibroplastic pleurisy. in another cat, an end stage kidney disease caused effusion, and one cat had thoracic effusion after subcutaneous urethral bypass placement, which resolved after treatment. the diseases of the cats of the control group (n = ) were definitively confirmed ante-mortem (n = ) or at necropsy with histopathological examination (n = ). ante-mortem diagnosis was established by echocardiography for cardiac diseases (n = ), and by cytology for neoplasia (n = ). immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, with three positive and eight negative results. all effusions of the cats in the control group were tested for fcov by rt-pcr, and all results were negative. the rt-pcr detection method has been described previously (felten et al., ) . a summary of the cases in the control group can be found in supplementary table . body cavity effusion samples of all cats were obtained ante mortem with ultrasound guidance for diagnostic purposes. the use of samples for this study was approved by the institutional animal care and use committee ('ethikkommission des zentrums für klinische tiermedizin'), permission number - - - . samples were stored at - °c in a . ml eppendorf safe-lock microcentrifuge tube until assayed. all samples were centrifuged for s at , × g. the supernatant of centrifuged thoracic and abdominal fluids was used for rna extraction. when using fresh fluid samples, omission of the centrifugation step should be considered to include intact cells with a high viral burden (pedersen et al., ) . in thawed samples, cell integrity is lost and cell debris can be removed. viral rna was isolated using the commercial zr viral rna kit™ (zymo research corp.) following the manufacturer's instructions. briefly, μl aliquots of samples were mixed with a buffer that facilitates viral particle lysis and allows for rna adsorption onto the matrix of the zymo-spin™ column. then the rna was washed and eluted with μl of rnase free water. extracted rna aliquots were stored at − °c in an eppendorf . ml safe-lock microcentrifuge tube until further processing. the rt-lamp primer design was assisted by the software primerexplorer (https://primerexplorer.jp/e/). based on sequence analysis, the gene for the membrane protein (m) was selected as a target because it is highly conserved among fcov strains. the dna sequence from position , to , of the fcov strain black (genbank accession number: eu . ) was used to design the rt-lamp primers used in this study. a set of six primers, including two outer primers (forward primer f and backward primer b ), two inner primers (forward inner primer fip and backward inner primer bip), and two loop primers (forward loop primer loopf and backward loop primer loopb) were selected as the target sequence ( fig. and table ). detection of fcov was performed using rt-lamp. two different commercial reaction mixtures (isothermal mastermix by optigene ltd., uk, pcrun™ molecular detection mix by biogal, israel,) were compared using the same set of primers. for the amplification following the isothermal mastermix protocol, the total volume of μl per reaction tube included μl isothermal master mix, μl template, μl primer mix and . μl superscript® iii reverse transcriptase (thermo scientific). the primer mix consisted of pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a real-time pcr system (applied biosystems). during rt-lamp, fluorescence of dna products was measured once every minute (fam detection channel, λ max nm), and the time to threshold crossing was analyzed. a positive sample (positive in rt-pcr and sequenced for mutations) was included in every run. all samples run in the realtime pcr system were subjected to a melt curve analysis after the run. a single sharp peak in the melt curve analysis demonstrates amplification of a single pcr product. the positive samples all showed a single peak and the same melting temperature as the positive control sample. following the pcrun™ molecular detection protocol the reaction mix contained . μl of luminescent reagent, μl of template, μl of primer mix and additional . μl superscript iii reverse transcriptase (thermo scientific). the primer mix consisted of . pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a pcrun™ reader (biogal). amplification was detected by measuring bioluminescence twice every minute (fig. ) . pcr products of both methods were verified on an agarose gel showing a typical pattern of multiple bands of different molecular weights (fig. ) . the results of the samples in the fcov rt-lamp assays using two different commercial reaction mixtures are shown in table . two samples tested false positive using the isothermal mastermix and one sample tested false positive with the pcrun™ molecular detection mix. sensitivity and specificity for the reported sample groups of both rt-lamp assays are shown in table . the pcrun™ molecular detection mix performed better both in sensitivity and specifity than the isothermal mastermix. the amplification times for the isothermal mastermix positives ranged between and min and for the pcrun™ molecular detection mix positives between - min. in the present study, rt-lamp assays were evaluated as a diagnostic tool for detection of fcov, in order to distinguish cats with and without fip that are presented with body cavity effusions. time from sample to result was kept to a minimum by isolating rna in about to min using a simple rna extraction kit. both commercially available reaction mixtures allowed an easy and fast preparation of the amplification reaction. with lamp assays, direct detection methods built into the amplification device are preferred, since opening lamp reaction tubes after amplification is not advisable to decrease the risk of carry-over contamination (parida et al., ; zanoli and spoto, ) . in the present study, a portable device results was used for the pcrun™ molecular detection mix. positive and negative amplification reactions were indicated by the device with '+' and '-', making it compatible as a point-of-care instrument. the isothermal mastermix is intended for use with a portable device, which was not part of this study. the machine used instead replicates the reaction conditions with a constant block temperature and uses a comparable fluorescence detection system. both assays tested have similar demands concerning handling skills and preparation time. detection of positive samples took about half the time with isothermal mastermix compared to the pcrun™ molecular detection mix, yet both tests require less than min. the specificity of both methods for the reported sample group was comparable, with . % and . %, respectively. however, false positive results in a test that diagnoses a fatal disease are very critical and should not occur. a cross-reaction of the lamp-primers or carry-over contamination might be the cause of these false positive results. however, the negative controls without sample material did not show any indication for carryover contamination and stayed negative in the lamp assays. another possibility for false positive results is the detection of fcov in cats without fip, since systemic spread of fcov does occur, but does not inadvertently result in fip (porter et al., ) . this explanation is not very likely for the three samples of cats without fip that were positive by rt-lamp, since all three were negative by rt-pcr. the sensitivity of the pcrun™ molecular detection mix was superior for the reported sample group with . % compared to . % of the isothermal mastermix, using the same primers and pcr conditions. since the samples for the rt-pcr and for the rt-lamp had the same preanalytical treatment (frozen, thawed, and centrifuged), the results can be directly compared and showed that the rt-pcr for fcov performed much better than the rt-lamp in our sample group. this is in agreement with a study on other coronaviruses, where rt-lamp also exhibited a lower analytical sensitivity compared to rt-pcr (bhadra et al., ) . false negative results can occur in samples with a lower viral burden. the fcov viral load determined by rt-pcr in effusions has been found to be quite low in some samples of fip-suspected cats (lorusso et al., ) . in our study, the results for rt-pcr were only returned as 'positive' or 'negative' without quantification, leaving open the question whether only effusions with a high viral load resulted in a positive rt-lamp detection. another possible reason for the lack of sensitivity might be that sequences of current fcov strains show sequence variations compared to the sequences deposited in the genbank database, which were used to design the rt-lamp primers. although the primers were chosen to bind in highly conserved regions, variations can occur, which might impair binding and eventually lead to low or no amplification, resulting in poor sensitivity. reliable primers for rt-pcr target the ′ utr of the fcov sequence, but the rt-lamp primers that were suggested by the primerexplorer software in this region included more sequence differences than the m region that we selected. modifications of the primers might enhance binding and improve sensitivity. two studies on lamp-based identification of fcov have been published to date. a study from thailand tested samples of body cavity effusions from cats that were suspected to have fip both by rt-pcr and by rt-lamp. more samples tested positive with the rt-lamp than with the rt-pcr ( % vs. %) (techangamsuwan et al., ) . however, the inclusion criteria for cats to be suspected of having fip were not described in that study. their control samples consisted of plasma and fecal samples from healthy cats without any contact to other cats. the samples from this healthy control group also had more positive results by rt-lamp than by rt-pcr ( % vs %). the authors mention high rates of false positives in the negative controls (no template controls) when using rt-lamp, so it remains unclear whether their rt-pcr was less sensitive or their rt-lamp was prone to unspecific amplification. in the second study, different sample types of cats with a clinical suspicion of fip and fecal samples for screening for fcov-shedding cats were tested (stranieri et al., ) . in most sample types, including effusions, their rt-pcr had about twice as many positive results as their rt-lamp, and none of the rt-pcr-negative samples was positive in the rt-lamp method. in agreement with our findings, the sensitivity of the rt-lamp appears to be inferior to the rt-pcr. for detection of amplification, both studies used gel electrophoresis and one study (stranieri et al., ) additionally used detection of color change from violet to blue with hydroxynaphtol blue. while gel electrophoresis is quite time-consuming, the color change can be difficult to detect in samples with low amplification. amplification detection with fluorescence or luminescence as used in the present study is preferable, since the results can be read immediately and could be easily converted to a quantitative format with a standard curve. as a perspective for the future, rt-lamp reactions for virus detection could be run on new devices that integrate nucleic acid extraction, amplification and detection in a miniature format to achieve a true point-ofcare diagnosis (stumpf et al., ) . in conclusion, the rt-lamp in the present study was relatively specific but not very sensitive. the sample type was restricted to effusions of cats unequivocally diagnosed with fip and of cats without fip but with clinical signs indicative of fip. this is a realistic setting in which a veterinarian would use a test for detection of fcov in the effusion sample. the rt-lamp assay with the pcrun™ molecular detection mix can be used in a clinical setting to a certain extent. however, before it can replace conventional rt-pcr methods, sensitivity and specificity have to be enhanced by optimizing primers and amplification conditions. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the pcrun™ molecular detection mix and the pcrun™ reader were provided by biogal free of charge. biogal was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the fcov rt-pcr and subsequent mutation detection was done by idexx free of charge. idexx was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. a study of naturally occurring feline coronavirus infections in kittens real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) spike 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the detection of feline coronavirus labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h n virus simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (lamp) development and application of reverse transcription loop-mediated isothermal amplification (rt-lamp) for feline coronavirus detection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses isothermal amplification methods for the detection of nucleic acids in microfluidic devices we thank biogal for providing the pcrun™ molecular detection mix and the pcrun™ reader for this study. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- -pk dl q authors: hu, xuejiao; deng, qianyun; li, junmin; chen, jierong; wang, zixia; zhang, xiqin; fang, zhixin; li, haijian; zhao, yunhu; yu, pan; li, wenmin; wang, xiaoming; li, shan; zhang, lei; hou, tieying title: development and clinical application of a rapid and sensitive loop-mediated isothermal amplification test for sars-cov- infection date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: pk dl q the severe acute respiratory syndrome coronavirus (sars-cov- ) outbreak urgently necessitates sensitive and convenient covid- diagnostics for the containment and timely treatment of patients. we aimed to develop and validate a novel reverse transcription–loop-mediated isothermal amplification (rt-lamp) assay to detect sars-cov- . patients with suspected covid- and close contacts were recruited from two hospitals between january and april . respiratory samples were collected and tested using rt-lamp, and the results were compared with those obtained by reverse transcription-quantitative pcr (rt-qpcr). samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. rt-lamp was also applied to an asymptomatic covid- carrier and patients with other respiratory viral infections. samples were collected from a cohort of cases ( nasopharyngeal swabs) and an independent cohort of patients ( nasopharyngeal swabs and sputum samples). the rt-lamp assay was validated to be accurate (overall sensitivity and specificity of . % and . %, respectively) and diagnostically useful (positive and negative likelihood ratios of . and . , respectively). rt-lamp showed increased sensitivity ( . % versus . %) and high consistency (kappa, . ) compared to those of rt-qpcr for sars-cov- screening while requiring only constant-temperature heating and visual inspection. the time required for rt-lamp was less than h from sample preparation to the result. in addition, rt-lamp was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. the developed rt-lamp assay offers rapid, sensitive, and straightforward detection of sars-cov- infection and may aid the expansion of covid- testing in the public domain and hospitals. importance we developed a visual and rapid reverse transcription–loop-mediated isothermal amplification (rt-lamp) assay targeting the s gene for sars-cov- infection. the strength of our study was that we validated the rt-lamp assay using clinical respiratory samples from two prospective cohorts of suspected covid- patients and on the serial samples from an asymptomatic carrier. the developed rt-lamp approach showed an increased sensitivity ( . %) and high consistency (kappa, . ) compared with those of reverse transcription-quantitative pcr (rt-qpcr) for sars-cov- screening while requiring only constant-temperature heating and visual inspection, facilitating sars-cov- screening in well-equipped labs as well as in the field. the time required for rt-lamp was less than h from sample preparation to the result (more than h for rt-qpcr). this study showed that the rt-lamp assay was a simple, rapid, and sensitive approach for sars-cov- infection and can facilitate covid- diagnosis, especially in resource-poor settings. preparation to the result (more than h for rt-qpcr). this study showed that the rt-lamp assay was a simple, rapid, and sensitive approach for sars-cov- infection and can facilitate covid- diagnosis, especially in resource-poor settings. keywords rt-lamp, sars-cov- , covid- , asymptomatic carriers, clinical diagnosis t he skyrocketing covid- outbreak has become a public health emergency of international concern. a total of , , confirmed cases and , deaths have been reported in countries since early december of , as of august , according to the who covid- report ( ) . at present, no effective drugs or vaccines have been reported for covid- , and prompt diagnosis, close contact tracking, and quarantine management are the hallmarks for the containment of this new pandemic. early and accurate diagnosis of severe acute respiratory syndrome coronavirus (sars-cov- ) infection is crucial to prevent virus transmission and provide appropriate treatment for patients. due to its nonspecific symptoms and radiological features overlapping those of the common cold and influenza, the confirmation of sars-cov- infection depends entirely on viral rna detection ( , ) . reverse transcriptionquantitative pcr (rt-qpcr) is the standard and most widely used method for sars-cov- rna detection in clinical laboratories ( ) . despite its outstanding analytical performance, rt-qpcr-based approaches to detect covid- still suffer from many limitations, such as long turnaround times ( to h), poor availability (it is currently restricted to public health laboratories), the need for expensive instrumentation, and a high proportion of false-negative results or equivocal values (up to %) ( , ) in upper respiratory samples due to insufficient viral materials. these limitations render the rt-qpcr test far from adequate to meet the current challenge of a tremendous undocumented infected population, asymptomatic transmission ( ) , and convalescence with viral rna conversion ( ) , highlighting the pressing need for a more rapid, simple, and sensitive approach to quickly identify infected patients in different settings. loop-mediated isothermal amplification (lamp) is regarded as a promising pointof-care test (poct) due to its advantages of high sensitivity and specificity, rapid reaction, and low laboratory infrastructure requirements ( ) . reverse transcription-lamp (rt-lamp) is a type of lamp method used to detect target rna with the avian myeloblastosis virus (amv) reverse transcriptase. this approach allows reverse transcription and dna amplification to be rapidly accomplished at a constant to °c temperature in less than h and in one step, and detailed amplification mechanisms were previously reported ( ) . rt-lamp results can be detected by visual turbidity or fluorescence in real time, rendering this method a practical near-patient assay. in recent years, rt-lamp has been widely used in specialized laboratory testing as well as field surveys to identify various pathogens, including mycobacterium tuberculosis ( ) , zika virus ( ) , middle east respiratory syndrome coronavirus (mers-cov) ( ) , and sars-cov ( ) . shirato et al. ( ) reported the development of a useful rt-lamp assay for the diagnosis of mers that was developed in this manner, with a detection limit of . copies per reaction and no cross-reactivity with other respiratory viruses. in addition, hong et al. ( ) developed a real-time quantitative rt-lamp assay for early and rapid diagnosis of sars-cov that demonstrated -fold greater sensitivity than conventional rt-qpcr assays. to accelerate clinical diagnostic testing for covid- , we conducted a prospective cohort study to develop and validate a novel rt-lamp assay capable of detecting sars-cov- rna for potential use in centralized facilities and point-of-care settings. moreover, we compared rt-qpcr and rt-lamp using clinical samples and demonstrated that rt-lamp had higher sensitivity and cost effectiveness for sars-cov- detection. to the best of our knowledge, this study is the first to comprehensively assess a rapid rt-lamp test for both covid- patients and an asymptomatic carrier, the results of which demonstrated that the test has improved diagnostic value over that of current diagnostics for sars-cov- infection. development of an rt-lamp assay. as described in materials and methods, in this study, we developed a rapid and simple rt-lamp assay to detect sars-cov- rna, where positive reactions resulted in a color change from purple to blue due to a decreased magnesium concentration in the presence of extensive bst dna polymerase activity, while negative reactions retained the purple color. figure s in the supplemental material shows the overall procedure of the rt-lamp assay. rt-lamp primers for covid- were specific and had a . % to . % nucleotide mismatching with sars, mers, and other coronavirus sequences (see table s ). furthermore, the crossreactivity experiment results demonstrated that the rt-lamp assay did not cross-react with other human-pathogenic coronaviruses and common viral pathogens, supporting the specificity of this assay for covid- (see fig. s ). dilution experiments with the synthetic sars-cov- s gene were performed to determine the limit of detection (lod) of rt-lamp relative to that of the rt-qpcr assay for the detection of sars-cov- (see fig. s ). the observed lod values for the rt-lamp and rt-qpcr assays were approximately . ϫ Ϫ ng per -l reaction solution (i.e., . copies/reaction) and . ϫ Ϫ ng/reaction solution (i.e., . copies/reaction), respectively. the rt-lamp assay exhibited a -fold higher sensitivity than the rt-qpcr assay currently being used in clinical settings, which is similar to the results of previous lamp-based assays ( , ) . characteristics of the subjects. we ultimately collected a prospective cohort of patients from guangdong provincial people's hospital (cohort i: the laboratory-confirmed covid- patients had a median age of . years (interquartile range [iqr], to years), . % ( / ) were male, and most of the patients reported an exposure history and presented primarily with fever, cough/expectoration, and muscle pain/fatigue ( table ) . most of the covid- patients ( . %) were identified as nonsevere cases, and only patients were severe cases on admission. forty of all covid- patients ( . %) manifested with chest computed tomography (ct) imaging abnormalities, with the most common chest ct patterns being ground-glass opacities ( . %) and bilateral patchy shadowing ( . %). the remaining ( . %) cases showed normal ct images. twenty-one ( . %) patients had comorbidities, . % of whom had hypertension and . % had diabetes. forty ( . %) patients presented with hematologic abnormalities. the demographic and initial clinical characteristics of the covid- patients in the two cohorts are provided in table . diagnostic potential of the rt-lamp assay for covid- patients and an asymptomatic carrier. we first evaluated the clinical application of the rt-lamp assay on nasopharyngeal specimens from cohort i. of these nasopharyngeal swabs, swabs were confirmed to be sars-cov- positive according to the combined criteria of positive test results ( samples) and next-generation sequencing (ngs) confirmation ( samples) ( table , see also table s and fig. s ). thirty-one of clinically positive samples were determined to be positive using the rt-lamp assay, and of clinically negative samples were observed to show a positive reaction, which were confirmed to be false-positive reactions by ngs. the performance of the rt-lamp assay was as follows: sensitivity, . % ( % confidence interval [ci], . % to . %); specificity, . % ( . % to . %); positive predictive value, . % ( . % to . %); negative predictive value, . % ( . % to . %); positive likelihood ratio, . ( . to . ); and negative likelihood ratio, . ( . to . ) ( table ) . compared with that of the rt-qpcr assay, the rt-lamp assay had significantly better sensitivity ( . % versus . %) and comparable specificity ( . % versus %) for the diagnosis of sars-cov- infection ( table ). the detection results obtained using the rt-lamp assay showed good concordance with those obtained using the rt-qpcr in cohort i, of nasopharyngeal swabs from covid- patients were confirmed to be sars-cov- positive according to the criteria of rt-qpcr ( samples) and ngs confirmation ( samples) (see table s in the supplemental material). c in cohort ii, of samples (paired nasopharyngeal swabs and sputum samples) from covid- patients were determined to be sars-cov- positive accordingly ( were rt-qpcr-positive, and were ngs-positive) ( table s ) . application of rt-lamp for sars-cov- assay, with a cohen's kappa of . ( . to . ), % positive predictive agreement, and . % negative predictive agreement. these observations are in line with data reported in studies by baek et al. ( ) and others ( , ) . in addition to exploring the diagnostic potential of rt-lamp in active covid- patients, we also tested the rt-lamp assay on an asymptomatic covid- carrier. a -year-old female patient presented to our hospital on january with a -year history of congenital heart disease and aggravation of shortness of breath symptoms for month. after admission, she tested positive for sars-cov- infection by rt-qpcr in our hospital without any covid- /viral pneumonia clinical symptoms or ct findings. her oropharyngeal swabs were also sent to the guangzhou cdc for repeat rt-qpcr testing and were confirmed to be sars-cov- positive on february and february. respiratory samples were collected throughout her illness from february to march and subjected to parallel rt-lamp and rt-qpcr assays for sars-cov- detection (fig. ) . ngs was simultaneously performed for samples yielding inconsistent results between the rt-lamp and rt-qpcr assays. the number of positive test results obtained by rt-lamp was . -fold higher than that observed by rt-pcr ( versus ), and rt-lamp-positive but rt-qpcr-negative samples were verified as sars-cov- positive using ngs ( fig. and table s ). during her hospitalization, the rt-qpcr threshold cycle (c t ) values fluctuated and became negative after february, suggesting a continuous viral shedding pattern and a decreased viral load over time (data not shown, available upon request). this case demonstrated that compared to that of rt-qpcr, rt-lamp has higher sensitivity in detecting sars-cov- , particularly in samples with a low viral load, and also suggested that rt-lamp can be used for the diagnosis of asymptomatic covid- carriers. validation of the rt-lamp assay. we next validated the rt-lamp assay in an independent cohort (cohort ii) of covid- patients and covid- exclusion cases. one nasopharyngeal swab and one sputum specimen were collected from every participant in cohort ii. the samples included positive samples ( swabs and sputum specimens) and negative samples (tables and s ). nasopharyngeal swabs from covid- patients showed a higher positive rate than sputum specimens in both the rt-qpcr and rt-lamp assays (rt-qpcr: swab, . % [ / ] [ / ] ). the rt-lamp assay had a sensitivity of . %, whereas that of the rt-qpcr assay was only . %. the specificity of the rt-lamp assay was roughly equivalent to that of the rt-qpcr assay ( . % versus . %) ( table ) , and the agreement between the two assays was excellent (kappa, . [ . to . ]) ( table ). these observations corroborate the results obtained from cohort i as well as previous rt-lamp findings ( ) ( ) ( ) , suggesting that the use of rt-lamp may improve the sensitivity of pathogenic diagnosis for covid- . to further assess whether the rt-lamp assay was specific for covid- , swab specimens from patients with influenza (n ϭ ) or other respiratory viral infections table s ). no positive results were observed, demonstrating that the rt-lamp-based detection approach can distinguish sars-cov- with no crossreactivity for other common respiratory viruses, similar to reports in recent studies ( ) ( ) ( ) . the rt-lamp assay results reported in this study for sars-cov- detection in the two cohorts are summarized as follows. the rt-lamp assay exhibited an overall sensitivity of . % (higher than the . % for rt-qpcr), an overall specificity of . %, high consistency (kappa, . ) with the rt-qpcr assay, and a median turnaround time less than h from sample preparation to the result in the detection of clinical specimens from two cohorts (fig. ) . additional advantages of rt-lamp include cost effectiveness, simple operation, and visual determination capability, which facilitate sars-cov- screening in well-equipped labs as well as in the field (fig. ) . rapid and reliable diagnosis is of particular importance for the containment of covid- outbreaks. in this study, we described a simple and sensitive rt-lamp approach to rapidly diagnose sars-cov- infection. the robustness of the present study was demonstrated, as the rt-lamp assay was useful for the diagnosis of active covid- patients and an asymptomatic carrier and was generally not confounded by other respiratory pathogen infections by using clinical samples from two hospitals. existing methods to detect sars-cov- are primarily based on rt-qpcr, ngs, and igm and igg immunological tests. comparing the results between the rt-lamp and rt-qpcr assays, rt-lamp provided better sensitivity ( . % versus . %) than rt-qpcr for sars-cov- . this added sensitivity is important considering that a significant number of covid- patients have presented with negative qpcr ( ) results or the "relapse after negative" phenomenon ( ) due to potentially large variability between clinical samples, low-viral-titer samples, and even disrupted binding of rt-qpcr primers due to variation in the viral genome ( ) . in this study, we used bst dna polymerase isolated in-house for the developed rt-lamp assay, which was demonstrated to have higher polymerization activity than the commercial bst dna polymerase ( ) and ensured the high sensitivity of this rt-lamp method. based on these findings, we propose that the rt-lamp assay can detect viral rna not only in samples testing positive by rt-qpcr but also in inconclusive samples. we observed that the rt-lamp assay was less sensitive and informative than multiplex pcr-based ngs in our study and other literature ( ) ( ) ( ) ( ) ( ) . ngs is a robust tool for obtaining extensive genetic information, allowing lod values as low as copies/ml for sars-cov- and serving as a reference test for covid- , especially for those challenging samples with a low viral content ( , ( ) ( ) ( ) . however, several experimental issues, such as erroneous barcode sequencing, the production of primer dimers, and potential cross-contamination between runs, may complicate sequence-based analyses and impact the validity of ngs results ( , ) . compared to the complex and costly ngs platform, rt-lamp has the advantages of low-threshold infrastructure, less data processing, and cost effectiveness, enabling this user-friendly assay to be immediately deployed in hospitals and communities. rt-lamp also showed no cross-reactivity with other viruses that manifest similar respiratory diseases such that the specificity of this assay was higher than that reported for igm-/igg-based detection methods ( ) . in addition, we described the accuracy of the rt-lamp assay in detecting sars-cov- by determining likelihood ratios. likelihood ratios are not affected by disease prevalence, and values higher than and lower than one strongly support the diagnostic value of a test ( ) . based on this metric, the near-patient rt-lamp assay used in this study is diagnostically useful for covid- . taken together, the rt-lamp assay established in this study may be a powerful complementary method for monitoring massive numbers of exposed individuals as well as facilitating screening efforts in hospitals and public domains, especially in areas with limited laboratory capacities. nasopharyngeal swabs from covid- patients had a higher positive rate than sputum specimens in both the rt-qpcr and rt-lamp assays. liu et al. ( ) reported that the detection rate of sars-cov- rna in nasopharyngeal swabs was lower than that observed in bronchoalveolar lavage fluid and sputum. this inconsistency is most likely due to poor sputum quality and fluctuations in viral rna levels during different stages of the disease course ( ) . despite this inconsistency, nasopharyngeal swabs are noninvasive and easy to acquire, and evidence has shown that sars-cov- replicates actively in upper respiratory tissue ( ) . therefore, we argue that nasopharyngeal swabs are suitable for the detection of sars-cov- detection at an early stage of infection. we note that four samples from non-covid- cases tested positive in the rt-lamp assay but negative by rt-qpcr (table ) , as reported previously ( ) . the four falsepositive results by rt-lamp were caused by aerosol contaminants, as we retested these samples in another clean room and obtained the expected negative rt-lamp result. contaminant issues are not uncommon for nucleic acid testing, even when the best available reference laboratory tests are used. precautions to prevent crosscontamination or aerosol contaminants during assays are highly recommended, including the use of a spray solution to eliminate potential rna fragments and changing gloves frequently. the rna extraction-free rt-lamp assay can address this important issue ( ) . since this study was completed, the sars-cov- rt-lamp test has been optimized further with the use of lyophilized reagents and the direct detection of sars-cov- without the need for rna extraction. this one-step single-tube rt-lamp assay decreases reaction time and minimizes false-positive reactions, making it an ideal poct for covid- if validated in future studies. one limitation of our study was the relatively small sample size of positive covid- cases, which resulted in widened confidence intervals for our estimates of diagnostic accuracy. we tested the samples using rt-lamp in a blind manner, and the designation of the actual status of sars-cov- infection in clinical samples was based on a set of combined criteria of rt-qpcr results and subsequent ngs confirmation to obviate potential false-negative or false-positive results. we further validated the diagnostic potential of rt-lamp in another independent cohort with nasopharyngeal swabs and sputum samples. therefore, despite our small sample size, our study was sufficiently robust for the rt-lamp assay. in summary, in this study, we developed a simple and rapid rt-lamp assay for sars-cov- detection and demonstrated its high diagnostic sensitivity and specificity among clinical samples. our findings suggest that rt-lamp can be an appropriate auxiliary assay for the diagnosis and epidemiologic surveillance of covid- in different hospital and community settings. this study was designed as a prospective observational cohort study with three sequential phases. in the initial stage, we developed a visual and rapid rt-lamp assay for sars-cov- detection and assessed its anti-cross-interface ability, stability, and detection limit. subsequently, we evaluated the rt-lamp and standard rt-qpcr assays on nasopharyngeal swabs from a cohort of suspected covid- patients and on serial upper respiratory samples from an asymptomatic carrier, and the inconsistent samples between rt-lamp and rt-qpcr were further subjected to next-generation sequencing (ngs) for sars-cov- confirmation. finally, we analyzed an additional patients with other viral infections, healthy individuals, and an independent cohort of cases suspected of having covid- to further validate the detection capacity of rt-lamp for sars-cov- . the overall study strategy is shown in fig. . subjects and sample enrollment. (i) cohort i. inpatients with clinical-radiological suspicion of covid- presenting to guangdong provincial people's hospital between january and april , were eligible for inclusion. close contacts with exposure to confirmed covid- cases were simultaneously enrolled in the present study. every participant underwent a standard set of sars-cov- investigations to test covid- . the patients' demographic, clinical, laboratory, and radiological findings were collected from their medical records. serial nasopharyngeal swabs were collected from patients during hospitalization and close contact screening. the sample sizes for swabs were defined by their availability. at least one nasopharyngeal swab from suspected covid- patients was simultaneously sent to the cdc for double checking as required, where rt-qpcr was routinely utilized for sars-cov- . covid- was diagnosed based on acute respiratory infection syndromes and/or the presence of chest imaging features consistent with viral pneumonia accompanied by confirmation of positive rt-qpcr test results for sars-cov- by the cdc, according to the criteria published in the updated covid- diagnostic criteria, th edition, china. suspected covid- patients from guangdong provincial people's hospital were defined as cohort i in this study and classified into two groups: covid- and non-covid- . covid- patients were further classified as nonsevere cases and severe cases; nonsevere cases included patients with mild and moderate pneumonia, and severe cases indicated patients with severe and critically severe acute respiratory distress syndrome (ards) or oxygen saturation at rest of Ͻ % who required mechanical ventilation or intensive care unit (icu) monitoring ( ) . application of rt-lamp for sars-cov- (ii) cohort ii. we enrolled an independent cohort of suspected covid- patients from guangdong second provincial general hospital for validation. sars-cov- testing and the diagnostic procedures for covid- were identical in the two hospitals. a nasopharyngeal swab and ml of morning sputum were collected from suspected covid- patients to validate the diagnostic performance of rt-lamp for sars-cov- . in addition, nasopharyngeal swab samples obtained from healthy subjects and patients with other respiratory viral infections were used to test the specificity of rt-lamp for sars-cov- detection. rna extraction. swabs were preserved in l of virus preservation solution (tianlong, china), which inactivates viruses and preserves all rna in the specimen. sputum samples were preprocessed by standard n-acetyl-l-cysteine (nalc)-naoh digestion. total rna was extracted from specimens within h of collection using a magnetic bead-based viral rna isolation kit with a da system instrument (daan gene, china) according to the manufacturer's instructions. the extracts were stored at Ϫ °c until use. rna extracted from each specimen was tested for sars-cov- in parallel by rt-qpcr and rt-lamp in a double-blind manner in a biosafety level laboratory. samples yielding inconsistent results between these two methods were further analyzed by ngs for verification. rt-qpcr amplification. rt-qpcr was performed using an officially approved clinical rt-qpcr kit for the abi covid- quantstudio dx real-time pcr system (applied biosystems, usa) according to the manufacturer's protocol (daan gene). primer and probe sets targeting the orf ab and n genes of sars-cov- are provided in table s in the supplemental material. for rt-qpcr, each -l reaction mixture comprised l of reaction buffer, l of enzyme solution, and l of template rna. the cycling program started at °c for min for reverse transcription, followed by °c for min for pcr initial activation and cycles of °c for s and °c for s. a cycle threshold value of less than was defined as a positive test. patients were defined as having laboratory-confirmed covid- when both targets (orf a/b and n genes) yielded positive results, and repeated tests using another approved rt-qpcr kit were necessary for single-target-positive (orf a/b-or n-positive) samples. rt-lamp assay. (i) rt-lamp primer design and testing. the complete genome sequence of sars-cov- (genbank accession number mn . ) was aligned and compared with the genbank nucleotide database gene sequences of all species, including other coronaviruses, to identify conserved sequences. a conserved sequence of the s gene (nucleotide to , no. mn . ) was selected as the target to design our rt-lamp primers because it is highly homologous among various covid- sequences and highly divergent from those of other coronaviruses examined. we designed sets of rt-lamp primers targeting the sars-cov- s gene sequence (no. mn . ) using the online primerexplorer v software (available at https://primerexplorer.jp/e/). one set of rt-lamp primers with the best parameters was selected, including two outer primers (f and b ), two inner primers (forward inner primer [fip] and backward inner primer [bip]), and two loop forward (lf) and backward (lb) primers (see fig. s ), all of which were synthesized by invitrogen (shanghai, china). primer specificity was verified with a blast search of the genbank nucleotide database via comparisons with other coronaviruses and published sars-cov- sequences, and the percent mismatch results are presented in table s . the reaction mixtures were incubated in a pcr thermocycler or dry bath at °c for min. the optimal incubation condition of °c for min was determined based on the banding pattern observed after gel electrophoresis and an absorption spectrum analysis of the rt-lamp reactions (see fig. s ). nontemplate controls (ntcs) were included in each run to ensure the absence of contamination. positive reactions could be observed by a visual color change from purple to blue, fluorescent light in response to uv excitation, or by the laddering pattern of bands after gel electrophoresis. cross-reactivity evaluation of the rt-lamp assay. synthesized plasmids of common viral pathogens, including sars, mers, influenza a h n /h n , influenza b, human parainfluenza viruses (hpiv- / / ), respiratory syncytial virus (rsv-a/b), epstein-barr virus, human cytomegalovirus, human mastadenovirus (hadv-b/e), enterovirus (eb-u/ ), human rhinovirus (hrv- / / ), and coxsackievirus (ca ), were used to test potential cross-reactivity in the developed rt-lamp assay. the rt-lamp products obtained using these plasmid templates were assayed by % agarose gel electrophoresis. detection limit of the rt-lamp assay. to determine the lower detection limit of the rt-lamp assay, samples from a -fold gradient dilution series of synthetic sars-cov- s gene cdna ( . ϫ to . ϫ Ϫ ng/reaction) were used as the template in rt-lamp reactions, and the minimum concentration of the positive reaction was recorded. this dilution series was assayed in parallel by rt-qpcr using primers targeting the same region of the sars-cov- genome. the detection limit of the rt-lamp assay was determined by comparing the lowest concentration of the positive reaction with that obtained by rt-qpcr. multiplex pcr-based next-generation sequencing. the samples yielding inconsistent results between the rt-lamp and rt-qpcr assays and those from covid- patients who tested negative by rt-qpcr were further analyzed by multiplex pcr-based enrichment and ngs to detect the sars-cov- genome. briefly, total rna was reverse transcribed to synthesize first-strand cdna with random hexamers and a superscript iii reverse transcriptase kit (vazyme, china). two-step sars-cov- genome amplification was performed with two pooled mixtures of primer sets (designed by genskey medical technology co., ltd.) designed to cover the entire sars-cov- genome. cdna was mixed with the components of the first pcr according to the manufacturer's instructions. the nd pcr was performed using the index primers, and the constructed libraries were sequenced on an illumina novaseq paired-end (pe) platform. data analysis was primarily performed based on an in-house pipeline produced by genskey medical technology. raw sequences were quality trimmed and subsequently filtered if shorter than bases using fastp v . . . sequence reads were first filtered against the human reference genome and then aligned to a reference genome of sars-cov- (nc_ . ) using bowtie v . . . the mapped reads were assembled with spades v . . with kmers ranging from to to obtain the coronavirus genome sequences. statistical analysis. the sensitivity, specificity, positive and negative predictive values, likelihood ratios, and their respective % confidence intervals for the rt-lamp and rt-qpcr assays of nasopharyngeal specimens were calculated, and agreement analysis was performed using kappa concordance coefficients (a value Ն . was deemed good) and percentage agreement (Ն . was considered good) ( ) . statistical analyses were performed in the r programming environment. ethics statement. written informed consent was obtained from all participants before the study, and the study was approved by the ethics committee of each participating institution. the analysis was conducted on samples collected during standard covid- tests, with no extra burden on patients. supplemental material is available online only. coronavirus disease (covid- ) situation report- diagnosis and treatment of novel coronavirus pneumonia (trial version six clinical management of severe acute respiratory infection (sari) when covid- disease is suspected genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention antibody responses to sars-cov- in patients of novel coronavirus disease sars-cov- viral load in upper respiratory specimens of infected patients positive rt-pcr test results in patients recovered from covid- diagnostic accuracy of loop-mediated isothermal amplification as a nearpatient 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syndrome-associated coronavirus infection enhancement of polymerase activity of the large fragment in dna polymerase i from geobacillus stearothermophilus by site-directed mutagenesis at the active site nanopore target sequencing for accurate and comprehensive detection of sars-cov- and other respiratory viruses genomic epidemiology reveals multiple introductions of zika virus into the united states multiplex pcr-based next-generation sequencing and global diversity of seoul virus in humans and rats multiple approaches for massively parallel sequencing of hcov- (sars-cov- ) genomes directly from clinical samples rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus advances in clinical next-generation sequencing: target enrichment and sequencing technologies understanding and overcoming the pitfalls and biases of next-generation sequencing (ngs) methods for use in the routine clinical microbiological diagnostic laboratory molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes diagnostic tests : likelihood ratios positive rate of rt-pcr detection of sars-cov- infection in cases from one hospital in viral load of sars-cov- in clinical samples virological assessment of hospitalized patients with covid- poor concordance between sequential transbronchial lung cryobiopsy and surgical lung biopsy in the diagnosis of diffuse interstitial lung diseases we thank xie mingzhou and li lifeng for assistance with processing and interpreting the sequencing data. we declare no competing interests. key: cord- - nu lz authors: jiang, kuiyu; zhu, ying; liu, wenxin; feng, yufei; he, lili; guan, weikun; hu, wenxia; shi, dongfang title: development of a loop-mediated isothermal amplification (lamp) for the detection of f fimbriae gene in enterotoxigenic escherichia coli (etec) date: - - journal: curr microbiol doi: . /s - - - sha: doc_id: cord_uid: nu lz the objective of this study was to establish a loop-mediated isothermal amplification (lamp) method for the detection of f fimbriae gene in enterotoxigenic escherichia coli. a set of four primers were designed based on the conservative sequence of coding f fimbriae. temperature and time condition, specificity test, and sensitivity test were performed with the dna of escherichia coli (f +). the results showed that the optimal reaction condition for lamp was achieved at °c for min in a water bath. ladder-like products were produced with those f -positive samples by lamp, while no product was generated with other negative samples. the assay of lamp had a detection limit equivalent to cfu/tube, which was more sensitive than pcr ( . × ( ) cfu/tube). the agreement rate between lamp and pcr was % in detecting simulation samples. thus, the lamp assay may be a new method for rapid detection of f fimbriae gene of etec. adhesion fimbria play an important role in causing young animal diarrhea by enterotoxigenic escherichia coli (etec), it enables etec to colonize the small intestinal cell surface, and etec produce toxins to cause the host diarrhea [ ] . the fimbrial adhesions most frequently diagnosed in etec strains isolated from piglets with diarrhea are f (k ), f (k ), f ( p), and f [ ] , which have different antigenicity. thus, vaccines developed from one specific fimbria could not provide protection against an etec strain expressing a different fimbria. therefore, it is necessary to detect fimbriae's type of etec-isolated strains and to select an appropriate vaccine prepared with right fimbriae antigen. the type of fimbriae is determined by the genes encoding fimbriae protein, thus detection of the genes encoding fimbriae protein can determine the type that the fimbriae has expressed. f fimbriae is one of the most common fimbriae antigen, it was established as a transmissible k antigen with adhesive properties in [ ] and encoded on a -megadalton plasmid [ ] . hybridization technique and pcr have been used for detecting f fimbriae gene as nucleic acid detection technologies [ , , ] , but the concentration of bacteria and purity may make an effect in accuracy and sensitivity with hybridization in practical applications, pcr requires special instruments and equipments, thus extensive application of these technologies are restricted. however, the invention of loop-mediated isothermal amplification (lamp) provides new ideas and technologies for establishing a rapid detection method of f fimbriae gene. lamp is a new technology of nucleic acid amplification reported first in by notomi. this method focus on an autocycling strand displacement dna synthesis performed by the bst dna polymerase large fragment, which is different from pcr in that four or six primers perform the amplification of the target gene, it is sensitive, specific, and rapid [ ] . at present, lamp has been successfully used to detect many pathogens, such as human malaria parasites, mycobacterium tuberculosis, and shiga toxin-producing escherichia coli, detection of the hclv against classical swine fever, and so on [ , , , ] . the objective of this study was to develop a lamp assay for detecting of f fimbriae gene more rapidly, accurately, and easily. e. coli c (f ?), c (f ab?), c (f ?), c (f ab?), and c (f ? f ?) were obtained from china institute of veterinary drugs control. recombinant e. coli tg (pmd t-f )-contained f fimbriae gene was structured in our laboratory; seven simulation samples of e. coli strains were prepared by a different lab in our school, two of the seven strains were f fimbria positive and the other five were negative. bst dna polymerase was purchased from neb. betaine was purchased from sigma, sybr green i was purchased from xiamen baiweixin company (china), and mgcl and dntps were purchased from takara. double-distilled water was used in all experiments. all other reagents were analytical grade. four primers targeting f fimbriae protein genes (genbank number: m ) were designed by means of the primer explorer v (http://primerexplorer.jp/e), including external primers f and b , and internal primers fip and bip. a pair of primers p and p was used for pcr of f fimbriae gene [ ] . information regarding the primer names and sequences is provided in table . e.coli c (f ?), c (f ab?), c (f ?), c (f ab?), and tg (pmd t-f ) were cultured using lb medium at °c overnight. one milliliter of each e. coli culture were transferred into . -ml eppendorf tube and centrifuged at , g. the supernatants were discarded, the cell pellets were suspended in ll te buffer ( mm tris, ph . ; mm edta; sigma-aldrich), and heated at °c for min in a dry heating block. after centrifugation at , g for min, the supernatants were stored at - °c until use as the dna template for lamp and pcr. according to the literature [ ] , the lamp assay was set up in a total volume of ll consisting of ll of lm fip and bip primers, . ll of lm f and b primers, . ll of mm dntps, ll of mm mgcl , . ll of m betaine, . ll of thermo buffer, . ll of bst dna polymerase, and ll of dna (c (f ?)), and the total volume was made up to ll with deionized water. the reaction was performed in a water bath for h at °c by stopping at °c for min. then, ll of the lamp products were separated on a . % agarose gel. in addition, one microliter of sybr green i was added to the reaction tube and the reaction was visualized directly under daylight or under uv light (wavelength: nm). according to the volume in the literature [ ] , the lamp reaction mixtures were incubated at , , or °c to determine the optimal reaction temperature. when reaction was finished, ll lamp products were separated on a . % agarose gel to obtain the desired reaction temperature. then, the lamp was performed at the desired reaction temperature for , , , , , and min to determine the optimal reaction time. the reactions were terminated by heat inactivation at °c for min. the amplified dna products from the lamp assays were visualized by . % agarose gel electrophoresis as above. the specificity of the lamp assay was tested with the dna of c (f ?) strain, c (f ab?) strain, c (f ?) strain, c (f ab?) strain, and tg (pmd t-f ) strain. the lamp system was done as described above and the reaction was performed at °c for min. three microlitre lamp products were separated on a . % agarose gel, one microliter of sybr green i was added to the reaction tube, and the reaction was monitored directly under daylight or under uv light (wavelength: nm). sensitivity analysis of the lamp template dna from c (f ?) ( . cfu/ml) was prepared as ''dna extraction'' section described and [ ] were used to detect limits of lamp and pcr. when reaction was finished, ll of the lamp products were separated on a . % agarose gel, one microliter of sybr green i was added to the reaction tube and the reaction was visualized directly under daylight or under uv light (wavelength: nm) as above. detecting seven simulation samples containing different fimbriae were provided by other staff of this lab by lamp and pcr methods [ ] respectively. when reaction finished, ll of the lamp products were separated on a . % agarose gel, one microliter of sybr green i was added to the reaction tube, and the reaction was visualized directly under daylight or under uv light (wavelength: nm) as above. specificity test, sensitivity test, and detection of simulation samples were conducted in triplicate under the optimized conditions to make sure the development of lamp is stable. the f dna and specific primers targeting f fimbriae genes were included in a lamp performed at °c in a water bath for min. product of positive reaction with lamp showed ladder-like pattern on gel electrophoresis, while negative control did not show the characteristic bands (fig. a) . the results showed that the primers were effective and specific. after the addition of sybr green i, the product under daylight showed green (fig. b) . in contrast, the negative control shows pale orange; else, the product showed green fluorescent under uv light and the negative did not change (fig. c) the optimal reaction temperature and time of the lamp were investigated. no significant difference was observed at different temperatures; however, the intensity of dna at °c showed strongest among all the test temperatures (fig. a) . reactions were then performed at , , , , , and min at °c. the subsequent results indicated that the dna products showed the ladder-like pattern literally, when the reaction was performed for min. the optimal reaction condition of the lamp for f fimbriae gene was °c for min (fig. b) . the result of detecting by established lamp were positive only for f fimbriae gene, and no positive dna products of the lamp assay were observed when these control strains (f ab, f , f , and f ab) were used as templates (fig. ) . the results showed that the detection limitation of lamp was cfu/tube (fig. a-c) ; in contrast, the pcr has a detection limit of . cfu/tube (fig. d) . the sensitivity of the lamp was more sensitive than pcr. seven simulation samples were tested by lamp and pcr assay [ ] , no. and no. were lamp-positive samples, as same as pcr results. test results were consistent with the simulated samples (fig a-d) . simulation samples' strain names and results were shown in table . specificity test, sensitivity test, and detection of simulation samples were conducted in triplicate under the optimized conditions. it is suggested that the lamp assay established has good repeatability and stability. lamp is a highly specific, sensitive, rapid, and reproducible gene amplification assay. it is easy to perform, can get a lot of specific amplification products at a constant temperature without the need for complex equipment, and the amplification reaction result can be visually determined by turbidity or by adding dye [ ] . lamp has also been used widely for the detection of pathogens [ , , , ] , but determining results by turbidity requires expensive equipment; thus it is not suitable for small-scale laboratory. in our lab, we added sybr green i in the reaction product, e. coli has a large-scale bacterial genome, about million base pairs, and some sequences are not published in genbank. therefore, lamp primers designed with known genome may result in false positive if they bind on some unknown genome. to guarantee primers' specificity, we appointed upper pcr primer of f fimbriae to f in fip of lamp primers [ ] , then designed following primers online, thus it reduced workload of detecting primers' specificity. in a lamp reaction, inner primer f in fip hybridizes to f c in the target dna and initiates complementary strand synthesis at first. outer primer f slowly hybridizes to f c in the target dna and initiates strand displacement dna synthesis, releasing a fip-linked complementary strand, which can form a looped out structure at one end. this single-stranded dna serves as template for bip-initiated dna synthesis and subsequent b -primed strand displacement dna synthesis, leading to the production of a dumb-bell form dna, which is quickly converted to a stem-loop dna by self-primed dna synthesis [ ] . so, f in fip and b in bip first combined with target dna in a reaction, their specificity have a direct effect on the reaction. adhesion fimbria is one of the causative agents of etec. it mediates etec to colonize the small intestinal cell surface and then etec produce toxins to cause the host diarrhea; thus, many vaccines about this used fimbriae as the immunogen. crouch, vaccinated cows with a combined vaccine against rotavirus, coronavirus and e. coli f (k ). there was a significant increase in the mean specific antibody titer against all three antigens in the serum of the vaccinated animals which was accompanied by increased levels of protective antibodies to rotavirus, coronavirus, and e. coli f (k ) in their colostrums and milk [ ] . rising [ ] experiments showed that sows vaccinated with a vaccine against neonatal e. coli diarrhea in piglets containing purified f ab, f ac, f , and f fimbriae and detoxified heat-labile toxin (lt) can make an effective protection of piglets against neonatal e. coli diarrhea. rapid detection and identification of fimbriae's type of etec strains isolated from animals with diarrhea contribute to select an appropriate vaccine prepared with right fimbriae antigen for improving the immune protective effect, also contribute for epidemiological survey more convenient. so, we can identify the fimbriae type of etec that caused young animals' diarrhea, and we can make prevention and treatment against etec diarrhea in the more targeted region [ ] . etec also causes diarrhea in humans, and the major virulence factors of etec strains in humans include colonization factor antigens (cfa) and toxins. some studies suggest that up to % of strains that cause diarrhea in humans express cfa/i, cfa/ii, or cfa/ iv [ , , ] , detection of the fimbriae type is also significant to choose or prepare an appropriate fimbriae vaccine. this study indicates that the lamp is sensitive, specific, and rapid for detecting f fimbriae gene, and the lamp assay have avoided low specificity, cumbersome operation, special and expensive instruments, and other shortcomings of conventional methods in detecting fimbriae gene. cfa of the etec strains in humans may also be detected by lamp assay. enterotoxins in acute infective diarrhoea detection of porcine parvovirus by loop-mediated isothermal amplification pcr detection of virulence factor genes in escherichia coli isolates from weaned piglets with edema disease and/or diarrhea in china serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f (k ) development of loop-mediated isothermal amplification (lamp) for detection of escherichia coli producing shiga toxin ii variant detection of four plasmodium species by genus-and species-specific loop-mediated isothermal amplification for clinical diagnosis detection of verotoxigenic escherichia coli o and o in food by plating methods and lamp method: a collaborative study regulation of expression of escherichia coli pilus k genotypic prevalence of the fimbrial adhesins (f , f , f , f and f ) and toxins (lt, sta, stb and stx e) in escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in korea loop-mediated isothermal amplification of dna development of an in-house loop-mediated isothermal amplification (lamp) assay for detection of mycobacterium tuberculosis and evaluation in sputum samples of nepalese patients prevalence of toxin types and colonization factors in enterotoxigenic escherichia coli isolated during a -year period from diarrheal patients in bangladesh protection against neonatal escherichia coli diarrhoea in pigs by vaccination of sows with a new vaccine that contains purified enterotoxic e. coli virulence factors f ac, f ab, f and f fimbrial antigens and heat-labile e. coli enterotoxin (lt) toxoid colonization factor different from k , k , f and p in enterotoxigenic escherichia coli strains isolated from postweaning diarrhoea in pigs phenotypic diversity of enterotoxigenic escherichia coli (etec) isolated from cases of travelers' diarrhoea in kenya hybridization of clinical escherichia coli isolates from calves and piglets in new york state with gene probes for enterotoxins virulence factors in escherichia coli strains isolated from swedish piglets with diarrhea loop-mediated isothermal amplification assays for detecting shiga toxin-producing escherichia coli in ground beef and human stools occurrence, distribution, and associations of o and h serogroups, colonization factor antigens, and toxins of enterotoxigenic escherichia coli rapid and sensitive detection of heat-labile and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by loop-mediated isothermal amplification detection of enteroaggregative escherichia coli by loop-mediated isothermal amplification development of a loop-mediated isothermal amplification for visual detection of the hclv vaccine against classical swine fever in china acknowledgments we thank two reviewers for their very valuable comments, which improved the manuscript. financial support for this study was provided by the grants national science and technology support project ( bad b , bad b ). key: cord- - kzr rq authors: parida, m. m. title: rapid and real-time detection technologies for emerging viruses of biomedical importance date: - - journal: j biosci doi: . /s - - - sha: doc_id: cord_uid: kzr rq the development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. the conventional identification methods require time-consuming culturing, and/ or detection of antibodies, which are not very sensitive and specific. the recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. we have developed two real-time assays ie., sybr green i based real time reverse transcription polymerase chain reaction (rt-pcr) and rt-loop-mediated isothermal amplification (lamp) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, japanese encephalitis, chikungunya, west nile, severe acute respiratory syndrome virus (sars) etc. both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specificity. one of the most important advantages of lamp is its field applicability, without requirement of any sophisticated equipments. both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of india. the establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future. the rapid diagnosis of virus diseases has assumed greater signifi cance owing to the direct benefi t of patient management in absence of suitable therapeutic and prophylactic measures. with the advancement of technologies, increasingly diverse methods are available which make it possible to detect and analyze any virus, including those, which cannot be cultured. the laboratory diagnosis of virus infection can be made by the detection of specifi c virus, viral antigen, genomic sequences and/or antibodies. nucleic acid amplifi cation is one of the most valuable tools in virtually all life science fi elds, including application-oriented fi elds such as clinical medicine in which diagnosis of infectious diseases, genetic disorders and genetic traits are particularly benefi ted by this new technique. now a days, several amplifi cation methods have been invented viz. nucleic acid sequence based amplifi cation (nasba), self-sustained sequence replication ( sr), strand displacement amplifi cation (sda) as well as polymerase chain reaction (pcr) (chan and fox ) . these methods can amplify target nucleic acids to a similar magnitude, all with a detection limit of less than copies and within a hour or so. although several amplifi cation methods have been developed, pcr is the most widely used because of its apparent high simplicity and reliability. routine use the development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. the conventional identifi cation methods require time-consuming culturing, and/ or detection of antibodies, which are not very sensitive and specifi c. the recent advances in molecular biology techniques in the fi eld of genomics and proteomics greatly facilitate the rapid identifi cation with more accuracy. we have developed two real-time assays ie., sybr green i based real time reverse transcription polymerase chain reaction (rt-pcr) and rt-loop-mediated isothermal amplifi cation (lamp) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, japanese encephalitis, chikungunya, west nile, severe acute respiratory syndrome virus (sars) etc. both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specifi city. one of the most important advantages of lamp is its fi eld applicability, without requirement of any sophisticated equipments. both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of india. the establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future. [parida m m rapid and real-time detection technologies for emerging viruses of biomedical importance; j. biosci. - ]] of pcr as a standard approach in biotechnology and medical diagnostic laboratories has been usually practiced. during the past decade, various forms of pcrs such as reverse transcription polymerase chain reaction (rt-pcr), nested pcr and multiplex pcr have been developed to address the need for rapid identifi cation of viruses to serotype level with more accuracy (ratcliff et al ) . despite the obtainable magnitude of amplifi cation, these pcr based methods require either high precision instruments for the amplifi cation or elaborate methods for detection of the amplifi ed products. in addition, these methods are often cumbersome to adapt for routine clinical use especially in peripheral health care settings and private clinics. in addition, the pcr method has several intrinsic disadvantages, such as the requirement of thermal cycling, insuffi cient specifi city and rather low amplifi cations effi ciency. more sensitive and real time based assays are therefore needed to complement the existing pcr based assay systems. the real-time pcr assay has many advantages over conventional pcr methods, including rapidity, quantitative measurement, lower contamination rate, higher sensitivity, higher specifi city, and easy standardization. thus, nucleic acid-based assays or real-time quantitative assay might eventually replace virus isolation and conventional rt-pcr as the new gold standard for the rapid diagnosis of virus infection in the acute-phase samples (ratcliff et al ) . real-time pcr has enhanced wider acceptance of the pcr due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. real-time pcr assays used for quantitative rt-pcr combine the best attributes of both relative and competitive (end-point) rt-pcr in that they are accurate, precise, capable of high throughput, and relatively easy to perform. the majority of diagnostic pcr assays reported to date have been used in a qualitative, or 'yes/no' format. the development of real-time pcr has brought true quantitation of target nucleic acids out of the pure research laboratory and into the diagnostic laboratory (espy et al ) . all real-time pcr systems rely upon the detection and quantitation of a fl uorescent reporter, the signal of which increases in direct proportion to the amount of pcr product in a reaction. in the simplest and most economical format, that reporter is the double-strand dna-specifi c dye sybr® green (molecular probes). sybr green binds to double-stranded dna, and upon excitation emits light. thus, as a pcr product accumulates, the fl uorescence increases. the advantages of sybr green are that it's inexpensive, easy to use, and sensitive. the disadvantage is that sybr green will bind to any double-stranded dna in the reaction, including primer-dimers and other non-specifi c reaction products, which results in an overestimation of the target concentration. for single pcr product reactions with well-designed primers, sybr green can work extremely well, with spurious non-specifi c background only showing up in very late cycles. the two most popular alternatives to sybr green are taqman® and molecular beacons, both of which are hybridization probes relying on fl uorescence resonance energy transfer (fret) for quantitation (fi gure ) taqman probes are oligonucleotides that contain a fl uorescent dye, typically on the ' base, and a quenching dye, typically located on the ' base. when irradiated, the excited fl uorescent dye transfers energy to the nearby quenching dye molecule rather than fl uorescing, resulting in a nonfl uorescent substrate. taqman probes are designed to hybridize to an internal region of a pcr product. during pcr, when the polymerase replicates a template on which a taqman probe is bound, the ' exonuclease activity of the polymerase cleaves the probe. this separates the fl uorescent and quenching dyes and fret no longer occurs. fluorescence increases in each cycle in proportion to the rate of probe cleavage fi gure ). molecular beacons also contain fl uorescent and quenching dyes, but fret only occurs when the quenching dye is directly adjacent to the fl uorescent dye. molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the fl uorescent dye and quencher in close proximity. when a molecular beacon hybridizes to a target, the fl uorescent dye and quenchers are separated, fret does not occur, and the fl uorescent dye emits light upon irradiation. unlike taqman probes, molecular beacons are designed to remain intact during the amplifi cation reaction, and must rebind to target in every cycle for signal measurement. taqman probes and molecular beacons allow multiple dna species to be measured in the same sample (multiplex pcr), since fl uorescent dyes with different emission spectra may be attached to the different probes. multiplex pcr allows internal controls to be co-amplifi ed and permits allele discrimination in single-tube, homogeneous assays. these hybridization probes afford a level of discrimination impossible to obtain with sybr green, since they will only hybridize to true targets in a pcr and not to primer-dimers or other spurious products. rt-pcr has become the benchmark for the detection and quantifi cation of viruses and is being utilized increasingly in novel clinical diagnostic assays. quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. quantitative pcr (qpcr) assays are most established for the detection of viral load and therapy monitoring (mackay et al ) . further, nucleotide sequence analysis of the amplifi cation products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. several investigators have reported fully automatic realtime pcr assays for the detection of viruses in acute-phase serum samples. with improved and automated nucleic acid sample isolation techniques, as well as real-time detection methods, a new generation of assays for most clinically important viruses is being developed (niesters ) . qpcr assays based on sybr green and taqman chemistries have been developed and validated and are beginning to reveal the virus's epidemiology and pathogenesis (gunson et al ; watzinger et al ) . the qpcr assay also provides critical prognostic information for clinical management. more recently, real-time pcr assays have provided additional major contributions, with the inclusion of an additional fl uorescent probe detection system resulting in an increase in sensitivity over conventional pcr, the ability to confi rm the amplifi cation product and to quantitate the target concentration and detection of multiple targets in a single tube. multiplex real-time quantitative rt-pcr assays have been developed for simultaneous detection, identifi cation and quantifi cation of hbv, hcv and hiv- in plasma or serum samples. genomic amplifi cation of one virus was unaffected by the simultaneous amplifi cation of the other two. although competition between hcv and hiv- amplifi cations slightly affected the yield of hiv- amplifi cation, quantifi cation of a single virus was possible. a novel qrtpcr assay consists of two multiplex reactions, one detecting infl uenza a and b and rsv, the other one piv - , and can generate a result within h. other real-time assays can differentiate between infl uenza a subtypes and rsv a and b facilitating diagnosis, patient management and strain identifi cation for vaccine production. qrtpcr based diagnostic assays have also been reported for the associated corona viruses and improved to give result in an assay with % specifi city in samples collected from day - of disease onset. the assay's potential for high throughput was also of critical importance in areas with outbreaks of sars in which large numbers of specimens had to be tested. in addition, the ability to quantify helped elucidate the pathogenesis of the disease. qrtpcr assays can now be used routinely to exclude sars-associated coronavirus in patients hospitalized with respiratory symptoms even in the presence of other respiratory viruses. qrtpcr assays have also been developed for the clinical diagnosis of viral meningitis and to detect enteroviruses in cerebrospinal fl uid and are signifi cantly more sensitive than viral culture (verstrepen et al ; beuret ) . the ability of qpcr to generate accurate quantitative data has had a huge impact on the study of viral agents of infectious disease and is helping to clarify disputed infectious disease processes and demonstrate links between specifi c viral sequences and patient clinical symptoms. however, the lack of commercially available validated reagent kits for most viruses remains a major problem, as does the absence of standardization of the existing tests (niesters ) . besides, all these nucleic acid amplifi cation methods have several intrinsic disadvantages of requiring either a high precision instrument for amplifi cation or an elaborate complicated method for detection of amplifi ed products. the high cost of instruments required for performing the real time assays restricted its use to laboratories with good fi nancial resources. loop-mediated isothermal amplifi cation (lamp) which stands for loop-mediated isothermal amplifi cation is a simple, rapid, specifi c and cost-effective nucleic acid amplifi cation method and is characterized by the use of different primers specifi cally designed to recognize distinct regions on the target gene (notomi et al ) . the amplifi cation proceeds at a constant temperature using strand displacement reaction. amplifi cation and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, dna polymerase with strand displacement activity and substrates at a constant temperature of °c. compared to pcr and real-time pcr, the lamp has the advantages of reaction simplicity and detection sensitivity. the higher sensitivity and specifi city of the lamp reaction is attributed to continuous amplifi cation under isothermal condition employing six primers recognizing eight distinct regions of the target. besides, the higher amplifi cation effi ciency of lamp reaction yields large amount of byproduct, pyrophosphate ion, leading to white precipitate of magnesium pyrophosphate in the reaction mixture. since the increase in turbidity of the reaction mixture according to the production of precipitate correlates with the amount of dna synthesized, real-time monitoring of the lamp reaction can be achieved by real-time measurement of turbidity (mori et al ) . being an isothermal amplifi cation, lamp does not require any thermal cycler and thus can be performed even with heating block and/or water bath. thus, lamp method has the characteristics of not requiring special reagents and sophisticated temperature control device. since it only requires simple equipments, cost effective point of care gene test can be achieved. both simple detection and real-time detection of the reaction are possible. in addition, in case of lamp, both amplifi cation and detection occur simultaneously during the exponential phase without going through the plateau phase where the non spurious amplifi cation leads to lower sensitivity and false positivity. designing of a highly sensitive and specifi c primer set is crucial for performing lamp amplifi cation. the target selection for primer designing can be accomplished by using the primer explore [lamp primer designing support software program, net laboratory, japan, http://venus.net laboratory.com)] after considering the base composition, gc contents and the formation of secondary structures. the primer set for lamp amplifi cation include a set of six primers comprising two outer, two inner and two loop primers that recognize eight distinct regions on the target sequence. the two outer primers were described as forward outer primer (f ) and backward outer primer (b ) and have a role in strand displacement during non-cyclic step only. the inner primers were described as forward inner primer (fip) and backward inner primer (bip) having both sense and antisense sequence in such a way that it helps in the formation of loop. further, two loop primers viz. forward loop primer (flp) and backward loop primer (blp) were designed to accelerate the amplifi cation reaction by binding to additional sites that are not accessed by internal primers. lamp amplifi cation can also be accomplished with the two outer (f and b ) and two internal primers (fip and bip) but by using the two loop primers (flp and blp), the amplifi cation is accelerated and thereby shortens amplifi cation time by one third to one half ( (notomi et al ) . the designing of the above mentioned types of primers are based on the following distinct regions of the target gene: the f c, f c and f c and flp regions at the ' side and the b , b , b and blp regions at the ' side. forward inner primer (fip) consists of the f region (at the ' end) that is complementary to the f c region, and the same sequence as the f c region at the ' end. forward outer primer consists of the f region that is complementary to the f c region. backward inner primer (bip) consists of the b region (at the ' end) that is complementary to the b c region, and the same sequence as the b c region at the ' end. backward outer primer consists of the b region that is complementary to the b c region. fip consists of a complementary sequence of f and a sense sequence of f . bip consists of a complementary sequence of b and a sense sequence of b (fi gure ). fip and bip were high performance liquid chromatography (hplc) purifi ed primers. the flp and blp primers were composed of the sequences that are complementary to the sequence between f and f and b and b regions respectively ( (notomi et al ) . the chemistry of lamp amplifi cation is based on the principle of auto cyclic strand displacement reaction being performed at a constant temperature. this method employs a dna polymerase and a set of six specially designed primers that recognize a total of eight distinct sequences on the target dna. there are two steps of lamp amplifi cation comprising non-cyclic and cyclic steps. in the non-cyclic step, there is the formation of stem loop dna with stem-loops at each end that serves as the starting structure for the amplifi cation by lamp cycling. as double stranded dna is in the condition of dynamic equilibrium at the temperature around °c, one of the lamp primers can anneal to the complimentary sequence of double stranded target dna, then initiates dna synthesis using the dna polymerase with strand displacement activity, displacing and releasing a single stranded dna ( (notomi et al ; ushikubo ). with the lamp method, unlike with pcr, there is no need for heat denaturation of the double stranded dna into a single strand. through the activity of dna polymerase with strand displacement activity, a dna strand complementary to the template dna is synthesized, starting from the ' end of the f region of the fip. the f primer anneals to the f c region, outside of fip, on the target dna and initiates strand displacement dna synthesis, releasing the fip-linked complementary strand. a double strand is formed from the dna strand synthesized from the f primer and the template dna strand. the fip-linked complementary strand is released as a single strand because of the displacement by the dna strand synthesized from the f primer. then, this released single strand forms a stem-loop structure at the ' end because of the complementary f c and f regions. this single strand dna in turn serves as a template for bip-initiated dna synthesis and subsequent b -primed strand displacement dna synthesis. the bip anneals to the dna strand produced by the above step. starting from the ' end of the bip, synthesis of complementary dna takes place. through this process, the dna reverts from a loop structure into a linear structure. the b primer anneals to the outside of the bip and then, through the activity of the dna polymerase and starting at the ' end, the dna synthesized from the bip is displaced and released as a single strand before dna synthesis from the b primer. the bip-linked complementary strand displaced forms a structure with stemloops at each end, which looks like a dumbbell structure. a dumbbell-like dna structure is quickly converted into a stem-loop dna by self-primed dna synthesis. this structure serves as the starting structure for the exponential amplifi cation in cyclic manner. in subsequent lamp cycling one inner primer hybridizes to the loop on the product and initiates displacement dna synthesis, yielding the original stem-loop dna and a new stem-loop dna with a stem twice as long. briefl y, fip anneals to the single stranded region in the stem-loop dna and primes strand displacement dna synthesis, releasing the previously synthesized strand. this released single strand forms a stem-loop structure at the ' end because of complementary b c and b regions. then, starting from the ' end of the b region, dna synthesis starts using self-structure as a template, and releases fiplinked complementary strand. the released single strand then forms a dumbbell-like structure as both ends have complementary f -f c and b c -b regions, respectively. furthermore, bip anneals to the b c region and primes strand displacement dna synthesis, releasing the b primed dna strand. as a result of this process, various sized structures consisting of alternately inverted repeats of the target sequence on the same strand are formed. the cycling reaction continues with accumulation of copies of target in less than an hour. the fi nal products are stem-loop dnas with several inverted repeats of the target and caulifl owerlike structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand (fi gure ). lamp amplifi cation can also be accomplished with the two outer (f and b ) and two internal primers (fip and bip) but by using the two loop primers (flp and blp), the amplifi cation is accelerated and thereby reducing the amplifi cation time to almost half (nagamine et al ) . the lamp reaction is usually carried out in a total μl reaction volume containing pmol each of the primers fip and bip, pmol each of the outer primers f and b , pmol each of loop primers flp and blp in a x reaction mixture having mm tris-hcl ph . , mm (nh ) so , mm mgso , mm kcl, . mm dntps, . m betaine, . % tween , units of the bst dna polymerase (new england biolabs), and μl of dna template. positive and negative controls should be included in each run, and all precautions to prevent cross-contamination should be observed. the studies temperature optima required for effi cient amplifi cation by lamp assay indicated that the optimum temperature for the lamp reaction was °c, which is optimum for the activity of bst dna polymerase. the amplifi cation of rna template was accomplished through rt-lamp assay by employing reverse transcriptase for reverse transcription step in addition to the bst dna polymerase. rt-lamp method can synthesize cdna from template rna and apply lamp technology to amplify and detect them. the real-time monitoring of-lamp amplifi cation can be accomplished through spectrophotometric analysis with the help of loop amp real-time turbidimeter (la- , teramecs, japan) that records the turbidity in the form of od at nm at every second ( mori et al ) (fi gures , a). on agarose gel analysis, the lamp amplicons revealed ladder like pattern in contrast to a single band as observed in pcr (fi gure b). this is due to the caulifl ower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. in order to facilitate the fi eld application of lamp assay, the monitoring of amplifi cation can also be accomplished with naked eye inspection either in the form of visual turbidity or visual fl uorescence. following amplifi cation, the tubes can be inspected for white turbidity through naked eye after a pulse spin to deposit the precipitate in the bottom of the tube. hence, the presence of turbidity can indicate the presence of target gene (fi gure c) .the tube containing the amplifi ed products can also be better visualized in the presence of fl uorescent intercalating dye viz. ethidium bromide, sybr green i and calcein etc. by illuminating with a uv lamp, the fl uorescence intensity increases. in practice, usually the visual inspection for amplifi cation is performed through observation of color change following addition of μl of sybr green i (a fl uorescent dsdna intercalating dye) to the tube. in case of positive amplifi cation, the original orange color of the dye will change into green that can be judged under natural light as well as under uv light ( nm) with the help of a hand held uv torch lamp. in case there is no amplifi cation, the original orange color of the dye will be retained. this change of color is permanent and thus can be kept for record purpose (fi gure d). in order to detect lamp products in a sequencespecifi c manner visually, an extremely simple method was reported by adding a small amount of low-molecular weight polyethylenimine (pei) to the lamp reaction solution. the biggest feature of this technique is the ability to visually present sequence information of amplicons without using an expensive source of light or a detector. the new detection method described above utilizes the unique nature of low-molecular-weight pei, i.e. it cannot form an insoluble complex with a single-stranded anionic polymer with a low molecular weight such as an oligo dna probe, but it can form an insoluble complex with dna with a high molecular weight such as lamp product (mori et al ) . capitalizing on its exquisite sensitivity, lamp has been designed to quantify the amount of gene copies in a person's blood (load) thereby allowing physicians to monitor their patients' disease progression and response to therapy. assessment of the load of the organisms/pathogens before, during and after therapy has tremendous potential for improving the clinical management of diseases. the quantifi cation of gene copy number and/or concentration of the organisms can be accomplished through generation of a standard curve by plotting a graph between known concentration of gene copy number or infectious unit of organisms and time of positivity to get the amplifi cation signal for that particular concentration. a linear relationship between various concentrations vs time of positivity is usually obtained through the real-time monitoring of the amplifi cation (fi gure ). the quantifi cation of gene copies in the clinical samples can be extrapolated from the standard curve on the basis of their time of positivity. lamp technology facilitates the detection of dna or rna of pathogenic organisms and, as such, is the basis for a broad range of clinical diagnostic tests for various infectious agents, including viruses and bacteria. these gene based tests have several advantages over traditional antibodybased diagnostic methods that measure the body's immune response to a pathogen. in particular, lamp is capable of detecting the presence of pathogenic agents earlier than pcr even on day one of fever where the amount of gene copy number is expected to be very low due to higher sensitivity with a detection limit of about - copies. earlier detection of infection can mean earlier treatment and an earlier return to good health. the loop mediated isothermal amplifi cation (lamp) assay is emerging as a simple, rapid and powerful gene amplifi cation technique for early detection of microbial diseases. although the inception of lamp refers back to but the popularity of lamp starts only after following emergence of west nile and sars viruses. since then, lamp assay is increasingly being adapted by researchers mostly from japan in clinical diagnosis of emerging diseases including bacteria, viruses and parasitic diseases. lamp has been successfully applied for rapid and real-time detection of both dna and rna viruses. however, most of the published researches have been directed for rna viruses may be due to the increased incidence of rna viruses in recent past in the form of major epidemic having signifi cant public health importance. a one-step single tube real-time accelerated reverse transcription loop mediated isothermal amplifi cation (rt-lamp) assays for rapid detection of some of the recently emerged human viral pathogens viz. west nile, sars, dengue, japanese encephalitis, chikungunya, norwalk, h n highly pathogenic avian infl uenza (hpai) viruses have been developed and evaluated ( parida et al ( parida et al , ( parida et al , ( parida et al , hong et al ; imai et al ; toriniwa et al ) on comparison to conventional rt-pcr, rt-lamp assay demonstrated to fold more sensitivity with a detection limit of . to pfu of virus in all these cases. the usefulness of lamp for amplifi cation of dna viruses was also reported for hbv, hpv (human papillomavirus) type - , , and , hsv, varivella zooster virus (vzv), cytomegalo virus (cmv) and found to be superior in terms of sensitivity, specifi city, rapidity, and simplicity, and can potentially be a valuable tool for the detection of hpv dna compared to pcr and real-time pcr ( the combination of excellent sensitivity and specifi city, low contamination risk, and speed has made real-time pcr technology an appealing alternative to culture-or immunoassay-based testing methods for diagnosing many infectious diseases. the recent advances in the development of fl uorophores, nucleotide labeling chemistries, and the novel applications of oligoprobe hybridization have provided real-time pcr technologies with a broad enough base to ensure their acceptance. the lamp is emerging as a new generation of cost effective and rapid gene amplifi cation tool having all the characteristics of rapidity and high sensitivity of real-time assays as well as easy adaptability under fi eld conditions due to its simple operation, rapid reaction, and easy detection. the rapidity and sensitivity of these real-time assays will assist in precise diagnosis, which is extremely useful to undertake suitable control measures and patient management at the earliest. simultaneous detection of enteric viruses by multiplex real-time rt-pcr nasba and other transcription-based amplifi cation methods for research and diagnostic microbiology development of real-time reverse transcriptase pcr assay to detect and serotype dengue viruses taqman reverse transcription polymerase chain reaction for the detection of japanese encephalitis virus infl uenza a h n detection rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplifi cation method real-time pcr in clinical microbiology: applications for routine laboratory testing rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplifi cation assay practical experience of high throughput real time pcr in the routine diagnostic virology setting loop-mediated isothermal amplifi cation method for detection of human papillomavirus type development and evaluation of a novel loop-mediated isothermal amplifi cation method for rapid detection of severe acute respiratory syndrome coronavirus rapid diagnosis of h n avian infl uenza virus infection by newly developed infl uenza h hemagglutinin gene-specifi c loop-mediated isothermal amplifi cation method sensitive and quantitative detection of severe acute respiratory syndrome coronavirus infection by real-time nested polymerase chain reaction sensitive and rapid detection of herpes simplex virus and varicella-zoster virus dna by loop-mediated isothermal amplifi cation real-time pcr in virology detection of loop mediated isothermal amplifi cation reaction by turbidity derived from magnesium pyrophosphate formation real-time turbidimetry of lamp reaction for quantifying template dna sequence specifi c visual detection of lamp reactions by addition of cationic polymers accelerated reaction by loop mediated isothermal amplifi cation using loop primers loop-mediated isothermal amplifi cation of dna rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplifi cation method real-time reverse transcription loop mediated isothermal amplifi cation for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplifi cation assay development and evaluation of reverse transcription loop mediated isothermal amplifi cation assay for rapid and real-time detection of japanese encephalitis virus rapid and realtime detection of chikungunya virus by reverse transcription loop mediated isothermal amplifi cation assay rapid and real-time assays for detection and quantifi cation of chikungunya virus early diagnosis of sars coronavirus infection by real time rt-pcr molecular diagnosis of medical viruses quantitative real-time pcr on lightcycler for the detection of human immunodefi ciency virus type development and evaluation of sybr green i based one step real time rt-pcr assay for detection and quantifi cation of chikungunya virus development and evaluation of sybr green i based one step real time rt-pcr assay for detection and quantifi cation of japanese encephalitis virus fast detection of noroviruses using a real-time pcr assay and automated sample preparation detection and quantitation of group a rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction development of group-and serotypespecifi c one-step sybr green i-based real-time reverse transcription-pcr assay for dengue virus comparison of loop-mediated isothermal amplifi cation, real-time pcr, and virus isolation for the detection of herpes simplex virus in genital lesions development of the loop-mediated isothermal amplifi cation method for rapid detection of cytomegalovirus dna rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by infl uenza a and infl uenza b viruses, respiratory syncytial virus rapid detection and quantifi cation of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplifi cation rapid and sensitive detection of mumps virus rna directly from clinical samples by real-time pcr principle of lamp method -a simple and rapid gene amplifi cation method rapid detection of enterovirus rna in cerebrospinal fl uid specimens with a novel single-tube realtime reverse transcription-pcr assay detection and monitoring of virus infections by real-time pcr key: cord- -ddcz zck authors: yang, jin; fang, mei-xin; li, jie; lou, guo-qiang; lu, hang-jun; wu, nan-ping title: detection of hepatitis c virus by an improved loop-mediated isothermal amplification assay date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ddcz zck an improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (lamp) assay targeting the ′ untranslated region (utr) was developed to detect hepatitis c virus (hcv) infection. based on an accelerating primer (ap), the present assay, named ap-lamp, has the advantages of rapidity and sensitivity over the routine lamp method. the possible ap-based amplification pathway during the reaction was revealed by restriction enzyme digestion and eletrophoresis. the detection limit of the ap-lamp assay was approximately iu/ml, and no cross-detection was observed. the assay was evaluated further with clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of hcv rna. the hepatitis c virus (hcv) pandemic has become a major public health concern, with such increasing prevalence that nearly million individuals are infected worldwide [ ] . the majority of acute hcv infections present an asymptomatic course. many infected individuals are therefore not seen in a medical setting [ ] . nearly % of infected people develop persistent infection and are at risk of longterm complications, ranging from mild liver damage to severe chronic hepatitis that can develop into cirrhosis, end-stage liver disease, or hepatocellular carcinomas [ ] . therefore, a rapid and accurate diagnosis of hcv is important for the prevention of viral transmission and management of disease progression. screening of antibodies against hcv, however, is not a reliable method of diagnosing acute hcv infection, since the appearance of antibodies against hcv can be delayed in up to % of patients at the onset of symptoms [ ] . moreover, the window period can be even longer in immunocompromised patients, who need to be screened routinely for hcv viremia [ ] . nucleic-acid-based detection techniques are currently the most reliable methods for detecting hcv infection. a variety of molecular diagnostic assays, such as reverse transcriptase pcr [ ] , nucleic-acid-sequence-based amplification [ ] , transcription-mediated amplification [ ] , branched-chain dna assay [ ] , and in-house realtime pcr [ ] , have been developed for the detection of hcv rna. these assays, whether qualitative or quantitative, are relatively time-consuming, labor-intensive, and dependent on specialized equipment. in resource-limited or point-of-care settings, the cost and technology requirement limit their universal application. since most hcv-infected individuals are asymptomatic, there are clear advantages to targeted screening for hcv in those who are at high risk. earlier detection of infection results in earlier treatment and thus earlier recovery [ ] . for this reason, there is still a great need for a tool to simplify the detection of hcv rna with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. loop-mediated isothermal amplification (lamp) is a novel rapid, accurate, and economical nucleic acid test [ ] . the method is characterized by employing a dna polymerase with strand-displacement activity, along with two inner primers (fip, bip) and two outer primers (f , b ) to form auto-cycling immediates. loop primers (lf, lb), first described by nagamine et al, could accelerate and enhance the sensitivity of the lamp assay [ ] . onestep lamp assays have been successfully applied to the rapid detection of a number of rna viruses, such as influenza virus [ ] , mumps virus [ ] , west nile virus [ ] , severe acute respiratory syndrome corona virus [ ] , and hiv- [ ] . lamp assays have also been developed to detect hepatitis viruses, such as hepatitis b virus [ ] , hepatitis a virus, and hepatitis e virus [ ] . at present, however, no hcv detection assay using this method has been reported. the attributes of the hcv target may interfere in complex ways with the lamp method. for instance, although the viral untranslated region ( -utr) is thought to be the most conserved portion of the hcv genome and is targeted by almost all of the commercial and in-house tests [ ] , one of the most important issues may be the existence of complex secondary structure across all of this region (fig. b) . also, the -utr is generally considered to be variable enough to distinguish all of the major types and many subtypes of hcv [ ] . in the study described here, we have developed a modified lamp method for rapid and economical detection of hcv rna. in contrast to the routine lamp (pre-lamp) method, we designed an accelerating primer (ap) and tested the performance of the ap-based lamp assay (ap-lamp). the new assay was further evaluated using clinical samples. clinical specimens and standard twenty-five blood samples collected from patients with confirmed chronic hcv infection and specimens obtained from patients suspected of having viral hepatitis who were admitted to hangzhou th and infectious hospital were used for evaluation in this study. confirmed cases of hcv infection were verified by a positive result in an enzyme-linked immunosorbent assay (kehua bio-engineering, shanghai, china) for antibodies against hcv or a quantitative real-time pcr for hcv rna. a panel of samples collected from healthy blood donors was also included as negative controls. in addition, five each of anti-hiv-antibody-, anti-hav-antibody-and hbv-dna-positive samples obtained from the corresponding patients were also tested. informed consent was obtained from all patients, and the study was approved by the local ethical committee, as per the declaration of helsinki ( ). five milliliters of blood was collected from each subject in a tube containing ll of . % edta. plasma was immediately separated after centrifugation at rpm for the in-house hcv rna standard was obtained by extracting rna from a hcv-rna-positive specimen with an hcv titer of iu/ml. this in-house standard was anti-hcv antibody positive and calibrated in triplicate in parallel with the national hcv rna reference material (gbw , . iu/ml - . iu/ml, genotype ) using several commercial real-time pcr tests (kehua bio-engineering, shanghai, china; daan biotech, guangzhou, china). the gbw panel was calibrated using the who hcv international standard (nibsc / ) [ ] . hcv genotyping was performed using sequencing of the ns b region of the hcv genome as described previously [ ] . sequence analysis indicated that the hcv genotype of the in-house standard was b. serial dilutions of the standard sample for experimental analysis were prepared in normal human plasma and stored at - °c until testing. viral rna was extracted from ll of plasma using a tianamp virus rna kit (tiangen biotech, beijing, china) as per manufacturer's instructions. this extraction protocol used a fast spin-column procedure. rna was eluted in ll of rnase-free water. the whole extraction procedure was done within an hour. to design an assay that can detect most of the genotypes of prevalent hcv strains, individual sequences of the utr region of hcv strains from the hcv database (http://hcv.lanl.gov/) were retrieved. through alignment analysis, the conserved fragments of the utr were used to design the primer set. the hcv genotype b sequence (genbank accession number ay ), chosen as a representative strain, was used as a reference for generating the set of primers. all of the primers, including two outer (f and b ), two inner (fip and bip) and two loop primers (lf and lb), were designed according to the guideline provided by primerexplorer v (http://loopamp.eiken.co.jp/). in this set, fip consisted of f c, complementary to the f sequence, and f sequence, and bip consisted of the b sequence and b c, complementary to the b sequence. f and b were located outside f and b , while the loop primers recognized the region between f and f , or b and b . to strengthen the power of lamp, an additional accelerating primer (ap), located between f and b , was added to the primer set. a schematic representation of the locations of the primers along with a representative alignment of the main hcv strains is shown in fig. . the details of the oligonucleotide primers used for the amplification are given in table . all primers were synthesized by invitrogen (invitrogen, shanghai, china). the routine one-step lamp (pre-lamp) reactions were carried out in a final volume of ll containing pmol each of the fip and bip inner primers, pmol each of the lf and lb loop primers, pmol each of the f and b outer primers, thermopol buffer (new england biolabs, beverly, ma), mm mgcl , m betaine (sigma aldrich, usa), . mm each deoxynucleotide triphosphate, u of bst dna polymerase (new england biolabs, beverly, ma), . u of amv reverse transcriptase (takara, dalian, china), and ll of template. the mixture was incubated at °c for min and then heated to °c for min to terminate the reaction. for the ap-lamp assay, the reaction mixture and the conditions were the same as those described for pre-lamp, except that pmol of ap was added to the reaction mixture. a negative control was included for each lamp run. serial dilutions of the standard were used as templates for the lamp assay to evaluate its sensitivity. the specificity of the assay was tested with samples from hav-, hbv-, and hiv-infected patients and healthy donors. after amplification, the amplified dna products were analyzed by electrophoresis in a . % agarose gel stained with ethidium bromide and visualized using a bio-rad transilluminator. the restriction enzymes nhei and smai (new england biolabs, beverly, ma) were used to digest amplified products to confirm amplification specificity. digested products were analyzed by gel electrophoresis on a . % agarose gel. for naked-eye visualization, one microlitre of diluted sybr green i (invitrogen, carlsbad, ca) was added to the reaction tube after amplification, and the reaction was observed visually. for a positive reaction, a change in the color of the reaction solution from orange to fluorescent green could be recognized. for real-time monitoring of the lamp reaction, the reaction was performed on an abi prism ht sequence detection system, with sybr-green i (invitrogen, carlsbad, ca) added to the reaction mixtures to provide the fluorescent signal. the run was set up as follows: cycles of min at °c ( cycle corresponding to min of reaction), with fluorescence reading at the end of each of these cycles. a commercial hcv rna real-time pcr detection kit (kehua bio-engineering, shanghai, china) was used according to the manufacturer's instructions. as the template, . ll of rna extract was used in a -ll reaction. the clinical sensitivity of this quantitation kit was iu/ml [ ] . to design a lamp primer set covering the main genotypes of hcv isolates, a multiple sequence alignment, including genotypes - , was generated by retrieving and aligning the sequences stored in the hcv databases (http://www.hcv. lanl.gov). the primers were designed to maintain maximum conservation for annealing to the target regions. mismatches at the ' or ' ends of fip/bip were substituted by degenerate bases (table ) . a database search using blast from ncbi showed that all of the primers were specific for the hcv genome. the routine lamp format (pre-lamp) for the hcvspecific assay was performed by using rna templates extracted from standard samples. the amplified dna products were subjected to electrophoresis on . % agarose gels and visualized under uv light after ethidium bromide staining. as a result, a typical lamp laddering pattern was observed, indicating the different replication intermediates of the stem-loop amplification process, while no bands were obtained from the no-template control. eletrophoresis-based monitoring of the pre-lamp product showed a sensitivity of iu/ml ( fig. a) . to test the specificity of the test, the amplified product was digested with the enzyme nhei, resulting in the detection of strong bands (fig. b, lanes ) . furthermore, the possibility of crossreactivity with other viruses known to cause similar clinical signs was also investigated. no amplification of any viral rna (or dna) extracted from hbv-, hav-, or hivpositive samples was detected (fig. e) . to improve the efficiency of detection, an accelerating primer (ap) was designed to form an additional synthesisstarting site. different from the loop primer, which binds the single strand in the f -f or b -b region in the classical lamp [ , ] , the ap designed here is complementary to one of the double-stranded regions between f and b (fig. a) . adding ap to the pre-lamp reaction, the assay, named ap-lamp, was carried out to evaluate its performance. serial dilutions of the templates were amplified by the ap-lamp assay. as shown in fig. , the one-step ap-lamp assay had a detection limit of a iu/ml of rna template. specificity tests, including restriction enzyme analysis and the use of negative samples, were also conducted, and these showed no crossreaction. since visualization of amplification products from lamp reactions without special equipment would make the assay widely applicable and available, sybr green i was added to the reaction mixtures, resulting in a color change from orange to green. the amplified products yielded a green color in positive ap-lamp reactions, demonstrating that the sensitivity of this assay is equal to that of eletrophoresis (fig. b ). given that the sensitivity of the ap-lamp assay for detecting hcv is higher than that of the pre-lamp method, the pathway of ap-based amplification was investigated. a dumbbell-like intermediate is the initial auto-cycling product for the subsequent amplification steps in the lamp assay. apart from the classical lamp pathway that is followed when using fip and bip, as described elsewhere [ ] , the ap pathway involves the synthesis via ap priming to promote elongation followed by fip self-priming. the characteristic feature of the products of the ap pathway is that the end products are partly derived from the concatenation of ap-fip fragments (fig. ). more importantly, if the ap pathway is followed in the assay, it is logical to speculate that the amplification would still occur without the outer primer (named ap-b lamp in this study), which is strictly required in the classical lamp [ ] . three lamp formats are shown in fig. a . to test this hypothesis, we compared the pre-lamp, ap-lamp, and ap-b lamp assays using the same templates. a panel of serial log dilutions of hcv rna of known concentration was tested using the ap-lamp, pre-lamp, and ap-b lamp assays on the real-time ht platform. the results for each dilution tested in batches of three replicates in two separate runs are given in table . for the ap-lamp assay, the average threshold time (tt) required to detect a positive signal ranged from . ± . min (mean ± sd) when iu rna was present to . ± . min when iu rna was present, compared to . ± . - . ± . min in the pre-lamp assay and . ± . - . ± . min in the ap-b lamp assay. the tt value was defined as the reaction time necessary to achieve a positive signal above the baseline [ ] . these results demonstrated that the ap-lamp assay was faster by - min than the pre-lamp reaction and was much faster than real-time pcr. furthermore, using the assays to analyze low-concentration standards ranging from to iu, no amplification was obtained when the template concentration was less than iu/ml in the pre-lamp assay. the real-time ap-lamp assay consistently detected hcv rna at levels of iu/ml, while only three of six ap-b lamp assays detected hcv rna. by probit regression analysis, the ap-lamp method detected iu/ml with [ % probability of a positive result, as compared to iu/ml and iu/ml for the pre-lamp and ap-b lamp assay, respectively. therefore, the sensitivity of the ap-lamp assay for detecting hcv is about two times higher than that of the ap-b lamp assay and nine times higher than that of the pre-lamp assay. when comparing the electrophoresis bands of the products of the ap-lamp, ap-b lamp and pre-lamp assays, the first two showed a similar pattern. the main difference between the ap-lamp and pre-lamp assays is that there were ladder-like bands between and bp. the bands in this region (in rectangles with a broken line, fig. ) are likely to correspond to the self-primed amplification product bounded by the ends of the ap and fip stemloop structure (* bp in size), which match the expected size of the ap-pathway product. moreover, the result of the ap-b lamp assay provided evidence that the ap pathway is used during amplification. several reports have indicated that the lamp assay strictly requires the strand displacement function of the outer primers [ ] . no lamp amplification occurs when fip, bip, f or b is absent [ ] . by using the ap in the assay, this study using real-time monitoring or agarose gel electrophoresis confirmed that the outer primer . the extracted rna template was prepared from the standard plasma and was subjected to analysis by the pre-lamp and ap-b lamp methods. a pre-lamp assay using serial dilutions from iu/ml to iu/ml. b ap-b lamp assay using template concentrations from iu to iu/ml. c lowconcentration standards ranging from to iu/ml, detected by the pre-lamp assay. d the same standards ranging from to iu/ml, detected by the ap-b lamp reaction. the dashed box indicates the band pattern with the most significant difference between the two assays is not required. the ap-b lamp assay has higher sensitivity than the pre-lamp assay, but it is less sensitive than ap-lamp. this assay type also showed lower stability than ap-lamp, especially when the samples were at low concentration. in addition, taking into account the similar band patterns between the ap-lamp and ap-b lamp assay, these results indicate that ap-priming-based amplification is inferior to the classical pathway in the lamp process. the amplified products were digested with two restriction endonucleases to confirm the specificity and structure of the amplified products from three different lamp assays. the restriction enzyme smai recognizes the sequence between f and b , while nhei cuts between b and b . if the products were amplified specifically and formed the structures shown in fig. a , nhei digestion would yield fragments of , , , and bp, and smai digestion would yield fragments of , and bp. since the amplified product is a concatenation of dna fragments of different sizes, the amplification kinetics probably affect the actual end products of the lamp reaction. due to cutting in the region between b and b , nhei-digestion bands were observed at approximately - ( ? ) bp in the pre-lamp product, in contrast to - bp in the ap-lamp and ap-b lamp products. while cutting the region between f and b , the sizes of the fragments generated by smai digestion were approximately and bp (fig. d) , which is in good agreement with the predicted sizes. the feasibility of the ap-lamp assay for detecting hcv in clinical material was assessed by using both positive and negative plasma specimens. the real-time pcr assays were performed simultaneously, and the results of both (table ) . of the samples collected from healthy volunteers, all tested negative in both ap-lamp and real-time pcr. a total of samples were obtained from chronic hcv patients (genotype b, n = ; genotype a, n = ; all anti-hcv positive), with the viral load ranging from . to . iu/ml. none of the samples were missed by the ap-lamp assay. of acute-phase samples collected from patients suspected of having viral hepatitis, three were anti-hcv positive, and these were also detected by ap-lamp. of the remaining anti-hcv-negative cases, only two were positive for hcv rna by ap-lamp. these two patients later seroconverted after and months, respectively ( table ). the ap-lamp gave a total of ( . %) positive results, and the same result was obtained by real-time pcr. in total, the ap-lamp method demonstrated % agreement with real-time pcr when used for analysis of clinical samples. these preliminary results suggest that the ap-lamp assay described here can be applied for the diagnosis of hcv infection in a clinical setting. among the nucleic acid amplification tests available to date, lamp method has many characteristics that make it suitable for the rapid, sensitive, and simple detection of pathogens [ ] . the adaptation of the lamp technology for hcv detection in point-of-care or resource-limited setting has many potential advantages. for examples, the reaction occurs under isothermal conditions and thus does not require special equipment. the powerful amplification efficiency of the lamp assay makes it extremely rapid, and it exhibits high analytical sensitivity. furthermore, the end product can be observed immediately by visual observation, through turbidity or dye staining [ ] . up to now, however, no hcv nucleic acid test has been available outside of the laboratory setting, possibly due to time, cost and technology limitations. the use of multiple primer combinations is one of the key features of the lamp method, but this can affect the primer selection for a given template, such as the hcv 'utr, which has a complex ordered secondary structure and genotypic variation sites simultaneously. we devised an accelerating primer to improve the efficiency of amplification. just like the loop primer in lamp, the ap provides an additional starting site for dna synthesis (fig. ) during the amplification, thereby reducing the overall reaction time and increasing the sensitivity. it should be mentioned, however, that in the lamp reaction, the loop region is always in a single-stranded state during the process. in contrast, the ap is located in the doublestranded region and is complementary to one of its strands. when comparing the performance of the ap-lamp and pre-lamp assays, higher amplification efficiency was found in the former. a possible explanation for this is that apart from the classical amplification process in lamp assay, there is an ap-based amplification pathway in the ap-lamp method. this notion is supported by the following evidence: first, by comparing the band patterns of ap-lamp and pre-lamp, the products of the ap pathway (ap-fip) were observed in the former. next, the ap-b lamp assay still amplified the target in the absence of outer primer b . in the classical lamp format, the outer primer is important for strand displacement to form an auto-cycling intermediate product. no amplification occurs without this primer [ ] . finally, by the digesting the end products with restriction enzymes that recognize different sites in the target, the most favorable structure of the amplified products was found by length polymorphism analysis, as shown in fig. . it is well known that primer design in lamp is more complex and difficult than that in pcr [ ] . since lamp reaction efficiency and sensitivity strongly depend on primer selection, a more flexible way for lamp primer design could promote the use of this method. summing up the above points, the ap strategy could be applied in lamp design to meet special demands under certain conditions. for instance, because both ends of the fip/bip secondary structure play a key role in amplification cycling in the lamp-based assay, using an ap could reduce the problem of selecting fip/bip. adding ap to the principle of ap in lamp amplification. a the three lamp formats in this study. the primers commonly used in the lamp assay (pre-lamp) include inner primers fip and bip, outer primers f and b , and loop primers lf and lb. the loop primer anneals the partially single-stranded portion. the ap devised in this study is the additional accelerating primer located between the f and b fragments. the ap-b lamp assay does not use the outer primer b . b the cyclic amplification step for the ap pathway is illustrated: the dsdna reaches a dynamic equilibrium at °c, and thus the ap can bind the partly free ' end of the template to initiate strand extension. complementation of the hairpin structure (f -f c) induces self-primed dna synthesis pre-optimized lamp assay could avoid the need to design different primer sets for optimization. the performance of ap-lamp was investigated in this study. running the ap-lamp assay in a real-time pcr machine consistently achieved a lower limit of detection of iu/ml by probit analysis. this sensitivity is comparable to a series of in-house tests published recently [ - , , ] . although this method is essentially qualitative at the outset, the sensitivity limits represent good performance, since the hcv plasma viremia in acute infections is generally higher than copies/ml [ ] . as expected, a specificity test using seronegative samples and other nontargeted virus samples demonstrated % exclusivity of the assay. by applying the ap-lamp assay to clinical specimens, there was % agreement between ap-lamp and the real-time pcr test. the ap-lamp method consistent detected hcv-infected samples with a broad range of viral loads. since the samples comprised the hcv genotypes b, a, which are prevalent in china [ ] , this ap-lamp assay is expected to work for the majority of hcv-infected individuals in the local region. because of the high mutation rate of the hcv 'utr, it is not easy to generate a single lamp primer set to detect every viral strain of an individual subtype. degenerate design is most the common way to address the issue of genotype inclusivity, but this may lower the diagnostic sensitivity due to a hybridization effect. for this reason, the ap strategy developed here would potentially be applicable in a situation like this. future evaluation of detection efficiency using an extensive collection of hcv genotypes and larger samples would be desired to validate the performance of the assay. in addition to the high sensitivity and specificity of the ap-lamp assay, its other major advantages are its rapidity and the flexibility of its detection method. the ap-lamp assay itself could be carried out in less than min. only . h (including the extraction step) was needed to perform the lamp assay, compared to . - h for the real-time pcr assay. amplification in the lamp assay can be detected with the naked eye in the form of visual fluorescence, e.g., the original orange color of the dye changes to green under natural light in the case of a positive amplification reaction [ ] , thus eliminating the need for gel electrophoresis or real-time monitoring. our results, as well as the results of previous studies using a fluorescent reagent to detect the lamp product visually [ , ] , showed a similar detection efficiency when compared to real-time pcr or electrophoresis. therefore, this hcvspecific lamp assay may be applicable under clinical or field conditions. in conclusion, as demonstrated using hcv, we have developed a lamp test using a novel principle based on an accelerating primer and have provided an alternative way to design a lamp assay for a complex target. this study presents a sensitive and specific lamp method for screening for or confirming infection with hcv in a simple, rapid, and cost-effective manner. loop-mediated isothermal amplification assay for hcv national institutes of health consensus development conference statement: management of hepatitis c real-time quantitative lamp (loop-mediated isothermal amplification of dna) as a simple method for monitoring ammonia-oxidizing bacteria natural history of chronic hepatitis c virus infection an in-house method for the detection and quantification of hcv in serum samples using a taqman assay real time pcr approach taqman amplification system with an internal positive control for hcv rna quantitation hiv- and hcv detection in dried blood spots by sybr green multiplex real time rt-pcr sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv- quantitation of hepatitis c virus using an in-house real-time reverse transcriptase polymerase chain reaction in plasma samples a novel diagnostic target in the hepatitis c virus genome loop-mediated isothermal amplification integrated on microfluidic chips for pointof-care quantitative detection of pathogens dynamics of viremia in early hepatitis c virus infection design of an antisense reverse-transcriptasepolymerase chain reaction primer efficient for all hepatitis c virus genotypes: comparison of its performance vs a commercial primer evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents sequence analysis of the ' untranslated region in isolates of at least four genotypes of hepatitis c virus in the netherlands reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis e virus detection and quantification of serum or plasma hcv rna: mini review of commercially available assays hepatitis c virus genotype distribution in china: predominance of closely related subtype b isolates and existence of new genotype variants a novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis c viral rna with armored rna as internal control loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases use of sequence analysis of the ns b region for routine genotyping of hepatitis c virus with reference to c/e and ' untranslated region sequences accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna acute hepatitis c: prevention and treatment realtime reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases comparison of conventional pcr with real-time pcr and branched dna-based assays for hepatitis c virus rna quantification and clinical significance for genotypes to loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products hcv screening to enable early treatment of hepatitis c: a mathematical model to analyse costs and outcomes in two populations rnase-resistant virus-like particles containing long chimeric rna sequences produced by two-plasmid coexpression system real-time quantitative assay of hcv rna using the duplex scorpion primer mumps virus reinfection is not a rare event confirmed by reverse transcription loop-mediated isothermal amplification the authors declare that they have no conflict of interest. key: cord- -lshsgex authors: yoda, tomoko; suzuki, yasuhiko; yamazaki, kenji; sakon, naomi; kanki, masashi; aoyama, ikuko; tsukamoto, teizo title: evaluation and application of reverse transcription loop‐mediated isothermal amplification for detection of noroviruses date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: lshsgex a one‐step reverse transcription loop‐mediated isothermal amplification (rt‐lamp) assay for the detection of norovirus (nv) was developed. in order to design primer sets for the detection of a wide range of nvs, nvs were categorized into three groups, that is, genogroup i (gi), prevalent gii, and minor gii; three sets of primers were developed for each group. clinical specimens of patients suffering from enteric rna viruses, such as nv, group a and c rotavirus, and sapovirus were examined using these primer sets. various genotypes of nvs were detected in clinical specimens from patients infected with nv where no false positive reaction was observed with other enteric rna viruses. additionally, samples of acute gastroenteritis outbreaks were analyzed by an rt‐lamp assay and compared with the results of routine rt‐pcr. the results of the rt‐lamp assay corresponded well to that of rt‐pcr. these findings suggest the practical application of the rt‐lamp assay for the detection of nvs in clinical specimens. consequently, the rt‐lamp system and conventional detection kits (nvgi and nvgii detection kits; eiken chemical co., ltd., japan) were compared. the detection rate of the prevalent and minor gii primer sets was similar to that of the conventional nvgii kit, while the detection rate of the gi primer set is different because it can detect several genotypes better than the conventional nvgi kit. this is an initial report that the rt‐lamp system is able to detect nvs in clinical specimens within a wide range. j. med. virol. : – , . © wiley‐liss, inc. the genus norovirus is a member of the virus family caliciviridae. noroviruses (nvs) have emerged as the single most common cause of outbreaks as well as sporadic cases of acute gastroenteritis in children and adults throughout the world [de wit et al., ; blanton et al., ; infectious agents surveillance report (iasr), (http: //idsc.nih.go.jp/iasr/); wheeler et al., ; kirkwood et al., ] . there is no consensus on a uniform classification scheme for nvs. however, five genogroups (g) including animal nvs (in pigs, cows, and mice) have been tentatively reported using the molecular characterization of complete capsid gene sequences [green et al., ; vinje et al., ; fankhauser et al., ; karst et al., ; oliver et al., ; zheng et al., ] . three of these five gs contain human strains (genogroup i (gi), gii, and giv) [zheng et al., ] . the national institute of infectious diseases (niid, tokyo, japan) group proposed slightly different nv genotyping using a precise scheme based on variability of the capsid n-terminal/shell (n/s) domain gene [kageyama et al., ] . they identified a total of genotypes ( genotypes in gi and genotypes in gii). frequently detected gii genotypes such as gii. , , , , and (prevalent gii) are classified as the same genotypes in gii for both classifications [kageyama et al., ; zheng et al., ] , whereas infrequently detected gii genotypes (minor gii) are classified mainly into different genotypes in gii according to both classifications. zheng et al. [ ] categorized gii. of kageyama's classification in giv. all genotypes used in this report follow kageyama's classification [kageyama et al., ] . routine laboratory diagnosis of nv infection is based primarily on rt-pcr using several pairs of primers. for interpretation of rt-pcr products, methods other than agarose gel electrophoresis are necessary in order to prevent false-positive results. dna sequencing and hybridization is used commonly for confirmation of nv rt-pcr products; however, both methods are timeconsuming and tedious, requiring more than days for completion. recently, rapid and sensitive nucleotide amplification-based detection systems other than routine rt-pcr, such as real-time rt-pcr using taqman probes [kageyama et al., ] have been reported. however, these methods require high-precision instrumentation. the loop-mediated isothermal amplification (lamp) assay originally described [notomi et al., ] is based on the principle of autocycling strand displacement dna synthesis for the detection of a specific dna sequence with specific characteristics: ( ) all reactions can be conducted under isothermal conditions ranging from to c; ( ) the specificity of the reaction is extremely high because it uses six primers [nagamine et al., ] recognizing eight distinct regions on the target nucleotides; and ( ) a simple detection method such as visual judgment using fluorescent detection reagent (eiken chemical co., ltd.) is possible. the lamp assay is also very effective for rna template detection using reverse transcriptase (rtase) together with dna polymerase. detection of rna viruses are reported as such, west nile virus , severe acute respiratory syndrome coronavirus [hong et al., ] , and dengue virus [parida et al., ] using a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay. therefore, the rt-lamp assay can be applied for the detection of nvs in many laboratories engaged in the detection of nv. this study describes (a) the evaluation of a newly developed rt-lamp assay for the detection of nvs and compares the results with that of the routine rt-pcr assay, (b) comparisons between the rt-lamp assay and conventional rt-lamp detection kits for nvs (eiken chemical co., ltd.), and (c) application of the rt-lamp assay for outbreaks of acute gastroenteritis. a total of samples from public health centers were submitted to osaka prefectural institute of public health (opiph) for investigation of the causative agent of acute gastroenteritis (outbreaks and sporadic cases) during - . ninety-four samples had been analyzed previously for bacterial incubation and enteric virus (such as nv, group a and c rotavirus, sapovirus, astrovirus, and enteric adenovirus). these samples were used to evaluate both of the rt-lamp systems (opiph system and the conventional rt-lamp kit) and the results were compared with routine rt-pcr assays (table ii) . subsequently, the newly developed rt-lamp assay was used as one of the methods for investigating acute outbreaks of gastroenteritis composed of clinical specimens. five of those outbreak incidents were suspected as cases of personto-person transmission and one incident was a foodborne illness. before the investigation, the causes of the other two cases were not clear (table iv) . the specimens had been stored as % suspensions in saline at c. using an automated rna extractor, magtration system gc or magtration system gc (precision science system co. ltd., matsudo city, chiba, japan), viral rna was extracted from ml of centrifuged samples according to the manufacturer's protocol. a ml volume of eluted solution was obtained after min and was stored at À c. nv-specific rt-lamp primers were designed in the relatively conserved region (rna polymerase region to n-terminal capsid region, orf to orf ). due to their diversity, the nvs were categorized into three categories, that is, gi (gi. , , , , , , , , and ) , prevalent gii (gii. , , , , and ), and minor gii (gii. , , , , , , and ) . the nucleotide sequences of strains including prototypes based on published sequences were aligned with the available sequences of other strains* (genbank accession numbers are described at the end of this paragraph) using dnasis software (hitachi software, tokyo, japan). a set of six primers was designed as shown in figure ; outer primers (f and b ), a forward inner primer (fip), a backward inner primer (bip), and loop primers (flp and blp). the fip primer consisted of the f c sequence, a tttt spacer, and the f sequence. in the same way, bip contained the b c sequence, a tttt spacer, and the b sequence. two additional loop primers were designed to accelerate the amplification reaction. the primers recognized eight distinct regions on the target sequence ( fig. ) and were designed by employing the lamp primer design support software program version (net laboratory, japan: http://venns.netlaboratory.com). in addition to the general criteria described [notomi et al., ] , terminal dimer formation, hairpin formation, and self-complementary were avoided. special attention was given to adjust the melting temperature (tms) of the primers in such a way that the tms were in the following order: f c and b c > f and b > f and b . all primer sets were composed of more than primers ( - primers) in order to address the variations of the nv sequences. table i lists the details of the oligonucleotide primers used for the lamp assay. all primers were opc cartridge purified grade and synthesized by nihon gene research laboratories, inc. (ngr, inc., sendai, japan). the scheme of kageyama et al. [ ] was used in this report for the nv classification. *gi. rt-pcr was performed using two sets of primer pairs (g skf and g skr for gi and g skf and g skr for gii) in accordance with the standard protocol [kojima et al., ] . briefly, following cdna synthesis with either of pmol reverse primers, g skr or g skr at c for min, with nmol dntp and u of rnasin (takara bio, inc., otsu city, shiga, japan) in  ex taq buffer (takara bio, inc.) using moloney murine leukemia virus (mulv) rtase (applied biosystems, foster city, ca), pcr consisted of cycles of denaturation ( c for sec), primer annealing ( c for min), and extension ( c for min) after the first denaturation at c for min with takara ex taq (takara bio, inc.) using -ml of cdna and pmol of each primer with pmol dntp in a -ml total reaction volume. after the rt-pcr was performed, ml of the product was analyzed by . % agarose gel electrophoresis and the products were visualized by ethidium bromide staining. all extracts that resulted in nv rt-pcr products of the appropriate size ( bp for gi and bp for gii) were sequenced with the same primers that were used for rt-pcr in both directions by using the bigdye terminator v . cycle sequencing kit (applied biosystems) as described previously [cauchi et al., ] . the phylogenetic category was determined by using the blast search at http://www.ddbj.nig.ac.jp/ search/blast-j.html. rt-pcr was performed in a similar way as the routine procedure, except with different forward primers (gii-fp: -caaccatgarracccvwcyga- or gii-fp : -ggactagrggvccyaaycatg- for gii and gi-fp: -acttagaarrmrdrtngatgg- for gi). the thermal profile for pcr was c for min, followed by cycles of c for sec, c for sec, and c for min except for the first five cycles. for the first five cycles, the annealing temperature was c instead of c. a final extension after the cycles was at c for min. the amplified products were analyzed by % agarose gel electrophoresis, and the positive product was used for sequencing (using the same primers as rt-pcr). the accession numbers of the sequences that were used for the primer design were: ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , ab , and ab . the rt-lamp reaction was preformed in a -ml total reaction mixture volume with a loopamp dna amplification kit (eiken chemical co., ltd., tokyo, japan) containing pmol each of inner primers fip(s) and bip(s), pmol each of outer primers f s and b s, pmol each of loop primers flp(s) and blp(s), . mm deoxynucleoside triphosphate, . m betaine, mm tris-hcl (ph . ), mm kcl, mm (nh ) so , mm mgso , . % tween , . u avian myeloblastosis virus (amv) rtase (invitrogen, carlsbad, ca), u bst dna polymerase large fragment (new england biolabs, ipswich, ma), and the specified amounts ( - ml) of target rna. the mixture was incubated at c for min in a loopamp real-time turbidimeter (la- ; teramecs, kyoto city, japan). real-time measurement of turbidity is available using an inexpensive photometer [mori et al., ] since the amplification of dna is directly correlated with the production of magnesium pyrophosphate leading to turbidity. the reaction was considered to be positive when the turbidity became . within min. to facilitate the field application of the rt-lamp assay, the results of amplification by the rt-lamp assay were obtained through naked-eye inspection using fluorescent detection reagent (fdr) (eiken chemical co., ltd.). briefly, the rt-lamp assay was performed in the same way as described above except with the addition of . ml of fdr to the mixture before the reaction. the reaction mixture was incubated at c for min and then heated at c for min to terminate the reaction using heat blocks and observed with the naked eye. fdr (calcein) binds with the manganese ion and remains quenched. when the lamp amplification reaction occurs, a by-product, pyrophosphate, deprives fdr (calcein) of the manganese ion, which results in the emission of fluorescence. when the free calcein binds to the magnesium ion in the reaction mixture, the fluorescence emission becomes stronger. all procedures were performed in a -ml total reaction mixture volume according to the manufacturer's protocol (eiken chemical co., ltd.). briefly, after mixing  buffer, primer mixture, and distilled water (dw), the tubes were heated at c for min and then kept on ice for min, followed by the addition of the enzyme mixture and ml of sample. amplification of the targeted dna fragments was performed at c for min in a loopamp real-time turbidimeter. cdna synthesis was performed as described in the rt-pcr section. real-time rt-pcr was conducted as described previously [kageyama et al., ] , with slight modifications. in brief, a -ml reaction volume was used containing ml cdna solution, ml taqman universal pcr master mix (applied biosystems), a set of primers, and probes. in the detection of nv gi, nm of each of primers cog f and cog r and a mixture of fluorogenic probes [ pmol of ring (a)-tp and pmol ring (b)-tp] were used. to detect nv gii, nm of each of primers cog f and cog r and a fluorogenic probe [ pmol of ring (a)] were used. pcr amplification was performed in triplicate with the abi prism sequence detector (applied biosystems), and data were collected and analyzed with sequence detector software version . . (applied biosystems). in g-specific operations, an nv gi-or gii-specific standard curve was generated by a -fold serial dilution ( - copies) of purified nv gi (gi. ) or gii (gii. ) cdna plasmids as previously described [kageyama et al., ] . the success of rt-lamp assay amplification relies on the specificities of the designed primer sets. the target range of the primers was selected on the basis of relatively conserved regions of the nvs. the potential target regions were selected in the rna polymerase and the n-terminal of the capsid regions according to nucleotide sequence comparison. because of the diversity of the nvs, the initial primer set for prevalent gii was targeted to gii. , minor gii was targeted to gii. and , and gi was targeted to gi. . each primer set was synthesized for each target genotype, and then improved for other genotypes within each group. the optimal reaction temperature was set to c. table i lists details of the final primer sets and locations. comparative evaluation of rt-lamp with routine rt-pcr was performed to examine the rate of genotype detection. genotypes were determined using rt-pcr followed by nucleotide sequencing. ninety-four samples were chosen from different incidents (table ii) . there are several minor sequence differences even within the same genotype. however, the results of the rt-lamp assay corresponded well with that of rt-pcr, as shown in table ii . the reactivity of the rt-lamp assay developed in this study was compared with that of ec nv gi and ec nv gii detection kits (eiken chemical co., ltd.); the results are listed in table ii . prevalent gii and minor gii primer sets have a detection rate similar to that of the ec nv gii detection kit. however, the rate of the gi primer set appeared to surpass that of the ec nv gi detection kit because the primer set can detect several genotypes (gi. , , and ) better than the ec nv gi detection kit. the specificity of nv rt-lamp was examined using other rna virus-infected specimens such as group a rotavirus, group c rotavirus, and sapovirus that had previously been tested for nvs by rt-pcr and had produced negative results for nvs. the specific reaction of the opiph rt-lamp assay was demonstrated as indicated in table ii . to compare sensitivity in detecting nvs among genotypes between the newly developed rt-lamp system (opiph system) and the ec nv detection kits, relatively often detected gi genotypes in opiph (gi. and gi. ) and prevalently detected gii genotypes (gii. , , , and ) were selected. since the sequence of the primer sets of the ec nv detection kits were not public, the clinical specimens were used and were estimated as nv copies of the specimens by real-time pcr (triplicated) before the comparison experiment. the entire sensitivity test was conducted in triplicate using serial -fold dilutions of ml of samples for the opiph and ec nv detection kits. the detection limit was defined the last positive copies (the sample was considered positive if all three samples tested positive) as shown in table iii . the representative results of the sensitivity test for detection of the nv genotypes are shown in figure a,b. figure a ,b indicates the newly developed rt-lamp reaction using gii. and gi. , respectively, and shows good reproducibility of both reactions within the detectable range. the opiph system required a slightly longer time to become positive than did that of the ec nv detection kits. however, the sensitivity of the newly developed primer sets for the detection of all genotypes is similar or better than that of the ec nv detection kits. the possibility of rt-lamp detection of nvs using fdr was examined. a sensitivity test using gii. was performed in triplicate in the same way as for the fdr test (i.e.,  ,  ,  ,  copies). the results of the representative tubes, that is, only positive and negative samples, are shown in figure . a handy uv lamp (blak-ray lamp, long wave uv- nm) was used. the degree of sensitivity to detect nvs using fdr is in agreement with that of real-time rt-lamp, that is, all three tubes became positive in up to  copies, and only one tube became positive in  copies. to examine the possibility that the rt-lamp assay is applicable for detecting outbreaks of nvs without non-specific reactions, the specimens of eight outbreaks were used, and the results of an rt-lamp assay were compared with that of a routine rt-pcr assay. there are three primer sets, all of which were used in this experiment, as shown in table iv . it became clear after the analysis that these eight outbreaks consisted of two non-nv incidents. however, the results of the rt-lamp assay for the detection of nvs corresponded well with those of rt-pcr. the third incident consisted of patients and asymptomatic persons who were responsible for cooking and serving food. the results of rt-lamp and rt-pcr indicated that this incident appeared to be a case of person-to-person infection rather than being foodborne. based on an epidemiological study, the fourth incident was thought to be a foodborne outbreak. as a result of rt-pcr (including sequence analysis), it was judged to be a case of foodborne transmission, as shown in table iv . the sixth case was an nv outbreak in an elementary school, and all five samples came from patients. in the seventh case, all the primer sets of the rt-lamp assay showed no non-specific reaction because this case was caused by group c rotavirus. the last case occurred at a restaurant serving broiled meat and was foodborne caused by campylobacter spp. in this case, the rt-lamp method also showed no non-specific reaction. in all cases, the results of rt-lamp agreed well with those of rt-pcr. cross-reactivity between the prevalent gii primer set and minor gii primer set were observed. however, there was no cross-reactivity between the gi primer set and either of the gii primer sets in any of the cases, as shown in table iv. j. med. virol. doi . /jmv fig. . sensitivity test to detect nv using rt-lamp assay (triplicated) measured by real-time turbidimeter (la- ; teramecs). the curves from left to right indicate decreasing concentrations of virus copies [ , to in (a), and , to in (b)]. a: detecting gii. using the newly developed prevalent gii primer set. b: detecting gi. using the newly developed gi primer set. *nc: negative control. **the turbidity of the third sample of eight copies in (b) was . , so it was judged as negative (see table iii ). establishment of simple, rapid, and reliable methods for diagnosis of nv infections enables appropriate medical treatment as well as prevention of secondary infection. the rt-pcr method usually requires days to confirm that a rt-pcr product is nv, while only hr is required for an rt-lamp assay. in this study, nvs were categorized initially into three groups for designing the appropriate primer sets. gi occupies about % of nv infection. among gii nvs, genotypes gii. , , , , and are frequently detected, so this group was categorized as prevalent gii. in contrast, genotypes such as gii. , , and are not often detected; gii. , , , , , , , , and were not detected in opiph during - . therefore, these gii genotypes were categorized as minor gii. to separate the target genotypes into three groups, well-designed primer sets were developed, as shown in table i . the detection rates of various genotype nvs between the opiph system and the conventional kit were compared using clinical specimens from patients infected with nvs. the specificity of the opiph system were examined using other enteric rna viruses in outbreaks and/or sporadic cases. the results showed that the opiph system and the conventional kit indicated similar detection rates for gii nvs. the striking difference between the two systems was that the detection rate of gi nvs was much greater in the opiph system than in the conventional kit (table ii) . regarding the specificity of the opiph system, no non-specific reactions were observed against other enteric rna viruses (table ii) . the reaction of the new primer set showed good reproducibility within the sensitivity range, as indicated in figure a ,b. these figures demonstrated the reproducibility of the rt-lamp reaction using more than primers ( - primers) as primer sets. there is a recent report of detecting nvs using rt-lamp [fukuda et al., ] , and a commercialized nv detection kit using lamp methods (eiken chemical ltd.) was developed based on this system. the sensitivity obtained by fukuda et al. [ ] was between and copies/tube, while those of a conventional kit and the opiph system showed À - copies/tube and À - copies/tube, respectively. when the sensitivities of the rt-lamp system, the opiph system, and the conventional kit were compared using clinical specimens, the opiph system showed better sensitivity than that of the conventional kit. one hundred and , times greater sensitivity for gii. and gi. , respectively, was observed for opiph system than for the conventional kit, where similar sensitivities were observed for other nvs in both systems (table iii) . in other features, the conventional kit from eiken chemical co. requires a heat treatment before min incubation at c to avoid non-specific reactions. in contrast, the rt-lamp assay in the opiph system showed no non-specific reactions without heat treatment. the advantages of using opiph rt-lamp systems compared with conventional nv detection kits are: ( ) sensitivity is higher in some nv genotypes (gi. and gii. ); ( ) specific gi genotypes (gi. , , and ) that cannot be detected by a conventional gi kit can be detected by the gi primer set developed in this study; and ( ) additional treatment, that is, heating at c for min and being kept on ice for min, is not required in the opiph system. practical applications of the rt-lamp assay were evaluated using specimens from outbreaks including non-nv incidents. the results of rt-lamp corresponded well with that of rt-pcr with one exception, case no. , as shown in table iv . in this case, the high sensitivity of rt-lamp enabled detection of nvs in the patient specimens that could not be detected by routine rt-pcr. the data indicated the possibility of field application of rt-lamp. the cross-reactivity between prevalent gii and minor gii (table iv) is not a significant problem because the one-step rt-lamp assay is for diagnosis and cannot be used to identify the nv genotype. in conclusion, the rt-lamp assay is a one-step, simple, and accurate method that does not require the use of expensive equipment. in addition to the above advantages, the availability of the results without opening the lid by either using fdr or a real-time turbidimeter reduces contamination. in this regard, it will be a valuable assay method for the accurate and rapid detection of nvs in laboratories under all sorts of conditions including use in developing countries. an automated rna extractor was used in this study, but a conventional rna extraction kit such as a qiaamp viral rna mini kit (qiagen) could be used for rna extraction. the rt-lamp assay will be the first candidate to be recommended for laboratories whose needs are the diagnosis or detection of nvs. rt-pcr assay should be used for subsequent molecular epidemiological analysis after the rt-lamp assay. this rt-lamp system was developed to meet the growing demand for detection of nvs. although a conventional rt-lamp kit for the detection of nvs has become available, the quality of this kit is unsatisfactory, especially for the detection of nv gi. comparison of the opiph system with the conventional kit proved that the opiph system has advantages in diagnosing outbreaks of acute gastroenteritis, especially those caused by gi. nevertheless, the rt-lamp assay method for nv detection has just been developed and there is great diversity among nvs. based on these factors, further studies are needed to evaluate the true value of the rt-lamp assay for detecting nvs compared with routine rt-pcr system. molecular and epidemiologic trends of caliciviruses associated with outbreaks of acute gastroenteritis in the united states molecular characterization of camberwell virus and sequence variation in orf of small round-structured (norwalk-like) viruses sensor, a population-based cohort study on gastroenteritis in the netherlands: incidence and etiology epidemiologic and molecular trends of norwalk-like viruses associated with outbreaks of gastroenteritis in the united states rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay capsid diversity in small round-structured viruses: molecular characterization of an antigenically distinct human enteric calicivirus outbreaks of norovirus infection broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to norovirus in japan stat -dependent innate immunity to a norwalk-like virus a -year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in genogroup-specific pcr primers for detection of norwalk-like viruses detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation accelerated reaction by loopmediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna molecular characterization of bovine enteric calicivirus: a distinct third genogroup of norovirus (norwalk-like viruses) unlikely to be of risk to humans real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus genetic polymorphism across regions of the three open reading frames of ''norwalk-like viruses study of infectious intestinal disease in england: rates in the community, presenting to general practice, and reported to national surveillance norovirus classification and proposed strain nomenclature our grateful thanks go to manmohan parida (division of virology, defense r & d establishment) and kouichi morita (institute of tropical medicine, nagasaki university, japan) for their help with the rt-lamp system in our early study. we thank osamu nishide (national institute of infectious diseases, tokyo) for supplying control plasmid dna for real-time pcr. we also thank all members of our department for their support during this study. key: cord- -zwvesrct authors: thiessen, lindsey d.; neill, tara m.; mahaffee, walter f. title: development of a quantitative loop-mediated isothermal amplification assay for the field detection of erysiphe necator date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: zwvesrct plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. more recently, a loop-mediated isothermal amplification (lamp) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low dna concentrations. a quantitative lamp (qlamp) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the willamette valley of oregon. custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. grower-conducted qlamp assays used a beta-version of the smart-dart handheld lamp reaction devices (diagenetix, inc., honolulu, hi, usa), connected to android . enabled, bluetooth-capable nexus tablets for output. quantification by a quantitative pcr assay was assumed correct to compare the lab and grower qlamp assay quantification. growers were able to conduct and interpret qlamp results; however, the erysiphe necator inoculum quantification was unreliable using the beta-smart-dart devices. the qlamp assay developed was sensitive to one spore in early testing of the assay, but decreased to > spores by the end of the trial. the qlamp assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools. molecular techniques, such as pcr, are capable of being used to detect specific pathogens in air samples with high sensitivity and specificity (carisse, bacon & lefebvre, ; carisse et al., b; falacy et al., ; thiessen et al., ; west et al., ) . the detection of airborne pathogen inoculum has been improved through the development of quantitative pcr (qpcr) assays that allow for near real-time monitoring of inoculum concentration (carisse et al., b; rogers, atkins & west, ; temple & johnson, ; thiessen et al., ) . despite the utility of qpcr to monitor pathogens, it is often impractical due to requirements for experienced laboratory staff and expensive equipment to accurately assess pathogen concentration (notomi et al., ; west et al., ) . loop-mediated isothermal amplification (lamp) assays could be an inexpensive alternative for detection in the field or at remote facilities. lamp can use relatively inexpensive and mobile equipment and utilizes the bst polymerase that has a high tolerance to reaction inhibitors , which allows for quick, minimal dna extraction protocols. these traits make lamp useful in field detection assays (harper, ward & clover, ; kubota et al., ; temple & johnson, ; tomlinson, barker & boonham, ; tomlinson, dickinson & boonham, ) . loop-mediated isothermal amplification has been developed for monitoring inoculum in numerous plant pathosystems, including grape powdery mildew (erysiphe necator), fire blight of pear (erwinia amylovora), and gray mold (botrytis cinerea) (temple & johnson, ; thiessen et al., ; tomlinson, dickinson & boonham, ) . traditional lamp assays produce a magnesium pyrophosphate precipitate when dna is amplified that can be detected with the human eye; however, in low concentrations of target dna, precipitate may be difficult to observe kubota et al., ; thiessen et al., ) or require expensive equipment (temple & johnson, ) . several dyes have been explored to improve detection including sybr green (notomi et al., ) , hydroxynaphthol blue (cardoso et al., ) , and other synthetic dyes (fischbach et al., ) , but the dyes have the potential to inhibit lamp reactions or require the use of spectrophotometers, which increase labor and equipment costs. the use of a fluorescence resonance energy transfer (fret)-based probe, allows for specific detection of lamp products and target quantification from field samples without inhibiting amplification , and several portable fluorescence-reading lamp devices have been made commercially available, such as the genie (optigene ltd., west sussex, uk) and bioranger (diagenetix, inc., honolulu, hi, usa). using a fluorescent probe also reduces potential classification error from visual detection of lamp products, which may improve the accuracy of pathogen detection and allow for quantification. grape powdery mildew, caused by e. necator, causes damages to grape (vitis vinifera l.) wherever it is produced. this disease requires numerous applications of fungicides, which are either applied on a calendar schedule from bud break (bbch ) until véraison (bbch ) or based on disease risk models (carisse et al., a; gadoury & pearson, ; thomas, gubler & leavitt, ) . more recently, fungicide applications have been reduced using inoculum detection systems (thiessen, neill & mahaffee, ; thiessen et al., ) ; however, these systems do not provide in-field inoculum concentration for producers. additionally, the lamp assay that was successfully designed for field use in the grape powdery mildew pathosystem had numerous false negatives or false positives, which may have been caused by difficulty in perceiving the magnesium pyrophosphate precipitate, reducing the predictive values of the lamp assay (thiessen et al., ) . a timely and cost-effective system that improves detection of e. necator inoculum throughout the growing season is needed to allow growers to accurately time fungicide applications early in the growing season and adjust application intervals based on inoculum concentration. the purpose of this research was to develop a quantitative molecular assay for commercial implementation that could be used by growers or vineyard consultants for the detection and quantification of airborne e. necator inoculum. the specific objectives of this project were to ( ) develop a real-time, quantitative lamp (qlamp) assay that was sensitive and specific to e. necator, and ( ) test field use of a mobile, qlamp device by growers. sample rods were created by cutting stainless-steel lsi welding rods ( . mm in diameter) (weldcote metals, kings mountain, nc, usa) to mm lengths, then sterilized and prepared according to thiessen et al. ( ) . to produce a standard curve, conidial suspensions were generated by suspending e. necator conidia from v. vinifera cv. "chardonnay" vines in a . % tween (sigma-aldrich, st. louis, mo, usa) and nuclease-free water solution then pipetting the conidial suspension onto rod sets resulting in rods with , , , or , conidia per sample. rods with one or spores were created by transferring individual spores with eyelash brush. a total of six independent spore dilution series were used to generate the standard curve for the quantitative assay. additionally, a set of sample rods containing conidia was also generated using the conidial suspension to act as a positive control for all dna extractions and molecular reactions. the rods were air dried prior to processing. quantitative lamp assay dna for qlamp analysis was extracted using a quick extraction method modified from thiessen et al. ( ) . spore rods were transferred to -ml screw-cap tubes containing ml of % chelex (sigma-aldrich, st. louis, mo, usa) in molecular grade, depc-treated water. tubes containing rods were vortexed for s then placed in boiling water for min. tubes were removed from boiling water and vortexed another s. the tubes were boiled for another min, and then removed and allowed to cool at room temperature for min. samples were centrifuged at , g for min to collect the contents in the tube. rods were aseptically removed in a laminar flow hood prior to the pellet being processed using the chelex dna extraction process (described below). after dna were extracted and amplified, samples were stored at - c for further analyses. the qlamp reaction is a modified assay from thiessen et al. ( ) and kubota et al. ( ) , which was optimized to generate a quantification standard curve (described above). a fret-based probe was designed using the forward loop primer region with a fam reporter ( -carboxyfluorescein) and a quencher strand . each reaction contained . ml of isothermal master mix with no dye (optigene ltd, west sussex, uk), internal primers fip en and bip en ( . mm), external primers f en and b en ( . mm), forward loop primer fam strand (fl-f, . mm), and quencher strand (q-strand, . mm) to create a ml reaction (table ) . lab-conducted qlamp (l-qlamp) reactions were carried out on an abi stepone plus qpcr machine (applied biosystems, grand island, ny, usa). reaction conditions were c for min followed by c for min. all reactions were run in triplicate. the reaction time threshold (r t ) values, measured in minutes, of the spore standards were averaged and used to create a log-linear standard curve against which unknown samples were compared (fig. ) . a log-linear curve is required to describe the assay because lamp amplification rate is faster than exponential due to concatenation of amplicon (mori et al., ) . a -conidia extraction control, and , -conidia positive controls, as well as non-template controls were included in all reaction setups. unknowns were compared to the standard curve to determine relative spore quantity. positive control samples were also compared to the standard curve to determine extraction efficiency and amplification efficiency. unknown sample r t values were adjusted based on positive control r t values if the positive controls showed poor alignment to the standard curve. to test the l-qlamp sensitivity to target dna, separate spore concentration series were created and tested for positive amplification. growers were provided with all equipment and supplies to conduct the dna extraction and the qlamp reaction protocol described above. dna extraction and qlamp assays were conducted in any location growers deemed appropriate (i.e., office space, winery hallway, tractor barn, kitchen table). for the grower-conducted qlamp assay (g-qlamp), frozen aliquots of qlamp master mix were stored in insulated cryoboxes (vwr north america, radnor, pa, usa) at - c until reactions were conducted. all reactions were conducted in beta-version smart-dart handheld lamp reaction devices (diagenetix, inc., honolulu, hi, usa), which connected to android . enabled, bluetooth-capable nexus tablets for output (google, mountain view, ca, usa). all g-qlamp reactions were conducted in duplicate including -conidia positive controls and non-template controls. reaction conditions followed the protocol described above. smart-dart lamp devices provided amplification curves and the r t values associated with amplification curves. growers were asked to determine if samples were positive, as indicated by the presence of a sigmoidal amplification curve, or negative, no amplification observed, based on the output from the handheld lamp device. the dna from collected spore sampler rod pairs was extracted using the powersoil Ò dna extraction kit (mo bio laboratories, inc., carlsbad, ca, usa) following the manufacturer's protocols. in each set of dna extractions, a set of positive control rods containing e. necator conidia was included as an extraction efficiency control. e. necator primers developed by falacy et al. ( ) were paired with a taqman Ò probe with a minor groove binder (thiessen et al., ) . all qpcr reactions contained . ml perfec t a Ò qpcr toughmix Ò (quanta biosciences, gaithersburg, md, usa), nm final concentrations of each e. necator forward and reverse primers and probe (table ) , and . ml extracted sample dna for a ml total volume. reactions were carried out using an abi stepone plus qpcr machine (applied biosystems, foster city, ca, usa). all qpcr reactions were performed in triplicate, and each reaction plate contained the conidia extraction control, and , conidia positive reaction controls, and template-free negative control. cycle threshold (c t ) analysis was conducted using abi stepone tm software according to protocols by thiessen et al. ( ) . spore concentrations were determined for field samples by identifying the average c t value for each triplicate reaction, and comparing this value to the standard curve described below. average c t values of positive controls figure sensitivity of qlamp assay to erysiphe necator as a function of percent amplification (y-axis) and spore + log concentrations (x-axis). each point represents the amplification of separate extractions created from different e. necator conidia dilution series ( , , and conidia concentrations), one and conidia eyelash transferred spore rods, and conidia-free spore rods (n = ). full-size  doi: . /peerj. / fig- ( , , and , conidia) from each set of qpcr reactions were used to confirm the efficiency and to the suitability of the standard curve for determining conidia concentration of unknowns. the standard curve was generated by creating five independent, -fold conidial dilution series on the stainless-steel sampling rods to  conidia (described above), dna was extracted using the powersoil kit (described above), and the average c t values for each conidia quantity from the five independent dna extractions was used to fit a linear curve. custom impaction spore samplers (thiessen et al., ) , were placed at a research vineyard and six commercial vineyard locations within the willamette valley of oregon. each spore sampler contained a pair of sample rods described above. spore samplers were run continuously, sampling l/min, and sample rods were replaced daily or every monday and thursday (bi-weekly). three spore samplers were placed at each of the six commercial vineyards that were collected by growers bi-weekly. the growers completely maintained one trap, processing all sample rods derived from that trap. sample rods from the other two traps were collected by the growers and transported to the lab for processing with the l-qlamp assay and the qpcr assay. at the oregon state university botany and plant pathology research farm vineyard, paired spore samplers, one for the qpcr assay and one for the qlamp assay, were collected and processed by laboratory personnel on a daily and bi-weekly schedule. spore samplers for the l-qlamp and the qpcr assays were deployed on april , and april , and sample rods were collected from bud break until véraison (bbch ). spore samplers for the g-qlamp assay were deployed april , and were collected until july , . estimates of airborne inoculum concentration derived using qpcr and qlamp were compared to assess the accuracy of the qlamp procedure. the g-qlamp assay detection results were compared to the l-qlamp assay and qpcr detection data as described below. data was analyzed using r . . . detections from samples collected and quantified with l-qlamp assay were compared to qpcr assay detections using a student's t-test. the g-qlamp detection results were compared to l-qlamp detection results using a  contingency table whereby the l-qlamp results were assumed correct. both the l-qlamp and g-qlamp spore detections were evaluated using a  contingency table whereby the qpcr assay results were assumed correct. the qlamp assay detection accuracy, true positive proportion, true negative proportion, fisher's exact test, and chi-squared test were assessed comparing the qlamp detection results to the qpcr detection results. the qlamp assay showed high sensitivity to e. necator conidia dna when separate spore dilution series were tested ( fig. ) with % of one conidia samples amplifying using the qlamp assay. all other spore quantities tested showed % amplification sensitivity within the qlamp assay. the qlamp assay standard curve development resulted in a standard curve (r = . ) when fit with a log-linear curve (fig. ) . a log-linear curve was fit to the log spore quantity to account for the number of primers used in the assay, and the amplicon produced concatenates resulting in greater than an exponential rate of amplification. this curve was used to quantify the l-qlamp samples collected from the botany and plant pathology research farm vineyard. the l-qlamp spore quantification was significantly lower than the qpcr quantification when daily samples were collected in (p < . ) (fig. a) , but the biweekly l-qlamp and qpcr sample quantification was not significantly different in (p = . ) (fig. b) . the l-qlamp assay significantly underrepresented spore levels for both the daily collections (p < . ) (fig. a ) and the biweekly collections (p = . ) (fig. b ) compared to the qpcr assay in . utilizing l-qlamp for detection of e. necator showed similar results to qpcr assay detections in both and (p < . ) ( table ). the l-qlamp assay detection results were % and % accurate in and , respectively compared to the qpcr assay detection results. the l-qlamp assay detection results showed true negative proportions of % and % and true positive proportions of % and % in in and , respectively. there was an unexplained loss of sensitivity in sample testing that was extensively examined (see below). grower-conducted qlamp assay the software provided with the mobile lamp device used auto-adjusting threshold values to account for noise of fluorescence readings which significantly reduced accurate quantification by growers. the g-qlamp assay for the detection of e. necator was not correlated to the qpcr detection results (p = . ) ( table ). the g-qlamp detection results showed % accuracy compared to the qpcr assay results, respectively. the g-qlamp detection results show true negative proportions of %, and true positive proportions of % compared to the qpcr detection results. due to loss of sensitivity of the qlamp assays to e. necator observed during assay testing in , extensive troubleshooting was conducted. primer purification, polymerase used (bst; new england biolabs, ipswich, ma or iso- ; optigene ltd, west sussex, uk), master mix distributer, assimilating probe removal, primer and assimilating probe manufacturer, inhibitor removal compounds in the master mix, dna extraction and clean up, adjustment of reaction temperature, and replacement of reagents and primers were all tested. primer purification was tested prior to the implementation of the experiment, and during the observed degradation of qlamp sensitivity with no observable difference between reaction efficiency of hplc or desalted primers. regardless of polymerase used, bst or iso- , reaction efficiency and sensitivity to e. necator dna was reduced compared to assays conducted prior to implementation of field testing. different distributers of the optigene isothermal mastermix were also tested to determine if the decreased sensitivity was caused by storage or shipping errors; however, there was no difference among master mix vendors. it was not possible to test previous lots of the master mix prior to the observed decrease in sensitivity. the assimilating probe was removed and gel electrophoresis was used to compare with and without probe presence, and no difference was observed in amplification. there was also no difference between different primer and probe manufacturers, which also suggests there were no differences in manufacturing process. the concentrations of inhibitor removal compounds within the master mix were assessed, including % polyvinylpyrrolidone (pvp) , edta, and bsa concentrations, to determine if inhibitor presence was causing decreased reaction efficiency, and no differences were observed for inhibitor removal compounds. in addition to testing master mix removal of inhibitors, three dna extraction methods (extractions with ph . , mm tris- . mm edta buffer (affymetrix, santa clara, ca, usa), pvp (sigma-aldrich, st. louis, mo, usa) in depc-treated water, and powersoil Ò dna extraction kit (mo bio laboratories, inc., carlsbad, ca, usa)) were assessed with separate field collected spore samples. no differences were observed in amplification time or efficiency when testing each side-by-side extraction method. to test the optimal reaction temperature of the polymerase, temperatures between and c were examined to find the optimal reaction temperature. lower spore quantities ( spores or less) amplified at c. a last effort to determine if the effect notes: a "positive" and "negative" indicate the number of samples for which e. necator dna was detected and not detected, respectively, as tested by l-qlamp (n = in and n = in ) assays as described in the text. b g-qlamp (n = in ) assessed by growers using mobile qlamp devices (diagenetix, inc., honolulu, hi, usa) as described in the text. c qpcr results based on taqman Ò probe with minor groove binder for detecting e. necator dna. "positive" and "negative" indicate the number of samples for which e. necator dna was detected and not detected, respectively. qpcr detection data based on quantitative data from (thiessen, neill & mahaffee, ) . d fisher's exact test was used to assess the null hypothesis that each lamp assay was significantly different from the qpcr assay. * significant chi-squared test at p < . of qlamp and qpcr assays. was due to degradation of reagents or primers during the growing season, all reagents, primers, and probe were replaced; however, the decreased sensitivity to e. necator dna was still observed. despite targeting various portions of the reaction and extraction, the cause for loss of qlamp assay sensitivity remains undetermined. a highly sensitive qlamp assay was successfully developed using a simple dna extraction method for use by growers or crop consultants to use as a decision aid for timing fungicide applications similar to thiessen et al. ( ) and thiessen, neill & mahaffee ( ) . the qlamp assay developed was sensitive to e. necator dna with one spore amplifying % (n = ) using the simplified dna extraction. this sensitivity indicated that the assay should be suitable to detect inoculum (i.e., ascospores) at low concentrations (< spores) and aid management decisions. however, the qlamp assay consistently underrepresented spore quantities later in the growing season compared to the qpcr assay, which may be due to an increase in the presence of pcr inhibitors (such as pollen, humic acids from soil, spider webs, etc.) found in air samples (wilson, ) that may not have been removed by the rapid chelex dna extraction. in early dna extraction testing prior to qlamp sensitivity loss, the powersoil Ò extracted dna showed more consistent amplification of field samples than the other extraction methods (thiessen et al., ) ; however, the powersoil Ò dna extraction kit requires a larger time commitment and several steps that may not be feasible for in-field dna extractions. the lamp assay has been widely described as more tolerant to inhibitors than qpcr (francois et al., ; kaneko et al., ) , but it appears that the lamp assay tolerates different inhibitors than the qpcr assay (nixon et al., ) . additionally, the qlamp r t variance from one to spore samples (fig. ) was so great that they cannot be distinguished. this variance is likely due to using dna extractions of each spore concentration as opposed a dilution from higher spore concentration as is typically done (mahaffee & stoll, ) . because the lamp assay is not limited by temperature cycles, annealing is reliant on proximity of dna to the polymerase and primer set (notomi et al., ) , and the improved sensitivity with lower annealing temperatures is likely the result of lower specificity of primers rather than optimal reaction temperature. in reactions with lower quantities of dna (e.g., one and spores), more time may be required for the polymerase, primers, and target dna to meet, which may explain the variability of r t values of low spore quantities (fig. ) . the inhibition of the field qlamp assay and the difficulty of differentiating low spore quantities indicates that the assay currently has more utility as a qualitative inoculum detection tool as opposed to quantitative assessment of inoculum availability. the g-qlamp results were significantly different from the qpcr detection results (p = . ) ( table ). this may be due to difficulty in assessing positive detections from the output of the mobile device. the curve smoothing algorithm used by the device application (g-qlamp) often produced curves that drifted linearly with r t values reported even though there was no detectable amplification using gel electrophoresis. growers conducting the q-lamp assay were directed to ignore curves that ascended linearly due to curve smoothing; however, this may have caused growers to be overlyconservative in determining positive detections. additionally, the grower-conducted q-lamp occurred in when the loss of q-lamp sensitivity was observed and there was very low disease. the l-qlamp assay detection results were similar to qpcr assay detection results in both and , but true positive and true negative proportions were variable between years. this variability may be due to the presence of inhibitors. in , the source of stainless-steel rod material was changed from previous testing, and significant inhibition of dna amplification was observed. after troubleshooting various rod cleaning processes and dna extraction techniques, a hexane soak was added to the steel rod cleaning protocol to remove oils prior to sterilization and % chelex was used as the extraction buffer. after the hexane wash step addition, the accuracy of samples was improved to %, and the misclassification rate was reduced from % to %. in addition to inhibitors from the rods, the variability of inhibitors from field collections may have caused inconsistencies in qlamp assay detection results compared to the qpcr assay detection results. early in the growing season, the weather in the region is characterized by frequent precipitation events that limit pollen and insect flight. later in the growing season, pollen, insects, birds, and soil particulates are abundant in the air, and subsequently on the sampling rods. rnases, dnases, humic acids, and other heavy metals may not be removed when using the chelex dna extraction (qlamp template), but are removed during the powersoil dna extraction (qpcr template). the results from the qlamp had lower true positive proportions and true negative proportions than that of turbidimetric lamp previously developed (thiessen et al., ) . these reductions may be due to other factors besides amplification inhibitors, such as manufacturer differences, degradation of polymerase, inclusion of probes, or buffering of the qlamp reaction (corless et al., ; roux, ). using the qlamp assay for field detection and quantification of fungal pathogens may not be as feasible as previously thought due to the random loss of assay sensitivity and potential inhibition of polymerase activity by environmental contaminants. redesigning primers was another potential approach to examining the cause of the reduced sensitivity; however, the primer set used here was the result of two previous redesigns during development and testing and there was not sufficient heterogeneity in other regions of the its. additionally, the lamp assay quantification was also affected by numerous inhibitors, such as soil, pollen, or insect debris, found in field collected samples. lamp is capable of tolerating some inhibitors that affect pcr assays (francois et al., ) ; however, to determine the extent that lamp assays are capable of tolerating inhibitors, each potential inhibitor should be tested (nixon et al., ) . other lamp assays developed have utilized more complex dna extractions to reduce the effect of inhibitors on amplification for quantitation of dna (harper, ward & clover, ; kubota et al., ; mori et al., ) ; however, complex dna extraction techniques are likely to preclude field implementation of lamp assays and increase assay costs. the observed inconsistency indicates that the developed qlamp assays might not be robust enough for commercial implementation. the lamp assay was developed due to reports of high sensitivity and specificity to target dna, tolerance of the reaction to the presence of reaction inhibitors, and the potential for use by growers or crop consultants using handheld lamp devices such as the bioranger (diagenetix, inc., honolulu, hi, usa) or the genie ii and iii (optigene ltd, west sussex, uk) (kubota et al., , mori et al., mori et al., , notomi et al., ; temple & johnson, ; tomlinson, dickinson & boonham, ) ; however, field testing of the qlamp assay for e. necator revealed an unidentifiable degradation of the sensitivity of the assay to the target dna. the qlamp assay may still be a useful tool for field inoculum detection, but further analysis of the system is required to determine the specific cause of the degradation of the assay. at the time this research was initiated the lamp technology was the most advanced for inexpensive field application and thus selected for investigation over other potentially suitable technologies. however, other dna amplification techniques have since become more accessible for field use (marx, ) , including qpcr (biomeme, inc., philadelphia, pa, usa), recombinase polymerase amplification (piepenburg et al., ) , and helicase-dependent isothermal dna amplification (vincent, xu & kong, ) . these assays require minimal dna preparation, are capable of real-time data, and may be easily adapted to the air samples used here but require evaluation. there are several reviews that discuss the advantages and disadvantages of these technologies (craw & balachandran, ; gill & ghaemi, ; mahaffee, ; niemz, ferguson & boyle, ; yan et al., ) . a highly sensitive qlamp assay was developed using a simple dna extraction method for use by growers or crop consultants utilizing inoculum detection; however, the qlamp assay consistently underrepresented spore quantities later in the growing season compared to the qpcr assay. additionally, the qlamp assay lost sensitivity to low spore quantities (< spores) in the sampling period, and the cause was not determined during the course of this study. grower-conducted inoculum monitoring technologies, like the qlamp assay developed in this study, may provide an inexpensive tool for producers to apply targeted fungicide applications based on inoculum presence and concentration. given the limitations described herein, more assessment of the qlamp assay degradation is necessary before utilizing it as a monitoring tool for e. necator inoculum concentrations. visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loopmediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye grape powdery mildew (erysiphe necator) risk assessment based on airborne conidium concentration a degree-day model to initiate fungicide spray programs for management of grape powdery mildew (erysiphe necator) development of a taqman real-time pcr assay for quantification of airborne conidia of botrytis squamosa and management of botrytis leaf blight of onion contamination and sensitivity issues with a real-time universal s rrna pcr isothermal nucleic acid amplification technologies for point-ofcare diagnostics: a critical review detection of erysiphe necator in air samples using the polymerase chain reaction and speciesspecific primers shining a light on lamp assays-a comparison of lamp visualization methods including the novel use of berberine robustness of a loop-mediated isothermal amplification reaction for diagnostic applications ascocarp dehiscence and ascospore discharge in uncinula necator nucleic acid isothermal 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cinerea by loop-mediated isothermal amplification helicase-dependent isothermal dna amplification pcr to predict risk of airborne disease inhibition and facilitation of nucleic acid amplification isothermal amplified detection of dna and rna we thank the technical support of andy albrecht, cole provence, chris gorman, and jim eynard. we also thank anonymous reviewers for their helpful suggestions to improve the manuscript. we especially thank the numerous vineyard managers that collaborated on the project. the use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. such use does not constitute an official endorsement or approval by the united states department of agriculture or the agricultural research service of any product or service to the exclusion of others that may be suitable. this work was supported by the american vineyard foundation, the oregon wine board, and usda-ars cris - - - d. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the authors: american vineyard foundation. oregon wine board. usda-ars cris: - - - d. the authors declare that they have no competing interests. lindsey d. thiessen conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. tara m. neill conceived and designed the experiments, performed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. walter f. mahaffee conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability:the raw ct values and positive negative values of detection events, as well as standard curve development and sensitivity of assay, are provided in supplemental dataset files. supplemental information for this article can be found online at http://dx.doi.org/ . /peerj. #supplemental-information. key: cord- -cq xe r authors: dao thi, viet loan; herbst, konrad; boerner, kathleen; meurer, matthias; kremer, lukas pm; kirrmaier, daniel; freistaedter, andrew; papagiannidis, dimitrios; galmozzi, carla; stanifer, megan l.; boulant, steeve; klein, steffen; chlanda, petr; khalid, dina; barreto miranda, isabel; schnitzler, paul; kräusslich, hans-georg; knop, michael; anders, simon title: a colorimetric rt-lamp assay and lamp-sequencing for detecting sars-cov- rna in clinical samples date: - - journal: sci transl med doi: . /scitranslmed.abc sha: doc_id: cord_uid: cq xe r the coronavirus disease (covid- ) pandemic caused by the sars-cov- (severe acute respiratory syndrome coronavirus ) coronavirus is a major public health challenge. rapid tests for detecting existing sars-cov- infections and assessing virus spread are critical. approaches to detect viral rna based on reverse transcription loop-mediated isothermal amplification (rt-lamp) have potential as simple, scalable, and broadly applicable testing methods. compared to rt quantitative polymerase chain reaction (rt-qpcr)–based methods, rt-lamp assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. here, we tested a two-color rt-lamp assay protocol for detecting sars-cov- viral rna using a primer set specific for the n gene. we tested our rt-lamp assay on surplus rna samples isolated from pharyngeal swab specimens collected from individuals being tested for covid- . we determined the sensitivity and specificity of the rt-lamp assay for detecting sars-cov- viral rna. compared to an rt-qpcr assay using a sensitive primer set, we found that the rt-lamp assay reliably detected sars-cov- rna with an rt-qpcr cycle threshold (ct) number of up to , with a sensitivity of . % and a specificity of . %. we also developed a swab–to–rt-lamp assay that did not require a prior rna isolation step, which retained excellent specificity ( . %) but showed lower sensitivity ( % for ct < ) than the rt-lamp assay. in addition, we developed a multiplexed sequencing protocol (lamp-sequencing) as a diagnostic validation procedure to detect and record the outcome of rt-lamp reactions. the coronavirus disease (covid- ) pandemic, caused by the sars-cov- (severe acute respiratory syndrome coronavirus ) coronavirus ( ), is a major global health threat. a still unknown proportion of people, especially the elderly and those with preexisting conditions, are at high risk of a severe course of covid- ( ), leading to a high burden on health care systems worldwide. further, because of limited testing capacity, only people with symptoms are usually tested for sars-cov- infection, although studies have confirmed that many individuals infected with sars-cov- are asymptomatic carriers of the virus ( , ) . this suggests that infection control strategies focusing on symptomatic patients are not sufficient to prevent virus spread. therefore, large-scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively. this would allow for the rapid isolation of infected persons during an existing wave of infection. in addition, continuous and repeated testing of large groups within a population may be required as a long-term strategy to contain new outbreaks while keeping societies and economies functional until effective vaccines become available. an active sars-cov- infection can be diagnosed by detecting either the viral genome or viral antigens in appropriate human samples. assays for detecting sars-cov- antigens are limited by the sensitivity, specificity, and production speed of diagnostic antibodies, whereas detecting viral rna only requires specific oligonucleotides. therefore, an assay that detects sars-cov- rna facilitates testing of large cohorts. the sars-cov- diagnostic pipeline that has proven to be successful and that is currently used in many test centers consists of three steps: collecting nasopharyngeal or oropharyngeal swab specimens, isolation of total rna, and specific detection of the viral genome by rt-qpcr. the latter comprises a reverse transcriptase (rt) step, which translates the viral rna into dna, followed by a semiquantitative dna polymerase chain reaction using oligonucleotides specific for the viral cdna (qpcr). as a result, a short piece of the viral genome is strongly amplified and then is detected by a sequence-specific oligonucleotide probe labeled with a fluorescent dye. this procedure includes several steps that require sample handling; therefore, the detection process in a clinical diagnostic laboratory takes about to hours or more, depending on the number of samples and process optimization of the test center. in addition, in the context of the covid- pandemic, many of the reagents required are only slowly being replenished due to insufficient production capacity or lack of international transport. therefore, increasing daily test capacities for rt-qpcr-based diagnostics for sars-cov- rna detection is currently limited. to accelerate and optimize such diagnostics, new scalable methods for rna isolation and the detection of viral genomes are needed. an alternative to rt-qpcr is reverse transcription loop-mediated isothermal amplification (rt-lamp) ( ) ( ) ( ) . rt-lamp reactions include a reverse transcriptase and a dna polymerase with strong strand displacement activity and tolerance for elevated temperatures and up to six dna oligonucleotides of a certain architecture. samples with potential template molecules are added to the reaction and incubated for to min at a constant temperature (e.g., °c). the oligonucleotides act as primers for the reverse transcriptase, and additional oligonucleotides for the dna polymerase are designed so the dna products loop back at their ends. these, in turn, serve as self-priming templates for the dna polymerase. in the presence of a few rna template molecules, a chain reaction is set in motion, which then runs until the added reagents (in particular, the deoxynucleotide triphosphates) are used up. to detect dna production in rt-lamp assays, various approaches have been described. one possibility is to use a ph indicator (e.g., phenol red) and run the reaction in a weakly buffered environment. as the chain reaction proceeds, the ph is lowered, which results in a visible color change from red to yellow making it an appealing assay for point-of-care diagnosis ( ) . previously, rt-lamp assays have been proposed for diagnostic detection of other rna viruses, such as influenza virus ( ) . also, several studies have demonstrated the use of isothermal dna amplification to detect small amounts of sars-cov- rna. the majority of these studies used in vitro transcribed (ivt) short fragments of the viral genomic rna ( - ) and showed a detection limit of somewhere between and rna molecules per reaction. for the detection of sars-cov- rna, a few commercial rapid tests have been developed [reviewed in ( ) ] using isothermal dna amplification reactions involving proprietary enzyme formulations that are not commercially available in a ready-to-go format. further, their exact sensitivity is still subject to discussion owing to a lack of studies using sufficiently large numbers of test samples. the performance of an rt-lamp assay does not require expensive special equipment such as a thermal cycler with real-time fluorescence measurement, because positive samples are determined by a color change from red to yellow within min after the start of the incubation at °c. for detection, simple mobile phone cameras, copy machines, office scanners, or plate scanners with spectrophotometric quantification can be used. during the early phase of the covid- pandemic (early march ) in germany, we tested the sensitivity and specificity of a colorimetric rt-lamp assay for detecting sars-cov- rna in clinical rna samples isolated from pharyngeal swab specimens collected from individuals being tested for covid- (and provided by the heidelberg university hospital's diagnostic laboratory after removal of an aliquot for sars-cov- rna testing by rt-qpcr) (fig. s ). we also developed a swab-to-rt-lamp assay that used naso/oropharyngeal swab specimens directly without the need for an rna isolation step. we tested > clinical rna samples with a wide range of viral loads, allowing us to determine accurately the sensitivity range of the colorimetric rt-lamp assay. we also developed a multiplexed lamp-sequencing protocol using barcoded tn transposase tagmentation that enabled rapid identification of positive results in thousands of rt-lamp reactions within the same next-generation sequencing run. establishing colorimetric rt-lamp assay sensitivity using an artificial sars-cov- rna template to detect sars-cov- rna with rt-lamp, we used the warmstart colorimetric rt-lamp x master mix (dna and rna) from new england biolabs. this mix contains two enzymes, an engineered reverse transcriptase (rtx) and a strand-displacing polymerase (bst . ). in addition, the reaction mixture contains oligonucleotide-based aptamers that function as reversible temperature-dependent inhibitors, ensuring that the reaction only runs at an elevated temperature (warmstart) to avoid nonspecific priming reactions. several primer sets were recently proposed for rt-lamp-based detection of sars-cov- rna by zhang et al. ( ) and by yu et al. ( ) , and these primer sets were subsequently validated with in vitro-translated rna. we prepared and tested two primer sets for different rna sections of the sars-cov- genome, the n-a set targeting the n gene and the a-a set targeting open reading frame (orf) a (table s ) ( ) . figure a shows that the oligonucleotide set for the n gene was capable of detecting ivt rna molecules in a test reaction with l of rna solution, as evidenced by the red-toyellow color change. the reaction was conducted for up to hour at °c. for time points > to min, the negative control frequently became yellowish (fig. a) . this was caused by spurious amplification products, which is a wellknown problem with rt-lamp ( ) . analysis by gel electrophoresis revealed clearly distinct banding patterns for the correct rt-lamp reaction products (lanes with ≥ molecules ivt rna input) and the spurious reaction products (fig. b) . to evaluate the colorimetric rt-lamp assay, we needed to compare its sensitivity and specificity to a validated rt-qpcr method. we first used rna samples and performed rt-lamp reactions using l of the isolated rna the rt-lamp reaction product ( . l) was analyzed on a % agarose gel. the typical band pattern of a successful rt-lamp reaction was visible in the samples with or more sars-cov- rna molecules, i.e., in those samples that showed a color change from red to yellow after min. in a reaction volume of . l. we detected a red-to-yellow color change in of the samples following an incubation of the reaction for min at °c ( fig. a ). to quantify the reaction, we used a plate scanner and measured the difference in absorbance (od) of the samples at and nm (corresponding to the absorbance maxima of the two forms of phenol red that were used in the assay as a ph-sensitive dye) at several time points. to visualize the data, we plotted the od values against incubation time and colored the time traces of individual samples according to the cycle threshold (ct) values obtained from the rt-qpcr test run in the clinical diagnostic laboratory (fig. b ). this rt-qpcr test was performed using a commercial diagnostic test kit containing a modified version of the e-sarbeco primer set for the viral e gene suggested by corman et al. ( ) and l of rna isolated with an automated platform (qiasymphony or qiacube). in a colorimetric rt-lamp reaction, positive samples with a ct < changed the color of the phenol-red dye within the first min of the reaction. samples with a ct > either did not change their color or did so at time points > min, simultaneously with a color change observed in some of the negative samples (fig. ) . on the basis of this observation, we used the od value at min to decide whether a sample was positive or negative. plotting the od measurements versus ct values at the -min time point revealed that all patient samples with a ct < showed a robust color change in the rt-lamp test, whereas for samples with ct values between and , a positive result was observed for only of samples (fig. c ). this suggested a detection limit of the colorimetric rt-lamp assay corresponding to a ct ≈ for rt-qpcr. the rt-qpcr kit used was calibrated and a ct ≈ corresponded to rna molecules present in the reaction according to the certificate provided by the manufacturer (see materials and methods). the performance of each rt-qpcr run was validated using this as a positive control. considering that l of isolated rna was used for rt-qpcr, but only l for the rt-lamp assay, a cutoff of ct ≈ agreed well with the observed experimental sensitivity of approximately rna molecules for the rt-lamp assay (fig. a ). therefore, it appeared that the n-a primer set used for the rt-lamp assay performed equally well with either ivt rna or rna samples isolated from the pharyngeal swab specimens. in march , at the beginning of the pandemic, the diagnostic laboratory that analyzed the pharyngeal swab samples by rt-qpcr validated all samples that tested positive with the e gene primer set in a second rt-qpcr using the n gene primer set, also of the sarbeco sets of corman et al. ( ) . when plotting rt-lamp assay results against the ct values for the n gene primer set, we observed a sensitivity cutoff of around ct ≈ ( fig. s a ). direct comparison of the ct values for the e gene and n gene primer sets for all samples revealed a difference of ~ . ct units (cycles) ( fig. s b ). this suggested that the n gene primers were less sensitive than the e gene primers for detecting sars-cov- rna by rt-qpcr. similar differences have been observed previously for other primer sets, e.g., between the e gene primers and the rdrp-sarsr primers ( ) . for the rt-lamp assay, we also tested the a-a primer set directed against orf a ( ) and found this primer set to be less sensitive than the n gene lamp primer set, with a sensitivity cutoff of ct ≈ when plotted against e gene rt-qpcr-derived ct values ( fig. s ). on the basis of these results, we decided to use the n-a primer set for the rt-lamp assay and to compare our results with rt-qpcr performed with the e-sarbeco primer set. to determine the specificity and sensitivity of the rt-lamp assay, we continued to analyze more rna samples. we assayed a total of rna samples obtained on different days ( fig. s ). visualization of the rt-lamp assay results min after the start of the incubation at °c showed comparable behavior of the samples in a total of ten -well test plates ( fig. a and table ), indicating that the rt-lamp assay was reproducible from day to day and from plate to plate. the consistency of the results during the analysis confirmed a threshold of od > + . as a robust measure to identify samples that were positive for sars-cov- rna (fig. a) . rt-qpcr-positive samples with a ct < scored positive in the rt-lamp assay ( of ), whereas almost all samples with ct values between and scored negative (only positive of ) (fig. b ). this confirmed the sensitivity of the rt-lamp assay for detection of sars-cov- rna in samples corresponding to a ct < . we observed small differences between different plates on the exact sensitivity threshold, probably caused by slight variability in plate or reagent handling. we found two rt-qpcr-negative samples that scored positive in the rt-lamp assay ( fig. a and table ) and one sample that scored just below the od cutoff of + . . the overall specificity of the rt-lamp test was . % (wilson's % confidence interval: . to . %), and the sensitivity for samples with ct < on rt-qpcr was . % (wilson's % confidence interval: . to . %) ( fig. b and table s ). our results indicated that the colorimetric rt-lamp assay enabled robust identification of positive samples after a -to -min incubation at °c. validation of positive results, however, required confirmation that the rt-lamp reaction led to the amplification of viral sequences. to analyze the sequences of many rt-lamp reaction products, we established multiplexed sequencing of rt-lamp products (lamp-sequencing). lamp-sequencing is based on tn transposase tagmentation ( ) and sample barcoding. tagmentation enables fragmentation and direct adapter ligation of dna samples for analysis by nextgeneration sequencing. we used a set of barcoded adapters for tagmentation to barcode the rt-lamp reaction products in each -well plate. after tagmentation, all barcoded fragments from each plate were pooled and sizeselected by bead purification to remove excess adapters. a second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented rt-lamp fragments. last, all amplified pools were combined for analysis using one next-generation sequencing run where the origin of each dna fragment was specified by the two barcodes (fig. a ). of the lamp-sequencing reads obtained, % mapped either to the part of the viral genome targeted by the rt-lamp primers ( . %) or contained short k-mers derived from primer sequences ( . %) ( fig. s ). this indicated that lamp-sequencing amplified the targeted sequences. reads containing only primer sequences were likely to be the result of spurious amplification products as these were also formed in the absence of input rna (fig. ) . for quantification of individual lamp reactions, we classified reads according to whether or not they contained viral sequences, which were not directly covered by the primers (orange segments in fig. s a ), and counted the reads for each sample (as specified by its barcode combination) ( fig. s b ). for of the samples, we obtained enough reads to make a call ( fig. s ). for the samples that underwent successful lamp-sequencing, the results confirmed all samples that scored positive on the rt-lamp assay with a ct < ( fig. b and table ). for the two samples with a negative rt-qpcr result that scored positive on the rt-lamp assay (fig. ) , the lamp-sequencing call agreed with the rt-qpcr result and thus corrected the rt-lamp result. lamp-sequencing was performed using the rt-lamp samples after a prolonged incubation of min at °c. at this time point, many of the negative samples and also samples with a ct between and had turned yellow. lamp-sequencing eliminated all of these samples (fig. c ). this indicated that even for the rt-qpcr-positive table shows numbers of samples stratified according to the results of the rt-lamp and the rt-qpcr assays. (b) sensitivity (right) and specificity (left) of the rt-lamp assay [derived from data in (a) and table ] are shown. the specificity is the fraction of rt-qpcr-negative samples correctly identified as negative by the rt-lamp assay. for sensitivity, the rt-qpcr-positive samples were stratified by ct values into three bins (as indicated by x axis labels), and for each bin, the sensitivity is given as the fraction of qpcr-positive samples in the respective ct bin that have also given a positive result in the rt-lamp assay. the thick black lines indicate the values of these fractions (i.e., the specificity and sensitivity estimates); the black boxes indicate the corresponding % confidence intervals (wilson's binomial confidence interval). (see also table s ) . samples with a ct between and , the color change that took place at time points > min was caused by spurious amplification products and not by late amplification of viral sequences. these results therefore confirmed that lamp-sequencing was able to assess the results of multiple rt-lamp reactions in parallel and to identify false-positive samples in the colorimetric rt-lamp assay. a swab-to-rt-lamp assay without rna isolation rna isolation is time consuming, costly, and depends on reagents with potentially limited supply during a pandemic. alternative, noncommercial solutions for rna isolation, e.g., using silica gel matrix or magnetic beads, require specialized knowledge and cannot be implemented easily for point-of-care or decentralized screening. several reports have indicated that rt-qpcr ( ) ( ) ( ) and rt-lamp assays ( , ) are compatible with direct testing of nasopharyngeal and oropharyngeal swab specimens without a prior rna purification or extraction step. to establish an rt-lamp assay that could test unprocessed specimens (swab-to-rt-lamp assay), we first assessed the stability of naked rna in swab specimens that were collected in amies medium. we titrated defined numbers of ivt rna molecules of the sars-cov- n gene into swab samples from covid- -negative control subjects. we tested different conditions, particularly the influence of detergent (to inactivate the virus) and heat (to denature the capsid and release the viral rna as well as inactivate the virus) (figs. s and s , and data file s ). consistent with previous reports about other rna viruses ( ) ( ) ( ) and tests using heat inactivation of swab specimens for direct rt-qpcr assays ( ) , these experiments established that native swab specimens and heat-treated swab specimens were compatible for detection of sars-cov- rna in swab samples from infected individuals. on the basis of these preliminary experiments, we decided to use swab samples either directly without any treatment (direct swab-to-rt-lamp assay) or after heat treatment for min at °c (hot swab-to-rt-lamp assay). as an additional precaution, we kept the samples in the cold (using an icecold metal block) whenever possible. for testing large numbers of clinical samples, we performed the rt-lamp assay using several -well plates. in total, we tested different samples using the hot swab-to-rt-lamp assay, and of these, samples also were tested by the direct swabto-rt-lamp assay. many samples were tested twice but using aliquots withdrawn at different time points (usually within hours) from the swab samples stored at °c. this resulted in direct swabto-rt-lamp assay measurements and hot swab-to-rt-lamp assay measurements (fig. a) . the hot swab-to-rt-lamp assay detected a color change in the majority of samples with a ct < with high sensitivity, whereas the direct swab-to-rt-lamp assay only exhibited a high sensitivity for samples with a ct < ( fig. and table ). the heat treatment rendered the rt-lamp assay more stringent as it reduced false positives and more sensitive for samples with a ct of to . we found that some positive samples did not induce a color change but did so when assayed a second time. we therefore would recommend running this assay using technical duplicates. comparison of the results of the direct swab-to-rt-lamp assay with the rt-lamp assay using isolated rna revealed a much broader distribution of the od measurements in negative samples (fig. a versus fig. a) . this was likely due to a sample-specific variability that influenced the starting ph in the lamp reaction. this might have affected the interpretability of the measurement at min (od min ). we investigated how this ph shift influenced the rt-lamp assay. for three plates, the data acquired for the rt-lamp assay also included measurements for the -min time point (od min ) (fig. a) . we plotted the change of the od between the -and -min time points (i.e., the difference od min -od min , corresponding to the slope of the lines) versus od min (fig. b) . table . summary of lamp-sequencing results. the cross tabulation of rt-qpcr and rt-lamp assay results shown in table have been split into samples where sequencing of rt-lamp reaction products (lamp-sequencing) was positive (pos), negative (neg), or inconclusive (too few reads) (see also fig. ). this removed the variability of the values for samples that did not change their color (negative samples) and permitted a better separation of the positive from the negative samples. we noticed that the ph variability depended on the sample volume used for the rt-lamp assay and the composition of the medium used for the swabs. for swabs in amies medium (which was used for the clinical samples in this study), an rt-lamp assay containing l of sample in a total volume of l was optimal. our results obtained using native and heat-treated swab specimens suggested better performance when using heat treatment of swab specimens before running the rt-lamp assay. here, we evaluated the use and suitability of the rt-lamp assay for the detection of sars-cov- infection. we also developed lamp-sequencing as a fully scalable alternative to colorimetric or fluorometric analysis of dna amplification reactions. our results indicate that whereas the rt-lamp assay using the n-a primer set is not sensitive enough to replace rt-qpcr in all applications, it does hold promise as a method for testing large numbers of samples. we tested the rt-lamp primer sets suggested by zhang et al. ( ) and found that the n-a primer set for the n gene worked better than the a-a primer set for orf a. for samples with a ct ≤ as measured by rt-qpcr with e-sarbeco primers, we found overall satisfactory sensitivity and specificity values for sars-cov- rna detection by the rt-lamp assay using rna samples isolated from pharyngeal swab specimens ( fig. and table ). for samples with ct > , the rt-lamp assay was much less sensitive. however, there is debate about which ct value for a positive rt-qpcr result should be considered clinically relevant. vogels et al. ( ) indicate that a ct value above corresponds to less than molecules of rna. on the basis of our data, we conclude that the colorimetric rt-lamp assay would be suitable for identifying individuals with a high or moderate sars-cov- viral load. on the other hand, for those with a low viral load (at the onset of illness or during later stages of the disease), the sensitivity of the rt-lamp assay, in its current implementation using the n-a primer set, is insufficient to detect a sars-cov- infection. a number of other lamp primer sets have been proposed and initially tested ( , , ) , showing that optimized primers and the use of combinations of primer sets hold promise to further increase the sensitivity of the rt-lamp assay for detecting viral genomes. furthermore, alternative sample types, e.g., sputum or stool ( ) , might be more reliable. one promising lead for future applications is the exploration of the hot swab-to-rt-lamp assay using saliva specimens, although the relative sensitivity compared to using pharyngeal swab specimens is currently unclear ( ) ( ) ( ) ( ) . compatibility of the rt-lamp assay with direct saliva specimens has been shown using spike-in experiments ( , ) . although faster and more convenient, the direct swab-to-rt-lamp assay was less sensitive and less robust than the rt-lamp assay using isolated rna. to increase robustness, various treatments of crude swab samples have been described previously [reviewed in ( ) ], many of which require additional processing of the samples, for example, by pipetting or by adding proteinase k to degrade contaminating proteins. rabe and cepko ( ) have suggested using cheap silica preparations and new sample inactivation protocols to enrich the rna before the rt-lamp assay, but this would complicate the simple swab-to-rt-lamp assay workflow. fig. to score the direct (left) and hot (right) swab-to-rt-lamp assays, namely, od at min, is shown on the y axis, and compared to an alternative score, namely, the difference between the od signals at min and at min after the start of incubation, shown on the x axis. the latter shows better separation between positive and negative samples. last, our analysis found that a short heat treatment of min at °c, which poses minimal additional handling steps, did not destroy the rna but rather stabilized it and this improved the sensitivity and specificity of the swab-to-rt-lamp assay (fig. ) . the heat likely helped to homogenize the sample, to inactivate ribonucleases (rnases), and to break up the viral capsid to release the viral rna. overall, our data demonstrate the feasibility of using a swab-to-rt-lamp test and suggest applications especially in scenarios where rna isolation is not available, e.g., in resource-poor settings. in such cases, the hot swab-to-rt-lamp assay seems a good option given that the direct swab-to-rt-lamp assay yields a number of false positives due to spurious amplification ( ) . although spike-in experiments with ivt rna can be informative, we have experienced clear differences when comparing such experiments to those using clinical rna samples isolated from swab specimens (figs. s and s , and data file s ). we therefore recommend validating any new proposed rapid sars-cov- diagnostic test using "real-life" clinical samples including a large fraction of negative clinical samples. to overcome the problem of spurious amplification, an expanded oligonucleotide set that incorporates sequence-specific probes ( ) or a crispr/cas a-based approach ( ) could be used. however, these applications have yet to be tested with large numbers of diverse clinical samples. there are several differences between the rt-lamp assay and rt-qpcr. first, rt-qpcr requires a thermocycler to conduct the dna amplification reaction, which is an expensive instrument, whereas isothermal incubation of rt-lamp reactions can be conducted using a simple water bath or a heating block. this makes the rt-lamp assay more amenable for point-of-care applications. second, the reagents for the rt-lamp assay are different from the ones used for rt-qpcr and are supplier independent. according to the supplier of the rt-lamp reagents used in this study (new england biolabs), production of rt-lamp reagents can be easily ramped up to satisfy high demand. third, the rt-lamp assay, when combined with lampsequencing, is suitable for analyzing large numbers of rt-lamp reactions owing to the fully scalable dna barcoding strategy. in contrast, there are several hurdles to scaling up rt-qpcr assays, the major hurdle being the need for a large number of thermocyclers. the rt-lamp assay overcomes this problem and therefore will be a more scalable method for mass testing. with its good sensitivity for samples up to ct ≈ , the colorimetric rt-lamp assay has several advantages: it is fast, inexpensive, and it can be evaluated without any equipment. rt-lamp reactions also appear to be less sensitive to contaminants in the samples than rt-qpcr, but care has to be taken that the samples used do not alter the ph as the colorimetric rt-lamp assay is performed under conditions of weak ph buffering. some clinical samples contain contaminants that can lead to acidification of the reaction independent of the presence of a template rna if too much sample is added. diagnostic rt-qpcr tests usually include a technical internal control, i.e., another rna species, which is spiked into all samples and which is detected independent of the gene of interest to safeguard against the possibility of a general reaction failure within a sample tube. it would be desirable to have a similar precaution for the rt-lamp assay. a multiplexed fluorescence readout might provide this ( ) but comes at the expense of the simplicity of a colorimetric readout. our particular implementation of deep sequencing to analyze many rt-lamp reactions simultaneously uses two sets of barcoded primers and is fully scalable so that, in one sequencing run, many thousands of lamp reactions can be quantitatively analyzed for the presence of viral genomic sequences. although we used illumina dye sequencing, more scalable sequencing technologies, such as oxford nanopore technologies sequencing, could be used for amplicon sequencing and counting ( ) . the workflow shown here uses lamp-sequencing as a validation and backup procedure to double check the results of the colorimetric rt-lamp assay. however, lamp-sequencing could also facilitate scale-up of the workflow for direct analysis of many thousands of samples in an efficient manner, provided that an infrastructure is established that allows the collection of such samples. thus, lampsequencing could become an important part of workflows for routine testing of large populations. schmid-burgk et al. ( ) proposed decentralized rt-lamp assays using combinatorial primer barcoding and centralized mass analysis of rt-lamp products by next-generation sequencing as a means to scale-up testing. although this poses additional challenges in generating the individualized rt-lamp assay reagents, it would simplify sample handling on the analytical side and it can be easily combined with the barcoding strategy shown here. there are several limitations to our study. we used surplus rna sample material from a diagnostic laboratory rather than newly collected clinical samples. the criteria for testing individuals may have influenced cohort characteristics and hence our findings. it is not clear yet how well viral load as indicated by ct values from rt-qpcr assays informs about the degree of infectivity of an individual with a sars-cov- infection. therefore, we cannot say how our findings on the sensitivity of the rt-lamp assay in comparison to rt-qpcr would translate into sensitivity for detecting infectious individuals who are shedding sars-cov- virus. moreover, the measured viral load does not indicate the course of a sars-cov- table . shown is rt-qpcr and rt-lamp testing of clinical samples stratified into ct value bins (see fig. a ). fig. a and table s show specificity and sensitivity values calculated from these numbers. infection, as even individuals with a very low measured viral load can still develop severe symptoms of covid- disease. this may be, in part, because the viral load in a clinical sample taken from a specific site such as the pharynx is not representative of the overall viral burden that an infected individual carries. we used lamp-sequencing to validate the rt-lamp assay results and did not use it as a diagnostic tool. lamp-sequencing is dependent on the sensitivity of the rt-lamp reaction as it cannot detect false negative results caused by a failure of the rt-lamp assay to amplify viral rna. also, reagents such as the primer sets for the rt-lamp assay may be subject to production-dependent quality fluctuations. therefore, all reagents must be precisely validated (batch control) before using an rt-lamp assay diagnostically. application of the rt-lamp assay has great potential, even more so as more sensitive primer sets become available. the rt-lamp assay and lamp-sequencing could offer scalable testing that would be difficult to achieve with conventional rt-qpcr-based tests. for example, the rt-lamp assay could be used for regular testing of a whole workforce or in sentinel testing, ideally combined with simplified sample collection, e.g., in the form of saliva samples. the rt-lamp assay and lamp-sequencing extend the range of available test methods and complement individual tests and pooled tests based on rt-qpcr ( ) with a faster, simpler, and potentially more costeffective test method. the intent of this study was to develop a clinical method for detecting sars-cov- rna in rna samples isolated from pharyngeal swab specimens from individuals being tested for covid- . we used pseudo-anonymized surplus rna sample material that had been collected for clinical diagnosis of sars-cov- infection by rt-qpcr carried out by the diagnostic laboratory of heidelberg university hospital. such reuse of material is in accordance with german regulations, which allow development and improvement of diagnostic assays using patient samples collected specifically to perform the testing in question. pharyngeal swab specimens provided to us were either collected through the nose (nasopharyngeal) or the mouth (oropharyngeal), or sometimes one swab was used to collect both. our study was designed to investigate the sensitivity and specificity of a colorimetric rt-lamp assay and to evaluate its suitability as an alternative to rt-qpcr testing for detecting sars-cov- viral rna in rna isolated from pharyngeal swab specimens. this study was conducted in heidelberg, germany in march and april of . the study was designed to first evaluate different existing primer sets for rt-lamp reactions and to use them for (i) detection of sars-cov- rna in rna isolated from pharyngeal swabs and (ii) detection of sars-cov- rna directly from swab specimens without prior rna isolation. all rna samples used were pseudoanonymized surplus material from the heidelberg university hospital diagnostic laboratory, and rt-qpcr results for these rna samples were retrieved from the laboratory's database only after the samples had been analyzed by the rt-lamp assay. the study design was to conduct rt-lamp testing until sufficient samples (at least several hundreds) had been analyzed to obtain a conclusive result. we also designed a deep sequencing-based method to validate the outcome of the rt-lamp reactions using a tn transposase-based fully scalable barcoding strategy (lamp-sequencing). specimens were collected as nasopharyngeal and oropharyngeal flocked swabs in amies medium (eswab, copan italia). the sample collection happened as part of the routine operation of heidelberg university hospital and at public testing stations set up by the city of heidelberg (fig. s ). collected samples were transported in sterile containers, delivered to the diagnostic laboratory within a few hours, and then examined directly or stored at °c until further processing. samples were processed in a biosafety level cabinet until inactivation by heat or mixing with a lysis buffer. the standard diagnostic pipeline of the hospital laboratory was as follows: rna was isolated from nasopharyngeal and oropharyngeal swab specimens using qiagen kits ( . the master mix ( l) was distributed per reaction into -well plates, and l of purified rna was added per well. the performance of the rt-qpcr was validated using a positive control for the e gene. a total of molecules of e gene rna per rt-qpcr reaction correspond to a ct ≈ . the rt-lamp primer sets used in this study have been designed by zhang et al. ( ) against orf a and n gene and were synthesized by sigma-aldrich (synthesis scale, . mol; purification, desalt; solution, water). the sequences and the concentrations of each oligonucleotide in the × primer mix used for the rt-lamp assay can be found in table s . an rna-positive control for the n gene was amplified from a short fragment from -ncov_n_positive control plasmid [integrated dna technologies (idt), ] with oligonucleotides t -genen-fragment.for and genen-fragment.rev including the t promoter and a subsequent ivt with the megascript t kit (invitrogen) purified using the rneasy minelute cleanup kit (qiagen). to prevent cross-contamination, we have taken several precautions. the × primer mix was prepared with nuclease-free water (am , ambion) and stored in aliquots at − °c. to set up an rt-lamp test, the rt-lamp master mix was prepared freshly immediately before the test at a separate workspace with a dedicated pipette set and filter tips. the -well pcr plate containing the rt-lamp mix was covered with an society for biomolecular screening (sbs) plate lid. to avoid mix-ups during sample addition through well-by-well pipetting, the rna or swab specimens were first collected into a -well seed plate. the rna was then added to the plate with the lamp reagents at a dedicated workspace with a manual -channel pipettor (liquidator l, mettler toledo) using filter tips. the rt-lamp and the rna seed plate were instantly sealed with an optically clear adhesive seal (gk -os, kisker biotech) and an adhesive aluminum foil seal (sl-am , steinbrenner laborsysteme, germany), respectively. if the product of an rt-lamp reaction had to be analyzed by gel electrophoresis, the plate was opened with extreme caution at a separated post-lamp workspace and loaded onto an agarose gel with a dedicated pipette. assays were assembled in total reaction volumes of either . l (for lamp assays using isolated rna) or l (for swab-to-rt-lamp assays). master mixes were prepared at room temperature for each reaction immediately before use with either . or l, respectively, of the warmstart colorimetric rt-lamp x master mix (m , new england biolabs) and . or l, respectively, of the × primer mix, filled up to . or l with nuclease-free water (am , ambion). values given are for one reaction: for a -well plate, times larger volumes were used, and the lamp mix was distributed to the wells of a -well plate ( ti- /c, brooks life sciences or , eppendorf) before pipetting l of sample into each well of the plate; for details, see previous paragraph. plates were prepared immediately before use to limit exposure of the lamp reagents to atmospheric co (to prevent acidification of the reaction) and kept on an ice-cold metal block. plates were sealed using a transparent adhesive foil (gk -os, kisker biotech), and the reactions were incubated in a pcr cycler at °c for to min with the lid heated to °c. to perform measurements at the indicated time points, the reactions were taken out of the pcr cycler and placed into an ice cold metal block for s. this intensifies the color before the measurement. photographs were taken with cell phone cameras or the scanner function of an office copying machine. absorbance measurements were performed with a spark cyto or infinite m (tecan) at and nm with flashes. these two peaks from phenol red are strongly changing during the acidification of the reaction ( nm absorbance is increased, nm absorbance is decreased). to obtain a good readout of the color change, absorbance at nm was substracted from the one at nm. this difference was denoted od. for direct and hot swab-to-rt-lamp assays, patient swab specimens were transferred first onto a -well seed plate. for the direct assay, we then transferred l of the specimen directly to l of lamp mix per well in a ready-made -well pcr plate ( , eppendorf). the plate was sealed using a transparent adhesive foil (gk -os, kisker biotech) and kept on an ice-cold metal block. for the hot assay, we sealed the seed plate with a pierceable lid ( ti- / , brooks life sciences) and heated it in a pcr cycler for min at °c (with the lid heated to °c). the seed plate was cooled down to °c on an ice-cold metal block. afterward, l of the heat-treated patient specimens was quickly added to a second ready-made plate with l of lamp mix per well. this plate was also sealed with transparent adhesive foil (gk -os, kisker biotech). both plates were then incubated at °c for the lamp reaction to proceed. for both swab-to-rt-lamp assays, the pcr plates were briefly spun down and then incubated in a pcr cycler at °c for to min (with the lid heated to °c). to perform measurements at the indicated time points, the reactions were taken out of the pcr cycler and placed into an ice-cold metal block for s. sequencing libraries for detecting viral sequences in rt-lamp products were prepared by a modified anchor-seq protocol ( , ) using tn transposase tagmentation instead of sonication for genomic dna fragmentation ( ) . the relevant primers are summarized in table s . in detail, transposon adapters containing well-defining barcodes and unique molecular identifiers (umis) were annealed by mixing m oligos (p -umi-xi … -me.fw, tn hy-rd -wat-sc ) in m tris-hcl (ph ), incubating at °c for min, and slowly cooling down to °c within min in a thermocycler. transposons were assembled by mixing tn (e k, l p) transposase ( ng/l) [purified according to ( ) ] with . m annealed adapters in mm tris-hcl (ph . ) and incubating the reaction for hour at °c. tagmentation was carried out by mixing . l of the rt-lamp product (~ ng dna) with . l of loaded transposase in freshly prepared tagmentation buffer [ mm [tris(hydroxymethyl)methylamino]propanesulfonic acid) (taps)] (ph . ), mm mgcl , and % (v/v) dimethylformamide] using a liquidator manual pipetting system (mettler toledo). the reactions were incubated at °c for min. reactions were stopped by adding sds to a final concentration of . %. tagmented dna of each plate was pooled and size-selected using a two-step ampurexp bead (beckman coulter) purification to target for fragments between and bp. first, l of pooled reaction was mixed with l of water and bound to l of beads to remove large fragments. to further remove small fragments, the supernatant of this reaction was added to l of fresh beads and further purified using two washes with % ethanol before the samples were finally eluted in l of mm tris-hcl (ph ). one pcr per plate with l of the eluate and rt-lamp-specific and tn -adapter-specific primers (p nxt-genen-a-lbrc and p -xi .. , p .fw) was performed using nebnext q hotstart polymerase (new england biolabs) with two cycles at °c for annealing and s elongation, followed by two cycles at °c for annealing and s elongation, and cycles at °c annealing and s elongation. all pcr reactions were combined and % of this pool was size-selected for to bp using a % agarose/tris-acetate-edta gel and column purification (macherey-nagel). the final sequencing library was quantified by qpcr (new england biolabs) and sequenced with a paired-end sequencing run on a nextseq machine (illumina) with % phix spike-in and cycles for the first read, cycles to read the -nt-long plate index (i ) and cycles to read the -nt-long well index (i ) and the -nt-long umi. for trimming of the reads (i.e., removal of p illumina adapter sequences), cutadapt (version . ) ( ) was used. for validation of the origin of the sequence of the lamp product ( fig. s a ), reads were randomly selected and used for the analysis. reads were mapped to the sars-cov- reference genome (nc_ . ) ( ), using bwa-mem with default settings (version . . -r ) ( ) . virus genome coverage was determined with the samtools depth command (version . ) ( ) . using bwa-mem, . % of reads could be mapped to the virus genome ( fig. s , b and c). to analyze the remaining sequences, a k-mer analysis using a custom script was performed. using -mers, this matched . % of the nonmapped reads with a maximal levenshtein distance of two to one of the lamp primers or their reverse complement sequences ( fig. s d ). this is explained by the fact that lamp products can consist of complex sequence rearrangements. for classification of samples by lamp-sequencing, reads were assigned to wells and counted using custom scripts. a read was considered as a match to sars-cov- n gene if at least one of three short sequences (~ nt, marked orange in fig. s a ) not covered by rt-lamp primers was found in the read, otherwise it was counted as unmatched. sequencing reads were grouped by umi and by position of the matched sequence with the aim of removing pcr duplicates. a sample was considered if more than total umis were observed and called positive if more than , virus-matching umis were observed. there is a very wide gap in the number of virus-matching reads between positive and negative samples ( fig. s a ): the count is either below umis or above , umis. this is why we placed the decision threshold for scoring a sample as lamp-sequencing positive within this gap. the fact that also rt-qpcr-negative samples give rise to some umi counts containing viral sequences is explained by template switching of unattached adapters that remain in the reaction after tagmentation, but no cause for concern due to the wide gap between negative and positive samples. for a few samples, we saw so few reads (less than umis) that we suspected that the multiplexing had failed and excluded them from the results. as most of these were in the same row of the same plate, we analyzed these samples after lamp-sequencing by gel electrophoresis ( fig. s b ) to check for dna content after rt-lamp. we found that the gel results agree with the rt-lamp outcome, indicating that the failure likely was caused later, probably during multiplexing. except where otherwise noted, all data were analyzed with r ( ) using the tidyverse ( ) and ggplot ( ) system or with graphpad prism. sensitivity and specificity values were obtained from count tables as follows: specificity of the rt-lamp assay was calculated as the fraction of rt-qpcr-negative samples that were also negative in the rt-lamp assay. sensitivity for a given ct interval was calculated as the fraction of all samples with an rt-qpcr ct value in that interval that was positive in the rt-lamp assay. in both cases, % confidence intervals were calculated by interpreting the fractions of counts as binomial rates and then using wilson's method for binomial confidence intervals as implemented in the r package binom ( ) . the r code used to perform analyses and produce figures can be found on github, together with all data tables: https://github.com/anders-biostat/lamp-paper-figures. stm.sciencemag.org/cgi/content/full/ / /eabc /dc materials and methods fig. s . design of the study. fig. s . comparison of the rt-lamp assay with ct values from rt-qpcr using primer sets e-sarbeco and n-sarbeco. fig. s . comparison of the rt-lamp assay using primer set a-a with ct values from rt-qpcr using primer set e-sarbeco. fig. s . analysis of lamp-sequencing reads. fig. s . sample classification with lamp-sequencing. fig. s . rna stability and detection limit of the rt-lamp assay in pharyngeal swab specimens with ivt rna. fig. s . swab-to-rt-lamp assay titration of positive covid- specimens. table s . sequences of primers and amplicons used in this study. table s . sensitivity and specificity of the rt-lamp test from fig. b . table s . sensitivity and specificity of the rt-lamp test from fig. b . table s . primers used for lamp-sequencing. table s . overview of time requirements for various sample handling steps. data file s . raw data for figs. s and s . view/request a protocol for this paper from bio-protocol. china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china intensive care management of coronavirus disease (covid- ): challenges and recommendations estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship jernigan; public health-seattle and king county and cdc covid-investigation team, presymptomatic sars-cov- infections and transmission in a skilled nursing facility loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products visual detection of isothermal nucleic acid amplification using ph-sensitive dyes rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation rapid detection of covid- coronavirus using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp a single and two-stage, closed-tube, molecular test for the novel coronavirus (covid- ) at home, clinic, and points of entry covid- diagnostics in context real-time detection and monitoring of loop mediated amplification (lamp) reaction using self-quenching and de-quenching fluorogenic probes detection of novel coronavirus ( -ncov) by real-time rt-pcr analytical sensitivity and efficiency comparisons of sars-cov- qrt-pcr primer-probe sets tn transposase and tagmentation procedures for massively scaled sequencing projects direct rt-qpcr detection of sars-cov- rna from patient nasopharyngeal swabs without an rna extraction step massive and rapid covid- testing is feasible by extraction-free sars-cov- rt-qpcr rapid direct nucleic acid amplification test without rna extraction for sars-cov- using a portable pcr thermocycler rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification sars-cov- detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification detection of noroviruses in fecal specimens by direct rt-pcr without rna purification evaluation of a direct reverse transcription loop-mediated isothermal amplification method without rna extraction for the detection of human enterovirus subgenotype c in nasopharyngeal swab specimens rapid and specific detection of asian-and african-lineage zika viruses an alternative workflow for molecular detection of sars-cov- -escape from the na extraction kit-shortage rapid sars-cov- testing in primary material based on a novel multiplex lamp assay enhancing colorimetric lamp amplification speed and sensitivity with guanidine chloride virological assessment of hospitalized patients with covid- consistent detection of novel coronavirus in saliva saliva is more sensitive for sars-cov- detection in covid- patients than nasopharyngeal swabs self-collected oral fluid and nasal swabs demonstrate comparable sensitivity to clinician collected nasopharyngeal swabs for covid- detection saliva is less sensitive than nasopharyngeal swabs for covid- detection in the community setting high-surety isothermal amplification and detection of sars-cov- , including with crude enzymes loop-mediated isothermal amplification (lamp) for the diagnosis of zika virus: a review crispr-cas -based detection of sars-cov- pooled clone collections by multiplexed crispr-cas a-assisted gene tagging in yeast lamp-seq: population-scale diagnostics using combinatorial barcoding efficient and practical sample pooling for high-throughput pcr diagnosis of genome-wide c-swat library for high-throughput yeast genome tagging large-scale low-cost ngs library preparation using a robust tn purification and tagmentation protocol cutadapt removes adapter sequences from high-throughput sequencing reads complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in wuhan aligning sequence reads, clone sequences and assembly contigs with bwa-mem genome project data processing subgroup, the sequence alignment/ map format and samtools r: a language and environment for statistical computing (r foundation for statistical computing) welcome to the tidyverse ggplot : elegant graphics for data analysis binomial confidence intervals for several parameterizations wrote the manuscript, with input from all authors. competing interests: p.s. has consulted for janssen. the other authors declare that they have no competing interests. data and materials availability: all data associated with this study are in the main text or the supplementary materials. the r code used to perform analyses and produce figures can be found on github a colorimetric rt-lamp assay and lamp-sequencing for detecting sars-cov- rna in clinical samples we thank v. pelechano, x. yin, and v. lohmann for helpful discussions and sharing unpublished work. we thank u. merle for providing specimens and the diagnostic laboratory team at heidelberg university hospital for providing rna samples isolated from pharyngeal swabs collected from patients being tested for covid- . we thank v. sonntag-buck, key: cord- -rl sdzd authors: lee, david; la mura, maurizio; allnutt, theo r; powell, wayne title: detection of genetically modified organisms (gmos) using isothermal amplification of target dna sequences date: - - journal: bmc biotechnol doi: . / - - - sha: doc_id: cord_uid: rl sdzd background: the most common method of gmo detection is based upon the amplification of gmo-specific dna amplicons using the polymerase chain reaction (pcr). here we have applied the loop-mediated isothermal amplification (lamp) method to amplify gmo-related dna sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. results: we have tested the specificity and sensitivity of the technique for use in gmo studies. results show that detection of . % gmo in equivalent background dna was possible and dilutions of template suggest that detection from single copies of the template may be possible using lamp. conclusion: this work shows that gmo detection can be carried out using lamp for routine screening as well as for specific events detection. moreover, the sensitivity and ability to amplify targets, even with a high background of dna, here demonstrated, highlights the advantages of this isothermal amplification when applied for gmo detection. the ability to detect the presence of gmo is pivotal for consumers to be able to exercise their lifestyle choice of whether to consume, or not, food containing gmos. though the detection and quantification of gmo proteins using immunoassay has been reported [ ] , denaturation of the protein during processing makes the method unsuitable for gmo testing and quantification of food. the durability of dna makes it a better substrate for testing and its amplification by pcr is the method of choice, as recommended by the ec ( / ), for detection and quantification of gmo in samples. an alternative dna amplification method was described by notomi and coworkers [ ] called 'loop mediated isothermal amplification' (lamp). the lamp assay relies on the design of a set of primers that generate stem looped (hairpin) structures during the early stage of dna synthesis. the hairpin structures form because two of the primers used contain, at their ' end, a reverse complement of a sequence that is present in the target further downstream of the initial binding site. displacement primers help the formation of these hairpins at the ends of the dna strands and once formed, these structures can be copied into a series of dna fragments containing multiple units of the target sequence under isothermal conditions utilizing the displacement properties of bst polymerase (see [ ] ). although lamp was first described using a set of four primers, enhanced sensitivity was reported using an additional pair of loop primers [ ] . as the reactions are performed at a single temperature, lamp assays can be performed very quickly since there are no separate denaturation, annealing and extension steps, and as such, reactions do not require thermalcyclers. here we assess the lamp protocol for the detection of gmos using primers that target event-specific sequences for transgenic ms and rf oilseed rape (brassica napus l.) and generic gmo sequences such as the cauliflower mosaic virus s promoter (p- s) and the promoter and terminator for the nopaline synthase gene (p-nos and t-nos, respectively) from agrobacterium spp. the lamp technique relies on the design of an interrelated set of primers. the orientation and positions are important for self-priming through stem-looped products that drive and perpetuate the reaction. the osr events ms and rf arise from the insertion of two closely related transgenes from the plasmids, pthw and pthw , respectively [ ] . the former encodes the barnase gene that give rise to male sterility, which is replaced in the latter by the barstar gene which restores fertility: both also have the selectable marker bar which confers tolerance to the herbicide glufosinate ammonium. though the left border of the rf insert has undergone rearrangement in the form of duplication and inversion [ ] , the right borders of both events are relatively intact (our data [ ] which agree with the two sequences in the database). even though the insertions of the transgenes have different breakpoints from the plasmids, they are very close so it was possible to design assays for the rf and ms events utilizing a common set of primers within the transgenes ( figure ). when used in conjunction with primers for the plant sequence at the border of each event, they are able to detect each event ( figure ). since they have % of primers in common, it was important to determine whether there was any cross reaction between the assays. specificity of the two assays was tested using plasmid dna for each event. no cross-reaction between the two targets was observed ( figure ). the sensitivity of the lamp reaction was assessed in two ways: copy number detection and background in which copies of the target could be detected. copy number detection was measured by serial dilutions of known amounts of dna containing the target sequences, either as genomic or plasmid dna ( figure ). reactions fail in both assays at dna molecule number of less than which is consistent with the stochastic probability of a target being present [ ] . we note that sometimes non-specific amplification can also take place, especially where the target dna is absent (see figure ) and there is low amounts of dna present in the reaction (cf figures a and ) . these do not form the specific banding patterns representing the different multimeric lamp products that are characteristic for each assay and thus can be easily distinguished on a gel. alternative banding patterns have been observed, also for low template reactions; analyses of these products show that they are formed by interactions of the primers used [ ] , to form lamp equivalents of primer-dimers. two factors seem to be important: in the absence of target, low background dna may aid the formation of non-specific products that go on to be amplified; and freeze-thaw repetitions may induce damage to the primers to permit the formation of 'primer intermediates' which can then be amplified. since lamp is capable of non-specific amplification, techniques that rely on the detection of by-products of dna synthesis, e.g., the use of magnesium pyrophosphate precipitation [ ] or the use of sybr green dye [ ] may not be able to distinguish between real and right border sequences of ms and rf figure right border sequences of ms and rf . sequences of the plant (above) and transgene (below) at the right border junctions for ms and rf . highlighted sequences are the targets of the lamp primers. the plant sequences are those shown in table : dark blue bases highlighted are the outer displacement primers, yellow and green sequences are the ' and ' ends of the lamp primers respectively, and light blue sequences depict the loop primers. the sensitivity of the lamp assay and its suitability for practical gmo detection was tested using assays for commonly-used sequence motifs, the camv s promoter (p- s) and the agrobacterium promoter and terminator for the nopaline synthase gene (p-nos and t-nos, respectively). these sequences are commonly used in constructs used to create approved gm events (see [ ] ). roundup ready™ soya construct contains both p- s and t-nos so provided convenient template for testing the assays. we used a sample where the copy number of the gm has been well characterized and thus control the number of template in each reaction. lamp sensitivity was assessed by the detection of ten roundup ready™ gmo targets in a background of ng of genomic oilseed rape dna (figure ). since the c value of both species is approximately pg [ ] , this background dna represents a gmo level of . % for both tnos and p- s assays. osr dna was used because we did not have any soya dna free from roundup ready™. dna extracted from our % crm was shown to contain . % gmo [ ] . the use of ng of this sample would be equivalent to adding copies of the transgene sequence, considerably more than the experimental input of copies. we believe the use of osr dna to be a valid substitute since none of the primer sequences for either assay will be present in non-transgenic soya or oilseed rape. we have tested the upper limits of dna that lamp reactions can tolerate and found that up to ng dna in a μl reaction, positive detection is reproducible. above this dna level, reactions become unreliable (data not shown). we have found that denaturation of template was a prerequisite step prior to amplification, unlike results found by nagamine and co-workers [ ] . this can be explained by the fact that we do not use a base pair destabiliser, such as betaine, in the reaction buffer. since we are detecting down to near single copies of templates, our results suggest no benefit to the sensitivity of the assay by their inclusion. the consistent amplification within all dilutions showed that lamp is an 'all or nothing' reaction, with little of the tailing off effect that is often observed in pcrs with diluting templates. this makes it easy to identify positive reactions. together with specificity and the speed at which reactions can be performed, lamp is an excellent method for diagnostics [ , , ] . the use of camv s promoter sequence in lamp has previously been reported as a screening method [ ] . here we demonstrate the sensitivity and reliability of the lamp method for gmo detection, both with generic and gmo-specific assays. the ability to perform reactions in a simple heated block or water bath without the need for thermal cycling makes testing using lamp more accessible. that lamp is able to detect very small amounts of target and do that even in high amounts of background dna makes it ideal for gmo detection. gmo testing can be performed in steps: routine screening for the presence of gmos using generic assays such as for s promoters and t-nos; and if required, identification of specific events can be performed using event-specific assays. equally, direct screening using event-specific assays is also feasible. the levels of sensitivity are orders of magnitude below the permissible threshold for gmo in food and feed (ec regulation / ), ensuring the detection of the presence of gmos at acceptable levels and the reliable detection of any presence of unauthorised gm events, for which at present there is no legally acceptable lower limit (according to ec regulations). conventional oilseed rape (osr) seed, variety 'hearty' was a gift from christine lewis, niab. the sample was originally purchased from monsanto uk ltd (cambridge, uk). dna was extracted by grinding g seed with ml extraction buffer [ . m nacl, . m edta ph and % (w/v) sds] in a mortar with a pestle. the sample was emulsified with ml of chloroform:isoamyl alcohol ( : ) and poured into a ml falcon polypropylene tube. after centrifugation at g for mins, the aqueous phase was transferred to a new tube and nucleic acids dna from the oilseed rape ms /rf was extracted from seedlings from a selfed ms /rf plant [ ] using dneasy plant dna extraction kit (qiagen, crawley, uk). the parent plant was genotyped to be ms ms /rf rf using real-time pcr (data not shown). the sample was quantified using picogreen fluorescence (molecular probes inc., invitrogen). dna containing roundup ready™ soya was extracted from soya roundup ready™ gmo reference material (fluka biochemika, sigma-aldrich, dorset, uk) and the gmo concentration of the sample has been accurately determined by dilutions of template combined with statistical analysis [ ] . the plasmid pgreenii was a gift from mark smedley and wendy harwood of the john innes centre. details of the plasmid can be found at the website: http:// www.pgreen.ac.uk/jit/pgreen _fr.htm sensitivity assessment of lamp detection figure sensitivity assessment of lamp detection. a. sensitivity of lamp using genomic target. dna from ms /rf sample ( ng.μl - ) was serially diluted and amplified by lamp, in triplicate, using primers to assay for the rf junction. the numbers in parenthesis represent the approximate copy numbers of the target assuming that the sample represents rf in a hemizygous state (determined using rt-pcr data not shown) for the transgene and using pg as the genome size for oilseed rape. c is the no dna control and m represents molecular size markers. the smear (*) shows an example of non-specific amplification. b. sensitivity of lamp using plasmid target. serial dilutions of the plasmid pgreenii were amplified using primers for the pnos target. numbers represent the calculated copy numbers of the plasmid derived from the dna value. c is the no dna control and m represents molecular sized markers. plasmids containing each event were constructed to test the specificity of the ms and rf assays separately. the junction at the right borders of the transgenes were amplified by pcr from the ms /rf dna sample using the displacement (outer) primers of the lamp reactions, ms -rf displr (b c) separately with ms displf (f ) and rf displf (f ), to amplify the ms and rf junctions, respectively (see figure and table ). the fragments were cloned into pgem-t vector (promega, southampton, uk) and transformed into dh α. clones containing the correct inserts were confirmed by sequencing. target sequences for p- s, p-nos and t-nos were chosen based upon common identity between different plasmids in the embl database containing the promoters and terminator. the sequences of the targets and positions of the primers are provided (see additional file ). primers for each target segment have tm's of - °c (calculated using the × at, × gc formula), except for the f and b regions ( ' of the lamp primers), where the tm was - °c. primer sequences are listed in table . for lamp reactions, primers were purchased from sigmagenosys (table ) . reactions were performed in μl containing × bst pol buffer (neb, ipswich, uk) with . mm each dntp with the appropriate primers listed in table : displacement primers were each used at a concentration of . μm; loop primers at . μm and lamp primers at . μm in the reactions. enough reaction reagents, without template and enzyme, were mixed together and split into two. template ( μl) was added to μl of the mix and the samples denatured at °c for mins and then cooled to °c. bst pol was added to the remaining reaction mix to a concentration of . u.μl - , mixed thoroughly, and μl was added to each reaction. reactions were incubated at °c for hours, followed by °c for mins to inactivate the enzyme and stored at °c until analysed. aliquots of the reactions ( μl) were run on . % (w/v) agarose gels, containing ethidium bromide validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study loop-mediated isothermal amplification of dna loop animation eiken genome site accelerated reaction by loopmediated isothermal amplification using loop primers report on the molecular characterisation of the genetic map of event ms × rf plasmid standards for real time pcr and gm enforcement testing the limits of gmo detection loop-mediated isothermal amplification for detection of african trypanosomes detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples genetically modified (gm) crops: molecular and regulatory details nuclear dna content of some important plant species. table i (nuclear dna content of a number of important plant species as determined by flow cytometry) quantitation using informative zeros (quiz): application for gmo detection and quantification without recourse to certified reference material loopmediated isothermal amplification reactions using a nondenatured template development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus real-time loop-mediated isothermal amplification for the camv- s promoter as a screening method for genetically modified organisms crop-to-crop gene flow using farm scale sites of oilseed rape (brassica napus) in the uk andy greenland and giacomo morreale are thanked for their comments. this work was carried out with the financial support of the co-extra project: gm and non-gm supply chains, their co-existence and traceability (contract ), funded by the european commission through the sixth framework programme under the food quality and safety priority. dr olga gandelman (lumora ltd, ely, uk) is thanked for help in primer design. mlm is the recipient of a ministero dell' universita' e della ricerca award from university of naples federico ii. dl, as project leader, coordinated the work and wrote the manuscript. mlm performed lab work. ta provided data and material for the experiments. wp, as group leader provided focus and direction. all authors contributed to discussions, read and approved the final manuscript. sequences of lamp targets. the sequences of the different genetic elements used to design the lamp assays are shown together with a genebank accession number that contains the sequence. the sequences targeted by the lamp primers are coloured. click here for file [http://www.biomedcentral.com/content/supplementary/ - - - -s .doc] sequence ( key: cord- -k q ir authors: liu, yi; chuang, ching-kai; chen, wei-june title: in situ reverse-transcription loop-mediated isothermal amplification (in situ rt-lamp) for detection of japanese encephalitis viral rna in host cells date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: k q ir background: clinical diagnosis of japanese encephalitis is usually difficult due to non-specific signs at the early and acute stages of the infection. virus isolation from peripheral blood is also not possible because of the short period and low level of transient viremia even in the acute stage of the disease. it is thus urgent to develop a feasible and convenient method for laboratory diagnosis of the infection. objectives: to establish a newly designed molecular approach that can be used to detect intracellular japanese encephalitis viral rna in host cells. study design: the method was firstly established and then was carried out to test its efficacy in cultured bhk- cells, subsequently in peripheral blood mononuclear cells (pbmcs) isolated from mice that have been inoculated with je virus suspension. results: in this study, in situ reverse-transcription loop-mediated isothermal amplification (in situ rt-lamp) was established; which combines merits of recently developed loop-mediated isothermal amplification (lamp) and in situ reverse-transcriptase polymerase chain reaction (in situ rt-pcr). conclusions: the newly designed method can detect viral rnas in peripheral blood mononuclear cells (pbmcs) in a short time with high sensitivity and efficiency. japanese encephalitis (je) is one of the very important mosquitoborne viral infectious diseases in the world, infecting approximately % of the susceptible population in southeast asian countries each year. the etiological agent of je belongs to the family flaviviridae which also includes many other related viruses such as yellow fever virus, dengue viruses, and west nile virus. the genome of the je virus is composed of a positive single-stranded rna of approximately ∼ , bases in length. infection rates of je virus in epidemic regions usually vary, ranging from a few cases to % of the population. clinically, at least , cases and , deaths occur each year, mostly among children in asia, in spite of mass vaccinations which have been implemented in many endemic countries. diagnosis of je by clinical symptoms is not feasible due to nonspecific signs at the early and acute stages of the infection. neither is it possible to isolate the je virus from peripheral blood because of the short period and low level of transient viremia even in the acute stage of the disease. nowadays, the laboratory serodiagnosis of je uses a versatile and convenient approach to detect immunoglobulin m (igm) or igg antibodies in serum or cerebrospinal fluid (csf) by enzyme-linked immunosorbent assays (elisas). , more recently, molecular techniques, e.g., the reverse-transcriptase polymerase chain reaction (rt-pcr), to amplify gene fragments of viral rnas are increasingly being used to make diagnoses. , nevertheless, the low copy number of viral rna in the blood of je patients makes it difficult to extract rna from this source. utilization of the rt-pcr to detect viral rnas in peripheral blood mononuclear cells (pbmcs), also called in situ rt-pcr, was thus developed as a molecular tool which does not require messenger (m)rna extraction. the technique has been applied to diagnosing je virus infection in pbmcs in a mouse model. the technique of loop-mediated isothermal amplification (lamp) is based on the principle of a strand displacement reaction and the stem-loop structure that amplifies the target gene fragment under isothermal conditions. , because the target is initially recognized by six distinct sequences, followed by another four distinct sequences, amplification of the target sequence is expected to be highly selective. furthermore, there is no need for any high-cost instrument (such as a thermocycler or gel imaging system), making the technique reliable, simple, rapid, and cost-effective for detecting and differentiating examined genomic nucleic acids. in this study, we aimed to establish a new approach, named as in situ reverse-transcription loop-mediated isothermal amplification (in situ rt-lamp), in order to detect je virus infection in cultured cells and in pbmcs isolated from infected mice. establishment of this approach is combined the techniques of rt-lamp and in situ rt-pcr which was previously described by this laboratory. je virus used in this study was the t p strain that was previously isolated from field-caught armigeres subalbatus. the virus was propagated in c / cells and titrated in bhk- cells based on previous reports. bhk- cells are being maintained in our laboratory at • c with % co in minimum essential medium (gibco tm brl life technologies, grand island, ny, usa) that contains % nonessential amino acids, . g/ml sodium bicarbonate (nahco ), . % antibiotic-antimycotic, and % fetal bovine serum. the plaque assay following our previous description was used for virus titration in this study. the virus titer was calculated and expressed as plaque-forming units per milliliter (pfu/ml). the oligonucleotide primers used for rt-lamp amplification used in this study were designed from the prm gene of the je virus. the sequence of the t p strain was retrieved from the genbank database (accession no. af ). sets of six primers comprising two outer (f and b ), two inner (fip and bip), and two loop primers (lf and lp) were selected based on the criteria shown in a previous description. fip consists of a complementary sequence of f and a sense sequence of f . bip consists of a complementary sequence of b and a sense sequence of b . the two loop primers were designed to accelerate the amplification reaction. the lf and lb primers were composed of sequences complementary to the sequences between the f and f and the b and b regions, respectively. details of the primers with regard to their positions in the genomic sequence are shown in table . genomic viral rna of the je virus was extracted from infected culture supernatant using viral nucleic acid extraction kit ii (geneaid biotech, taipei, taiwan) in accordance with the manufacturer's protocol. for total rna extraction from je virus-infected bhk- cells, the standard trizol (gibcobrl ® life technologies)chloroform-isopropyl-ethanol wash method was used as described previously. in this study, the rt-pcr was used to compare with the rt-lamp assay described below. the primer pair ( f and r) was used to amplify a specific genomic fragment of nt - of the je virus, covering the non-coding region, c, and prm genes. the sequences of the f sense primer and the complementary r primer respectively were -ctgtgtgaacttcttggcttagtatcg- and -tcagttttcatgagatatcgtgtgtggc- . in this study, rt was performed with the bip primer at • c for min. then the lamp reaction was carried out in a -l total reaction mixture containing l cdna mixture, . in this study, bhk- cells were infected with the t p strain of the je virus for h at a multiplicity of infection (moi) of and then transferred from a flask to microtubes. cells were fixed with % paraformaldehyde for min, and then permeabilized with . % triton x- for min at • c. for the in situ reaction, after rt, cells were precipitated by centrifugation at × g. after removing the rt reaction mixture, cells were re-suspended with the lamp reaction mixture, and the lamp reaction was performed as mentioned above. in the following step, cells were treated with a blocking solution ( % bovine serum albumin; bsa) for min. in order to visualize the target cdna inside cells, we added . m digoxigenin (dig)-labeled primer (the -end of the inner fip primer labeled with dig) or . mm dig-labeled dctp to the lamp reaction. after cells were washed with phosphatebuffered saline (pbs), l of the diluted goat anti-dig antibody conjugated with alkaline phosphatase (roche, mannheim, germany) ( : in pbs) was added to the microtubes for a -h incubation. subsequently, cells were washed twice with washing solution which contained mm maleic acid and mm nacl (ph . ) for min followed by washing solution ( mm tris-hcl, mm nacl, and mm mgcl ; ph . ). subsequently, the substrate nitroblue tetrazolium chloride/ -bromo- -chloro- indolyl-phosphate (nbt/bcip) was added to the microtubes and incubated for min. cells were washed again with pbs, removed from the tubes, and distributed onto -well slides. in this study, each female icr mouse was intravenously injected with l of pbs (the control group) or × pfu of the je virus suspension. pbmcs were then isolated from to ml of whole blood, taken at , , or d post-inoculation, and pooled from seven or eight inoculated mice, with the ficoll-hypaque solution (ge healthcare biosciences, uppsala, sweden). dig-labeling of the primer or dctp was used in this study to visualize amplified gene fragments in rt-lamp; which was carried out by the method of southern blotting. briefly, a -l aliquot of rt-lamp products was first run by electrophoresis on a % agarose gel in tae buffer. the gel was treated with denaturing solution ( . m nacl and . n naoh) twice for min each and then with neutralizing solution ( . m tris-hcl and m nacl) for min. after being transferred onto a nylon membrane, the membrane was exposed to uv for min for cross-linking (spectrolinker xl- uv crosslinker). the membrane was then immersed in blocking solution ( . % bsa, mm maleic acid, mm nacl; at ph . ; then autoclaved) for min, incubated with the anti-dig-ap antibody ( : in blocking solution) for min, and ultimately in the solution containing the nbt/bcip substrate until the color could be visualized. the success of the rt-lamp method relies on the specificity of the designed primer sets. the primers were selected to target the prm gene derived from the t p strain of the je virus. in order to validate the sensitivity and specificity of the primer set, . and . g of total rna extracted from je virus-or mock-infected bhk- cells were separately subjected to the rt-lamp reaction at • c and min for rt and subsequently at • c and min for the lamp reaction. in the meantime, the rt-pcr was used in the same experiment as a positive control. the results indicated that the viral rna could be detected from the extract of je virus-infected bhk- cells, but not that from mock-infected cells, at both concentrations, shown as dna ladder-like products (fig. a) . in the rt-pcr, viral rna was also detected and showed a single band representing the -bp fragment (fig. b) . to examine the limit of the reaction time, lamp was performed at -min intervals ( - min). while the viral gene was detected as early as min, a time course of min for amplification was shown to be the optimal condition (fig. ) . in this study, dig-labeling was used for better visualization after the reaction. when the in situ rt-lamp was run with the diglabeled primer, amplified dna ladder-like products were clearly detected in both groups using the unlabeled or dig-labeled primer, although the signal when using the latter primer was relatively weak (fig. a) . however, no signal appeared on the nylon membrane on which amplified dna fragments had been transferred, indicating that dig molecules had not been incorporated into the amplified fragments (fig. b) . on the other hand, using the in situ rt-lamp with the diglabeled dctp, dna ladder-like amplified products were shown in the unlabeled group and in two groups labeled with dctp ( . and . mm), but not in that from mock-infected bhk- cells ( fig. a ). when the rt-lamp products were transferred onto nylon membranes and visualized with the relevant antibody and substrate, as mentioned above, dna ladders were actually present on the membrane in a dose-dependent manner (fig. b ). when the in situ rt-lamp was run with the dig-labeled primer, no positive reaction was shown in either je virus-infected (fig. a ) or uninfected (fig. b ) bhk- cells. however, a positive reaction with a deep-brown color was obvious in je virus-infected bhk- cells (fig. c ), but not in uninfected cells (fig. d) , when the reaction was run with the dig-labeled dctp. using the dig-labeled dctp, results showed that the je virus was detected in all pbmcs isolated from mice that had been inoc- ulated with the virus suspension for , , and d (fig. a-c) , but not from mock-inoculated mice (fig. d ). in addition, the results revealed that the highest infection rate of pbmcs occurred at d post-inoculation, while a relatively low rate of pbmcs was shown at d post-inoculation (fig. b ). the known pathogenesis of je reveals that the virus enters pbmcs including monocytes/macrophages as the primary site for replication in vertebrate hosts. subsequently, the virus migrates to the central nervous system (cns) where brain inflammation or encephalitis may occur. je can be fatal; fatality rates of the infection statistically differ in different localities from % to %. those who recover from the infection with clinical symptoms often display sequelae with serious neurological impairment and/or mental retardation. , as a result, rapid and efficient techniques for the early diagnosis of je are urgently and critically needed. in past years, virus isolation from nerve tissues and antibody (igm and igg) detection from serum and/or csf have extensively been used to detect je virus in most laboratories. however, molecular techniques including rt-pcr and real-time rt-pcr assays are believed to be more useful due to their characteristics of rapidity, high sensitivity, and high specificity. , the techniques have been routinely used for identifying the je virus in acute-phase serum or csf samples from patients. drawbacks of these molecular techniques actually include requirements for delicate labor and experienced techniques as well as a relatively expensive instrument, resulting in restricted application in a number of laboratories in developing countries. various gene fragment-amplification techniques, such as rt-pcr, real-time rt-pcr (taqman or sybr green), and nucleic acid sequence-based amplification (nasba), have been developed during the past decade, with the goal of rapidly identifying the je virus with greater accuracy. [ ] [ ] [ ] despite the high magnitude of amplification, these pcr-based methods require either high-precision instruments for amplification or elaborate methods for detection of the amplified products. in the meantime, these methods are often cumbersome to adapt for routine clinical use. after the development of the lamp technique, a new era of the rapid and efficient identification of microbial infections has been initiated. it is particularly useful as its reaction can be monitored through real-time detection of changes in turbidity. , moreover, the high sensitivity and specificity facilitates its potential use in the laboratory diagnosis of a number of infections, covering all kinds of organisms. thus far, the technique has been applied to detect or identify viruses, [ ] [ ] [ ] [ ] fungi, bacteria, protozoa, [ ] [ ] [ ] and even vertebrates. recently, the technique of rt-lamp was modified from the conventional lamp; this is specifically useful in efficiently and rapidly detecting or identifying various rna viruses, including flaviviruses, , , togaviruses, the severe acute respiratory syndrome (sars) coronavirus, and the h n avian influenza virus. application of rt-lamp to je virus detection has been shown to be relatively rapid and allows for virus quantification. it was demonstrated that the technique can be used for the rapid diagnosis of je using acute-phase csf samples during an epidemic. in spite of rt-lamp detection of the je virus being rapid and quantitative, this approach requires a larger number of serum or csf specimens to achieve a clinical diagnosis of je patients. it was noted that the je virus can persist in mice for - weeks following intraperitoneal inoculation and may later be reactivated. in addition, the je virus is also reported to persist in the nervous system of some patients. as a matter of fact, flaviviruses including the je virus and other flaviviruses have been identified to exist in pbmcs of convalescent blood samples, indicating the possibility of persistent viral infection in leukocytes. moreover, je viral rna fragment has ever been amplified from white blood cells during the acute phase of infection. this suggests that detection of such viruses in leukocytes may help make a diagnosis even though the blood was collected in the acute viremia stage. in order to achieve this goal, we herein established a technique called in situ rt-lamp, which was successfully applied to detect je virus in both cultured cells and pbmcs isolated from infected mice. the technique of in situ rt-lamp can avoid the drawback from the potential inefficiency of rna extraction that is a necessary step for conventional rt-pcr as well as rt-lamp. furthermore, one advantage is that the experiment can be run at a constant temperature. thus there is no need for a thermocycler that is necessary when running this technique with a conventional rt-pcr. although in situ rt-pcr can actually skip the process of rna extraction, it still needs an expensive instrument, a thermocycler. in other words, in situ rt-lamp actually is more convenient than both in situ rt-pcr and lamp. to build up a higher-efficiency protocol, we initially carried out dig-labeling of the primers. however, no satisfactory signal was shown either in gel electrophoresis or on blotted nc membranes in the experiment using infected bhk- cells. this suggested that the large size of dig molecules labeled on the outer primer fip probably interrupted their binding to stem-loop dna. thus we shifted the dig-labeling to dctp. it turns out that the amplification of nucleic acids was successfully detected; it could clearly be differentiated from that in the control group (mock-infection). this indicated that dig-labeling of dctp is relatively efficient, compared to primer labeling, and is much more efficient, resulting in better visualization and easier differentiation from negative results. in addition to detecting the je virus in cultured bhk- cells, the technique was also applied to detect the virus in pbmcs isolated from whole blood of infected mice. eventually, the virus was detected at , , and d post-inoculation, consistent with our previous work using in situ rt-pcr. nearly half of the inoculated mice dying of the disease (data not shown) suggested that most infected pbmcs may have migrated into brain tissues. taken together, this newly designed technique of in situ rt-lamp is a sensitive (a higher detection rate), rapid (no more than h), efficient (under isothermal conditions), cheap (no need for expensive instrument), and convenient (not labor-intensive) method. this new diagnostic design can serve as a robust tool for diagnosing je virus infection, especially in remote and developing countries. it is particularly useful to detect viruses in blood samples, usually the genomic copy number is low, collected at the acute stage or which are persistently infected. theoretically, this delicate technique can also be applied to diagnosing other viruses that normally infect leukocytes or pbmcs, e.g., human immunodeficiency virus (hiv), with advantages of economy, convenience, and high efficiency. we would like to declare that there is no financial or personal relationship with other people or organizations that could inappropriately influence our work during the submission process. the epidemiology of japanese encephalitis: prospects for prevention flavivirus genome organization, expression, and replication monath tp. fields virology japanese encephalitis: current worldwide status new initiatives for the control of japanese encephalitis by vaccination viral infections of humans an intranasal challenge model for testing japanese encephalitis vaccines in rhesus monkeys rapid diagnosis of japanese encephalitis by using an immunoglobulin m dot enzyme immunoassay identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood rapid identification of flavivirus using the polymerase chain reaction detection of japanese encephalitis virus in mouse peripheral blood mononuclear cells using an in situ reverse transcriptase polymerase chain reaction accelerated reaction by loop mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification potential role of armigeres subalbatus (diptera: culcidae) in the transmission of japanese encephalitis virus in the absence of rice culture on liu-chiu islet a model to study neurotropism and persistency of japanese encephalitis virus infection in human neuroblastoma cells and leukocytes analysis of japanese encephalitis epidemic in western nepal in a survey of the clinical sequelae of japanese encephalitis a hospital-based surveillance for japanese encephalitis in bali sensitive and specific detection of strains of japanese encephalitis using a one-step taqman rt-pcr technique development and evaluation of sybr green i-based one-step real-time rt-pcr assay for detection and quantization of japanese encephalitis virus detection of west nile and japanese encephalitis viral genome sequences in cerebrospinal fluid from acute encephalitis cases in karachi nasba and other transcription-based amplification methods for research and diagnostic microbiology taqman reverse transcription polymerase chain reaction for the detection of japanese encephalitis virus detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection rapid detection and differentiation of dengue virus serotypes by real-time reverse transcription loop-mediated isothermal amplification assay rapid and real-time detection of hepatitis a virus by reverse transcription loop-mediated isothermal amplification assay evaluation of loop-mediated isothermal amplification (lamp) to rapidly detect arbuscular mycorrhizal fungi specific and rapid detection of food-borne salmonella by loop-mediated isothermal amplification method development of a multiplex loop-mediated isothermal amplification (mlamp) method for the simultaneous detection of bovine babesia parasites species-specific loop-mediated isothermal amplification (lamp) for diagnosis of trypanosomosis evaluation of loop-mediated isothermal amplification (lamp), pcr and parasitological tests for detection of trypanosoma evansi in experimentally infected pigs rapid sexing of water buffalo (bubalus bubalis) embryos using loop-mediated isothermal amplification real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus rapid and real-time detection of chikungunya virus by reverse transcription loop-mediated isothermal amplification assay persistence, latency and reactivation of japanese encephalitis virus infection in mice persistence of japanese encephalitis virus in the human nervous system japanese encephalitis virus latency in peripheral blood lymphocytes and recurrence of infection in children detection and isolation of japanese encephalitis virus from blood clots collected during the acute phase of infection the blood-brain barrier in the cerebrum is the initial site for the japanese encephalitis virus entering the central nervous system funding: this work was financially supported by grants from chang gung memorial hospital (cmrpd and emrepd ). key: cord- - w g qk authors: walker, faye m.; hsieh, kuangwen title: advances in directly amplifying nucleic acids from complex samples date: - - journal: biosensors (basel) doi: . /bios sha: doc_id: cord_uid: w g qk advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. this requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (naats) at the point of care (poc), though advances in “lab-on-chip” platforms that integrate sample preparation and naats have made great strides in this space. alternatively, direct naats—techniques that minimize or even bypass sample preparation—present promising strategies for developing poc diagnostic tools for analyzing real-world samples. in this review, we discuss the current status of direct naats. specifically, we surveyed potential testing systems published from to , and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for poc diagnostics. we introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct naats. through our review, we hope to initiate an in-depth examination of direct naats and their potential for realizing poc diagnostics, and ultimately transformative technologies that can further enhance healthcare. nucleic acid amplification tests (naats) have become indispensable tools in biology and medicine. for example, for infectious diseases diagnostics, naats are generally faster, more sensitive, and more specific than the current gold standard of culture-based techniques. in fact, a number of dna-and rna-based diagnostics are now recommended by the us food and drug administration (fda) for infectious diseases such as human immunodeficiency virus (hiv) [ , ] . bringing naats to the point of care (poc), and particularly to resource-poor settings, is envisioned to revolutionize healthcare. unfortunately, many naats require access to expensive, specialized equipment and a degree of expertise that is highly unlikely to be found in decentralized laboratories. as an additional challenge, these tests typically require an extraction step to isolate dna or rna from blood, urine or sputum, and a purification step to eliminate contaminants from the sample matrix that can confound the actual detection procedure (figure ). these procedures necessitate expensive instrumentation and can add up to several hours to sample-to-answer results, which further restricts the use of naats within centralized laboratories. direct nucleic acid testing is much more convenient and streamlined than the three-step method with preparatory techniques. in a typical extraction experiment, buffer with lytic agents is added to dilute the sample and homogenized with a mixer. sonication creates pressure waves that burst the cells in mechanical lysis. lysozyme enzymatically destroys cells, and is removed from the reaction with vortexing and centrifugation in a phenol/chloroform phase separation. the dna is precipitated in fresh ethanol and the resulting mixture is washed to remove excess contaminants. excess liquid is removed so that the dna can be resuspended in an appropriate buffer. many groups have attempted to develop portable, integrated, microfluidics-based platforms to increase the functionality of diagnostic sensing and analysis [ ] [ ] [ ] , and some of these have even been commercialized (e.g., biomerieux's nuclisens easyq tests, twistdx's twistamp kits, and enigma diagnostic's minilab). these platforms present breakthrough technologies for rapid, cost-effective, and user-friendly diagnostics. while it remains to be seen whether these systems are simple and error-free enough for developed and developing settings, they demonstrate the feasibility of implementing existing nucleic acid amplification methods for poc use [ ] [ ] [ ] [ ] . an alternative approach to time-consuming and cumbersome sample preparation is performing naats directly from complex samples ( figure ). the advantage of traditional amplification technologies, such as pcr with real-time spectroscopic or mass spectrometry detection, is that the results are highly specific and quantitative. however, these sensing platforms are expensive and require prior extraction of genetic material from the sample. direct naats are advantageous when complicated, costly laboratory apparatuses are not available. they not only reduce the time, labor, and technical constraints of molecular testing, but also bring the additional benefit of standardizing results [ ] . indeed, a growing number of groups are developing such "direct" naats. most notably, the alere i influenza a&b assay became the first fda clinical laboratory improvement amendments (clia)-waived nucleic acid-based test [ ] in january . as the alere i system requires no front-end nucleic acid extraction, and can be used outside of traditional laboratory sites [ ] [ ] [ ] [ ] [ ] , its development and clia-waived status provide strong support for further development of direct assays that can minimize or even bypass sample preparation. thus motivated, we present the current state of direct assays and platforms that achieve nucleic acids detection and analysis from clinically-relevant, complex samples but with either minimal or biosensors , , of even no sample preparation procedures. we surveyed the literature from - and came across a significant number of works that reported naats from bodily samples (e.g., blood-based liquids, oral samples, swabs) without the complex steps generally involved in sample preparation. this meant discarding the works that depended on sophisticated instruments and operations that are labor-, time-, and cost-intensive, such as enzymatic (proteinases), chemical (acids, detergents), or physical (temperature shock, mechanical disruptions) treatments. then, we describe examples whereby data visualization can be used to reveal the connections between the robustness, sensitivity, and efficacy of technologies developed for direct dna-and rna-based tests. it is our hope that in reviewing technologies such as these, and presenting these promising early findings in an information-rich and accessible fashion, we can help to accelerate the development of approaches that make poc nucleic acid testing rapid, accurate, simple, and affordable. in order to find relevant articles with data on naat parameters, we performed literature searches from december to february . we searched google scholar with a combination of search terms. these followed a formula of combining a descriptor (e.g., "point-of-care"), an amplification technology (e.g., "lamp" or "loop-mediated isothermal amplification") and a sample matrix (e.g., "blood"). references of previously published reviews, as well as those included in original studies, were checked for possible candidate articles. articles were initially screened on the title, and secondly on the abstract. any articles that relied on microfluidic platforms or commercialized extraction devices were excluded. publications that required complex pre-processing with enzymatic treatment or chemical purification were not selected. studies were included if they involved direct amplification and detection of genetic material from one of six representative sample types: blood, dried blood spot, serum and plasma, saliva and sputum, swabs, urine, and stool. the full text of appropriate articles was read to extract the necessary information. from each of the published works surveyed, we extracted and recorded data that corresponded to test performance. there are many parameters that cannot be ignored when considering naats: accuracy, specificity, user-friendliness, training requirements, and so on. as such, we provide an extensive examination of nucleic acid template specificity (including single or multiplexed reactions), amplification methodologies (enzymes, operating temperatures, and amplification technology), and user-friendliness (storage considerations, pretreatment requirements, and physical involvement) in supplementary table s . in addition, we have classified assays that can feasibly be completed without extensive training or high-end instrumentation as "direct," whereas those with greater labor or equipment demands (e.g., freezers, high-speed centrifugation, or incubation for multiple hours) are deemed "semi-direct." specifically, the "semi-direct" assays have the following exceptions to a simple laboratory setup: alternating between two or more incubation temperatures (other than room temperature), relying on enzymatic activity, or requiring more than a brief, low-speed (< × g) centrifugation. methods categorized as "semi-direct" face some hurdles to implementation as an on-site service for patient care. what these tests do offer is a way to deliver actionable results that can link diagnosis to treatment. with appropriate conversion from requirements for highly trained staff and sophisticated tools to easy-to-use methods, "semi-direct" procedures will meet the requirements for poc diagnostic devices. we found certain parameters could be distilled into numerical data, yielding particularly useful insights when examining different tests. we have devised three major criteria that are indicative of each platform's robustness, sensitivity, and clinical efficacy: tolerance to the sample of interest-ideally, the assay should be able to detect its target against a high concentration of background contaminants. we note that, although sample dilution sometimes provides a convenient way of permitting amplification, doing so inevitably reduces the limit of detection (lod). most naats analyze only a fraction of the sample volume. sample dilution therefore increases the likelihood of false negative results, especially when the samples already have low target concentrations. . lod-by foregoing sample preparation, one generally sacrifices the opportunity to concentrate bulk samples, reducing the limit of detection and making sensitivity an important consideration. clinical evaluation-recognizing assays that have been validated with clinical samples. finally, we sought to devise a visual strategy that would clearly and quickly communicate the importance of our criteria, compare the wide range of assays, discover trends in the data, and reveal patterns in a single glance. specifically, the essential information of the reviewed publications is presented quantitatively in a single plot. relevant values are standardized and communicated in terms of visual attributes of position, size, shape, and color. we have found it particularly useful to visualize the data as "bubble plots." in a bubble plot, numerical values from three parameters are simultaneously visualized via the two axes and the size of the circular marker. different categories can also be grouped according to the color of the markers. in our case, we can readily display the essential information (e.g., sample tolerance, lod, and instances of clinical testing) of related procedures to discern those that enhance test performance. in our survey, we came across eight dna-and rna-based testing techniques. as expected, pcr (and reverse transcription pcr, or rt-pcr) has been the predominant technique. notably, a number of isothermal amplification techniques have also been used to develop direct naats. herein, we provide brief overviews of these lesser known isothermal amplification techniques. while pcr is the most commonly reported method of amplification, there is an increasing number of isothermal amplification technologies that can be truly used at the poc. the single reaction temperature enables the use of less costly, complicated instruments than for thermal cycling tests. loop-mediated isothermal amplification (lamp) is one such widely researched, developed, and characterized method [ ] . amplification employs a strand-displacing polymerase and two or three pairs of primers: one that is sacrificed to linearize the template, and one or two others that prime the dna synthesis to produce concatenated, cauliflower-like products [ ] . as with pcr, lamp has been modified to target rna as reverse-transcription (rt)-lamp [ ] . lamp has been compared to pcr in other ways as well, including applications with bacterial, viral, fungal, and parasitic assays. not only has the specificity and sensitivity been equivalent to that of pcr, the robustness of lamp to certain preparations of serum, swabs, and blood has shown it to be more tolerant to inhibitors than pcr [ ] . the nucleic acid sequence-based amplification (nasba) method is unique in its ability to amplify single-stranded rna directly [ ] . this is most desirable for targeting rna viruses and for transcriptome analysis [ ] . the continuous, homogeneous, isothermal process relies on rna polymerase, rnase, and reverse transcriptase. first, the reverse transcriptase creates a double stranded rna:dna hybrid from the rna template; next, the original rna is destroyed; a dna duplex is synthesized; then, the polymerase can transcribe rna from the dna. each new rna molecule can repeat the cycle for exponential amplification. nasba has been applied to a wide-ranging set of research problems, including hiv diagnosis during the aids epidemic of the s and automated, real-time, clinical tests in blood with the modern nuclisens (biomerieux, inc., durham, nc, usa) or in urine with the aptima assay (hologic, san diego, ca, usa). nasba is also used outside of the commercial sector with systems to monitor viruses in serum [ ] . the strand displacement amplification (sda) technique is based upon the abilities of a restriction enzyme and a dna polymerase. a primer containing a recognition sequence for the restriction enzyme binds to its complementary, single stranded dna target. after extension by the polymerase, the restriction enzyme nicks the unmodified strand of the double-stranded hemiphosphorothioate recognition site. dna polymerase then extends the end of the nick, displacing the downstream strand. the end result is exponential target amplification from the displaced strands, which serve as targets for new reactions. sda is not complex, but it does suffer from sensitivity issues in the presence of background dna. the best way to overcome off-target amplification, and hence reduce false-positives, is to use simple pretreatment procedures like those that have been developed for detection with the bdprobe-tec (becton dickinson microbiology systems, sparks, md, usa) and in-house systems for urine [ ] . recombinase polymerase amplification (rpa) avoids thermal cycling by using three core proteins that operate optimally between - • c [ ] . the first protein, recombinase, binds to primers that recombine with a duplex target for strand displacement. the second, a single-stranded dna binding protein, attaches to the displaced strand before a strand-displacing polymerase copies the dna from the primer onwards for exponential amplification. one of the requirements for rpa technology is sequence-specific detection. this avoids the problem of primer artifacts that add to background fluorescence with nonspecific, intercalating dyes. with its specific readout and rapidity (< min to results) as two main features, rpa provides an alternative to the time-consuming processes of culturing and bacterial genotyping when testing for pathogens [ ] . strand invasion based amplification (siba) is another amplification process that relies on recombinase activity. in siba, there is a separate recombinase substrate that is inserted between two primer-binding sites. the duplex peripheral to this insertion site is separated, enabling the primers to bind. dna polymerase can then extend the template from the bound primers. this use of an invading substrate, one that is neither consumed nor included in the extension of dna, is advantageous because it abolishes primer artifacts. siba can therefore be used to reliably detect low copy numbers of pathogens-other isothermal methods generate non-specific amplification products in the absence of target dna [ ] . going further, the specificity of siba enables multiplexing for the detection of templates that differ by as little as two bases [ ] . multiple displacement amplification (mda) is a technique that exploits the strand displacement, proofreading, and polymerase activity of the φ bacteriophage dna polymerase [ ] . the highly processive polymerase uses random primers to amplify an entire genome. mda is therefore well-suited for whole genome amplification from crude biological samples, which can be followed by single nucleotide polymorphism (snp) testing and genotyping [ ] . the concept of hybridization chain reaction (hcr) [ ] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type (hiv- ) in serum [ ] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from to ( figure ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure , black) or isothermal amplification techniques ( figure , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as , several years after the advent of lamp in , and twelve years after the introduction of nasba [ ] . pcr-based systems emerged within five years of the technique's inception in [ ] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. the concept of hybridization chain reaction (hcr) [ ] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type (hiv- ) in serum [ ] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from to ( figure ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure , black) or isothermal amplification techniques (figure , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as , several years after the advent of lamp in , and twelve years after the introduction of nasba [ ] . pcr-based systems emerged within five years of the technique's inception in [ ] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. because blood contains circulating nucleic acids, cells, and over , different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [ ] , staphylococcus aureus [ ] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [ ] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [ ] or chelating necessary cofactors [ , ] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. because blood contains circulating nucleic acids, cells, and over , different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [ ] , staphylococcus aureus [ ] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [ ] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [ ] or chelating necessary cofactors [ , ] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. bubble plot of nucleic acid diagnostics performed in whole blood. percent concentration (v/v) of blood per reaction in a given procedure is displayed as a function of the limit of detection (lod) in g of template. the number of patient samples tested is proportional to the log of the marker area, as shown at top, and the testing methodology is indicated by marker color. cases shown with × instead of bubble markers illustrate that patient testing was not reported. by assessing the pcr-and isothermal-based data, we could obtain some insight into how to optimize these techniques to better tolerate blood as a sample matrix. several of the semi-direct works with pcr have employed over % blood in a reaction after heat-cold shock [ ] . more noteworthy is a truly direct example that relied on the specificity and efficacy of the phusion polymerase (new england biolab, ipswich, ma, usa) to perform pcr in % blood [ ] . pcr typically employs the taq polymerase from thermus aquaticus. chemical additives, whether commercially-available cocktails [ , ] or in-house buffers [ - ], allow the taq family of polymerases to amplify dna from whole blood. pcr can likewise be optimized through the use of more unconventional polymerases [ - ] and physical heating steps [ , [ ] [ ] [ ] [ ] [ ] [ ] to reduce the inhibitory effect of blood components. these referenced works offer expedited methods to obtain amplifiable templates with similar sensitivities to chemical-based extraction kits [ ] . though several authors include the use of a centrifuge in the extraction process, these semi-direct methods of template preparation could likely be completed by relying on careful pipette-based transfer of supernatants rather than high-speed centrifugation [ , ] . most isothermal amplification-based diagnostics in blood make use of lamp [ ] , which offers a highly tolerant means of amplification [ ] . simple treatments with heat [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or chemicals [ , [ ] [ ] [ ] [ ] [ ] can increase the sensitivity of the lamp or rt-lamp reaction. most impressive are the examples of direct amplification of dna in blood with lamp-based technologies [ , ] and other isothermal amplification methods like mda [ ] . some of these assays employ a post-heating centrifugation step, but since poon et al. have demonstrated that lamp can be performed directly on heat-treated blood without a spin-down process, this step could likely be avoided in semi-direct processes [ ] . even though these successful examples of simple, direct nucleic acid testing methods highlight the promise of dna amplification in whole blood, there is an ongoing need for further improvements. no assay has come close to reaching the capacity of burckhardt et al.'s pcr amplification with taq polymerase in nearly % whole blood, as demonstrated over years ago in [ ] . the associated treatment method is one of the more technically-involved and time-intensive, demanding cycles of heating and cooling. it remains to be seen whether an isothermal amplification method could equal this tolerance. perhaps these techniques will make up for their decreased level of tolerance in their ease of use, as evidenced by suzuki et al. achieving % incorporation of whole blood in lamp with only a five-minute heating [ ] . dried blood spots offer a convenient alternative for screening for genetic disorders, testing for infectious diseases, and profiling drug metabolism in settings with limited laboratory or storage capabilities. such samples are typically prepared by spotting whole blood, either from venous blood or a finger prick, onto filter paper [ ] . sampling time is quick, temperature-controlled storage is unnecessary, and biohazard risks are minimized for health care workers [ ] . the downside of such samples is that the dna in the dried blood must be eluted from the paper-based cellular components before it can be amplifiable. filter paper has been used as medium to test blood for infectious diseases since the s [ ] . from syphilis diagnosis during world war ii [ ] , to infant screening in the s [ ] , to hiv detection and monitoring in the modern day [ , ] , there are important assays with dried blood spots in naats. commercial technologies are even becoming widely available to map, monitor, and survey blood spots from patients infected with malaria or other neglected tropical diseases [ , ] . in a similar manner, the preparation and processing techniques for dried blood samples presented below could open new avenues for disease control and elimination when combined with well-standardized assays for detecting bloodborne pathogens. as shown in figure , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example ( clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [ ] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. biosensors , , x for peer review of as shown in figure , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example ( clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [ ] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [ , ] . pretreatments, such as fixing [ , [ ] [ ] [ ] [ ] or heating [ , [ ] [ ] [ ] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [ ] can be streamlined into a five-minute methanol fix [ ] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [ , ] , in-house buffers [ , , - ], or water [ , ] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [ ] . similar preparatory approaches are used for lamp, wherein heating in water [ , ] , phosphate buffered saline (pbs) [ ] , or sodium dodecyl sulfate (sds) buffer [ ] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [ , , ] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to resuspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of % for patient to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [ , ] . pretreatments, such as fixing [ , [ ] [ ] [ ] [ ] or heating [ , [ ] [ ] [ ] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [ ] can be streamlined into a five-minute methanol fix [ ] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [ , ] , in-house buffers [ , , - ], or water [ , ] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [ ] . similar preparatory approaches are used for lamp, wherein heating in water [ , ] , phosphate buffered saline (pbs) [ ] , or sodium dodecyl sulfate (sds) buffer [ ] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [ , , ] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to re-suspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of % for patient samples [ ] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [ ] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [ ] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [ ] and hepatitis b virus [ ] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [ ] [ ] [ ] . more recently, however, paper-or card-based devices [ , ] , membrane-based sedimentation [ ] , and microscale devices for cell differentiation and filtration [ ] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the % range ( figure ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in % (v/v) serum, they achieved a very low lod of pg [ ] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented- out of the cases examined here included testing with patient samples. samples [ ] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [ ] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [ ] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [ ] and hepatitis b virus [ ] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [ ] [ ] [ ] . more recently, however, paper-or card-based devices [ , ] , membrane-based sedimentation [ ] , and microscale devices for cell differentiation and filtration [ ] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the % range ( figure ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in % (v/v) serum, they achieved a very low lod of pg [ ] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented- out of the cases examined here included testing with patient samples. researchers have developed convenient pcr assays for both direct and semi-direct testing in blood-based fluids. by using enhanced enzymes, dna [ , , , ] and rna [ , ] targets have been successfully amplified in plasma and serum. additionally, heat-based pretreatment can be used to release nucleic acids prior to carrying out amplification [ , , [ ] [ ] [ ] [ ] . the effect of preheating can be seen in lamp as well. lamp is generally tolerant to the serum or plasma environment [ , ] , but preheating the input sample has been found to have a favorable effect [ ] [ ] [ ] [ ] that produces up to a -fold improvement in sensitivity [ ] . this heating also enabled pardee et al. to detect zika virus rna in serum with high sensitivity using nasba [ ] . hcr performs especially well in serum without any pretreatment [ , , [ ] [ ] [ ] , presumably because the reaction relies on cascaded hybridization events instead of polymerases. because plasma and serum contain very low-abundance analytes, nucleic acid tests need to operate with high sensitivity. fortunately, lamp-based applications are achieving increasingly low limits of detection. nijru et al., for instance, demonstrated that their lod of trypanozoan parasite/l serum in hat diagnosis was -fold more sensitive than pcr testing. such methods could still benefit from user-friendly techniques for large-scale processing. some semi-direct examples presented above include a centrifugation step to collect condensate formed after heating, but could just as easily rely on pipette collection to obviate the need for a high-speed centrifuge. others might benefit from certain stand-alone modules for plasma and serum separation that could be integrated into a poc workflow [ , ] . saliva and sputum are abundant and easy to obtain, and are thus attractive samples for diagnostics. saliva flows into the oral cavities through salivary glands, where blood vessels secrete the same protein and nucleic acid biomarkers as in peripheral blood. in contrast with blood-based samples, saliva sampling does not require trained technicians, presents fewer antigen-associated risks, and can be more easily purified (saliva is % water) [ ] . sputum, a necessary sample for respiratory infections, is mucus from the lower airways. unfortunately, saliva and sputum are very heterogeneous with respect to the distribution of organisms, chemical composition, and the presence of outside contaminants such as toothpaste, cigarette smoke, coffee, or mouthwash. technical extraction kits such as rnaqueous and magmax (life technologies, grand island, ny, usa) are often used to eliminate inhibitors and nucleases from oral samples. the viscosity of sputum requires particularly cumbersome protocols for sample preparation: full processing begins with mucolytic agents such as n-acetyl-l-cysteine (nalc) and dithiothreitol (dtt), disruption of mycobacteria by detergents and proteolytic enzymes, then isolation of target dna by organic solvents or capture reagents [ , ] . the human salivary microbiome has importance as a diagnostic indicator of oral cancer, oral diseases such as periodontitis, and systemic diseases such as pneumonia [ ] . as for sputum, it has become the specimen of choice for detecting tuberculosis [ , ] . naats for mycobacterium tuberculosis have been endorsed by the who (world health organization) and the fda for their high accuracy [ , ] . the systems introduced below extend the practical usage of sputum for poc testing by reducing the requirements for sputum manipulation. most reported nucleic acid testing methods for saliva and sputum show fairly high numbers for patient samples tested, with only two of cases that did not examine clinical specimens ( figure ). the approaches we examined generally employ dilutions of % or less, and achieve low detection limits. this is critical for avoiding false positives, as the target concentrations in sputum and saliva are small. the lowest lod achieved- fg of acinetobacter baumannii bacterial gdna in sputum-required pretreatment with sputazyme (kyokuto, tokyo, japan) and heat before lamp analysis [ ] . in contrast with blood, the high water content of saliva should make it relatively easy to augment the concentration of matrix that can be employed in an amplification reaction. there are several examples of amplification directly on dried sputum collected via filter cards, which is an especially promising direction for direct testing at the poc if samples need to be stored, handled by multiple clinicians, or reevaluated at later dates [ , , ] . amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [ ] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [ ] [ ] [ ] , although heat [ ] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [ , , , ] . with the broad range of bacterial species present in the mouth (over inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [ ] . several other groups have followed suit in detecting bacterial taxa in healthy [ , ] and diseased saliva samples [ ] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [ ] and malaria [ ] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [ ] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [ ] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [ ] . several semi-direct examples based on lamp [ ] [ ] [ ] , recombinase polymerase amplification (rpa) [ , ] , or pcr [ ] [ ] [ ] [ ] [ ] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [ ] . furthermore, tarhan et al. showed amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [ ] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [ ] [ ] [ ] , although heat [ ] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [ , , , ] . with the broad range of bacterial species present in the mouth (over inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [ ] . several other groups have followed suit in detecting bacterial taxa in healthy [ , ] and diseased saliva samples [ ] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [ ] and malaria [ ] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [ ] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [ ] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [ ] . several semi-direct examples based on lamp [ ] [ ] [ ] , recombinase polymerase amplification (rpa) [ , ] , or pcr [ ] [ ] [ ] [ ] [ ] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [ ] . furthermore, tarhan et al. showed that the sensitivity in tb testing was better for sputum samples that were measured directly, rather than after extraction with naoh-nalc [ ] . in pursuing alternatives to the lengthy naoh-nalc method, sputum has been used in pcr after adding mucolytic agents [ , ] , diluting in buffer [ ] , or bead-beating [ ] to reduce viscosity. a more aggressive pretreatment, relying on chemical, thermal, and mechanical means of disruption, used heating and centrifugation to detect tb in sputum samples with comparable performance to more expensive molecular-based systems [ ] . one particularly noteworthy example of direct testing used lamp to diagnose tuberculosis at three peripheral laboratories [ ] . in the study, lamp had a sensitivity of . %, detecting out of smear-negative, culture-positive sputum samples. the authors canvassed the laboratory personnel after they implemented the heating, washing, and filter-tip capture steps before direct amplification to verify that the assay had significant potential to be adopted for routine use. several early examples of pcr on sputum samples with mycobacterium spp. reported sensitivities in the single-digit copy number range. however, the pretreatment methods were quite divergent. sjobring et al. used long centrifugation and sonication steps, in addition to boiling, to detect down to eight organisms [ ] . sritharan et al. was able to cut down the steps to a min boiling period, with a resulting lod of organism [ ] . since these examples in the s, only one technique with a pre-amplification wash and an rpa reaction has been able to match this performance in detecting a single mycobacterium [ ] . as far as combining specificity and sensitivity without adding technical difficulty, priye et al.'s recent multiplexed rt-lamp detection system for zika, dengue, and chikungunya achieved lods of copies/reaction with no need for lysis or extraction [ ] . these impressive outcomes from isothermal technologies like rpa and lamp illustrate that fancy hardware is not necessary for testing modalities to achieve high specificity directly in human samples. swabs have become a mainstay in testing for viral pathogens. molecular systems that identify respiratory tract infections in nasal swabs [ ] or stis (sexually transmitted infections) in dermal, genital, and conjunctival swabs [ ] are used for rapid, accurate patient diagnosis. dna collection from swabs is attractive because it is simple, minimally invasive, and even enables self-sampling. however, swab-collected specimens are likely to contain polymerase inhibitors such as secreted minerals, electrolytes, hormones, enzymes, immunoglobulins, and cytokines, as well as topical medications [ , [ ] [ ] [ ] [ ] . as a result, many swab tests now on the market remove these inhibitors via extraction methods that are too involved and complex to be suitable for the poc [ ] . all of the swab sample studies we examined employed at least one patient sample, and most achieved high sensitivity at a reasonable level of dilution in the buffer used for dna elution from the solid swab (figure ) . one remarkable study examined patient swabs in direct pcr for str (short tandem repeat) analysis [ ] -unfortunately, the authors did not report the yields of dna obtained or the lowest amounts detected. this is a particularly common problem amongst these references-when the lods are not reported, it is especially difficult to replicate these procedures or compare the manipulations used in sample storage, dna replication, and detection [ , ] . special attention should therefore be paid to reproducibility in future efforts at direct amplification of swab samples. typically, elution either at room temperature [ , , [ ] [ ] [ ] [ ] [ ] or with heating [ , , [ ] [ ] [ ] [ ] [ ] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [ ] . lamp-based testing of swab samples can also be performed at room-temperature [ ] [ ] [ ] [ ] [ ] or while heated [ , [ ] [ ] [ ] [ ] [ ] [ ] , as can mda [ ] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [ ] . moving towards instrument-free molecular diagnostics systems, a lateral-flow stranddisplacement amplification (sda) [ ] assay could directly detect mrsa from nasal swabs with a sensitivity of copies/reaction [ ] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h n in patient nasopharyngeal specimens. their sensitivity of copies/reaction was well below the mean viral load for h n patients [ ] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [ ] could directly test clinical genital swabs in transport medium for hsv types and [ ] . the nucleic acid assays had lods of . and . copies/reaction for hsv- and hsv- , respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [ ] , m-swab diluent (copan diagnostics inc., murrieta, ca) [ ] , or water [ ] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [ , ] . rodriguez et al. used a similar approach in detecting clinical levels of h n by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [ ] . future developments could focus on direct reactions with swabs that integrate extraction, figure . swab-based procedures for nucleic acid testing, with data presented as in figure . typically, elution either at room temperature [ , , [ ] [ ] [ ] [ ] [ ] or with heating [ , , [ ] [ ] [ ] [ ] [ ] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [ ] . lamp-based testing of swab samples can also be performed at room-temperature [ ] [ ] [ ] [ ] [ ] or while heated [ , [ ] [ ] [ ] [ ] [ ] [ ] , as can mda [ ] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [ ] . moving towards instrument-free molecular diagnostics systems, a lateral-flow strand-displacement amplification (sda) [ ] assay could directly detect mrsa from nasal swabs with a sensitivity of copies/reaction [ ] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h n in patient nasopharyngeal specimens. their sensitivity of copies/reaction was well below the mean viral load for h n patients [ ] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [ ] could directly test clinical genital swabs in transport medium for hsv types and [ ] . the nucleic acid assays had lods of . and . copies/reaction for hsv- and hsv- , respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [ ] , m-swab diluent (copan diagnostics inc., murrieta, ca, usa) [ ] , or water [ ] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [ , ] . rodriguez et al. used a similar approach in detecting clinical levels of h n by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [ ] . future developments could focus on direct reactions with swabs that integrate extraction, amplification, and detection in a single tube for poc usage. this would give low-resource settings alternatives to instrument-dependent assays like the alere i platform for detecting influenza a & b from nasal swabs. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [ ] [ ] [ ] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [ , ] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [ ] [ ] [ ] [ ] [ ] . in surveying direct nucleic acid testing methods (figure ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of . pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [ ] [ ] [ ] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [ , ] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [ ] [ ] [ ] [ ] [ ] . in surveying direct nucleic acid testing methods (figure ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of . pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogelencased reaction [ ] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogel-encased reaction [ ] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated biosensors , , of by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters biological inhibitors out of the amplification reaction, enabling detection of mycoplasma homonis and ureaplasma urealyticum. for feces, the options are to employ inhibitor-resistant polymerases [ ] and buffer additives [ ] . in the case of one large-scale characterization by hall et al., the combination of both phire polymerase (new england biolabs, ipswich, ma, usa) and ampdirect (biomatrica, san diego, ca) gave an lod of nearly one copy/reaction in pcr for francisella tularensis in . % stool [ ] . one can also lyse bacteria in urine [ ] or stool [ , ] through heating to release an amplifiable amount of target dna with minimal levels of inhibitors. moore et al. managed to detect human norovirus repeatedly in out of outbreak stool samples after boiling the diluted feces in pbs [ ] . although centrifugation is employed in some semi-direct methods to create a supernatant from the collected stool, we believe a dilution step could accomplish the same feat by allowing solids to settle at the base of a highly aqueous, non-viscous sample. isothermal amplification techniques like lamp and rpa generally demonstrate a higher tolerance than pcr for urine, as they can be carried out directly. as such, lamp-based assays without any pre-processing steps or chemical enhancements have detected the causative agents of viral infections [ , , ] or stis [ , ] , and pathogenic bacteria such as escherichia coli [ ] . the developers of the recently established isothermal method siba showed the utility of this amplification technique by detecting chlamydia trachomatis and neisseria gonorrhoeae in a low-copy urine sample [ ] . the landscape of molecular diagnostics is constantly advancing, as is the current paradigm of healthcare. the advent of mobile health and telemedicine has decentralized patient care. it has also put a new emphasis on usability and non-invasiveness in disease testing. nucleic acid diagnostics that reduce the difficulties and expenditures of standard multi-step procedures by direct amplification can expedite patient testing in poc, hospital, and laboratory situations [ ] . in this review, we are thus motivated to discuss the current state of the art for direct naats: assays and platforms that require minimal or no sample preparation procedures. we search the literature from to and find published works that we consider direct naats. we first categorize these works based on the type of complex samples. we subsequently employ bubble plots to facilitate the comparison of the amplification method, robustness in complex media, sensitivity to target, and clinical usage. our findings indicate that the majority of direct naats exhibit a tolerance of less than % for their sample of interest, and fewer than patient-based evaluations. still, there are diagnostic procedures that far surpass these averages. sim et al.'s study of direct pcr on buccal swabs [ ] , for instance, is robust and carried out directly on the entire sample for maximal ease. improvements must continue to be made for all sample types in terms of facilitating this level of evaluation with clinical samples. despite significant developments to date, there remain several challenges for realizing direct naats with user-friendliness, consistency, and generalizability. in furthering the development of direct testing, it is important to take a holistic approach and consider the type of sample to be analyzed, the method of sample acquisition, the throughput and volume, and any chemical or mechanical requirements, and the amplification technique. another key point to note is that different matrices will function best in different environments. an improved understanding of the mechanisms behind emerging nucleic acid amplification reactions and mutant polymerases will enable the rationalization of how inhibitory compounds can make or break an amplification system. furthermore, molecular assays have much to learn from diagnostics that are continuously being developed in the commercial pipeline. proprietary technology will always hold knowledge at a cost to the user, but the implementation of new ideas can lead researchers towards better and more successful ways in which to modernize the ever-changing field of disease testing. as we enter the age of electronic, mobile, and personalized medicine, there remains much room for creativity and innovation in the design of naats and poc diagnostics. molecular diagnostics, as the highest-growing segment of all in vitro diagnostic products [ ] , truly have great potential for both developed and low-resource areas. the diagnostic community continues to strive for tests that are reliable against variable electrical resources, water quality, trained staff, or harsh environmental conditions [ , ] . researchers continue to seek approvals such as the fda's clia waivers or the who's assured (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and delivered to end-users) criteria. in this regard, direct naats present a promising approach. through this review, it is our hope to stimulate the discussion on direct naats and their potential as poc diagnostics. ultimately, we seek to help accelerate the development of poc diagnostics that can be clia waived and/or meet the who assured criteria, thereby ushering in the next revolution in healthcare. the following are available online at http://www.mdpi.com/ - / / / /s , table s : detailed properties of amplification assays. point of care diagnostics for sexually transmitted infections: perspectives and advances advances in developing hiv- viral load assays for resource-limited settings commercialization of microfluidic point-of-care diagnostic devices micro total analysis systems for cell biology and biochemical assays microfluidic dna amplification-a review nucleic acid isothermal amplification technologies: a review point-of-care nucleic acid testing for infectious diseases isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings fully automated quantification of cytomegalovirus (cmv) in whole blood with the new sensitive abbott realtime cmv assay in the era of the cmv international standard fda grants first clia waiver for nucleic acid-based flu diagnostic test; food and drug administration focus diagnostics receives fda clearance for moderate complexity simplexa hsv & direct molecular test for aiding the diagnosis of encephalitis performance of the molecular alere i influenza a&b test compared to that of the xpert flu a/b assay detection of influenza a and b with the alere tm i influenza a&b: a novel isothermal nucleic acid amplification assay evaluation of the alere i influenza a&b nucleic acid amplification test by use of respiratory specimens collected in viral transport medium evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus nucleic acid sequence-based amplification low-cost detection of zika virus using programmable biomolecular components reliability of nucleic acid amplification methods for detection of chlamydia trachomatis in urine: results of the first international collaborative quality control study among laboratories reliability of nucleic acid amplification methods for detectio dna detection using recombination proteins invasion based amplification (siba): a novel isothermal dna amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte multiplex strand invasion based amplification (msiba) assay for detection of chlamydia trachomatis and neisseria gonorrhoeae comprehensive human genome amplification using multiple displacement amplification triggered amplification by hybridization chain reaction highly sensitive electrogenerated chemiluminescence biosensor based on hybridization chain reaction and amplification of gold nanoparticles for dna detection specific enzymatic amplification of dna in vitro: the polymerase chain reaction. cold spring harb standardization of nucleic acid tests for clinical measurements of bacteria and viruses highly sensitive detection of staphylococcus aureus directly from patient blood a potent inhibitor of taq polymerase copurifies with human genomic dna identification of the heme compound copurified with deoxyribonucleic acid (dna) from bloodstains, a major inhibitor of polymerase chain reaction (pcr) amplification capacity of nine thermostable dna polymerases to mediate dna amplification in the presence of pcr-inhibiting samples pcr based diagnosis in the presence of % (v/v) blood a reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification detection of toxoplasma gondii dna by pcr following microwave treatment of serum and whole blood comparison between toxoplasma gondii dna and specific immunoglobulins during pregnancy. la rev whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method by microwave irradiation improved method for direct pcr amplification from whole blood direct pcr from whole blood, without dna extraction rapid point-of-care isothermal amplification assay for the detection of malaria without nucleic acid purification rapid and sensitive detection of orientia tsutsugamushi by loop-isothermal dna amplification african trypanosomiasis: sensitive and rapid detection of the sub-genus trypanozoon by loop-mediated isothermal amplification (lamp) of parasite dna sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification a simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (htlamp) assay for malaria elimination highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in uganda clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria short report: evaluation of loop-mediated isothermal amplification (lamp) for malaria diagnosis in a field setting rapid detection of haptoglobin gene deletion in alkaline-denatured blood by loop-mediated isothermal amplification reaction mitochondrial dna targets increase sensitivity of malaria detection using loop-mediated isothermal amplification detection of plasmodium vivax infection in the republic of korea by loop-mediated isothermal amplification (lamp) heat denaturation increases the sensitivity of the cytomegalovirus loop-mediated isothermal amplification method molecular diagnostic field test for point-of-care detection of ebola virus directly from blood direct blood dry lamp: a rapid, stable, and easy diagnostic tool for human african trypanosomiasis direct loop-mediated isothermal amplification from plasmodium chabaudi infected blood samples: inability to discriminate genomic and cdna sequences sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv- a smartphone-based diagnostic platform for rapid detection of zika, chikungunya, and dengue viruses quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of rna viruses unbiased whole-genome amplification directly from clinical samples dried blood spots in hiv monitoring: applications in resource-limited settings early infant human immunodeficiency virus type detection suitable for resource-limited settings with multiple circulating subtypes by use of nested three-monoplex dna pcr and dried blood spots review article: an overview of the clinical use of filter paper in the diagnosis of tropical diseases the diagnosis of syphilis on a dessicated and defibrinated blood drop a simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants dried blood spots for hiv- drug resistance and viral load testing: a review of current knowledge and who efforts for global hiv drug resistance surveillance a diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles cystic fibrosis genotyping by direct pcr analysis of guthrle blood spots an evaluation of direct pcr amplification enhanced direct amplification of guthrie card dna following selective elution of pcr inhibitors dna analysis of cystic fibrosis in brazil by direct pcr amplification from guthrie cards screening for cystic fibrosis: feasibility of molecular genetic analysis of dried blood specimens polymerase chain reaction amplification from dried blood spots on guthrie cards rapid, efficient method for multiplex amplification from filter paper gene amplification directly from guthrie blood spots molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening dna extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction validation of a fast and low-cost alkaline lysis method for gdna extraction in a pharmacogenetic context a reliable and effective method of dna isolation from old human blood paper cards comparison of six commercially-available dna polymerases for direct pcr short report: rapid dna extraction from archive blood spots on filter paper for genotyping of plasmodium falciparum polymerase chain reaction amplification of dna from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media improved procedure for eluting dna from dried blood spots adaptation of a visualized loop-mediated isothermal amplification technique for field detection of plasmodium vivax infection point-of-care technologies for molecular diagnostics using a drop of blood multilaboratory evaluation of real-time pcr tests for hepatitis b virus dna quantification cell-free nucleic acids as biomarkers in cancer patients characterization of circulating dna in healthy human plasma isolation and characterization of dna from the plasma of cancer patients blood separation on microfluidic paper-based analytical devices simple, miniaturized blood plasma extraction method membrane-based, sedimentation-assisted plasma separator for point-of-care applications micro-scale blood plasma separation: from acoustophoresis to egg-beaters enzyme-free and ultrasensitive electrochemical detection of nucleic acids by target catalyzed hairpin assembly followed with hybridization chain reaction comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus direct serum assay for cell-free bmi- mrna and its potential diagnostic and prognostic value for colorectal cancer direct serum assay for microrna- concentrations in early and advanced breast cancer circulating dna of hotair in serum is a novel biomarker for breast cancer simple and rapid identification of low level hepatitis b virus dna by the nested polymerase chain reaction microwave treatment of serum facilitates detection of hepatitis b virus dna by the polymerase chain reaction. results of a study in anti-hbe positive chronic hepatitis b improved detection of hbv dna by pcr after microwave treatment of serum tolerance of loop-mediated isothermal amplification to a culture medium and biological substances direct detection of human herpesvirus b by the lamp method using newly developed dry-reagents direct detection of human herpesvirus dna in serum by variant specific loop-mediated isothermal amplification in hematopoietic stem cell transplant recipients rapid detection of hiv- by reverse-transcription, loop-mediated isothermal amplification (rt-lamp) development of a loop-mediated isothermal amplification assay for rapid detection of bk virus direct detection of human herpesvirus dna in serum by the loop-mediated isothermal amplification method enzyme-free and label-free ultrasensitive electrochemical detection of dna and adenosine triphosphate by dendritic dna concatamer-based signal amplification g-quadruplex based two-stage isothermal exponential amplification reaction for label-free dna colorimetric detection pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings saliva: physiology and diagnostic potential in health and disease inhibition of the polymerase chain reaction by mucolytic agents direct detection of mycobacterium tuberculosis in sputum by polymerase chain reaction and dna hybridization defining the normal bacterial flora of the oral cavity defining the normal bacterial flora of the oral cavity analytical and clinical evaluation of the epistem genedrive assay for detection of mycobacterium tuberculosis exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in hiv-negative patients by use of the genexpert mtb/rif assay determinants of pcr performance (xpert mtb/rif), including bacterial load and inhibition, for tb diagnosis using specimens from different body compartments rapid molecular detection of tuberculosis and rifampin resistance clinical specimen-direct lamp: a useful tool for the surveillance of blaoxa- -positive carbapenem-resistant acinetobacter baumannii flinders technology associates (fta) filter paper-based dna extraction with polymerase chain reaction (pcr) for detection of pneumocystis jirovecii from respiratory specimens of immunocompromised patients operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries successful use of saliva without dna extraction for detection of macrolide-resistant 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difficile detection of toxigenic clostridium difficile: comparison of the cell culture neutralization, xpert c. difficile, xpert c. difficile/epi, and illumigene c. difficile assays evaluation of the gen-probe chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women sensitive and rapid detection of chlamydia trachomatis by recombinase polymerase amplification directly from urine samples mohd-zain, z. pentaplex pcr assay for detection of hemorrhagic bacteria from stool samples removal of pcr inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to pcr development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses a novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods loop-mediated isothermal amplification test for detection of neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea loop-mediated isothermal amplification assay for rapid detection of common strains of escherichia coli molecular diagnostics of infectious diseases the global molecular diagnostics market report #a point-of-care tests for diagnosing infections in the developing world simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv- testing this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the support of h.t. soh in the initial stages of manuscript preparation. the authors declare no conflict of interest. key: cord- -e tdb v authors: schermer, bernhard; fabretti, francesca; damagnez, maximilian; di cristanziano, veronica; heger, eva; arjune, sita; tanner, nathan a.; imhof, thomas; koch, manuel; ladha, alim; joung, julia; gootenberg, jonathan s.; abudayyeh, omar o.; burst, volker; zhang, feng; klein, florian; benzing, thomas; müller, roman-ulrich title: rapid sars-cov- testing in primary material based on a novel multiplex rt-lamp assay date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: e tdb v background: rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus (sars-cov- ) infections and decision-making in times of a pandemic outbreak. however, point-of-care (poc) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. the need for thermal cycling and nucleic acid isolation hampers the use of standard pcr-based methods for this purpose. methods: to avoid these obstacles, we tested pcr-independent methods for the detection of sars-cov- rna from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (rt-lamp) and specific high-sensitivity enzymatic reporter unlocking (sherlock). results: whilst specificity of standard rt-lamp assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qpcr) assays yet. we describe a novel multiplexed rt-lamp approach and validate its sensitivity on primary samples. this approach allows for fast and reliable identification of infected individuals. primer optimization and multiplexing helps to increase sensitivity significantly. in addition, we directly compare and combine our novel rt-lamp assays with sherlock. conclusion: in summary, this approach reveals one-step multiplexed rt-lamp assays as a prime-option for the development of easy and cheap poc test kits. a a a a a the recent pandemic of sars-cov- is a major challenge for healthcare systems worldwide. lacking an effective and approved vaccine, reliable screening of samples from nasopharyngeal swabs for viral rna is a fundamental pillar for effective disease control. governmental measures (e.g. shutdown of social life) largely depend on such data to allow for a rapid adaptation to changes in epidemiologic indicators. furthermore, isolation and quarantine of infected and exposed individuals are key for effective outbreak control. here, the prompt establishment of a diagnostic real-time pcr based testing [ ] has been instrumental already in the early phases of the pandemic. such qpcr-based tests are extremely powerful due to their high sensitivity and specificity. however, these assays require specific lab equipment and expertise which is typically not widely available directly at the point-of-care making transportation to a specialized facility necessary. furthermore, the material required for the different steps involved may become subject to shortages during a pandemic making alternative approaches an important goal. even though rna isolation and subsequent qpcr is performed in only a few hours, actual turnaround times from sample collection to diagnostic test results are often much longer. decision-making in both healthcare facilities and other spheres of public life would be facilitated enormously by direct testing on site with short turn-around times. such an approach could also limit the need for quarantine, allow healthcare workers after exposure to continue their work upon a daily negative swab and avoid shortages in systemically relevant personnel. recently, a number of pcr-independent methods have been proposed for this purpose including isothermal amplification (rpa, lamp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and their combination with genome editing tools such as cas a [ ] or cas a [ , ] for improved performance. the majority of these studies, however, have not been carried out on direct primary material but on rna isolated from patient samples or generated in vitro [ ] [ ] [ ] [ ] [ ] [ ] [ ] ]. in the study at hand, we use previously described rt-lamp and sherlock assays on both isolated rna and primary material from patients [ , ] . in addition, we describe a newly designed multiplexed rt-lamp assay targeting orf a and orf a of sars-cov- . orf a and orf a have been selected since these targets have not been used for any available diagnostic qpcr assay, to avoid the risk of amplicon cross-contamination between different types of assays. nasopharyngeal swabs were taken from symptomatic patients presenting at university hospital of cologne from march to april . swabs were directly transferred in - ml of universal transfer medium (utm) or pbs. for diagnostic qpcr, rna was extracted from μl utm / pbs of the swab samples using the automated magna pure system (roche). diagnostic qpcr was performed using a realstar sars-cov- rt-pcr kit . , with primers targeting e and s gene (altona diagnostics) on a lightcycler (roche). positively and negatively tested utm/pbs samples were randomly selected for rt-lamp or sherlock assays. here, . to . μl isolated rna or . to . μl utm/pbs was used for subsequent rt-lamp/sherlock assays. this study was performed exclusively with surplus diagnostic material that was analyzed anonymously. no specific approval number from the irb (ethikkommission der medizinischen fakultaet der universitaet zu koeln (irb of university of cologne)) was required. for assays from direct material μl of utm/pbs were incubated at ˚c for minutes in a pcr cycler with heated lid placed inside a safety cabinet. treatment of min at ˚c has been shown to inactivate sars-cov- [ ] and ˚c was found to be preferable for downstream rna-based applications. for orf a and gene n we used previously described primer sets [ ] . additional primer sets were designed for orf a, orf a and the m gene using the primerexplorer v tool (http:// primerexplorer.jp/e/). all primers were ordered from idt, purified with standard desalting, as page purification did not improve the performance of the assays. all primer sequences are listed in s table. all rt-lamp reactions were assembled on ice. in brief, each μl reaction contained μl warmstart colorimetric rt-lamp mix (neb), μl primer mix (f /b μm each; fip/bip μm each; lf/lb μm each), mm guanidine hydrochloride (from a m stock solution, ph ), dnase/rnase free water and , μl sample. for multiplexed rt-lamp the additional primer mix replaced μl of dnase/rnase free water. initially, the assays were performed without guanidine hydrochloride at ˚c for minutes, while the multiplexed reaction was done at ˚c for to minutes, as indicated in the figures. each assay was performed including several negative controls. positive reactions were identified due to a clear change in color from pink/red to orange/yellow. in two cases out of almost , the mere addition of . μl sample to the reaction resulted in a color change. these samples are not included in the data shown. the two step sherlock assay for orf a and s -gene was performed according to the protocol established in feng zhang's lab at mit (https://zlab.bio/s/covid- -detection-v .pdf) [ , ] . in brief, an rpa reaction was set up using . μl rpa mix (twist amp), . μl protoscript rt (neb), μl rpa primer mix ( μm each), . μl sample (isolated rna or utm/pbs) and . μl magnesium acetate ( mm stock solution). the reaction was mixed, spun down and incubated ˚c for min. the cas a-based detection step was performed in a μl reaction as follows: μl tris-hcl buffer ( mm, ph . ), . μl dnase/ rnase free water, μl lwacas a (corresponding to ng of protein), μl crrna ( ng/μl), μl lateral-flow-reporter ( μm), μl superase in rnase inhibitor (thermofisher scientific), . μl t polymerase (lucigen), . μl rntps ( μm each; neb), μl mgcl ( mm stock solution) and μl of previous rpa reaction. the reaction was mixed and spun down, then incubated at ˚c for min. next, the reaction was diluted with μl of hybridetect buffer (milenia), mixed, and then a hybridetect dipstick (milenia) was placed in the reaction tube and incubated at rt. results were visible within minutes. to combine the rt-lamp amplification with cas a detection we added a t promoter in the loop region of the fip primers for the rt-lamp assay (s table) . after the rt-lamp reaction at ˚c for min, . μl were used in the cas a detection assay assembled as described above, using a specific crrna targeting the rt-lamp gene n amplicon. to allow for the comparison of different nucleic acid detection methods for sars-cov- we collected redundant material from nasopharyngeal swabs obtained for qpcr testing in clinical routine due to suspected covid- . samples were selected randomly from samples collected in the period from march to april . the cohort included individuals between the age of month and years with a close to equal distribution of men and women (s table) . we first tested two recently described assays for sars-cov- detection on isolated rna from patient samples. specifically, we performed colorimetric rt-lamp assays using primers targeting orf a and gene -n [ ] and two-step sherlock assays combined with lateral flow detection [ , ] targeting orf a and s gene (s a-s c fig) . even though both assays worked well on these samples, they failed to detect the virus in specimen that were positive in diagnostic qpcr at cycle threshold (ct) values > for e and s gene (s fig). moreover, rt-lamp appeared to be slightly more sensitive and specific (s c fig) . due to limited availability of the rpa reagents required for the first step of sherlock at that time paired with the results described above, we focused our efforts on validation of the rt-lamp assays. since detection worked on isolated rna, we went on to test this approach using primary material (i.e. transport medium from nasopharyngeal swabs without rna isolation) strikingly, both rt-lamp assays performed well on these specimens. using as little as . μl utm as input for each reaction to avoid inhibitory effects of the medium or of tissue contaminants on the reaction, detection worked in samples tested positive by qpcr (see ct as reference) (fig a) . in parallel, additional sets of rt-lamp primers targeting additional genes of the sars--cov- genome (nc_ ) were designed, tested and validated. here, we preferred genes that are not target of any standard diagnostic qpcr assay, to minimize and avoid any possible interference of such point of care assays with other routine diagnostic pipelines. among these new primer sets, the oligos targeting orf a showed the highest sensitivity and specificity in several tests on diluted isolated rna and was thus selected for further testing in primary material. in this second set of experiments we screened a total of samples, of which had been tested positive for sars-cov- by diagnostic qpcr. fig b displays representative results of the rt-lamp assays targeting gene n, orf a and orf a. assays targeting gene n and orf a were more sensitive than the one targeting orf a as indicated by the mean ct value of rt-lamppositive samples (fig c) , by the total number of samples that were correctly identified ( fig d and e ). out of specimens turned out positive for gene n and out of for orf a and orf respectively, that were negative by qpcr. in order to increase the sensitivity of rt-lamp assays to a comparable level with qpcr we performed a "two-step" lamp reaction, either using a small amount ( . to . μl) of a first rt-lamp reaction as template for a second one or by refreshing the first reaction with new enzymes and dntps. all of these attempts resulted consistently in negative controls (water, empty utm or samples from negative patients) turning positive, either because of unspecific amplification or because of the necessary re-opening of the reaction tubes after the first amplification step. the use of mineral oil on top of every reaction-performed to avoid cross-contamination-did not improve these results. in an additional set of experiments, we combined the rt-lamp assays targeting gene n with lwacas a mediated detection. to this end, we to increase accessibility of rna and efficiently inactivate the virus we incubated utm from swabs at ˚c for min. treatment at ˚c for minutes has been demonstrated to efficiently inactivate sars-cov- [ ] , while ˚c appeared a good choice for downstream rna applications in case of sars-cov- [ ] . after incubation at ˚c, some of the utm samples showed a gel-like consistency. this was observed in only a minor fraction of the samples and was most likely caused by protein and sugar supplements contained in one specific type of utm. this utm was easily recognizable since it was the only one containing phenol red as ph indicator. in these cases, pipetting about times up and down with a p pipette allowed for complete homogenization and subsequently accurate pipetting. again, we only used . μl utm for each reaction. in addition, we added guanidine hydrochloride as a classical rnase inhibitor and lamp enhancer [ ] to the multiplexed reaction at ˚c. these modifications in sample preparation combined with our new multiplexed assay were used to analyze a set of clinical samples consisting of sars-cov- positive and negative swabs (fig ) . sars-cov- positive samples with ct values up to were positive in the rt-lamp assay (fig b) , while still out of qpcr positive samples were not detected in our assay on direct material. however, the vast majority of samples up to a ct of were correctly identified ( %; out of ). meanwhile, rpa reagents arrived and we performed the two step sherlock assays on some of the very same direct samples. however, sensitivity was much lower with a cut-off threshold of cycles (fig d; representative assays shown in s fig). in summary, our multiplex rt-lamp protocol is a simple and sensitive way to detect sars-cov- rna from clinical samples. based on our data we conclude, that multiplexing primers in rt-lamp reactions is a highly promising way to further increase sensitivity of these assays and to quickly develop a rapid poc test. currently, a test based on our multiplexed rt-lamp assay would-in contrast to a good specificity-most likely miss to identify those infected patients with very low amounts of viral rna in the nose or throat and would not yet reach the sensitivity of the gold-standard qpcr assays. however, the rt-lamp approach comes with several clear-cut advantages. in contrast to classical qpcr, it can be used in primary material without the need for rna isolation and yields results rapidly. furthermore, only little equipment is required (thermoblock) and could easily be made available in e.g. emergency rooms. the estimated costs for reagents are below . € per reaction with the advantage-compared to qpcr-that additional expenses for nucleic acid isolation are not required. regarding the target setting of primary screening, one could hypothesize, that identifying patients with high viral loads would detect those individuals that are highly infectious. consequently, in this setting, even a poc test with a lower sensitivity as compared to diagnostic qpcr would be extremely useful and could be backed up by a combination with qpcr the results of which get available later. currently, a relation between the level of viral rna in swabs and the infectivity of a patient has not yet been definitively established. detection of viral rna is not equivalent to detection of infectious virus. one retrospective study, however, suggested low infectivity of patients with ct-values > in e gene qpcr from swabs since the authors did not observe viral growth in exposed vero cells [ ] . besides, a statement paper of the national centre for infectious diseases and the chapter of infectious disease physicians (singapore) refers to a study with covid- patients where a ct > was found to be the threshold of infectivity [ ] . additional studies report ct values between and as cut-off for infectivity in different set-up [ , ] . a recently updated meta-analysis provides a very comprehensive overview on this important topic supporting the conclusion that infectivity is related to the cycle threshold level, but a clear cut-off can currently not yet be defined [ ] . nonetheless, the ultimate goal would be a poc test that reaches the sensitivity of qpcr and can completely replace the current approach where favorable. studies in the near future will clarify, whether patients with high viral rna loads are indeed the most contagious individuals. besides, this knowledge will also be of greatest importance for the actual clinical interpretation of qpcr results in the future. by now, it has been shown that the viral load is already high before and maybe highest at onset of symptoms [ ] and at the time point of presentation to the clinic followed by a steady decline [ ] . consequently, when focusing the screening on pre-symptomatic individuals, sensitivity of the rt-lamp assay may actually be higher than in the current cohort. additionally, one thing to be kept in mind when directly comparing sensitivity between the different methods, is the fact that qpcr reaches this standard only in isolated rna whilst the multiplex rt-lamp assay attains an optimized detection rate already in primary material. of note, further addition of a step concentrating rna using bead-based pulldown to an rt-lamp-based protocol has also been successful [ ] . remarkably, here the authors move from swabs, that require trained personnel and personal protection equipment, to a home-based gargle [ ] and a very recent report using qpcr demonstrated, that even saliva could be used as a valid source for viral diagnostics [ ] . optimizing also the collection of samples and including such specimens will be an important step towards broadly applicable poc testing. regarding specificity, only few specimens turned out to be positive in the multiplex rt-lamp assay that were negative in qpcr. since qpcr is the gold-standard this can be interpreted as a minor limitation in specificity. however, qpcr itself does not reach a sensitivity of % [ ] . consequently, it is not clear yet whether these individuals were truly negative or missed by the qpcr assay. crispr/ cas [ ] or cas a [ ] based assays are another promising way to detect rna in a pcr-independent manner. comparison of this approach with rt-lamp demonstrates a surprisingly high sensitivity even of the colorimetric one step rt-lamp assay. very recently, a novel protocol for cas a -called stop ('sherlock testing in one pot')-has been described [ ] . as this replaces the isothermal rpa reaction of the original sherlock protocol by a rt-lamp reaction, the authors use a thermo-stable cas a enzyme to enable performing the entire reaction at the same temperature. whilst being a very interesting approach, nonetheless, this comes with the difficulty the reaction tube has to be opened for the final lateral flow assay used for detection. in the real-world poc testing setting, this would require the establishment of a 'pre-amp' and 'post-amp' area to avoid cross-contamination, which may limit its use. alternatively, lateral flow could be replaced by using a fluorescent probe together with an appropriate simple detection device. however, the highest ct value resulting in a positive stop assay is-at about cycles-in a similar range compared to a recently described cas a-based method (detectr) [ ] . the rt-lamp assay described in our study works without re-opening the test tube after amplification and provides detection at higher ct values. on the other side, both sherlock and detectr add an additional level of specificity to the detection due to the crrna directing the cas / enzyme. both sherlock and detectr require a considerable number of pipetting steps. in contrast, the multiplexed rt-lamp assay demonstrates a similar or even higher sensitivity and requires only two simple pipetting steps at the poc: ( ) taking a aliquot of the utm for 'boiling' and ( ) the addition of the . μl sample to the reaction tube, which could also be reached without a pipet by an inoculating loop. reaction tubes can be prepared in anticipation elsewhere (e.g. in any nearby central facility) and stored for hours at ˚c. the preparation of these reaction tubes, is also done with only four simple pipetting steps ( . primer mix pre-diluted in water, . rt-lamp mix, . guanidine hydrochloride, ! aliquot in μl tubes). this could even be further simplified by adding guanidine to the primer mix. after amplification, the tube and the according controls are photo-documented and discarded. in summary, we are convinced that systematically combining and testing different multiplex rt-lamp primer sets on primary swab material is one of the most promising approaches to develop a powerful poc test. transferring such assays to automated microfluidic formats [ ] can become an important tool to support disease control strategies. detection of novel coronavirus ( -ncov) by real-time rt-pcr clinical assessment and validation of a rapid and sensitive sars-cov- test using reverse-transcription loop-mediated isothermal amplification development of a reverse transcription-loopmediated isothermal amplification as a rapid early-detection method for novel sars-cov- rt-lamp for rapid diagnosis of coronavirus sars-cov- a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- . virol sin. development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus (sars-cov- ) rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp sars-cov- on-the-spot virus detection directly from patients rapid isothermal amplification and portable detection system for sars-cov- journal of clinical virology: the official publication of the pan american society for clinical virology crispr-cas -based detection of sars-cov- a protocol for detection of covid- using crispr diagnostics sherlock: nucleic acid detection with crispr nucleases evaluation of heating and chemical protocols for inactivating sars-cov- an alternative workflow for molecular detection of sars-cov- -escape from the na extraction kit-shortage enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride predicting infectious sars-cov- from diagnostic samples national centre for infectious diseases and the chapter of infectious disease physicians, academy of medicine presymptomatic sars-cov- infections and transmission in a skilled nursing facility viral rna load as determined by cell culture as a management tool for discharge of sars-cov- patients from infectious disease wards viral cultures for covid- infectivity assessment. systematic review temporal dynamics in viral shedding and transmissibility of covid- temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study rapid and highly sensitive isothermal detection of sars-cov- for laboratory and home testing saliva or nasopharyngeal swab specimens for detection of sars-cov- . the new england journal of medicine correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases point-of-care testing for covid- using sherlock diagnostics we thank stefanie keller for her outstanding technical support during very special and challenging times. special thanks to the team of benchling for revolutionizing the work with dna/rna sequences. venn diagrams were generated using "venny . " by j.c. oliveros (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed in may ). key: cord- -go cw q authors: huang, wei e.; lim, boon; hsu, chia‐chen; xiong, dan; wu, wei; yu, yejiong; jia, huidong; wang, yun; zeng, yida; ji, mengmeng; chang, hong; zhang, xiuming; wang, hui; cui, zhanfeng title: rt‐lamp for rapid diagnosis of coronavirus sars‐cov‐ date: - - journal: microb biotechnol doi: . / - . sha: doc_id: cord_uid: go cw q the pandemic coronavirus sars‐cov‐ in the world has caused a large infected population suffering from covid‐ . to curb the spreading of the virus, who urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. we applied a reverse transcription‐loop‐mediated isothermal amplification (rt‐lamp) to achieve the detection of sars‐cov‐ in min. we designed four sets of lamp primers ( primers in each set), targeting the viral rna of sars‐cov‐ in the regions of orf ab, s gene and n gene. a colorimetric change was used to report the results, which enables the outcome of viral rna amplification to be read by the naked eye without the need of expensive or dedicated instrument. the sensitivity can be copies of viral rna per ml in a sample. we validated the rt‐lamp method in a hospital in china, employing clinic samples with positives and negatives. the testing results are consistent with the conventional rt‐qpcr. in addition, we also show that one‐step process without rna extraction is feasible to achieve rna amplification directly from a sample. this rapid, simple and sensitive rt‐lamp method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas. there is an urgent need for rapid diagnosis of sars-cov- infected covid- patients even before an immune response can occur and for asymptomatic carriers. this is critical in making decisions on public health measures, such as movement restrictions, and quarantine duration. nucleic acid testing, that is detecting the viral rna, is a feasible and practical method. currently, the most common method for nucleic acid diagnosis is based on real time rt-pcr (drosten et al., ; mackay et al., ; espy et al., ) . for example, bgi in china (https://www.bgi.com/kit) and the us cdc (https://www.cdc.gov/coronavirus/ -ncov/about/testing.html) provide reagents, primers and probes to support rt-pcr diagnosis for sars-cov- . although real-time rt-pcr is sensitive and reliable, it is time-consuming (~ h) and requires a specific detection device or instrument, which limits its broad application to current huge demand for the global pandemic of to address this challenge, a fast and simple-to-operate test kit would be highly desirable. with such a test, virus infected patients could be identified at an early stage and quarantined to prevent the spread of the infection, whilst the non-infected individuals could carry on with their life as usual. ideally, the test kit is mobile without the need of any complicated instrument and the test result can be read by the naked eye. hence, it can be used at airports, railway stations and hospitals, particularly regional hospitals and medical centres in rural areas. loop-mediated isothermal amplification (lamp) is a rapid technology of dna amplification (notomi et al., ; tomita et al., ) , which has been applied to pathogen detection such as virus, bacteria and malaria (thai et al., ; boehme et al., ; mori and notomi, ; law et al., ; reboud et al., ) . the lamp reaction generally takes place in a constant temperature, and the target dna can be amplified in min (tomita et al., ) . the lamp method employs or primers to bind six regions of a target dna, and the specificity is extremely high (notomi et al., ; tomita et al., ) . since the lamp method only needs one constant temperature (usually °c), the device can be simple. initially, the lamp uses primers (notomi et al., ; tomita et al., ) , later it is found that the addition of two loop primers can shorten half of the time required for the original lamp reaction (nagamine et al., ) . the availability of warmstart rtx reverse transcriptase (new england biolabs, uk) makes it possible to combine both reverse transcription and lamp in one reaction. since sars-cov- is a rna virus with the length about kb (wang et al., ) , the single reaction of reverse transcription (rt) and lamp together can significantly shorten the reaction time without the dna purification step from rt, thus a rapid detection of sars-cov- can be achieved. in this work, we developed a covid- diagnosis kit for the rapid detection of sars-cov- , using one-step reverse transcription and loop-mediated isothermal amplification (rt-lamp). the whole reaction can be as short as min at a constant °c. the detection limit is copies of viral rna per ml sample. a simple colour change indication can be visualized by the naked eye to confirm the result of viral rna amplification. the kits were validated by clinical covid- samples. this new technique can also provide a rapid and feasible approach for the detection of various virus. specificity of o , s , n and n primers to amplify synthesized dna of sars-cov- we designed four sets of lamp primers o , s , n and n , which target rna encoding orf ab, spike glycoprotein and two regions in nucleocapsid protein of sars-cov- respectively (fig. s ). each set has primers (table ) . sars-cov- is a single stranded rna virus with the length of about kb. orf ab is about kb long, encoding the replicase polyprotein (woo et al., ) . o primers were designed to cover the conversed region of '-end of the viral rna in orf ab (fig. s ). s primers target the s gene, which encodes spike glycoprotein, a key for this sars-cov- virus to bind human ace protein and to invade human cells (menachery et al., ; cui et al., ; walls et al., ) . the n gene for nucleocapsid protein is at the ʹ-end of the viral rna, conserved to sars-like coronavirus (cui et al., ) . during sampling and rna extraction processes, the viral rna might be attacked by rnase, degrading from ʹ-to ʹ-, so we designed n and n to ensure the detection of ʹ-end of the viral rna of sars-cov- , even though it is partially degraded. the targeted sars-cov- rna fragments of these four sets of primers ( fig. s and table ) are about - bp, long enough to be targeted by primers and short enough to achieve a rapid amplification. in all experiments of lamp and rt-lamp, the experiments were carried out with more than three biological replicates and the results were consistent. to verify their specificity, we first tested these primers using synthetic target dna of sars-cov- . n and n primers were able to amplify dna fragments of the n gene, but not human genomic dna ( fig. a and b). by adding the fluorescent dye provided by neb (new england biolabs, uk), positive results can be visualized directly from the pcr tubes under uv exposure. the fluorescent intensity in the amplificationpositive tubes was stronger than that in the amplification-negative tubes, although fluorescence background was also present in the negative tubes ( fig. a and b) . the fluorescent read-outs are consistent to the results by electrophoresis gel. however, the fluorescent signal background might cause ambiguous reading and it requires fluorescent sensitive instrument to analyse the result. so we changed it to colorimetric reading later in this study. similarly, o and s primers can also specifically amplify the synthesized dna fragments of orf ab gene and s gene, respectively, but they were unable to amplify human genomic dna ( fig. c and d) . the addition of human genomic dna to the viral dna in the reaction mixture did not interfere with the amplification performance (lane in fig. a and b, lane in fig. c and d), which is essential for using rt-lamp to detect viral rna in a human sample. the lamp reaction time for all experiments was min. positive amplification products were obtained even for copies of the synthetic viral dna fragment template in min when using the s primers (lane in fig. d ), demonstrating that the lamp reaction was rapid and sensitive. rt-lamp and sensitivity to amplify synthesized rna of sars-cov- to assess the potential of rt-lamp in detecting rna virus of sars-cov- , we then tested the performance of these primers with synthesized rna fragments of the n gene, s gene and orf ab gene obtained from in vitro transcription (appendix s ). it shows that reverse transcription and lamp can be integrated into one single rt-lamp reaction to specifically amplify viral rna fragments (fig. ) . we evaluated the sensitivity of rt-lamp by diluting the copy number of rna target from to copies per reaction (in total ll reaction solution) and examined the efficiency by sampling the reaction mixture at , and min after the reaction started. it shows that copies of target rna can be amplified to detectable level using n primers within min, whilst it requires min using n primers ( fig. a-c) . the sensitivity and efficiency of rt-lamp were also tested on s and o primers. as shown in figure , copies of s gene rna can be detected using s primers in min, whilst the detection limit using o primers is copies of orf ab gene rna in min. the results suggest that rt-lamp is sensitive enough to detect viral rna within a -min reaction and the sensitivity can be copies in ll reaction ( copies viral rna per ml) by employing primers n and s . it is also observed that the higher copies of rna, the shorter time is needed to obtain the products (fig. ) . when the copy number of rna is per reaction, the amplification result can be observed as short as min (fig. ) . since result-reporting using fluorescent dye requires a specific instrument (fig. ) , we also tried to read the results using the naked eye. since the nucleic acid amplification releases pyrophosphate and hydrogen ion, which decreases the ph of reaction solution, a sensitive ph indicator can be used to show the positive or negative result of rt-lamp (tanner et al., ; poole et al., ) . hence, rt-lamp result can (fig. ) . we show that the colorimetric rt-lamp assay can reliably detect n genes of sars-cov- viral rna, and the colour change could indicate the number of target sequence semi-quantitatively ( fig. ) . colorimetric reading is dependent on ph change during rt-lamp. however, in clinical application, many factors might interfere ph change. for example, patient sample conditions can be different, introducing uncertain factors to ph change in the rt-lamp reaction. in addition, viral rna of sars-cov- might be eluted by various buffers, which might be compatible to rt-lamp reaction but could significantly affect the colorimetric reading (fig. s ) . to overcome the uncertainty and ensure the reliability of the diagnosis results, we also designed another set of fip primers, which ʹ-end was conjugated with fluorescent fam ( -carboxyfluorescein; table ). figure shows that the application of ʹ-fam-fip primers was able to display both colorimetric and fluorescent readings, and both results were consistent. comparing the results between rt-qpcr and rt-lamp, it shows that the rt-lamp was able to detect viral rna with c t = by rt-qpcr (table ) , suggesting that rt-lamp with visually colour change reading is very sensitive. one-step process of nucleic acid detection using rt-lamp cell samples can be processed in one single step to obtain nucleic acid amplification. heating cells at the same temperature of rt-lamp °c should lyse cells or virus and release rna. in this study, we tested the onestep process using human episomal induced pluripotent stem cells (ipscs), which were suspended in . % saline. figure shows that the nucleic acids of b-actin gene in ipsc cells can be directly amplified using rt-lamp within min. after min, only the purified human rna can be amplified, showing the colour change (fig. a) . however, after min, the tube with human cells changed the colour, whilst the tube without human cells and blank water control remained unchanged pink colour (fig. b) . the one-step process is sensitive enough to detect human cells per -ll reaction. this one-step process took min longer than rt-lamp to rna, because it takes about - min time to lyse cells and release nucleic acids. the result suggests that it should be possible to use one single step to achieve the detection of specific nucleic acids in cells. further test is needed to validate its practical feasibility on clinical swab samples containing sars-cov- . here, we described a fast and simple-to-operate method to diagnose covid- . we applied rt-lamp to achieve a rapid testing of sars-cov- , using different primers (o , s , n and n ) to target orf ab gene, s gene and n gene. we showed that a min reaction could detect down to copies of target rna per ll reaction ( copies per ml; figs and ). however, this extreme sensitivity is also a double-edged sword: carry-over contamination was common in lamp reactions, which usually cause false positive results (hsieh et al., ; ma et al., ) . we also observed carry-over contamination after the rt-lamp reactions were performed several times in the laboratory. we suspect the aerosol formed from the rt-lamp products were able to spread everywhere in the laboratory, contaminating lab-coats, pipettes, reagents, materials and equipment used for the next reaction, thus for clinical use all components required in the rt-lamp reaction need to be prepared in a separate laboratory space with good practice of molecular biology. the colorimetric display of the testing outcome is easy without the need of expensive or complicated instrument. however, the colour change of rt-lamp reaction is based on ph indicator phenol red (tanner et al., ; poole et al., ) . the elution buffers of the various rna extraction kits will significantly affect the result. hence, in all of our experiments including the clinical tests, the rna samples were eluted in dnase/rnasefree water to ensure the rna elution buffer has a minimal impact of ph indication in rt-lamp reactions. the viral rna clinical tests work well to match the results of rt-qpcr. to tube # , all samples were negative, suggesting that rna of human b-actin gene transcript was not amplified in the samples (fig. ) . the four-primer design for human b-actin (poon et al., ) might require a longer reaction time than min in rt-lamp assay. in the future, the better human control should be applied. for example, the primers for human rnase gene can be used as a positive control. the ultimate aim is to develop an enclosed device that integrates rna extraction, purification, reverse transcription (rt) and loop-mediated isothermal amplification (lamp) to detect the sars-cov- virus directly from a throat swab sample. it has been well-known that colony pcr can be done directly using cells (huang et al., ) . heating cells can cause cell lysis and nucleic acid release, which can be used as template for the amplification of nucleic acids. we tested one-step rt-lamp for nucleic acid amplification and it worked well (fig. ) . the advantage is that the same temperature °c for rt-lamp was able to lyse cells and the cell lysis in pure water (or with low salt) did not affect the reaction of rt-lamp. this preliminary work suggests that it is possible to combine rna extraction, rt and lamp in one single step, which will significantly simplify the detection process and shorten the detection time. current criteria of the test in china are copies ml À ; the preliminary results of one-step rt-lamp should meet this requirement. proving the practicability of rt-lamp in diagnosing covid- is the first step towards developing a more accessible diagnostic kit. testing is crucial not just for getting treatment to those in need, but also to curb the spread of disease within a region. as we face a global shortage of testing kits amid this global pandemic, our work will be vital to address this need in clinical practice as well as to extend towards a home-based or personal diagnostic system. with this proof-of-concept work, it is possible to develop a simple, low cost and one-step testing kit which can be used to screen individuals for sars-cov- virus (table ) , and eight positive samples were labelled 'p', and eight negative samples were labelled 'n'. the components of each test kit are listed in experimental procedures. three tubes in each rt-lamp assay are # , # , # in order from left to right. tube # and # separately contain o and n primers targeting the orf ab gene and n gene of sars-cov- . tube # contain human b-actin primers. to tube # and , all positive samples turned yellow, whilst all negative samples remained pink after -minute reaction. the results of rt-lamp method are consistent to the results of conventional rt-pcr (table and fig. s ). at the point-of-care testing (poct). there is a need for an on-site rapid test for the infection which can be operated easily with minimal training and without the risk of getting infected. existing methods to detect sars-cov- are either based on detecting the virus rna itself using reverse transcription real-time polymerase chain reaction (rt-qpcr) technologies, or based on specific igm and igg from patients' blood generated several days after the infection. currently, these tests would require a laboratory with specific machines operated by skilled scientists and technicians that take at least h to perform. the ultimate goal will be development of a rapid ( - min) enclosed test device with one-step operation for the detection of sars-cov- virus. multiple sites on the viral rna are detected to ensure specificity and sensitivity, and an internal control is embedded to prevent false positives and false negatives. this study will pave the way to move forward for rapid and large-scale screening and diagnosis, where patients exhibiting suspected symptoms can all be tested in the early stages. human episomal ipsc line was purchased from thermo fisher (uk) and used as the source of human genomic dna and rna. shenzhen luohu people's hospital is authorized by chinese cdc for the detection of clinical samples for sars-cov- virus. all clinical samples were sampled by clinical throat swab, stored and transported by virus transport media (vtm). virus deactivation steps were done following the standard guidelines for detection of nucleic acid of sars-cov- in clinical samples. the research on rapid diagnostic technique for covid- using clinical samples has been approved by the ethical committee at shenzhen luohu people's hospital. four sets of lamp primers were designed targeting the orf ab, s and n genes of sars-cov- published at ncbi --- . À À ct, cycle threshold; e, e gene for envelop protein; ic, rnase gene as internal control; n, n gene for nucleocapsid protein; orf ab, orf ab gene. (genbank nc_ . ). primers were designed using lamp primer designing software, primerexplorer (http:// primerexplorer.jp/e/; tomita et al., ) . six primers including forward primer f , backward primer b , forward inner primer fip, backward inner primer bip, forward loop primer lf and backward loop primer lb were designed to accelerate the lamp reaction (nagamine et al., ) . table shows all the primer sequences used in this work. in some experiments, ʹ-end of fip primers were labelled by fam. to carry out mock experiment, we also designed four dna fragments, each containing a t promoter for in vitro transcription (appendix s ). all designed primers and dna fragments, as well as a -ncov_n_positive control plasmid, were ordered from integrated dna technologies (idt, uk). the dna fragments of n, s and orf ab target genes were synthesized by idt and reconstituted to ng µl À according to the manufacturer's instruction. the copy number of each target gene was determined from their molecular weight and diluted into , , and copies µl À for subsequent experiments. t _n, t _s, and t _o dna synthesized by idt were reconstituted to ng µl À according to the manufacturer's instruction. these dna sequences were subjected to in vitro transcription using hiscribe tm t high yield rna synthesis kit (new england biolabs, uk). transcription products were purified using rneasy mini kit (qiagen, uk), and the concentration and quality of rna were measured using nanodrop. the copy number of each target rna was determined from their molecular weight and diluted into , , and copies µl À for subsequent experiments. human whole-genome was purified from human ipsc cells using fast dna tm spin kit from mp biomedicals followed by followed by dna cleanup kit (new england biolabs, uk). human whole-rna was purified from human ipscs using rneasy mini kit (qiagen, uk). all equipment, laminar-flow cabinet and working bench were sprayed with rnasezap tm prior to experimental work. filtered pipette tips were used to prevent crosscontamination. all experiments of lamp and rt-lamp were run for at least three replicates. a x primer mix (fip, µm; bip, µm; f , µm; b , µm; lf, µm; lb, µm) was made before the reactions. both lamp and rt-lamp reactions were carried out using warmstart tm a µl reaction mixture ( mastermix, . µl; primer mix, . µl; dna/rna target, µl; dnase & rnase-free molecular grade water, µl) was mixed homogeneously and centrifuged for s. lamp or rt-lamp was performed in a thermocycler at °c for min. colour change can be observed directly by the naked eye, and gel electrophoresis was performed to confirm the result. lamp and rt-lamp for human cells. all equipment, laminar-flow cabinet and working bench were sprayed with rnasezap tm prior to experimental work. filtered pipette tips were used to prevent cross-contamination. human episomal ipscs obtained from thermo fisher scientific (uk) were dissociated with . mm edta (ph . ; thermo fisher scientific) in sterile pbs. the cells were then counted, resuspended and diluted into , , and cells µl À in sterile . % nacl (sigma-aldrich) solution. a b-actin primer mix (fip, µm; bip, µm; f , µm; b , µm) was made before the reactions, and lamp and rt-lamp were carried out using warmstart tm colorimetric lamp master mix (dna & rna) from new england biolabs (neb). for the onestep amplification of nucleic acids, cells in µl . % nacl were first heated at °c for min and a -µl reaction mixture ( mastermix, . µl; primer mix, . µl; the boiled cell sample, µl; dnase & rnase-free molecular grade water, µl) was prepared. on the other hand, for the cell in lamp experiments, cells were directly added into the lamp reaction reagents, where a -µl reaction mixture ( mastermix, . µl; primer mix, . µl; . % nacl solution with or without ipscs , µl; dnase & rnase-free molecular grade water, µl) was prepared. lamp/rt-lamp was performed in a thermocycler at °c for or min. colour change was observed directly by the naked eye. respiratory specimens (throat swabs) collected from patients were immediately placed into sterile tubes containing ml of vtm (health gene technologies, ningbo, china). in total, clinic samples were collected from patients. the swab samples were deactivated by heating at °c for min in a biosafety level (bsl ) medical laboratory of shenzhen luohu people's hospital in china. rna was extracted from swab samples using rna extraction kit (health biomed, china) on a smart labassist- platform (taiwan advanced nanotech inc, taoyuan, china). to avoid the interference of te buffer to rt-lamp reaction, rna was eluted by rnase-free and dnase water here. conventional rt-qpcr for sars-cov- a commercial -ncov rt-pcr kit (shanghai zj bio-tech, china) was used to determine if the samples are positive or negative to sars-cov- virus. in an abi real-time pcr system (thermo fisher scientific inc., waltham, usa) according to the manufacturer's instructions. a ll reaction mixture contained ll of rna as the template, ll of -ncov rt -pcr buffer, ll of rt-qpcr enzyme mix. the thermal cycling condition was min at °c for reverse transcription, min at °c for pcr initial activation and cycles of s at °c and s at °c. rt-lamp assays on clinical samples were performed in pre-mixed test kits. each test kit consists of three tubes # , # and # , which contain o- , n- and human bactin primers respectively (table ). the three tubes were first filled up with ll of rnase-free water (sigma-aldrich), . ll of warmstart colorimetric lamp master mix (new england biolabs, uk), and . ll of x primer mix. the test kits were first prepared in oscar and delivered to shenzhen luohu people's hospital in an ice box. for the detection of sars-cov- virus, ll of extracted rna from each patient sample was added into tube # , # and # respectively. the test kit was incubated at °c for min. sequences and simple visual detection of products. nat protoc : - . walls, a.c., park, y., tortorici, m.a., wall, a., mcguire, a.t., and veesler, d. ( ) structure, function, and antigenicity of the sars-cov- spike glycoprotein. cell : - . wang, c., horby, p.w., hayden, f.g., and gao, g.f. ( ) a novel coronavirus outbreak of global health concern. lancet : - . woo, p.c.y., huang, y., lau, s.k.p., and yuen, k.y. ( ) coronavirus genomics and bioinformatics analysis. viruses-basel : - . additional supporting information may be found online in the supporting information section at the end of the article. appendix s . synthesised dna with t promoter. fig. s . the rna map of sars-cov- and the locations of four sets of primers: o- , s- , n and n- , which target the regions encoding orf ab, spike protein, and n protein. fig. s . the impact of virus transport media to the reading of rt-lamp results. all tubes contain n primers and warmstart colorimetric lamp master mix. the tubes were incubated at °c for min. operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries origin and evolution of pathogenic coronaviruses rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr real-time pcr in clinical microbiology: applications for a routine laboratory testing simultaneous elimination of carryover contamination and detection of dna with uracil-dna-glycosylase-supplemented loop-mediated isothermal amplification (udg-lamp) chromosomally located gene fusions constructed in acinetobacter sp. adp for the detection of salicylate rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations a novel method to control carryover contamination in isothermal nucleic acid amplification real-time pcr in virology a sars-like cluster of circulating bat coronaviruses shows potential for human emergence loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (nina-lamp) detection of human influenza a viruses by loop-mediated isothermal amplification paper-based microfluidics for dna diagnostics of malaria in low resource underserved rural communities visual detection of isothermal nucleic acid amplification using ph-sensitive dyes development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus loop-mediated isothermal amplification (lamp) of gene none declared. key: cord- -s t qxj authors: soliman, h.; el-matbouli, m. title: reverse transcription loop-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: s t qxj a one step reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for detection of viral hemorrhagic septicaemia virus (vhs). a set of six primers were designed, based on the g-protein sequence of the vhs virus serotypes (he, f , . , klapmolle and rindsholm). the assay was optimised to amplify vhs rna by incubation at °c for only h, and required only a simple water bath or heating block to provide a constant temperature of °c. rt-lamp amplification products were detected by visual inspection using sybr green i stain and had a ladder-like appearance when electrophoresed on an agarose gel. the detection limit of the rt-lamp assay was found to be similar to the commonly used rt-pcr method: both methods detected vhs rna at a dilution of ( ). the assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for vhs virus. reverse transcription loop-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) animal rhabdoviruses, belonging to the family rhabdoviridae, infect a broad range of hosts throughout the animal kingdom, including insects, mammals and fish. aquatic rhabdoviruses cause significant diseases in fish reared as part of the worldwide salmonid farming industry (rocha et al., ) . among fish rhabdoviruses, viral hemorrhagic septicaemia virus (vhs), of the genus novirhabdovirus, often causes pronounced cumulative mortality of salmonids, especially rainbow trout oncorhynchus mykiss (miller et al., ) . the virus is enzootic in much of continental europe (wolf, ) and has been found in north america (king et al., ; snow and smail, ) . vhs is a notifiable disease, included in list ii of the european union directive / ( ), and is known by several names including egtved disease and infectious kidney-swelling and liver degeneration. www.elsevier.com/locate/vetmic veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the virus' aetiology was established by jensen ( ) , and it is known to infect primarily rainbow trout, oncorhynchus mykiss, brown trout, salmo trutta, and to a lesser extent northern pike, esox lucius (jorgensen, ; meier and jorgensen, ) , grayling, thymallus thymallus, and whitefish, coregonus sp. (wizigmann et al., ; ahne and thomsen, ; meier et al., ) . the virus has also been isolated from free-living marine fish in the northern pacific and atlantic oceans, the north sea and the baltic sea (meyers and winton, ; mortensen et al., ) . virus isolates from wild marine fish are serologically indistinguishable from normal fresh water isolates (jorgensen and olesen, ) . vhs virus is transmitted by direct contact with either infected fish or contaminated water (vestergard jorgensen, ) . survivors from infection become carriers and shed the virus with urine and sexual fluids (neukirch, ) . although the virus can be present on the surface of eggs, it is readily dissipated and there is no true vertical transmission (vestergard jorgensen, ) . vhs infection in susceptible fish species is often lethal, due to impairment of osmotic balance; clinical signs include oedema and red haemorrhagic spots around the eyes and on the gills, and blood clots in body fat, viscera and muscle (wolf, ) . rapid diagnosis of vhs is necessary to prevent further spread of the disease (miller et al., ) . serological tests have been used to diagnose vhs virus, and include the virus neutralisation test (jorgensen, (jorgensen, , , a fluorescent antibody technique (jorgensen, ; enzmann, ) and enzyme linked immunosorbent assay (elisa) (olesen and jorgensen, ). yet serological tests along with other traditional means of diagnosing viral infection, including epizootiological data, direct light or electron microscopic observation and isolation of the virus in cell culture, are often time-consuming and expensive (amos, ; sanz and coll, ) . recently, molecular biological techniques have been developed to improve diagnosis of fish pathogens by rapid and sensitive detection of pathogen rna and dna. a reverse transcriptase-polymerase chain reaction (rt-pcr) has been developed to detect vhs (bruchhof et al., ; einer-jensen et al., ; miller et al., ; strommen and stone, ; guillou et al., ; williams et al., ) . however, diagnosis of vhs using this pcr test requires expensive equipment and reagents, rendering it unfavourable for use on a large-scale basis, or onsite. a novel, rapid and sensitive technique called loop-mediated isothermal amplification (lamp) has been developed by notomi et al. ( ) and is capable of amplifying dna or rna under isothermal conditions with high specificity and efficiency. lamp is based on the principle of autocycling strand displacement dna synthesis. the reaction is performed by a dna polymerase with high strand displacement activity and a set of two specific inner primers and two outer primers (notomi et al., ) . a reverse transcriptase coupled lamp assay has been used to detect west nile virus and for rapid detection of sars (severe acute respiratory syndrome) coronavirus (thai et al., ) . the aim of the present study was to develop a onestep, single-tube, accelerated rt-lamp reaction for rapid detection of different serotypes of vhs virus. the specificity and sensitivity of the method were assessed, and its applicability as a diagnostic test was evaluated. vhs virus serotypes (he, klapmolle, rindsholm, and . ) were kindly provided by dr. fichtner, federal research institute for animal health, insel riems, germany. the vhs virus serotype (f ) and infectious hematopoietic necrosis virus (ihnv) were provided by dr. widemann, department of virology, central institute of animal health, bavaria, germany. fish tissues suspected of being infected with vhs (spleen, kidney and brain) were preserved in rna later. these were then processed by grinding thoroughly in liquid nitrogen with a mortar and pestle. twenty milligram of tissue powder was then placed into a rnase-free, liquid nitrogen-cooled ml microcentrifuge tube with ml lysis buffer. the lysate was transferred directly into a qiashredder spin column (qiagen gmbh, hilden, germany) and centrifuged at maximum speed ( ,  g) for min. one volume ( ml) of % ethanol was added to the supernatant and extraction completed as per the manufacture's instructions. rna was eluted in rnase-free water ( c) and stored at À c. genomic viral rna was extracted from ml of infected culture supernatant using the qiaamp viral rna kit (qiagen gmbh, hilden, germany) according to the manufacture's instructions. after elution of the rna in elution buffer, it was stored at À c until required. six lamp primers were designed, based on the gprotein sequence of the vhs virus serotypes: f , klapmolle, rindsholm, he and . (genbank accession number af , af , af , u and u , respectively). the forward inner primer, fip, consisted of complementary sequence of f ( nt), a tttt linker and the sense sequence of the f ( nt): -gatccaccga-tactgtttttggggttttcccgttcttc cctg aaccc- . the backward inner primer, bip, contained a sense sequence of b ( nt), a tttt linker and the complementary sequence of the b ( nt): -argggg tytgcacarcctcgc tttt cgack-ygggrcaakgggc- . the outer primers consisted of the sense sequence of f ( ) -ggsaagcaaggaycacgag- ; and complementary sequence of b ( nt) -caggtgtc-cytctagtgtttc- . to accelerate amplification, two loop primers were used: a loop forward primer (loop-f, nt): -gttatgtccttatggacattg- ; and a loop back-ward primer (loop-b; nt): -gtcaaact-cattggcaggg- . the location of the primers within the gene fragment is shown in (fig. ) . the reverse transcriptase-polymerase chain reaction used specific primers vg and vgr, which amplified a bp segment, and seminested primers vd and vd , which amplified a bp segment of vhs cdna (bruchhof et al., ) : vg : -atggaatggaacacttttttc- , vgr: -tcagaccgtctgacttctgga- ; vd : -tcccgctatcagtcaccag- ; vd : -tgtgatcatgggtcctggtg- . rt-pcr was performed using a titanium tm onestep rt-pcr kit (bd clontech, heidelberg, germany). for a ml reaction volume, mg of rna was mixed with  one-step buffer, dntps mix, recombinant rnase inhibitor, thermostabilising reagent, gc-melt, oligo (dt) primer, mm of each vg and vgr vhs specific primers, and rt-titanium tm taq enzyme mix. the reaction conditions comprised: incubation at c for h, then denaturation at c for min; followed by cycles of c for s; c for s and c for s. there was a terminal extension step of c for min. following rt-pcr, ml of the product was added to ml of semi-nested pcr reaction mixture: comprising .  reddymix pcr master mix (abgene, hamburg, germany: mm tris-hcl (ph . ), mm (nh ) so , . mm mgcl , . % tween , . mm each of datp, dctp, dgtp, dttp; . u taq dna polymerase and red dye for electrophoresis), and pmol of each vhs specific primers vd and vd . the reaction entailed initial denaturation at c for min, followed by cycles of c for s, c for s and c s. there was a terminal extension step of c for min. the lamp reaction was performed in a ml reaction volume containing: mm tris-hcl (ph . ), mm kcl, . mm mgso , mm (nh ) À so , . % triton x- , . m betaine, deoxynucleotide triphosphates . mm each, . mm each fip and bip, . mm each loop-f and loop-b, . mm each f and b primers, u bst dna polymerase (new england biolabs, gmbh, frankfurt, germany), . u avian myeloblastosis virus (amv) reverse transcriptase (invitrogen, groningen, the netherlands) and ml rna template. rna template was omitted from one reaction as a negative control. the mixture was incubated at c for min and then heated at c for min to terminate the reaction. rt-pcr and rt-lamp reaction products were analysed by gel electrophoresis with . % agarose in tris acetate-edta buffer, tae ( . m tris acetate, mm edta), stained with ethidium bromide and visualised on a uv transilluminator. a bp dna molecular weight standard ladder (cambrex bio science, inc., rockland, me, usa) was used. success of the rt-lamp reaction was also gauged by visual inspection: ml of : diluted sybr green i nucleic acid gel stain, ,  concentration in demso (cambrex bio science, rockland, inc., me, usa) was added to the reaction tube and any colour change noted. the ability of the assay to amplify only vhs rna was appraised by testing it with rna from infectious hematopoietic necrosis virus a related rhabdoviruses. rna from non-infected fish was used as a negative control to determine any non-specific amplification. ten-fold serial dilutions of the used vhs serotypes rna were tested by both rt-lamp and rt-pcr to determine the detection limits of each assay. the suitability of the assay to diagnose vhs was evaluated by comparing detection results of rt-lamp assay on experimentally infected rainbow trout with vhs serotype f (mattes, ) against rt-pcr results on the same samples. different primer concentrations, primer combinations, amplification temperatures, and amounts of reverse transcriptase were tested to determine the optimal rt-lamp conditions. optimal amplification of vhs rna was achieved by incubation of fip, bip, f , b primers, u of bst dna polymerase and u of amv reverse transcriptase enzyme with the target rna at c for h. the amplification product appeared as a ladder-like pattern (many bands of different molecular weights) on the gel (fig. ) . there was no amplification of the negative control. the assay was able to amplify all five serotypes of vhs rna (fig. ) . two additional primers, loop-f and loop-b, were subsequently added to enhance and accelerate the reaction down to min (fig. ) . there was no difference in amplification pattern using the additional primers. colour changes were noted on visual inspection of rt-lamp reaction tubes after addition of diluted sybr green i: positive samples turned green, while negative samples and no template control reactions remained orange (fig. ) . these observations agreed with gel electrophoresis results. the specificity of the vhs-lamp primers and conditions to vhs virus was confirmed by its aptitude to amplify only rna from vhs virus serotypes, with no amplification of ihnvor non-infected fish (fig. ) . amplification of -fold serial dilutions of vhs rna by rt-pcr and rt-lamp revealed that both tests could detect viral rna to a dilution of in ( figs. and ) . the same result was obtained with the all tested vhs serotypes. rt-lamp detected vhs rna from infected samples, which also tested positive by the rt-pcr test. this result indicates that the developed rt-lamp assay can feasibly be used as a diagnostic test of vhs infection. viral infections pose a serious threat to the aquaculture industry and are responsible for significant financial losses (caipang et al., ) . it is clearly important to rapidly identify and differentiate the causative agents of fish diseases to prevent further disease transmission or outbreaks (bruchhof et al., ) . vhs is one of several rhabdoviruses that fig. . agarose gel highlighting the specificity of the rt-lamp primers to vhs virus rna. the reaction was carried out at c for h using the primer set. lanes: f , he, . , klapm; and rinds, amplification products of vhs virus rna serotypes (f , he, . , klapmolle and rindsholm); ihnv, no amplification product was detected for ihn virus rna; nf, no amplification product was detected using rna from noninfected fish; mar = bp molecular weight marker. fig. . agarose gel showing rt-lamp reactions using -fold serial dilutions of template rna. the assay detected vhs rna at a dilution one in . lanes: À = template concentration À ; À = À ; À = À ; À = À ; À = À ; À = À ; À = À ; mar = bp dna molecular weight standard. affects the aquaculture industry, and causes disease with high mortality among salmonids (miller et al., ) . difficulties associated with isolation of viruses from clinical specimens has lead to development of rapid and reliable virus detection assays including many rt-pcr assays developed for diagnosis of vhs (bruchhof et al., ; einer-jensen et al., ; miller et al., ; strommen and stone, ; guillou et al., ; williams et al., ) . despite the simplicity of the rt-pcr reaction, its requirements for a high precision thermacycler and elaborate methods for detecting the amplified products have restricted its wide use as a routine diagnostic tool (thai et al., ) . to overcome these issues, notomi et al. ( ) developed a novel nucleic acid amplification assay: loop-mediated isothermal amplification. the lamp reaction has been used successfully to detect fish viral infections (caipang et al., ; gunimaladevi et al., gunimaladevi et al., , , and has been applied to the detection of vhs in the present study. the rt-lamp technique is rapid, straightforward and includes a simple visual method for detecting positive results. the reaction is executed in a single tube and only requires a simple water bath or heating block to provide a constant temperature of c for h. the lamp reaction relies on autocycling strand displacement dna synthesis and utilises a set of four specially designated inner and outer primers (notomi et al., ) which are used during the initial steps of the reaction. during the subsequent cycling reactions, only the inner primers are used for strand displacement dna synthesis . each inner primer contains two distinct sequences corresponding to the sense and antisense sequences of the target dna. the primers form stem-loop structures which initiate self-priming dna synthesis, and serve as the starting material for the subsequent lamp cycling reaction (kuboki et al., ) . to further reduce the reaction time, two additional loop primers were used to hybridise to the stem-loops formed by the outer primers. the loops hybridised by the inner primers subsequently prime strand displacement dna synthesis (nagamine et al., ) . due to the variation in the glycoprotein gene sequence of the vhs serotypes, degenerative primers were designed from an alignment of the g-protein sequences of vhs serotypes he, f , . , klapmolle and rindsholm. these primers were then aligned against g-protein sequences of ihnv serotypes, wrac, srcv and rb, to check that they would not bind; which was confirmed by testing in a reaction with ihnv rna (fig. ) . the lamp primers are suitable for amplification of at least five vhs serotypes (fig. ) . the specificity of the reaction was extremely high because it uses six primers that recognise eight distinct regions on the target dna (notomi et al., ; nagamine et al., ) . the amplified products appeared as a ladder-like pattern on the gel due to the formation of a mixture of stem-loop dna products with varying stem lengths, and cauliflower-like structures of multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand (thai et al., ) . the assay used sybr green i dna staining as a rapid, fig. . agarose gel showing vhs rt-pcr using -fold serial dilutions of template rna. the rt-pcr produced a bp amplification product and detected vhs rna at a dilution of in . lanes: À = template concentration À ; À = À ; À = À ; À = À ; À = À ; À = À ; À = À ; mar = bp dna molecular weight standard. specific method for detection of amplification products. visual detection is possible due to the high specificity and amplification efficiency of lamp (iwamoto et al., ) , and to the high binding affinity of sybr green i to dna (karlsen et al., ) . visual inspection is a superior detection method as there is no need for gel electrophoresis and staining with ethidium bromide; only ml of diluted sybr green i was required to visualise the reaction products: a distinct green colour indicated a positive result while orange indicated negative. all samples which tested positive by visual inspection were positive also when analysed by electrophoresis; there were no samples which were negative by visual examination but which tested positive by electrophoresis. the rt-lamp assay detected vhs rna at a dilution of in , which was equivalent to the limit of rt-pcr. the amplification efficiency of the lamp method was extremely high under isothermal conditions at the optimal temperature for the polymerase; any inhibition reactions at the latter stages of amplification (kalinina et al., ) were less likely to occur compared with pcr (mori et al., ) . the suitability of the rt-lamp assay for detection of vhs rna in infected samples was successfully validated. in conclusion, the developed rt-lamp assay is extremely rapid, cost effective, sensitive and specific for detection of vhs rna. the test requires only a water bath and is completed in h compared with - h for rt-pcr. considerably less time is required to obtain a result using sybr green i stain, compared with traditional gel electrophoresis. the assay is suitable to be used under field conditions for the rapid diagnosis of vhs, which would allow expedited control and hygiene measures to be implemented to prevent spread of infection. occurrence of viral hemorrhagic septicaemia virus in wild whitefish coregonus sp procedures for the detection and identification of certain fish pathogens differential diagnosis of fish pathogenic rhabdoviruses by reverse transcriptase-dependent polymerase chain reaction rapid detection of a fish iridovirus using loop-mediated isothermal amplification (lamp) use of polymerase chain reaction (pcr) to differentiate serologically similar vhs virus isolates from europe and america rapid identification of vhvs from trout by immunofluorescence detection of viral hemorrhagic septicaemia virus (vhs) in rainbow trout (oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. diagnostic validation detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification a loop mediated isothermal amplification (lamp) method for detection of infectious hematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples research on the virus of egtved disease serological identification of egtved virus egtved virus: antigenic variation in virus isolates examined in neutralization tests and by means of the fluorescent antibody technique egtved virus: the susceptibility of brown trout and rainbow trout to eight virus isolates and the significance of the findings for the vhs control cod ulcus syndrome rabdovirus is indistinguishable from the egtved (vhs) virus nanoliter scale pcr with taqman detection sybr green i dann staining increases the detection sensitivity of viruses by polymerase chain reaction distribution of viral hemorrhagic septicaemia virus in wild fish species of the north sea, north east atlantic ocean and irish sea loop-mediated isothermal amplification for detection of african trypanosomes untersuchungen zur empfänglichkeit zweier regenbogenforellen-stämme gegenüber tetracapsuloides bryosalmonae, yersinia ruckeri und dem viralen hämorrhagischen septikämie-virus isolation of vhs virus from pike fry (esox lucius) with hemorrhagic symptoms fish viruses: viral hemorrhagic septicaemia in whitefish (coregonus sp vhs-virus in north america rapid and sensitive reverse transcriptase polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation isolation of vhsv from wild marine fish species in the baltic sea, kattegat, shagerrak and the north sea accelerated reaction by loop-mediated isothermal amplification using loop primers uptake, multiplication and excretion of vhsv in rainbow trout loop-mediated isothermal amplification of dna rapid detection of viral hemorrhagic septicaemia virus in fish by elisa real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus conformation-and fusion-defective mutations in the hypothetical phospholipidsbinding and fusion peptides of viral hemorrhagic septicaemia salmonid rhabdovirus protein g techniques for diagnosing viral diseases of salmonid fish experimental susceptibility of turbot scophthalmus maximus to viral hemorrhagic septicaemia virus isolated from cultivated turbot detection of vhs virus in fish tissues by semi-nested polymerase chain reaction (pcr) development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of sever acute respiratory syndrome coronavirus the survival of vhs virus associated with trout eggs artificial transmission of vhs of rainbow trout multiplex reverse transcriptase pcr assay for simultaneous detection of three fish viruses isolation of viral hemorrhagic septicemia virus from fry of rainbow trout, pike, and grayling fish viruses and fish viral diseases we would like to express our grateful thanks to dr. d. fichtner and dr. bergmann, federal research institute for animal health, insel riems, germany and dr. widemann, department of virology, central institute of animal health, bavaria, germany for providing us with the vhs and ihn viruses. we also thank dr. marianne mattes for providing samples of the experimentally infected rainbow trout. key: cord- -kftffes authors: mohon, abu naser; oberding, lisa; hundt, jana; van marle, guido; pabbaraju, kanti; berenger, byron; lisboa, luiz; griener, thomas; czub, markus; doolan, cody; servelitta, venice; chiu, charles; greninger, alexander; jerome, keith; pillai, dylan r. title: optimization and clinical validation of dual-target rt-lamp for sars-cov- date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: kftffes a novel reverse-transcriptase loop mediated amplification (rt-lamp) method targeting genes encoding the spike (s) protein and rna-dependent rna polymerase (rdrp) of sars-cov- has been developed. the lamp assay achieves a comparable limit of detection ( copies per reaction) as commonly used rt-pcr protocols using clinical samples quantified by digital droplet pcr. precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. clinical validation of dual-target rt-lamp (s and rdrp gene) achieved a ppa of . % ( % ci . % to . %) and npa . % ( % ci . % to . %) based on the e gene and n gene reference rt-pcr methods. the method has implications for development of point of care technology using isothermal amplification. over the last several decades, we have witnessed the rise of both known and novel viruses, areas. in december and early january , a cluster of pneumonia cases from a novel coronavirus, sars-cov- , was reported in wuhan, china ( ) ( ) ( ) . sars-cov- has now resulted in a global pandemic with the epicentre at the time of writing in europe and north america ( ) . a common theme in the public health response to covid and similar threats is the lack of rapidly deployable testing in the field to screen large numbers of individuals in exposed areas, international ports of entry, and testing in quarantine locations such as the home residences, as well as low-resourced areas ( ) . this hampers case finding and increases the number of individuals at risk of exposure and infection. with the ease of travel across continents, delayed testing and lack of screening programs in the field, global human-tohuman transmission will continue at high rates. these factors make a pandemic very difficult to contain. early identification of the virus and rapid deployment of a targeted point of care test (poct) can stem the spread through immediate quarantine of infected persons ( ) . we used existing viral genome sequences to develop a sars-cov- loop mediated amplification (lamp) assay for clinical use ( , ) . lamp relies on an alternate set of reagent chemistry that does not depend on or hinder critical elements of the rt-pcr supply chain which is now under duress ( ) . our group has previously demonstrated the utility of lamp for other infectious agents like malaria and dengue ( ) ( ) ( ) . clinical samples used in this study were standard archived nasopharyngeal (np) in viral transport medium (vtm) stored at - o c at alberta precision laboratories (calgary, canada), j o u r n a l p r e -p r o o f university of washington, and university of california, san francisco. ethical approval for use of the archived samples was obtained from the conjoint health research ethics board (chreb) of the university of calgary (reb - ). this study was approved by the institutional review board (irb) at university of california, san francisco (ucsf irb # - ) as a no subject contact study with waiver of consent. remnant np swab after clinical testing and pcr cycle threshold results were collected for analysis. the use of de-identified specimens were deemed non-human subject work by the university of washington institutional review board (irb). genomic sequences (cdna) of the sars-cov- were retrieved from the genbank database (https://www.ncbi.nlm.nih.gov/genbank/sars-cov- -seqs/) and multiple sequence alignment analysis (https://www.ebi.ac.uk/tools/msa/clustalo/) was conducted with other related viruses. from the multiple sequence alignment, several regions unique to the sars cov- were identified. the primers were designed using the primer explorer v software (http://primerexplorer.jp/lampv e/) by uploading the sequences of the s and rdrp gene. initially, primers were designed with the default settings of the software and then screened based on the optimal self-dimer, ' end, and 'end g values. additionally, the intervening sequence between f /f and b /b primer binding sites was manually set at a minimum of nucleotides. other lamp primer design software (eg. http://www.optigene.co.uk/lampdesigner/) are available commercially but were not used in this study. initial screening experiments were performed on several potential primer sets and two chosen based on amplification efficiency (data not shown). lamp primer sets were designed targeting unique regions of the spike (s) protein gene and rna-dependent rna polymerase gene (rdrp) were ultimately used (table ) . for the external lamp amplification control, primers were used against bacteriophage ms as previously described ( ). in silico analysis of primer combinations to determine cross-reactivity and inclusivity j o u r n a l p r e -p r o o f a blast search alignment (https://blast.ncbi.nlm.nih.gov/blast.cgi) for primers in set (spike gene) and set (rdrp gene) were performed against a critical list of infectious agents that cause upper respiratory tract infections. a nucleotide local alignment using blastn with the default parameters was performed against the national center of biotechnology information (ncbi) nucleotide database (see supplementary data). the twelve rt-lamp primers were aligned against , sars-cov- (taxid: ) viral sequences in the ncbi nucleotide database available on august th, . the output of the "discontiguous megablast" algorithm showed no divergence available between the primers and the sars-cov- database and all of the query sequences showed a % identity against the expected sequences from the sars-cov- virus ( genomes). four fragments of specific sars-co-v regions (orf ab (nsp , - ), rdrp (nsp ), and spike (s)) were synthesized by sgi-dna inc. (san diego, ca). fragments were ligated to make one large concatenated dna template using the bioxp (sgi-dna, san diego, ca) automated gibson assembly system. the final template was base pairs long containing concatenated single artificial construct together with flanking plasmid sequence in that order ( figure ). to create a recombinant virus expressing the relevant rna, the template containing the targeted sequences of interest was cloned into sindbis virus (sv) viral vector system (sinrep ) containing green fluorescent protein (egfp) and then transfected into bhk cell lines ( , ) . the number of rna genome copies was based on the number of fluorescent focus forming units generated by the recombinant sv vector. the dual-target lamp reaction was conducted using a combination of warmstart rtx reverse limit of detection of the lamp assay was evaluated by using a nasopharyngeal (np) swab sample infected with sars-cov- for which the viral load was quantified using digital droplet pcr (see supplementary methods). the np sample was serially diluted to achieve the described copies of virus per lamp reaction. calculation of viral copy number using digital droplet pcr is j o u r n a l p r e -p r o o f the workflow used to conduct rt-lamp is depicted in figure . the primer sequences used to target the spike gene (set s ) and rdrp gene (set ) are listed in table . the limit of detection was evaluated using a patient sample (np swab in vtm viral load confirmed by digital droplet pcr). the quantified sample was serially diluted to achieve a range from to . copies per reaction. the limit of detection was confirmed at copies per reaction when using mm guanidine hydrochloride (ph . ) in the reaction mix ( table ). twenty four replicates from a serial dilution containing - copies of sars-cov- which equates to x lod (patient sample np swab in vtm viral load confirmed by digital droplet pcr) per reaction were tested using dual-target rt-lamp (table ) . twenty-three of samples ( . %) were positive. in silico analysis confirmed that no significant cross-reactivity that affects lamp reactions that rely on six primers per reaction were present (supplementary table ) . finally, further studies were performed for precision on a daily basis ( replicates for days twice day and replicates on instruments daily for days) that demonstrated % concordance (supplementary table and ). a clinical validation sample set of nasopharyngeal swabs were used in the analysis. given no gold standard exists, percent positive agreement (ppa) and negative percent agreement (npa) were calculated. reference methods included rt-pcr (e gene and n gene) methods employed by reference laboratories. dual-target rt-lamp dual-target rt-lamp (s and rdrp gene) achieved a ppa of . % ( % ci . % to . %) and npa . % ( % ci . % to . %) based on the e gene and n gene reference rt-pcr methods (table ) . one false negative sample by rt-lamp was positive by both e gene and n gene. one sample out of j o u r n a l p r e -p r o o f was strongly positive by the n gene and negative by e gene and considered a true positive. no cross-reactivity was observed with known circulating respiratory viruses, namely (hcov) oc , e, nl , and hku or influenza virus a (h n ) pdm (data not shown). the global pandemic with sars-cov- has resulted in the need for diagnostic test development at a scale never seen before. rapid deployment of validated laboratory-developed diagnostic tests or commercial tests is essential to the containment of the virus as it allows for selfquarantine measures to be imposed in a strategic fashion before widespread community transmission occurs ( , ) . diagnostic tests have to be analytically sensitive in order to not to miss any cases in the acute phase of viremia ( ) . as such, nucleic acid amplification tests serve this purpose. in particular, rt-pcr has been employed as the primary diagnostic countermeasure ( ) . however, reagent supply chains for key items are under immense pressure. local solutions to reagent sources have become paramount because barriers to trade of these selected items have been a concern. the dual-target rt-lamp test for sars-cov- developed in this study has comparable analytical sensitivity and specificity, limit of detection, precision, and achieved excellent agreement compared to the reference rt-pcr methods used internationally. the addition of guanidine hydrochloride (ph . ) at mm in the lamp reaction improves the limit of detection ( copies per reaction) to a level comparable to rt-pcr methods. lamp does not rely on the same reagents as rt-pcr and thus alleviates pressure on key supply chain items. the lamp method is amenable to high throughput testing in either -well or -well. other groups have j o u r n a l p r e -p r o o f presented rt-lamp solutions in the literature ( ) ( ) ( ) ( ) ( ) ( ) . the lamp solutions differ in several ways: first, the target genes of choice vary between studies as do the specific primer sequences chosen; second, the limits of detection reported vary in terms of sars-cov- copies detected per reaction; third, the detection systems vary from thermocycler based detection for laboratory developed test (ldt) solutions to near-patient solutions based on visual detection of dyes or fluorophores; fourth, the extent which data reflect requirements for clinical validation. the rt-lamp assay described is unique in that it offers the most thorough clinical validation to date meeting regulatory standards which include precision studies on several instruments, reproducibility studies over days, a robust clinical validation sample set, and a limit of detection equal or superior to other lamp studies ( copies per reaction). these data should enable a clinical laboratory to perform this assay as a ldt. additionally, the lamp assay chemistry presented in this work is able to detect sars-cov- in vtm without the need for a kit-based rna extraction method using lyophilized reagents and visual detection (manuscript in preparation). this format may be of particular interest to resource-limited settings. limitations of the study include not testing other sample types such as alternate swabs, nasal washes, saliva, sputum, or stool. this work is ongoing with a special emphasis on swab-free testing and direct visualization. lamp presents a much needed alternative approach to sars-cov- diagnostic testing that is available for deployment immediately in a laboratory-developed test format as it relies on other key reagents that do not cannibalize rt-pcr reagents. in the future, the lamp chemistry has potential to be adapted to a microfluidic device poct to be deployed in the community, either at ports of entry, homes, pharmacies, or workplaces. cd is an employee of illucidx inc. (a start-up company of the university of calgary) which retains patents related to lamp technology. drp is a scientific advisor to illucidx inc. nasopharyngeal and oropharyngeal swabs at hampshire hospitals nhs foundation trust. preprint, infectious diseases (except hiv/aids). figure : map of the gene fragments from sars-co-v (genbank id mt . ) that were used for synthesizing the genetic construct template. four fragments of specific sars-co-v regions (orf ab (nsp , - ), rdrp (nsp ), and spike (s)) were concatenated into a single artificial construct base pairs long. j o u r n a l p r e -p r o o f figure : workflow used to analyze samples in this study. images were obtained from the centers for disease control (www.cdc.gov) and bio-rad laboratories (www.bio-rad.com). abu naser mohon: conceptualization; data curation; formal analysis; methodology data curation; resources; writing -review & editing data curation; resources; writing -review & editing writing -review & editing funding acquisition; investigation; supervision; writing -review & editing funding acquisition; investigation; supervision; writing -review & editing funding acquisition; investigation; supervision; writing -review & editing pillai: conceptualization; data curation; formal analysis; funding acquisition; investigation roles/writing -original draft avian flu, sars, mers, ebola, zika… what next? feng z. . early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a new coronavirus associated with human respiratory disease in china severe outcomes among patients with coronavirus disease (covid- ) -united states diagnostic testing for severe acute respiratory syndrome-related coronavirus- : a narrative review novel coronavirus disease (covid- ): paving the road for rapid detection and point-of-care diagnostics phylogenetic network analysis of sars-cov- genomes genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding predicting the impacts of epidemic outbreaks on global supply chains: a simulation-based analysis on the coronavirus outbreak (covid- /sars-cov- ) case risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the colombo dengue study ultrasensitive loop mediated isothermal amplification (us-lamp) to detect malaria for elimination prevalence and epidemiological characteristics of asymptomatic malaria based on ultrasensitive diagnostics: a cross-sectional study molecular diagnostic field test for point-of-care detection of ebola virus directly from blood detection of novel coronavirus ( -ncov) by realtime rt-pcr comparative performance of sars-cov- detection assays using seven different primer/probe sets and one assay kit rt-lamp for rapid diagnosis of coronavirus sars-cov- a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- development of reverse transcription loop-mediated isothermal amplification (rt-lamp) assays targeting sars-cov- rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification a reverse-transcription loop-mediated isothermal amplification dr. ranmalee amarasekara, dr. tara winstone, and barbara chow for expert technical assistance, daniel castaneda mogollon for performing bioinformatics analysis, and omar abdullah and noah toppings for research analytical support. key: cord- - qs ov authors: hagiwara, masanori; sasaki, hajime; matsuo, koma; honda, mariko; kawase, masaaki; nakagawa, hidemi title: loop‐mediated isothermal amplification method for detection of human papillomavirus type , , , and date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: qs ov a new method was developed for detection of human papillomavirus (hpv) by loop‐mediated isothermal amplification (lamp), which was compared with the polymerase chain reaction (pcr), and real‐time pcr for specificity and sensitivity. all initial validation studies with the control dna proved to be type‐specific. in order to evaluate the reliability of hpv type‐specific lamp detecting hpv dna from clinical samples, tissue specimens were obtained from patients with external genital polypoid lesions. the histologic diagnoses included condyloma acuminatum (n = ), bowenoid papulosis (n = ), seborrheic keratosis (n = ), epidermolytic acanthoma (n = ), and hairy nymphae (n = ). hpv‐ dna and hpv‐ dna were detected in and of condylomata acuminata, respectively, and there was no simultaneous infection. hpv‐ dna was detected in one of two bowenoid papuloses. hpv dna was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. these results correlated perfectly with those from real‐time pcr analysis. most positive samples contained high copy numbers of hpv dna. hpv‐ dna was detected in one case that could not be detected by pcr. the average reaction time was about min. there was a linear correlation between the genome quantity and reaction time to reach the threshold. the lamp method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of hpv dna. j. med. virol. : – , . © wiley‐liss, inc. human papillomavirus (hpv) is a small dna virus which belongs to the family papovaviridae, and to date, more than types of hpv have been identified [syrjanen, ] . condyloma acuminatum is a benign tumor caused by low risk/mucous membranes type of hpv such as hpv- or infecting the cutaneomucous regions of the genital and anal areas [wang, ] . a diagnosis is usually made clinically, but there are many tumors that can occur in the skin and mucous membranes. therefore, clinical diagnosis may not be sufficient, and histopathological diagnosis and virological testing are necessary. moreover, since high-risk types of hpv associated with cervical cancer and bowenoid papulosis have been detected, identification of genotypes is preferable [zur hausen, ] . virological testing includes in situ hybridization (ish), southern blot hybridization method, dot blot hybridization, polymerase chain reaction (pcr), and real-time pcr [lindh et al., ; brown et al., ; oliveira et al., ; qu et al., ; tucker et al., ] . however, detectability using ish is low. in addition, pcr and real-time pcr need specific expensive equipment such as a thermal cycler, and these methods have not yet become common procedures in hospital laboratories. the loop-mediated isothermal amplification method (lamp) is a cheap, rapid, and simple gene amplification method that was developed originally by notomi et al. [ ] as an amplification method instead of pcr, attaining amplification efficiency and sensitivity similar to or higher than pcr. the reaction proceed between and c without thermocycling, and all procedures are completed in one step in about an hour when detecting the amplification by the appearance of turbidity in the reaction process. recently published reports have suggested that lamp could be an effective method for the rapid diagnosis of infectious diseases [iwamoto et al., ; kuboki et al., ; maruyama et al., ; parida et al., ] . in this study, a lamp-based hpv typespecific dna amplification method was developed and were compared its specificity and sensitivity with pcr and real-time pcr. biopsy specimens measuring mm in diameter were taken from two lesions. one was processed for routine histopathological diagnosis. the other was stored À c for dna extraction. dna was extracted from the specimen using qiaamp dna kit (qiagen, chatsworth, ca). after extraction, dna was eluted in ml distilled water and was stored at À c. j. med. virol. doi . /jmv fig. . the locations and names of the target sequences used as primers for hpv type-specific lamp within the e region of hpv- , the e region of hpv- , the e region of hpv- , and the e region of hpv- (a). names and sequences of each primer for hpv type-specific lamp are shown (b). b c, sequence complementary to b ; f c, sequence complementary to f . to determine the specificity of type-specific lamp method, types of cloned hpv dnas, were integrated in pbr and were cloned by colon bacillus hb . the plasmids were refined and extracted using qiaplep spin miniplep kit (qiagen). after extraction, dna was eluted in ml distilled water and was stored at À c. in addition, to determine the sensitivity of typespecific lamp method, the dna concentrations of hpv- , - , - , and - were analysed on a dyna quant fluorometer (amesham pharmacia biotech, piscataway, nj), and the serial dilutions of each standard were prepared to cover the range of to copies/tube. the lamp reaction was conducted as described by notomi et al. [ ] and nagamine et al. [ ] . specifically, the lamp method requires a set of four primers (b , f , bip, and fip) to recognize a total of six distinct target dna sequences (b -b , f -f ) within the target dna. lamp primers for e region of hpv- , e region of hpv- , e region of hpv- , and e region of hpv- were designed, using the primer explorer v software (fujitsu, tokyo, japan). the location and sequence of each primer in the target dna sequences are shown in figure . lamp reactions were undertaken with a loopamp dna amplification kit (eiken chemical, tochigi, japan). reaction mixtures ( ml) contained . mm each of inner primer (fip and bip), . mm each of outer primer (f and b ),  reaction mix ( . ml), bst dna polymerase ( ml), and ml of each sample. the mixtures were incubated at c for min. next, turbidity was measured by termamecs la (teramecs, kyoto, japan). the cutoff value of turbidity used to distinguish negative from positive samples was at . , higher than the mean plus three sd of the turbidity of five negative samples. after turbidimetry, the lamp products were subjected to electrophoresis on a . % seakem tm me agarose (cambrex bio science, rockland, me) gel stained with ethidium bromide. to avoid contamination between the samples, dna extraction and lamp were carried out in different rooms, and pipette tips with filters for aerosol prevention were used. pcr was used for typing of hpv dna as described by yoshikawa et al. [ ] . the consensus primers amplifying at least nine hpv types, hpv- , - , - , - , - , - , - , - , and - were used for this assay, including l c ( -cgtaaacgttttccctattttttt- ), l c ( -taccctaaatactctgtattg- ), and l c m ( -taccctaaataccctatattg- ). pcr was performed using . u taq dna polymerase (takarabio-medicals, tokyo, japan) by takara thermal cycler (takarabiomedicals) with rounds of thermal cycling conditions; degeneration at c for . min, annealing at c for . min, extension at c for min. pcr products were confirmed by electrophoresis through % nusieve tm gtg agarose (cambrex bio science) gel stained with ethidium bromide. next, they were typed on the basis of restriction fragment length polymorphisms (rflps) by dde i and rsa i. real-time pcr for hpv- , - , - , and - type-specific real-time pcr was used to measure the quantity of the dnas of hpv- , - , - , and - in each sample. the sequences of primers and probes for e /e region used have been described by tucker et al. [ ] . pcr reactions were carried out using the taqman pcr kit (pe applied biosystems, foster city, ca) according to the manufacture's directions. standard curves measuring hpv- , - , - , and - dna concentrations were constructed using c t values obtained from serially diluted plasmids, pbr , respectively, which contain the target dna sequences. the c t value from each sample was plotted on a standard curve, allowing automatic calculation of the copy number using sequence detector v . software (pe applied biosystems). each sample was tested in duplicate; the copy number of each sample is represented as the mean of the two values. the specificity of hpv- , - , - , and - type-specific primers was evaluated. hpv type-specific lamp was performed by each primer on control dna of different hpv types. each tube contained copies of hpv dna. as lamp products contained several sizes of invertedrepeat structures, positive samples demonstrate a ladder pattern upon agarose gel electrophoresis. hpv type-specific lamp primers amplified only the respective type of hpv dna; no lamp products were detected in reactions carried out with other type of hpv dnas (fig. ) the sensitivity of pcr with consensus primer was reported to detect . pg in the previous report [yoshikawa et al., ] . the sensitivity of hpv- , - , - , and - type-specific lamp methods were determined. serial dilutions of the pbr plasmid to cover the range of to copies/tube were used to determine the detection limit of hpv type-specific lamp. the sensitivity of hpv- , - , - , and - type-specific lamp determined by turbidity assay were , copies/tube (fig. ) . in contrast, the sensitivity of hpv- , - , - , and - type-specific lamp determined by agarose gel electrophoresis were copies/tube, copies/tube, , copies/tube, and copies/tube, respectively (data not shown). twenty-seven biopsy tissue specimens (sample numbers - ) collected from patients with genital polypoid lesions were examined (table i) . the histologic diagnoses included condyloma acuminatum (n ¼ ), bowe-j. med. virol. doi . /jmv fig. . to determine the sensitivities of each assay, serial dilutions of hpv- , - , - , and - dnas were amplified by hpv- (a), hpv- (b), hpv- (c), and hpv- (d) type-specific lamp, respectively. the detection of lamp products was assessed by turbidity assay using a la- . the numbers in the figures are the dilution of n copies/tube. noid papulosis (n ¼ ), seborrheic keratosis (n ¼ ), epidermolytic acanthoma (n ¼ ), and hairy nymphae (n ¼ ). the results of hpv type-specific lamp were compared with those of pcr and hpv type-specific realtime pcr. the average reaction time was min sec. among codylomata acuminata samples, ( %) were positive for hpv- , and the remaining ( %) were positive for hpv- by type-specific lamp. one bowenoid papulosis specimen was positive for hpv- by type-specific lamp. no hpv- dna was detected in any samples. hpv type-specific lamp amplified the respective hpv genomes and showed no cross-reactivity. hpv dnas were detected from of ( %) in total, while they were detected from of ( %) by pcr, consisting of hpv- s, hpv- s, and hpv- by rflps of pcr, identical to the results of lamp. no hpv dna was detected in samples that were negative by lamp. among the three hpv- positive samples by lamp, only sample no. was negative by pcr. furthermore, the results of real-time pcr correlated perfectly with those of lamp. most of the positive samples by lamp contained high copy numbers of hpv dna. the correlation between the time (in sec) to reach the threshold > . of turbidity and copy number was analyzed. the result is shown in figure . there was a linear correlation between the genome quantity and reaction time to reach the threshold: y (copy numbers) ¼ À . x (sec) þ . . j. med. virol. doi . /jmv hpv is detectable in condyloma acuminatum and bowenoid papulosis, and it often becomes a problem as a sexually transmitted disease [schwartz and janniger, ; beutner and tyring, ; beutner et al., ; leung et al., ] . in addition, there is a high-risk type hpv that is associated with cervical cancer. therefore, it is important to identify the genital hpv type from both an epidemiological and a public health standpoint. virologic testings include ish, southern blot hybridization, dot blot hybridization, and pcr [yoshikawa et al., ; lindh et al., ; brown et al., ; oliveira et al., ] . recently, several investigations described real-time pcr, which has the additional advantage of being a quantitative method [cubie et al., ; seth et al., ] . however, these methods are time-consuming, require special expensive equipment such as thermal cyclers, and may not be appropriate for testing in a clinical setting. lamp developed by notomi et al. [ ] has been applied for the detection of various types of infectious agents, mainly varicella-zoster virus [okamoto et al., ] , sars coronavirus [poon et al., ] , herpes simplex virus , measles virus , mumps virus [okafuji et al., ] , and influenza virus [ito et al., ] . lamp proved to be rapid, highly sensitive, highly specific, and simple, suggesting that it might be used for rapid diagnosis. lamp for the detection of hpv was developed and the sensitivity and specificity of lamp were compared with those of pcr and real-time pcr for the detection of hpv. a pair of primers in the e region was designed at first, but since there is % homology between the pcr products of hpv- and hpv- , cross-hybridization occurred between hpv- and hpv- . in addition, portions of the e gene show greater homology and may be deleted often during hpv integration. therefore, a pair of primers in the e /e region was designed, since portions of the e /e show smaller homology, and are retained and are expressed usually even when hpv integrates into cellular chromosomes. hpv type-specific lamp amplified only the respective type of hpv dna with no cross-reactivity. this specificity was confirmed by two independent detection methods, turbidity assay and agarose gel electrophoresis. the detection limit for hpv type-specific lamp by the turbidity assay was , copies/tube. in contrast, the detection limit for hpv- , - , - , and - type-specific lamp by agarose gel electrophoresis was copies/tube, copies/tube, , copies/tube, and copies/tube, respectively. although the turbidity assay is less sensitive than agarose gel electrophoresis , the ease and rapidity of the turbidity assay make it more appropriate for clinical monitoring. furthermore, this system also minimizes potential contamination because tubes are closed during amplification and detection of amplified dna. the reliability of hpv lamp was evaluated for the detection of viral dna from clinical samples. since the turbidity analysis seemed to be the most appropriate test for clinical laboratory use, this assay was used for clinical sample analysis. hpv- dna and hpv- dna were detected in and of condylomata acuminata, respectively, without concomitant infection. hpv- dna was detected in one of two bowenoid papuloses. hpv dna was not detected in seborrheic keratosis, epidermolytic acanthoma, and hairy nymphae. these results correlated perfectly with those from real-time pcr analysis. most of the positive samples contained high copy numbers of viral dna. hpv dna was not detected in samples that were negative by lamp. among three hpv- positives by lamp, one was negative by pcr. therefore, the sensitivity of hpv type-specific lamp was nearly the same as that of realtime pcr and was greater than that of pcr, for the detection of hpv infection, demonstrating the high sensitivity and specificity of hpv type-specific lamp in the analysis of clinical samples. as for sample no. of bowenoid papulosis, hpv was not detected by the lamp method, pcr, and real-time pcr, but hpv- was detected by the hybrid capture ii assay. recently, the detection of various types of hpv has been reported [zur hausen and de villiers, ]. this lamp method may not be useful for detection of broad hpv genotypes. however, there was a linear correlation between the genome quantity and reaction time to reach the threshold by the lamp method, making quantitation of hpv dna in clinical samples possible. several studies showed that an increased hpv- viral load correlated with the risk for cervical carcinoma [peitsaro et al., ; ho et al., ] . the presence and quantity of hpv dna are likely to be a reflection of metastasis and may have a prognostic value. therefore, this assay may be useful to study the epidemiology, pathogenesis, and monitoring vaccine trials of hpv. the average reaction time of hpv type-specific lamp was about min, not more rapid than that of other viruses, mainly measles virus , influenza virus [ito et al., ] , or herpes simplex virus [sugiyama et al., ] . therefore, modification of primer design or induction of loop primers may be necessary to shorten the reaction time in future analyses, as additional loop primers increase the amplification efficiency [nagamine et al., ] . in summary, by setting a type-specific primer in the e /e region, a new type-specific method for the detection of hpv- , - , - , and - was developed. the sensitivity of amplification of lamp for detection of viral dna was nearly the same as that of real-time pcr. cross-reactivity was not observed, and reliability of testing with clinical specimens was demonstrated. the lamp method is superior in terms of sensitivity, specificity, speed, and simplicity, and can potentially be a valuable tool for the detection of hpv dna in laboratories. we are grateful to eiken chemical for their contribution to the study. human papillomavirus and human disease external genital warts analysis of human papillomavirus types in exophytic condylomata acuminata 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assay my /my and gp þ/gp þ primer systems bowenoid papulosis detection and quantitation of hpv gene expression using real-time pcr comparison of loop-mediated isothermal amplification, realtime pcr, and virus isolation for the detection of herpes simplex virus in genital lesions human papillomavirus infections and oral tumors real-time pcrbased fluorescent assay for quantitation of human papillomavirus types , , , and condyloma acuminatum: histopathology and detection of human papilloma virus detection and typing of multiple genital human papillomaviruses by dna amplification with consensus primers detection of human herpesvirus dna by loop-mediated isothermal amplification cervical carcinoma and human papillomavirus: on the road to preventing a major human cancer human papillomaviruses key: cord- -cv qgno authors: zhang, yinhua; odiwuor, nelson; xiong, jin; sun, luo; nyaruaba, raphael ohuru; wei, hongping; tanner, nathan a title: rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: cv qgno the ability to detect an infectious agent in a widespread epidemic is crucial to the success of quarantine efforts in addition to sensitive and accurate screening of potential cases of infection from patients in a clinical setting. enabling testing outside of sophisticated laboratories broadens the scope of control and surveillance efforts, but also requires robust and simple methods that can be used without expensive instrumentation. here we report a method to identify sars-cov- (covid- ) virus rna from purified rna or cell lysis using loop-mediated isothermal amplification (lamp) using a visual, colorimetric detection. this test was additionally verified using rna samples purified from respiratory swabs collected from covid- patients in wuhan, china with equivalent performance to a commercial rt-qpcr test while requiring only heating and visual inspection. this simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure. the emergence of a new coronavirus ( -ncov, now named sars-cov- ) has infected tens of thousands of people in china, with cases in at least other countries and prompting a worldwide response. the diagnostics industry has responded rapidly, with emergency use authorization granted for pcr-based tests from the us cdc (https://www.cdc.gov/coronavirus/ -ncov/about/testing.html), seegene in korea and bgi in china (https://www.bgi.com/global/company/news/bgi-develops-realtime-dna-based-kit-for-detecting-the- -novel-coronavirus/) among many other providers releasing reagents, primers and probes, and other materials to support this urgent public health need. the current diagnostic standard combines clinical symptoms and molecular method, and as many of the symptoms resembles those of common cold and influenza, an accurate molecular result is critical for final diagnosis. these molecular methods include metagenomics sequencing mngs ( ) and rt-qpcr( ), both are excellent and sensitive techniques, but approaches not without limitations. mngs is restricted by throughput, turnover time, formidable high cost and requirement for high technical expertise. as with most molecular diagnostics, rt-qpcr is the most widely used method, but it requires expensive laboratory instruments and is difficult to utilize outside of well-equipped facilities. in combination to the different patient samples containing variable number of virus, a high proportion of patients were diagnosed as false negatives ( , ) . here we describe a molecular diagnostic approach for sars-cov- rna detection using loop-mediated isothermal amplification (lamp) and simple visual detection of amplification for potential use in rapid, field applications. lamp was developed as a rapid and reliable method to amplify from a small amount target sequence at a single reaction temperature, obviating the need for sophisticated thermal cycling equipment ( ) . since the initial description of lamp, a number of advancements in detection technology have helped establish lamp as a standard method for simple isothermal diagnostics. these detection methods have allowed detection by visual examination without instrumentation using dyes that utilize inherent by-products of the extensive dna synthesis, such as malachite green ( ), calcein ( ) and hydroxynaphthol . cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint blue ( ) . we recently developed a method for visual detection of lamp amplification ( ) using ph-sensitive dyes to exploit the change of ph that resulting from proton accumulation due to dntp incorporation. this method has been used in a large scale field survey of wolbachia-containing mosquitos ( ), grapevine red blotch virus without dna extraction ( ) , testing urine samples for zika virus ( ) and even amplification detection on the international space station ( ) . the breadth of application highlights the applicability of visual detection methods to provide an advantage in simplicity and portability for enabling new, rapid diagnostics. this study describes testing and validation of sets lamp primers targeting two fragments of the sars-cov- genome using short (~ bp) rna fragments made with in vitro transcription and rna samples from patients. we also demonstrate compatibility with simple approaches to rna purification to simplify the detection procedure and avoid complex rna extraction, and with clinical swab samples taken from covid- patients. our aim is to share this information in order to help develop a reliable and easy method to detect this viral rna outside of sophisticated diagnostic laboratories and expand the toolbox of molecular tests used to combat and surveil this growing public health threat. we designed sets of lamp primers targeting two fragments (table ) of sars-cov- sequence (genbank accession number mn ) using the online software primer explorer v (available for free use at: https://primerexplorer.jp/e/). dna fragments containing these two regions were synthesized as gblocks (integrated dna technologies) and t rna polymerase promoters were added by pcr (neb m , numbers indicate neb catalog id unless otherwise noted) using primer pairs where one primer containing the promoter sequence. rnas were then synthesized by in vitro transcription (e ) using these pcr products as templates and purified using rna clean up columns (t ). the resulting rnas as well as the gblocks were serially diluted in -fold increments using . x te buffer containing . % tween . rt-. cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. were allowed to cool at room temperature before further processing. if the samples were not for use immediately, they were stored at °c. total viral rna was extracted from the deactivated samples using automated extraction instrument, purifier tm modesty (genfine biotech, beijing-china, ltd), according to the . cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we designed full lamp primers sets targeting sars-cov- rna, with amplicon regions designed to the ′ region of the orf a gene and gene n. each set was tested with synthetic dna substrates and rna transcribed from that dna substrate before consideration for use clinically. to evaluate detection sensitivity, synthetic rnas were serially diluted from ~ million copies down to ~ copies (per μ l reaction) at fold intervals in lamp reactions. all five primer sets showed similar detection sensitivity and could consistently detect as low as a few hundred copies, with sporadic detection of copies (or . copies/μl; fig. ). the results from colorimetric detection were % in agreement with the real time detection. to estimate the relative efficiency using rna or dna templates, we compared synthetic rna with similarly diluted gblock dsdna on real-time lamp signal (fig. ) . for the primer sets we compared, one showed slightly slower amplification and detection with rna template while the other appeared slightly faster, confirming the rna is efficiently converted to cdna by the reverse transcriptase (warmstart rtx) and subsequently amplified via lamp by the dna-dependent dna polymerase (bst . warmstart). . cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint with current diagnostic methods, e.g. rt-qpcr, purified rna is used in the input. this will of course enable maximum efficiency and sensitivity but requires skill and instrumentation in addition to adding extra time to the diagnostic workflow. we investigated whether it is possible to perform detection using crude cell lysate in order to avoid this rna purification step. the results indicated that ~ copies were detected with all four primer sets, a similar sensitivity as the detection sensitivity with synthetic rna alone (fig. a) with no interference by the lysate to either the amplification efficiency or visual color change. we also tested whether we could recover the synthetic rna spiked into biological sample with a mock experiment during purification of total rna. we spiked various amount of synthetic rna into whole human blood and purified the total blood rna. we were able to recover and detect the spiked rna (fig. b) , indicating the total rna has no interference during the purification or the detection process. while the column-based approach is less compatible with the simple, field detection enabled by colorimetric lamp, this is a typical laboratory workflow and can be used with simple isothermal amplification in a similar fashion to more expensive and involved qpcr detection workflows. with this preliminary evaluation of potential lamp primer sets, the best performing sets (orf a-a, genen-a) were shared and synthesized in wuhan for testing with actual covid- samples. rna was extracted from swabs using the standard laboratory protocol (see materials and methods) and rna tested in colorimetric rt-lamp alongside a commercial rt-qpcr assay. in addition to controls, a total of patient rna samples were tested, of which were determined to be positive by rt-qpcr using orf a primers (c q - . , table ) and positive with gene n primers. one sample was negative by both rt-qpcr primer sets. when these samples were tested in the colorimetric lamp assay, all rt-qpcr positive samples showed visible color change indicating positive amplification, while the single rt-qpcr negative samples maintained pink color and was judged negative (figure ). thus the colorimetric lamp assay showed % agreement with the rt-qpcr results across a range of c q values. although a small number of samples were tested here, the colorimetric lamp assay enables reliable sars-cov- detection without sophisticated instrumentation, matching the rt-qpcr performance in field and point-of-care settings. . cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org / . / . / in conclusion, colorimetric lamp provides a simple, rapid method for sars-cov- rna detection. not only purified rna can be used as the sample input, but also direct tissue or cell lysate may be used without an rna purification step. this combination of a quick sample preparation method with an easy detection process may allow the development of portable, field detection in addition to a rapid screening for point-of-need testing applications. this virus represents an emerging significant public health concern and expanding the scope of diagnostic utility to applications outside of traditional laboratories will enable greater prevention and surveillance approaches. the efforts made here will serve as a model for inevitable future outbreaks where the use of next generation portable diagnostics will dramatically expand the reach of our testing capabilities for better healthcare outcomes. we thank dr. guoping ren for advice and assistance using the luna cell ready lysis module. . cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . comparison of rna and gblock dsdna templates in lamp amplification using real time amplification curves. two primer sets (orf a-a and gene n-a) were used to amplify either rna (green curves, dilutions from x to copies) or gdna (blue curves, x to copies). for orf a-a primer set, the gblock is faster than rna template; for gene n-a primer set, the rna is slightly faster. each "cycle" represents seconds, with minute timepoint noted by dashed line. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . lamp detection of target rna spiked in whole blood. the maximal possible copies of the target rna that could be recovered were shown. in a control, ng of jurkat total rna was added, which is similar to the total rna present in the reaction with blood samples. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . ( ) with commercial rt-qpcr tests were assayed using colorimetric lamp assay with primer set targeting orf a (a) and genen (b). yellow indicates a positive detection after min incubation, and pink a negative reaction with results compared to the negative control (n). b, blank control without template. p, samples containing the plasmid used as positive control for qpcr. cc-by . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in wuhan outbreak clinical features of patients infected with novel coronavirus in wuhan consistent detection of novel coronavirus in saliva loop-mediated isothermal amplification of dna development of a loopmediated isothermal amplification method for rapid mass-screening of sand flies for leishmania infection loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue visual detection of isothermal nucleic acid amplification using ph-sensitive dyes detecting wmel wolbachia in field-collected aedes aegypti mosquitoes using loop-mediated isothermal amplification (lamp) a rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification rapid colorimetric detection of zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (rt-lamp) nucleic acid detection aboard the international space station by colorimetric loop-mediated isothermal amplification (lamp) key: cord- -a p ma r authors: khan, pavana; aufdembrink, lauren m.; engelhart, aaron e. title: isothermal sars-cov- diagnostics: tools for enabling distributed pandemic testing as a means of supporting safe reopenings date: - - journal: acs synth biol doi: . /acssynbio. c sha: doc_id: cord_uid: a p ma r [image: see text] the covid- pandemic, caused by the sars-cov- virus, poses grave threats to both the global economy and health. the predominant diagnostic screens in use for sars-cov- detection are molecular techniques such as nucleic acid amplification tests. in this review, we compare current and emerging isothermal diagnostic methods for covid- . we outline the molecular and serological techniques currently being used to detect sars-cov- infection, past or present, in patients. we also discuss ongoing research on isothermal techniques, crispr-mediated detection assays, and point-of-care diagnostics that have potential for use in sars-cov- detection. large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. the low-cost isothermal technologies described in this review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks. c ovid- is the pandemic disease caused by a novel coronavirus first isolated in late , sars-cov- . belonging to the same family of viruses that caused the sars (severe acute respiratory syndrome) epidemic of and the mers (middle east respiratory syndrome) outbreak of , sars-cov- has proven to be highly transmissible. − just as sars-cov and mers-cov have animal reservoirs, sars-cov- is also thought to be of zoonotic origin, jumping from an animal to human host. both a bat and pangolin coronavirus have been identified as the closest genetic relatives to sars-cov- , but the exact route from animals to humans has yet to be elucidated. , on march , , the world health organization designated the outbreak to be a pandemic, and as of july , there have been over . m cases and k deaths spanning the globe in countries, demonstrating that the virus is a serious threat to global health. sars-cov- is an rna virus with a ca. kb genome (ncbi reference sequence: nc_ . ). the genome encodes four structural proteins: the spike (s) protein, the envelope (e) protein, the membrane (m) protein, and the nucleocapsid (n) protein. around two-thirds of the genome consists of the orf ab region, which codes for orf ab polyproteins that are cleaved into various nonstructural proteins, composed of many proteins important for replication, such as an rna dependent rna polymerase (rdrp), replicase−transcriptase complex, and rna helicase. − sars-cov- infects by binding the angiotensin converting enzyme- (ace ), present on the surface of a variety of cell types, with the s protein. the virus is transmitted from person to person through aerosol droplets from an infected persons' cough, sneeze, or saliva. the pathological symptoms of the disease can vary but most commonly include respiratory illness, dry cough, fever, and cytokine-storm related complications. further, many sars-cov- infected individuals are asymptomatic and capable of inadvertently spreading sars-cov- , increasing the spread of the disease. − with no available vaccine and high transmissibility, proper detection and containment are key to helping mitigate the spread and devastation caused by sars-cov- . the current most common diagnostic method used to identify sars-cov- infection is a molecular technique for detecting viral rna through nucleic acid amplification, rt-pcr. around the world, a variety of rt-pcr kits are in use, each with varying specificity. rt-pcr is a ubiquitous method for a myriad of disease detection, but the ability to employ this method in low resource areas lacking centralized care facilities or at the point of care (poc) is limited due to equipment and energy needs. oftentimes, it is required to obtain a rapid result at the poc, allowing for easier containment of a pathogenic virus such as sars-cov- . this review discusses current molecular and serological methods in use for sars-cov- detection, with particular interest in isothermal detection platforms. we discuss their advantages and disadvantages relative to pcr-based and serological techniques (table ) as well as isothermal techniques not yet used in sars-cov- diagnostics that could allow for the expansion of testing capabilities as a means of supporting safe reopenings of businesses and schools as well as allowing for routine household testing. nucleic acid amplification tests (naats) are the most common diagnostic tests used to detect pathogens, and many of the current sars-cov- detection techniques are primarily based on naats. naats involve nucleic acid amplification, a process that initiates with a small quantity of starting nucleic acids and uses primers that target specific, short nucleic acid sequences in conjunction with enzymes to amplify or increase the quantity of starting nucleic acids. the most common type of naat is the polymerase chain reaction (pcr), which amplifies dna and can be used in reverse transcription pcr (rt-pcr) for detection of rna or in quantitative pcr (qpcr) for measurement of copy number, and reverse transcription pcr (rt-pcr) for detection of rna. recently, ellington and coworkers described a single-enzyme approach to rt-pcr detection of sars-cov- . this approach uses an engineered thermostable reverse transcriptase/dna polymerase based on a reverse transcription xenopolymerase (rtx) generated by directed evolution of archaeal family-b dna polymerases (polb). , advantages of pcr. pcr is the current "gold standard" in molecular diagnostics, and the multiple temperatures employed in the reaction allow for tuning a range of properties, including primer annealing temperature, denaturation temperature, and extension temperature. these can be adjusted at each successive cycle (e.g., touchdown pcr), and instruments exist that can screen a range of temperatures for a given step (e.g., gradient pcr). fluorescence-monitored pcr instruments (e.g., realtime pcr instruments) exist, which can monitor reaction progress by either nonspecific dyes (e.g., sybr green) or duallabeled probes (e.g., taqman). disadvantages of pcr. owing to its requirement for repeated excursions to multiple annealing, extension, and denaturation temperatures, pcr requires bidirectional temperature control. this necessitates expensive instrumentation, typically using a peltier-effect device as implemented in most thermal cyclers. similarly, readout requires either electrophoresis followed by gel readout, requiring a skilled technician, or real-time fluorescence readout, requiring a real-time pcr instrument with associated excitation source(s), optics, and emission detectors. this necessarily increases costs. ■ isothermal sars-cov- detection platforms isothermal detection platforms make use of isothermal nucleic acid amplification methods that allow amplification at constant temperatures. this type of naat can avoid the high temperatures associated with thermal cycling in pcr and are thus more applicable to low-resource environments, field applications, and laboratories lacking expensive, energy intensive pcr equipment. isothermal detection techniques can undertake rapid sample and reagent preparation and can also be coupled with a variety of readouts, enhancing their ease-ofuse and accessibility (table , figure ). the loop-mediated isothermal amplification (lamp) method uses a dna polymerase enzyme in conjunction with − different primers, each recognizing different regions of the target dna ( figure ). the dna polymerase used in lamp has high strand displacement activity and can be used in conjunction with multiplexed reactions and reverse transcription techniques to detect pathogenic agents. reverse transcription loopmediated isothermal amplification (rt-lamp) is useful for detecting rna-based viruses as it combines conventional lamp with a reverse transcriptase enzyme, allowing for simultaneous reverse transcription and amplification. rt-lamp is a one-step amplification reaction, allowing all the primers and necessary enzymes to be incubated isothermally in a single step. the final readout for lamp can be real-time detection using fluorescence or absorbance instrumentation. lamp and sars-cov- . rt-lamp was recently shown to detect sars-cov- in < min, using patient samples such as urine, saliva, and oropharyngeal and nasopharyngeal swabs spiked with various concentrations of synthetic covid- amplicon. the low detection time is promising for the potential use of this technique in poc applications. this rapid in another report, lu et al. designed a rt-lamp assay targeting the rdrp gene exhibiting a colorimetric readout based on the ph of the sample. this assay has a limit of detection of copies of rna and detection takes place in min. specificity was tested against clinical samples for other common respiratory illnesses, and no off-target amplification was seen. a unique adaptation of lamp was implemented in a barcoded rt-lamp method called lamp-seq that uses three isothermal steps to barcode amplicons. the protocol incorporates sample-specific barcodes by inserting barcode sequences into the lamp primers, followed by pooling of reactions into batches, which are then subjected to a secondary amplification and barcoding with pcr. these amplicons are then purified and deepsequenced, and analyzed using a software package, lamp-seq-inspector, also reported in the manuscript. with a limit of detection of rna molecules/μl and a cost of less than $ per test, lamp-seq has the ability to pool and process these barcoded amplicons through mass sequencing, making the process scalable for millions of tests every day. this is especially relevant in combating virus spread through asymptomatic carriers. high-throughput techniques such as lamp-seq could enable population-scale testing, which will be beneficial for monitoring cases in areas of sars-cov- resurgence and will enhance surveillance, limiting exponential spread. rt-lamp-based ilaco (isothermal lamp-based method for covid- ) is another recently reported lamp-based technique developed for detection of sars-cov- . ilaco is as sensitive as qpcr, with a detection limit of copies of sars-cov- , and a detection time of between and min. since dna polymerase-led nucleotide incorporation is accompanied by hydrogen ion release, ilaco, like the assay developed by lu et al., combines rt-lamp with a ph-based readout to enable colorimetric output. ilaco was tested on viral rna extracted from patient samples and reverse transcribed using an engineered reverse transcriptase (superscript (iii). primer design involved analysis of a region of the orf ab gene. using five sets of different primers, samples were tested in both a thermal pcr cycler and a water bath maintained at °c for − min. primer specificity was ascertained with sequence comparison to other related coronaviruses and influenza viruses. the colorimetric detection entailed that negative reactions would remain at higher ph, resulting in the phenol red ph indicator remaining pink, while a reaction positive for sars-cov- would decrease in ph, resulting in a color change from pink to yellow. ilaco is rapid; a signal for positive reactions can be detected in about min. the test is also sensitive to up to copies of the orf ab gene, and the test detection time correlated with qpcr cycle number; a qpcr ct of corresponded to a ilaco color change point of min. the study also used several alternative detection methods such as fluorescence detection and gel imaging. furthermore, the use of clinical samples that were confirmed positive by rt-pcr methods is promising for the clinical use of the ilaco system. forty-two of the known-positive samples were detected successfully by ilaco within min of incubation, when the rna concentration in the sample was around . − ng/μl. with a short detection time of ca. min, this test shows efficacy in detecting sars-cov- in both rna and cdna samples. another recent preprint from ellington and coworkers uses rt-lamp and seeks to overcome potential false positives due to aberrant amplification. this is a problem with nonspecific readouts, seen with ph probes or intercalating dyes. to overcome this, they used oligonucleotide strand exchange (osd) probes. in the technique described, sars-cov- lamp-osd, fluorescent probes are used that are mostly double-stranded and contain a fluorescent probe-quencher pair at one end. strand exchange occurs when a toehold in the probe base pairs with the product of the lamp reaction, initiating branch migration until the probe's fluorophore and quencher are separated. bhadra et al. demonstrates this to work with probes previously designed in assays using nonspecific read outs. , , building on these assays, bhadra et al. created a one-pot reaction capable of detecting two targets, the n gene and the orf ab gene. with primers targeting both regions and probes for each amplicon, a positive signal was seen with as little as genomic rna copies/reaction, and no signal was seen when using ng of human genomic dna as the negative control. handyfuge-lamp was recently developed as an optimized lamp assay that is especially suited for poc diagnostics due to its low-cost and electricity-free centrifugation methods. the group established a hardware system called "handyfuge" that is cheap (<$ per unit) and easily assembled for achieving the high speed centrifugation needed for separation of inhibitory components from inactivated saliva samples. the reagents also include a chaotropic salt binding solution and a silica binding solution that enables viral rna capture. this allows the whole procedure of sample processing and amplification to be carried out with an inexpensive handyfuge and a water bath, along with lamp reagents. it is thus able to avoid multiple-step sample handling and expensive centrifugation machines. the preprint claims a detection limit of − copies per μl in saliva, shown by colorimetric change to indicate presence of synthetic sars-cov- rna. advantages of lamp. the primary advantage of this rapid detection technique is that it is performed isothermally and hence is amenable to use with low-cost instrumentation. the use of lamp with a ph-based readout showcases the adaptability of this reaction. another important advantage is that the detection procedure can avoid the nucleic-acid extraction step, a procedure that is time-consuming and potentially contamination-inducing. lamp has been shown to function with cell lysates. however, extraction steps concentrates the nucleic acid as well, enhancing overall assay sensitivity. by using multiple primer sets in a single reaction, lamp has been shown to be extremely specific for the target amplicon. furthermore, lamp reagent kits are amenable to lyophilization and have been disadvantages of lamp. due to the number of primers needed, primer design is complicated, making it difficult to design new assays. it is also important to note that isothermal detection techniques can exhibit false positives and thus more stringent controls may need to be in place than with rt-pcr methods. recombinase polymerase amplification (rpa) is an isothermal amplification technique that involves two primers binding a double-stranded template with assistance of single-stranded acs synthetic biology pubs.acs.org/synthbio review binding proteins and a recombinase, followed by extension with dna polymerase, to allow isothermal amplification on dna targets of interest. the process involves recombinase−primer complexes that are able to scan dsdna, identify a primerbinding site, and enable strand invasion by the primer. rpa circumvents the need for thermocycling by employing a recombinase enzyme and a strand displacing dna polymerase. via the use of loading factor proteins, primers anneal to their complementary region by the recombinase enzyme. singlestranded binding proteins then complex the strand displaced by the primer, preventing reannealing and allowing for a strand displacing dna polymerase to polymerize a new strand. as in pcr, the amplicon is comprised of the region spanning the two primers ( figure ). penn-ramp is a novel isothermal poc diagnostic method that combines the lamp strategy with rpa. this test incorporates a nested nucleic acid amplification technique showing higher sensitivity of detection than lamp alone and conventional rt-pcr for minimally processed sars-cov- samples. penn-ramp avoids false negatives by implementing two stages of isothermal amplification -a first stage of rpa at °c and a second stage of lamp at °c. in this closed-tube molecular test, rpa is conducted in the tube cap while lamp is conducted inside the tube. the rpa reaction mix has primers, buffers and salts, and this mix is loaded in the tube cap alongside samples with various target concentrations. after the preliminary − min incubation at °c for the rpa process, the tube is centrifuged or inverted multiple times to mix the rpa and lamp reactions, followed by this creates an amplification duplex for near amplification, with recognition sites for the nicking endonuclease. (ii) nicking endonucleases conduct cleavage and allow for strand displacement amplification, forming a complex with a single-stranded target region, a stabilizing duplex region, and a site for nicking endonuclease activity. (iii) a full-length duplex is formed again, which undergoes subsequent amplification through rounds of nicking, extension, and primer binding (iv−v). with primers targeting the orf ab gene in sars-cov- , penn-ramp has a limit of detection of copies per reaction and is highly specific to sars-cov- . with the use of purified rna, the authors obtained -fold lower detection limits than conventional pcr for sars-cov- . specificity was tested using other related coronaviruses such as alphacoronaviruses, gammacoronaviruses and deltacoronaviruses as negative controls. for colorimetric detection, a leuco crystal violet (lcv) solution was used, which can be lyophilized. the lamp mix contained an aliquot of lcv solution along with the lamp primers and polymerase, allowing for colorimetric detection at the end of the rpa and lamp phases. the color change can be observed by the naked eye and also monitored in real time using a camera or smartphone. the penn-ramp study used synthesized dna to mimic the target sequence for sars-cov- due to lack of access to real patient samples. thus, more research on clinical specimens may be needed to validate the specificity and sensitivity. furthermore, a platform for quantification needs to be assimilated into the penn-ramp testing method, enabling quantification in real-time and increased ease of use. advantages of rpa. primer design for rpa reactions is similar to pcr, and it is thus fairly straightforward to design new assays. due to the reaction's isothermal nature, the melting temperature of the primer is not a factor, in contrast to pcr. twistdx, a united kingdom-based company, commercially produces reagents, with lyophilized kits available making figure . dna endonuclease-targeted crispr trans reporter (detectr). detectr is a one-pot detection method that combines isothermal amplification with crispr-cas-based detection. the first step is reverse-transcriptase lamp (rt-lamp)-based isothermal amplification of dna at °c for − min. this is followed by a cas a detection reaction at °c for min. upon recognition of a thymine-rich pam sequence, cas a is able to bind to the double-stranded dna (dsdna) template and becomes catalytically activated. cas then uses its ruvc nuclease catalytic domain to generate a staggered cut with a ′-overhang in the dsdna, downstream of thymine-rich pam sequences and complementary to the guide rna sequence. next, cas a participates in indiscriminate trans cleavage of ssdna, and it releases the pam-distal ssdna cleavage products of the dna strand from the active site of the ruvc. addition of an ssdna fluorescence-quencher reporter (ssdna-fq) allows for production of a fluorescent signal upon cleavage. acs synthetic biology pubs.acs.org/synthbio review transport of reagents possible without a cold chain. no initial thermal denaturation step is required for rpa as well. rpa can be coupled to multiple different types of readout (e.g., fluorescent and colorimetric), allowing for adaptation to poc diagnostics. for example, a colorimetric readout technique that combines rpa with cell free transcription-translation systems could be modified for sars-cov- detection. the lucks group recently showed successful detection of plant pathogens following this method by incorporating small transcriptional activators into the amplification reaction and subsequently activating transcription and translation of catechol , dioxygenase (cdo) for the conversion of catechol into hydroxymuconic semialdehyde, producing a yellow positive signal. , disadvantages of rpa. multiple enzymes are needed to perform rpa, including strand displacing polymerase, singlestranded binding proteins, and recombinase, as well as cofactors and accessory proteins. this makes rpa more difficult from a manufacturing perspective than other methods employing fewer enzymes. in penn-ramp, rpa was coupled with a second isothermal amplification (lamp) reaction to overcome false positives and increase sensitivity, further complicating primer design, increasing the reaction cost, and adding an additional isothermal step and temperature. rpa amplicons are typically − nt, with larger products amplifying more poorly due to the commercially available twistamp rpa kits being optimized for amplification speed. ■ nicking and extension amplification the nicking and extension amplification reaction (near) system amplifies short sequences of nucleic acids. , the first use of near was in , and it was later amended for in vitro diagnostics in . two primers, a dna polymerase and a nicking enzyme, are used. the reaction temperature can vary depending on the polymerase and nicking enzyme used. each primer consists of a binding region and a nicking enzyme recognition region. the first primer can bind the sequence of interest, enabling a dna polymerase to extend it, creating a dsdna product. the nicking enzyme then nicks only a single strand of the sequence allowing for dna polymerase to elongate the primer. the second primer can bind the displaced product of the first strand and undergo extension by dna polymerase. , this continues to allow amplification of the region between the two primers, similar to pcr. near is able to amplify − mers to fold in under min (figure ) . near and sars-cov- . the abbott id now covid- test is an automated, instrument-based, poc sars-cov- test using a qualitative fluorescence readout technique enabled by near. this test isothermally amplifies a portion of the rdrp gene. one study found abbott id now detected ca. % ( out of ) known positive samples, whereas another study found abbott id now detected only one-third of the positive samples detected by an rt-pcr method, with dilution associated with the use of viral transport media providing one possible explanation for the diminished sensitivity. advantages of near. the id now test is rapid and takes place inside the manufacturer's instrument, with a reaction time of ca. min, making this the fastest time-to-completion among approved tests. this speed is in part due to the small size of the amplicon compared to other naats. fluorescently labeled molecular beacon probes provide a real-time readout. this reaction can be adapted to different temperatures by the use of various primers, polymerases, and nicking enzymes. disadvantages of near. as discussed above, near has exhibited false negatives under some conditions. some studies have suggested that dilution associated with the use of viral transport media prior to amplification could play a role. ■ isothermal amplification coupled to crispr/cas-based detection clustered regularly interspaced short palindromic repeats (crispr)-based disease detection methods utilize enzymes with nonspecific dnase or rnase activity, such as cas or cas from the bacterial immune system, along with guide rnas to direct enzyme binding to specific target areas on pathogenic dna or rna sequences. addition of fluorescent reporter sequences to this ribonucleoprotein complex enables readout of enzyme activity which can be used for pathogen detection. crispr/cas and sars-cov- . a novel crispr-cas based sars-cov- detection system called sars-cov- dna endonuclease-targeted crispr trans reporter (detectr) uses a lateral flow assay for detection of sars-cov- in under min with respiratory swab rna extracts. the technique first uses rt-lamp for reverse transcription and isothermal amplification of viral rna, and then employs the cas a enzyme to identify sequences of sars-cov- , allowing cleavage of a reporter molecule ( figure ). sars-cov- detectr uses primers that amplify regions of overlap in the e gene and n gene of sars-cov- and takes advantage of the nonspecific dnase activity of cas a for indiscriminate ssdna cleavage and degradation. this cas a activity allows for cleavage and activation of an ssdna reporter. design of the cas a guide rnas (grnas) entailed designing three e gene grnas for identification of three sars-related coronaviruses and one n gene grna that specifically targeted the n gene for sars-cov- detection, thus requiring identification of both e and n genes for detection. the specificity analysis showed that the test was specific for sars-cov- and not related coronaviruses such as bat-sl-covzc and sars-cov. the human rnase p gene was used as a control. the detection reaction involved an isothermal rt-lamp phase at °c for min, followed by cas a-grna guided detection reaction at °c for min, the output from which could be visualized using a lateral flow assay in the form of an easily interpretable, qualitative, visual signal. the limit of detection for this test was found to be copies/μl of reaction, with a % negative predictive value and a % positive predictive value. importantly, this assay has been tested on rna extracted from sars-cov- pcr-positive clinical patient samples. ding et al. recently described the use of rpa with cas a to detect sars-cov- in a single reaction, which they have termed all-in-one dual crispr-cas a, aiod-crispr. this assay exhibits a limit of detection of . copies of the n gene synthetically derived from a plasmid in min. the specificity was tested against sars-cov and mers-cov control plasmids. this assay was also tested against hiv- rna. another crispr-cas -based assay developed by lucia et al. uses rpa in conjunction with reverse transcription and subsequent detection by the cas a endonuclease collateral activity. with primers targeting the orf ab region, the authors' test reached a limit of detection of viral copies/μl. this test was compatible with both fluorescence readouts and a paper-based strip assay. furthermore, successful testing on spiked clinical saliva samples shows that the method is valid for easily obtainable saliva specimens and thus suitable for poc operations. another study on a crispr-based detection system, called the casdetec (crispr-assisted detection) strategy, shows improved sensitivity compared to cas a-based detection systems owing to the accuracy and specificity of dsdna transcleavage by the cas b enzyme. casdetec uses reverse transcriptase recombinase aided amplification (rt-raa) for a preamplification step, followed by the use of cas b with grna targeted to the rdrp gene, with a limit of detection of sars-cov- pseudovirus copies/ml. the raa assay is performed between and °c and employs single-stranded binding proteins, a recombinase and a dna polymerase. to simplify the reaction, it was split within one vessel, with rt-raa conducted inside the reaction tube while the cas- b-based detection reagents were kept in the cap of the tube, followed by a spin-down mixing of the reagents and subsequent fluorescent readout with a total reaction time of about min. the authors demonstrated that their readout was compatible with led excitation, increasing usability in poc settings. a novel crispr-based diagnostic for covid- detection with recent u.s. food and drug administration (fda) approval is called stopcovid (sherlock testing in one pot covid) and uses the sherlock (specific high sensitivity enzymatic reporter unlocking) technique. stopcovid targets the sars-cov- n gene and aims to be a poc assay, taking about an hour to process results and needing very little instrumentation. similar to detectr, stopcovid uses the reaction can be conducted using a water bath or a heat block and uses taurine to confer thermostability to reaction components. with an lod of copies of the sars-cov- genome per reaction, the test has a sensitivity of % and a specificity of %. showing successful detection in healthy saliva samples spiked with standard concentrations of sars-cov- rna, the test is highly amenable to poc assays with easily obtained saliva samples and does not require trained personnel. another recent study from myhrvold and colleagues proposes assay designs for surveillance of sars-cov- and other related viruses and uses crispr-based nucleic acid detection. this study is unique in that it addresses common challenges with virus surveillance such as false positives due to cross-reactivity of detection assay reagents with related coronavirus species, higher demand for tests than supply, and the complicating occurrence of coinfections in patients with sars-cov- . the study developed a machine learning model and concurrent algorithms to evaluate the comprehensiveness, predicted sensitivity and predicted specificity of nucleic acid detection techniques. comparing different sherlock methods, the model identified a rpa-cas a-based sherlock detection system to have the best performance and highest specificity. , , the study subsequently conducted this sherlock assay to show detection of synthetic sars-cov- rna at copies/μl. employing the nuclease specificity of the crispr system for diagnostic readout allows the detection system to be designed for single base specificity. detecting rna viruses can be a challenge due to their high rate of evolution and single nucleotide changes. being able to detect such changes with crispr-based methods would allow for determining the path of infection a virus takes without the need for sequencing. crispr-based detection uses both primer-specific amplification and guide rna directed detection, thus increasing sequence specificity in two different ways. furthermore, the current readout for sars-cov- detectr and stopcovid employs lateral flow detection, which has numerous advantages for poc applications. crispr-based detection techniques can also be combined with a variety of isothermal detection methods, as showcased by the described methods using rt-lamp and rpa with sensitivity compared to rt-pcr. disadvantages of crispr-based detection. rt-lamp or another isothermal amplification method is required to amplify rna of interest to generate sufficient signal. accordingly, any difficulties associated with the chosen isothermal method would all apply crispr-based detection. as these assays rely on collateral activity of cas enzymes, their readout is also qualitative. the aptamer-coding primer binds and reverse transcriptase extends the aptamer-coding primer, generating a dna duplex corresponding to the amplicon, with a t rnap promoter (red) and an aptamer coding sequence (purple). (v) t rnap generates an rna fusion construct that contains the sequence antisense to the target sequence, fused at the ′ end with the aptamer corresponding to the sequence to the aptamer-coding sequence (green). (vi) aptamer-coding primer binds this rna, which again enters the rt-rnase h-t rnap cycle. at each step, rna is generated with multiple turnovers, enabling exponential amplification. in the conventional nasba cycle, the aptamer coding sequence is omitted from the second, nonpromoter-containing primer. many useful isothermal amplification techniques exist which are not currently adapted to detect sars-cov- , but have the potential to be beneficial for addressing the global pandemic. isothermal amplification techniques such as rolling circle amplification (rca), nucleic acid sequence based amplification (nasba), multiple displacement amplification (mda), helicase dependent amplification (hda), and signal mediated amplification of rna technology (smart) would allow for efficient amplification of nucleic acids, followed by easily interpretable readouts (table ) . rolling circle amplification. rca and rolling circle transcription (rct) techniques developed based upon the discovery that dna and rna polymerases could use small (less than nucleotides) circular constructs as their template. polymerases have been shown to work on templates as small as nucleotides; templates that have a smaller diameter than the polymerase's footprint suffice for amplification. only a single primer is needed, and due to the primer seeding multiple copies of the same sequence, the ratios of template to primer differ greatly from those in pcr. the primer binds to the circular template and allows for a strand displacing dna polymerase to continually elongate the circular template ( figure ). in the case of rct, the primer binds, creating a double-stranded promoter sequence capable of recruiting the selected rna polymerase. the resulting product is a long, repetitive nucleotide sequence. with the discovery of highly processive enzymes from bacteria, products can reach greater than bases. by adding a ligase and a specifically designed padlock probe, this technology has been used to detect micrornas. rct only amplifies a small part of the target, accompanied by an easily detectable sequence. the technique has been used in cells to detect let- a microrna, which is associated in regulation of development and tumor suppression, with femtomolar sensitivity. similar to the methods used previously, incorporating a hemin-binding g-quadruplex into a rolling circle amplification (rca) reaction advantages of rca. the process of rca is simpler than other most isothermal techniques due to simple primer design. the phi dna polymerase has catalytic activity at °c or room temperature, making the process energy-efficient without the need for thermostable enzymes. furthermore, since amplification is preceded by a padlock-probe approach where specific ligase activity is needed to make the circular dna template there is a high degree of specificity associated with rca. this makes it suitable for scoring single-nucleotide polymorphisms (snps) and for high-throughput assays. disadvantages of rca. rca requires an additional ligase step, since it amplifies circular dna templates and the circularization of the dna template can be low-yielding if folding is not optimal. nucleic acid sequence based amplification. nasba is an isothermal amplification reaction that can use either dna or rna as a template. nasba employs three enzymes: a reverse transcriptase, rnase h, and an rna polymerase. two primers are used, and in addition to a complementary region to the sequence of interest, one of the primers also encodes for an rna polymerase promoter sequence. through reverse transcriptase and rnase h activity, a dsdna is created with an rna polymerase promoter sequence. rna polymerase transcribes this sequence, creating a pool of product rna. primers can bind this product rna, again generating dsdna with t promoter sequences through reverse transcriptase and rnase h activity providing exponential amplification (figure ) . recently, two groups developed a method for tagging nasba reactions with fluorescent aptamers, enabling genetically encoded fluorescent readout. unrau and coworkers recently demonstrated the use of the mango aptamer in nested mango nasba, , and engelhart and coworkers showed multiplexed detection with cell phone camera-based readout using the broccoli, corn, and malachite green aptamers in apta-nasba. these techniques enable an inexpensive fluorogenic real-time readout. nasba has also been detected by an aptamer-based molecular beacon approach. advantages of nasba. nasba has the capability of starting with either dna or rna, with rna as the main reaction product, enabling the use of functional nucleic acids such as aptamers and ribozymes in the reaction product, opening the door to using a variety of detectable signals. , by applying fluorescent rna aptamers for the readout, it is possible to obtain comparable specificity as a fluorescent probe, but with the cost of an intercalating dye such as sybr green. the optimal reaction temperature is − °c, lower than that of lamp or rpa. the reaction is also compatible with lyophilization, mitigating the need for a cold chain in field applications. disadvantages of nasba. false positives, as with many isothermal techniques, can complicate detection. recently, abdolahzadeh et al. mitigated this issue through a nested procedure, and aufdembrink et al. used a competitor dna duplex to do so. , as with other multienzyme isothermal reactions, the use of multiple enzymes necessarily adds complexity to manufacturing processes. helicase dependent amplification. hda mimics the replication process in a cell, which isothermally separates dna using helicase enzymes. in hda, a helicase unwinds dsdna, allowing for primers to bind, initiating elongation with a dna polymerase. single-stranded binding proteins are included, as in rpa, to stabilize the ssdna unwound by the helicase (figure ). two primers are used to amplify specific sequences of dna, but due to the isothermal nature of the reaction, a lower concentration of primers is typically used compared to pcr to minimize formation of primer dimers. altered dntps have been shown to be successful in mitigating primer dimers in selfavoiding molecular recognition systems (smars) where, due to the base changes, primers can only pair with naturally occurring nucleotides and not themselves. advantages of hda. hda has been shown to work on a variety of samples, including genomic dna, plasmid dna, blood samples, bacteria cultures, and fecal samples. it is also amenable to readouts used with pcr. amplivue assays have been commercialized for certain pathogens with a lateral flow device where the only additional equipment required beyond a heat source for the reaction is a heated lid. disadvantages of hda. as with most isothermal reactions, false positives are an issue. the use of smars, as described above, helps diminish this. while there are many published methods using hda for pathogen detection, there are a limited amount of validated tests. a reverse transcription reaction must be performed if trying to detect rna, as is required to detect retroviruses such as sars-cov- . multiple displacement amplification (mda). mda is a technique used in whole genome amplification which has proved useful in single-cell sequencing. it works by using a strand displacing polymerase and multiple primers. a denaturing step is used at the beginning to obtain single-stranded dna. primers bind their complementary region and a highly processive, strand displacement-capable dna polymerase, typically phi dna polymerase, synthesizes new dna strands. additional primers bind to the newly synthesized strand, or displaced strand, allowing for amplification of specific regions (figure ). based on the primers used, certain sequences have preferential amplification. by using multiple primers specific toward human papillomaviruses (hpv), this technique has been used on patient samples to identify novel hpv and thus has the potential to be used in detecting sars-cov- and novel mutations in the sars-cov- genome. advantages of mda. mda is capable of identifying novel viruses in patient samples through the use of many primers. as in lamp, the use of multiple primers increases the specificity of the reaction compared to other isothermal methods. disadvantages of mda. mda requires a reverse transcription step to detect rna. multiple primers are needed for this reaction, adding complexity to primer design when setting up a new assay. signal mediated amplification of rna technology. smart takes advantage of the requirement for t rna polymerase to bind a double-stranded promoter to transcribe rna. targeting rna, two probes create a three-way junction with the template of interest. the two probes each contain a sequence complementary to adjacent regions of the target of interest and a second mutually complementary sequence, enabling the formation of a three-way junction. the sequence complementarity between the two probes is not sufficient to allow probe annealing, and the template brings probes together via three-way junction formation, enabling primer extension. the first probe, termed the extension probe, is shorter and acts as a primer for elongation using the second, longer probe, termed the template probe, as the template. the template probe contains the antisense template sequence, a t rna polymerase promoter, and a sequence complementary to the sequence to be detected. when sequences hybridize, forming a three-way acs synthetic biology pubs.acs.org/synthbio review junction, dna polymerase is used to create a dsdna template for t rna polymerase to transcribe. t rna polymerase can transcribe many copies of the specified sequence, giving an amplified signal ( figure ). if further sensitivity is required, a third probe can be used for greater amplification. the third probe contains a single-stranded t rna polymerase promoter region and complementarity to the product formed via the three way junction transcription. this product acts as a primer for the dna polymerase, allowing dna polymerase to create another dst rna polymerase promoter. t rna polymerase is then able to come in and create multiple copies of a second detectable sequence ( figure ). while this step can increase sensitivity, it also increases complexity and hands on time, as it is done in two separate steps. this technique has the advantage of being easily amenable to many target sequences by altering only the sequence complementary to the target. , advantages of smart. this technique has the advantage of being easily amenable to many target sequences. like nasba, rna is the main product and there is potential for a variety of functional sequences to be used for varying read outs. disadvantages of smart. smart lacks an exponential step, making it a linear amplification reaction. the product produced by smart does not seed another reaction allowing for increased amplification. ■ serology-based sars-cov- rapid serological tests use serum as a specimen for diagnosing whether an individual has been affected by a certain disease causing pathogen. in contrast to naats, these tests' detection modalities are based on recognition of circulating antibodies in blood serum produced by the immune system in response to an invading foreign pathogen. tests for sars-cov- mainly detect immunoglobulin m (igm) and immunoglobulin g (igg) antibodies, where igm antibodies are first detectable in blood around day of infection, while igg antibodies first appear around day of infection. the rapid diagnostic test (rdt) is a serology test that comes in the form of a poc assay employing a lateral flow device for testing serum samples or blood samples collected from a finger prick. these tests are set up to test the presence of igm and igg antibodies in the blood and are not suitable for quantitative results on the levels of these antibodies. there are currently serological tests with fda emergency use authorization that use a variety of lateral flow techniques, chemiluminescent immunoassays, or enzyme linked immunosorbent assays. advantages of serological tests. most serological tests are easy to use for poc diagnostics. they exhibit rapid results with high sensitivity and specificity. they are also capable of indicating whether a person has been infected with sars-cov- even if no symptoms were ever present, a highly desirable attribute when transmission is possible from asymptomatic carriers. disadvantages of serological tests. serological tests depend on the response of the infected individual's immune system. for each disease, this differs. thus far, the tests designed for sars-cov- have been developed using the disease progression timeline of sars-cov. if tests are used too early in infection, a false positive could be obtained, as sufficient immune response may not yet have been mounted. less than a month after the first reported case in wuhan, china, sars-cov- was designated a world health emergency, and now months laterover deaths have been attributed to the virus. differing approaches taken by countries to contain sars-cov- have shown various levels of success. while the response on how to best contain the virus has varied acs synthetic biology pubs.acs.org/synthbio review around the world (strict immediate lockdowns, stay at home orders, mandated usage of masks in public, ban of international travel, access to testing resources, etc. ), a common theme between countries that have a smaller case count is the speed with which they responded. a correlation exists between countries that immediately implemented tactics to stop the spread of sars-cov- and the total number of cases reported. having the ability to detect such diseases quickly is imperative to helping mitigate the spread of devastating pathogens. the ability to respond swiftly to changing diagnostic demand is especially critical in the current environment. reopenings present unique challenges, in that the spread of infections must be monitored while individuals resume normal daily activities. low-cost isothermal technologies such as those described in this review provide a uniquely well-suited means of performing large-scale testing, as well as distributed testing, including by end-users (e.g., clia-waived testing in the united states). multiple arenas of interaction exist that possess a unique combination of both ( ) high economic and social benefits associated with reopening and ( ) increased risk of disease spread. these include universities, airports, and essential workplaces in which human contact is difficult, at present, to avoid, such as retail locations and food processing sites. the use of rapid, frequent, distributed tests to determine infectiousness in these arenas provides a powerful potential means of realizing these benefits while simultaneously mitigating associated risks of viral transmission. a new coronavirus associated with human respiratory disease in china viral evolution and the emergence of sars coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia projecting the transmission dynamics of sars-cov- through the postpandemic period signal mediated amplification of rna technology (smart). (i) an extension probe and a template probe bind to the target dna, allowing dna polymerase to extend the extension probe. (ii) a newly synthesized double-stranded t (dst ) rna polymerase promoter region is formed, allowing for rna polymerase to start transcription. (iii) rna polymerase transcribes multiple rna transcripts containing the detectable signal. (iv) for greater sensitivity, a third probe can be added with a region complementary to step iii rna transcripts, allowing for the rna transcripts to bind. 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systems for rapid pathogen detection trueprime is a novel method for whole-genome amplification from single cells based on tthprimpol whole-genome multiple displacement amplification from single cells multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers use of signal-mediated amplification of rna technology (smart) to detect marine cyanophage dna detection of virus mrna within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within synechococcus sp isothermal amplification and quantification of nucleic acids and its use in microsystems clinical validation of a commercial lamp test for ruling out malaria in returning travelers: a prospective diagnostic trial review: a comprehensive summary of a decade development of the recombinase polymerase amplification detection of rotavirus using padlock probes and rolling circle amplification effect of the concentration difference between magnesium ions and total ribonucleotide triphosphates in governing the specificity of t rna polymerase-based rolling circle transcription for quantitative detection improvements of rolling circle amplification (rca) efficiency and accuracy using thermus thermophilus ssb mutant protein label-free pathogen detection by a deoxyribozyme cascade with visual signal readout evaluation of the combination of the nuclisens easymag and the easyq applications for the detection of mycoplasma pneumoniae and chlamydia pneumoniae in respiratory tract specimens isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review isothermal multiple displacement amplification of dna templates in minimally buffered conditions using phi polymerase serologic and molecular biologic methods for sars-associated coronavirus infection serological and molecular findings during sars-cov- infection: the first case study in finland developing a national strategy for serology (antibody testing) in the united states serologic testing for igg antibodies against sars-cov- -insights identifying airborne transmission as the dominant route for the spread of covid- the best global responses to the covid- acs synthetic biology pubs.acs.org/synthbio review key: cord- -ebr rm authors: zhang, qingli; liu, shuang; yang, haolin; zhu, luoluo; wan, xiaoyuan; li, xiaoping; huang, jie title: reverse transcription loop-mediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp date: - - journal: journal of invertebrate pathology doi: . /j.jip. . . sha: doc_id: cord_uid: ebr rm abstract a disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in china and several countries of southeast asia, leading to serious losses in production. litopenaeus vannamei (also known as pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. a new nodavirus, termed covert mortality nodavirus (cmnv), has been shown to be the etiological agent. in this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for the rapid and quantitative detection of cmnv. the optimal conditions for this newly developed rt-lamp reaction were found to be mm mgcl and . mm dntps, an incubation temperature of °c and a reaction time of min. the analytical sensitivity of the rt-lamp assay was estimated to be . pg total rna of cmnv-infected shrimp and copies of the target plasmid. the diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription pcr (rt-pcr) assay was . % and . %, respectively. the reaction products were detected by visual inspection after staining with an in-tube dna fluorescent dye, a measure taken to eliminate the risk of contamination. the quantitative rt-lamp assay for cmnv showed high correlation coefficient (r = . ) when the initial templates were above copies, however the correlation coefficient decreased when the initial templates were lower than copies. test of viral load in shrimp indicated that the viral loads varied from . × to . × copies per mg of cephalothorax tissue. thus, the cmnv rt-lamp assay is a sensitive and specific new tool for the field detection and quantification of cmnv in the diagnosis and surveillance of covert mortality disease. litopenaeus vannamei, also known as pacific white shrimp, is an economically important variety of shrimp. however, production of l. vannamei is limited by its susceptibility to various diseases. in china, covert mortality disease (cmd) has been reported in pond cultures of l. vannamei recently (xu and ji, ; zhang et al., ) . the epidemiology study proved the prevalence of cmd in shrimp ponds in thailand, indonesia, india and ecuador (flegel, ) . typical clinical signs of cmd in the shrimp include hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage (gu, ; huang, ; zhang, ; zhang et al., ) . the symptom of abdominal muscle whitening and necrosis is similar to the symptoms reported in the early stages of disease caused by infectious myonecrosis virus (imnv) and penaeus vannamei nodavirus (pvnv) in the shrimp. however, these diseases can be distinguished by rt-pcr (zhang et al., ) . cmd-related mortality has been found to be sporadic, and may be increased by environmental stress, such as high temperature. when the water temperature rises above °c, survival in infected ponds decreases sharply and the cumulative mortality rate of shrimps infected by cmd has been reported to be up to % (gu, ; xing, ; xu and ji, a range of techniques were employed to identify the etiological agent of cmd including histopathology, virion purification, sequence analysis, experimental infection, and fluorescence in situ hybridization. a new nodavirus, designated as covert mortality nodavirus (cmnv) (zhang et al., ) , was found to be the causative agent of cmd. like other members of the genus alphanodavirus, the genome of cmnv is a positive single-stranded rna. histological examination of the diseased l. vannamei shrimp revealed atrophied, structurally discorded, hepatopancreas tubules and coagulative necrosis of skeletal muscle. eosinophilic inclusions were detected in the hepatopancreas and lymphoid organ (zhang et al., ) . specimens of the main economic species of shrimp in china, fenneropenaeus chinensis (chinese white shrimp) and marsupenaeus japonicus (japanese tiger prawn), were also found to be infected by cmnv in an epidemiological investigation conducted using histological examination and rt-pcr for virus detection (zhang et al., ) . therefore, the risk of a cmnv pandemic in both farmed and wild shrimp is substantial. in an attempt to control the spread of cmnv, rapid and effective detection methods for cmnv are urgently required. in this study, we developed a rapid and sensitive reverse transcription loopmediated isothermal amplification (rt-lamp) method for the detection of cmnv in infected shrimp. the newly developed assay also has the advantage of quantifying cmnv in infected samples. fifty-two living l. vannamei samples, ranging from to g in weight, were obtained from ponds of shrimp farms in hebei, shandong, jiangsu and zhejiang provinces and samples were determined as cmnv positive by using the previously described rt-pcr method (zhang et al., ) . the rest of samples were cmnv negative by the rt-pcr method. in addition, to validate of the newly developed rt-lamp method, experimentally infected shrimp individuals with typical syndrome of cmd were collected. total rna of the shrimp was extracted using the rnaprep tm pure tissue kit (tiangen, beijing, china) following the manufacturer's protocol and was stored at À °c prior to use. for rt-pcr, cmnv cdna was synthesized using the reverse transcription system (promega, madison, wi, usa) and random primers. a partial sequence of the rna-dependent rna polymerase gene in the rna genome of cmnv (genbank accession number km ) was used as a template to design the rt-lamp primer set using the primer explorer software, version . (http://primerexplorer.jp/elamp . . /index.html). the details of the primers are shown in table . the rt-lamp reaction was carried out in a -ll reaction mix- tomita et al. ( ) . the mixture was prepared on ice and then immediately incubated for min (one circle per minute) in a gradient pcr machine at , , or °c, followed by °c for min to terminate the reaction. changes in fluorescence were monitored every min at nm. to determine the optimum concentration of mgcl and dntps, the rt-lamp reaction was optimized by taguchi's l ( ( )) orthogonal design with two elements (dntps and mgcl ) at four concentration levels ( table ). the analytical specificity of the rt-lamp primers was tested under the optimized conditions described above. nucleic acids from other shrimp pathogenic viruses, such as macrobrachium rosenbergii nodavirus (mrnv), taura syndrome virus (tsv), yellow head virus (yhv) and white spot syndrome virus (wssv), were used in this analysis. alignment of the target gene sequence of bp of cmnv against the corresponding sequences of pvnv (genbank accession number hq . ) and mrnv (genbank accession number fj . ) was performed for comparison using the software of bioedit . and blast. the detection limit of the rt-lamp assay was analyzed using two different templates: ( ) the total rna extracted from the shrimp infected by cmnv, ( ) a plasmid vector (pmd -t) containing the target fragment from the rna-dependent rna polymerase gene of cmnv (designated pmd -t-cmnv). a -fold serial dilution of the total rna ( . pg  - À ) from cmnv-infected shrimp and plasmid pmd -t-cmnv ( .  - copies) was used as a template for rt-lamp under the predetermined conditions. the validity of the cmnv rt-lamp was determined by testing clinical samples, including: ( ) cephalothoraxes tissue samples collected from spontaneously infected l. vannamei from shrimp farms in , ( ) cephalothoraxes tissue samples collected from artificially infected l. vannamei in the laboratory. total rna of the samples was extracted and the optimized cmnv rt-lamp was performed as described in section . . rt-pcr assays were also performed on the same total rna samples according to zhang et al. ( ) . the diagnostic sensitivity (dse) and diagnostic specificity (dsp) of the two methods, as defined by the world organization for animal health ( ), were calculated according to zhang et al. ( ) . the cmnv rt-lamp was performed according to the optimal protocol described in section . , except that calcein and mncl were omitted. one microliter of fluorescent dye genefinder tm (bio-v, xiamen, china) was sealed into the cap of the reaction tube in advance using paraffin wax. at the end of the reaction, fluorescence development was initiated by incubating the tube at °c for min and then immediately mixing the genefinder tm with the reaction mixture. different fluorescent colors appeared in the reaction mixtures of the positive and negative samples. to determine viral load in the shrimp tissues, the rna extracted from the cephalothorax of the shrimp clinical samples described in section . was amplified by quantitative rt-lamp (qrt-lamp). the rt-lamp assay was quantified using -fold dilutions of cmnv plasmid (pmd -t-cmnv) as standards, in a final reaction volume of ll. for real-time monitoring, the qrt-lamp reactions were incubated at °c for cycles ( min per cycle) with a cfx connect tm real-time pcr detection system (bio-rad, foster city, ca, usa). for cmnv quantitative detection of samples, a standard curve was generated for cmnv qrt-lamp by plotting a graph between different concentrations of pmd -t-cmnv plasmids, ranging from to copy numbers, to cycle threshold (ct) values, obtained through real-time monitoring of the amplification. rt-lamp reactions were performed using mm mgcl and . mm dntps for min at , , , or °c to determine the optical reaction temperature. the results showed that the amplification occurred earlier when the reaction was incubated at °c compared with the other incubation temperatures (fig. a) . experiments to optimize the concentrations of mgcl and dntps showed that the smallest average ct value ( . ) was generated when the concentrations of mgcl and dntps were . mm and . mm, respectively. however, the smallest average ct value was accompanied by a standard error of . , indicating substantial fluctuations in amplification efficiency. meanwhile, the third smallest ct value ( . ), with the smallest standard error of . , was obtained when the concentrations of mgcl and dntps were . mm and . mm, respectively. therefore, the optimal concentrations of mgcl and dntps were determined to be . mm and . mm, respectively. the real-time kinetics of the rt-lamp reaction, with or without loop primers, was analyzed with . mm of mgcl , . mm of dntps and incubation at °c. the results indi-cated that the time required for initiation of amplification was . min with loop primers or . min without loop primers, which indicated that using loop primers accelerated the amplification, thereby reducing the detection time, compared with rt-lamp without loop primers. based on these findings, further rt-lamp assays were incubated for min at °c with mm mgcl and . mm dntps. alignment of the cmnv-lamp target sequence ( bp) with the corresponding sequences from the closely related viruses, pvnv and mrnv, indicated that the eight cmnv rt-lamp primers covered or more mutation sites in the corresponding sequences of pvnv and mrnv (fig. ) . amplification only occurred when the template was the rna from cmnv; no amplification was observed when the template was the nucleic acid from mrnv, yhv, tsv or wssv (fig. b) . furthermore, both blast and embl searches revealed that the cmnv-lamp target sequence did not align with any other viral sequences available in these databases, including all members of the nodaviridae, such as the flock house virus (fhv), black beetle virus (bbv), mrnv and pvnv. taken together, these results indicate that the rt-lamp primer set is specific for amplification of cmnv nucleic acid. the lowest detection limit of the newly developed cmnv rt-lamp method was . pg of total rna when the reactions were tested using ll of -fold serially diluted rna from shrimp artificially infected with cmnv (fig. a) . when the reaction was tested using ll of -fold serially diluted pmd -t-cmnv dna ( . ng/ll equivalent to .  copies/ll), the analytical sensitivity of the rt-lamp method was estimated to be as low as copies of the plasmid (fig. b) . rna from clinical samples was used to compare the newly developed rt-lamp assay with conventional rt-pcr methods. rt-pcr results indicated that of the samples were positive for cmnv. the rt-lamp assay showed that samples from the cmnv-positive samples determined by rt-pcr gave positive results. moreover, two of the samples that were cmnv negative by rt-pcr yielded a positive result with the rt-lamp assay (table ) . therefore, the dse and dsp values for the rt-lamp method compared with the rt-pcr method were . % and . %, respectively. visual inspection of cmnv rt-lamp products was performed by adding a fluorescent dye to the reaction mixture. green fluorescence was observed clearly with the naked eye in the reaction tubes of the cmnv-infected shrimp and the positive control, whereas an orange color was observed in the tubes of the negative control and the cmnv-free shrimp (fig. ) . a high correlation coefficient (r = . ) was obtained for the cmnv quantitative rt-lamp (qrt-lamp) assay when the initial template was above copies (fig. ) . quantitation of viral copies in the cephalothorax of the clinical samples was calculated based on the ct value of all rna samples using the generated . sensitivity of the rt-lamp assay for the detection of cmnv rna. a. amplification plots - (from left to right), reaction conducted using -fold serial dilutions of rna from shrimp infected by cmnv: .  , .  , .  , .  , .  , . and . pg of rna, respectively. b. sensitivity of rt-lamp detection of pmd -t-cmnv plasmid containing the target dna fragments (the rna-dependent rna polymerase gene of cmnv). amplification plots - (from left to right), reaction conducted using -fold serial dilutions of the plasmid (pmd -t-cmnv): .  , .  , .  , .  and . plasmid copies, respectively. calcein indicated the fluorescent indicator used in the qrt-lamp assay. standard curve. the viral loads of the positive clinical samples varied within the range .  - .  copy number per mg of tissue. the optimal reaction temperature was determined to be °c and the optimal concentration of mgcl and dntps was determined to be mm and . mm, respectively. these conditions are similar to those reported for lamp assays for the detection of other organisms such as hepatitis b virus and the toxic dinoflagellate alexandrium (wang et al., ) . the results of the temperature optimization showed that bst dna polymerase effectively amplified the nucleic acid templates at temperatures from to °c, which is consistent with other classic lamp assays (mori et al., (mori et al., , notomi et al., ) . the effectiveness of this assay across a wide temperature range would greatly benefit the future application of this method under field conditions. with regard to reaction time, when ng of rna (approximately copies of the virus, as determined by quantitative rt-lamp) from cmnv-infected shrimp was used as template, initiation of amplification took . min with loop primers and saved . min comparing with the reaction without loop primers, indicating that the presence of loop primers accelerated the lamp reaction (nagamine et al., ; parida et al., ; yang et al., ) . the development of a rapid rt-lamp assay for cmdv detection would be advantageous in the field. to develop a detection assay with high specificity for the target species, it is important that the primers employed do not cross react with other viruses. in this study, we needed to ensure that the primers used for the rt-lamp assay allowed for the detection of cmnv but did not cross react with other shrimp virus species, especially with other members of the genus alphanodavirus. the -bp target sequence using for primer designing in this study shared no similarity with any other viral sequences available in the genbank database. specificity testing confirmed that amplification only occurred when nucleic acid from cmnv was used as template; the assay developed in this study can therefore be used for the specific detection of cmnv. the analytical sensitivities of the rt-lamp method were . pg of rna from cmnv-infected shrimp. this degree of analytical sensitivity is similar to that reported by hirayama et al. ( ) for sexing water buffalo, by chen et al. ( ) for the detection of swine-transmissible gastroenteritis coronavirus and by li and ling ( ) for the detection of tomato necrotic stunt virus. in comparison to the reference method of nested rt-pcr, the dse and dsp values for the cmnv rt-lamp assay were . % and . %, respectively. one spontaneously infected l. vannamei sample was determined as negative of cmnv by rt-pcr; however it showed positive of cmnv by qrt-lamp and was quantified as .  viral copies. the inconsistent result tested by rt-pcr and qrt-lamp might attribute to pcr inhibitors that could affect the taq dna polymerase used in conventional rt-pcr (alhassan et al., ; bakheit et al., ; liang et al., ) . in contrast, such inhibitors may not affect the bst polymerase used in lamp (enosawa et al., ; alhassan et al., ) . this characteristic of bst polymerase makes it be appropriate for field application. for visual inspection of the cmnv rt-lamp assay result and to eliminate the risk of contamination caused by reopening the reaction tube, a method of dna fluorescent dye pre-setting was developed in the study. at the end of the reaction, the dye was incorporated into the reaction mixture by shaking, allowing for the visual inspection of the rt-lamp products. the use of a fluorescent dye can therefore substitute for gel electrophoresis or realtime pcr as a method of viral detection, simplifying the assay and reducing the contamination risk. a standard curve was constructed using -fold serial dilutions of the pmd -t-cmnv plasmid with reference to the ct value. based on the standard curve, an equation was calculated using regression analysis comparing ct values with the standard copy number. in the range of - plasmid copies, the correlation coefficient was high (r = . ), which indicates that qrt-lamp is appropriate as a quantitation tool. however, when the plasmid copy number decreased to less than copies, the correlation coefficient also decreased significantly, which confirmed the findings of previous reports that it is difficult to determine the exact correlation of the initial template quantity and the ct value at very low concentrations of template (mori et al., ; suzuki et al., ; wei et al., ) . in summary, this report describes a rapid, highly sensitive, highly specific, financially economical, quantitative rt-lamp method for cmnv detection. this assay could therefore become the assay of choice for the routine detection and quantification of cmnv in the laboratory and in the field. which was sealed into the cap of the tube in advance using paraffin wax. negative reactions appeared orange and positive reactions appeared green in daylight against a black background. tubes and : samples from spontaneously infected shrimps; tubes and : samples from shrimps without signs of cmd; pc: positive control (pmd -t-cmnv plasmid); nc: negative control (sterile water). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) table the comparison of detection results of the newly developed rt-lamp and rt-pcr. rt-pcr positive rt-pcr negative total cmnv-rt-lamp positive cmnv-rt-lamp negative total development of loop-mediated isothermal amplification (lamp) method for diagnosis of equine piroplasmosis sensitive and specific detection of cryptosporidium species in pcr-negative samples by loop-mediated isothermal dna amplification and confirmation of generated lamp products by sequencing detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification use of loop-mediated isothermal amplification of the is sequence for rapid detection of cultured mycobacterium avium subsp. paratuberculosis ems/ahpnd: a game changer for the future development of aquaculture analysis of causes of the covert mortality disease of pacific white shrimp and its control strategies rapid sexing of water buffalo (bubalus bubalis) embryos using loop-mediated isothermal amplification the asia pacific emergency regional consultation on the emerging shrimp disease: early mortality syndrome (ems)/acute hepatopancreatic necrosis syndrome (ahpns) development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus development of loop-mediated isothermal amplification assay for detection of entamoeba histolytica real-time turbidimetry of lamp reaction for quantifying template dna development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation loop-mediated isothermal amplification reaction using a nondenatured template accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus comparison of real-time reverse transcription loopmediated isothermal amplification and real-time reverse transcription polymerase chain reaction for detection of noroviruses in municipal wastewater loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products loop-mediated isothermal amplification method for rapid detection of the toxic dinoflagellate alexandrium, which causes algal blooms and poisoning of shellfish a novel method of real-time reverse transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus principles and methods of validation of diagnostic assays for infectious diseases discussion of the control measures for the ''bottom death" (covert mortality disease) of pacific white shrimp comprehensive control of the covert mortality disease of pacific white shrimp development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of porcine cytomegalovirus under field conditions to be cautious of ''bottom death" in the intensive farming of pacific white shrimp a new nodavirus is associated with covert mortality disease of shrimp development of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of grass carp reovirus this work was supported by the following projects: special scientific research funds for central non-profit institutes, the chinese academy of fishery sciences ( a and a xk ), the special fund for agro-scientific research in the public interest (grant: ), the china agriculture research system (cars- ), the construction programme for ''taishan scholarship" of shandong province of china, and the programme for chinese outstanding talents in agricultural scientific research. key: cord- -qt jhg authors: lakshmi, vemu; neeraja, mamidi; subbalaxmi, m. v. s.; parida, m. m.; dash, p. k.; santhosh, s. r.; rao, p. v. l. title: clinical features and molecular diagnosis of chikungunya fever from south india date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: qt jhg an epidemic of chikungunya fever of unprecedented magnitude occurred in many parts of india in early after an interval of years, and there has been a resurgence in some parts of south india since june . the article highlights clinical manifestations of infection and various molecular tests that were used for diagnoses of chikungunya virus infection. of particular interest is the real-time loop-mediated isothermal amplification (rt lamp) assay, which is rapid and cost-effective and can be adopted at ill-equipped laboratories. clinical symptoms were characterized by a triad of fever, rash, and severe rheumatic manifestations. rt lamp identified additional chikungunya virus—positive cases, compared with reverse-transcriptase polymerase chain reaction. chikungunya virus was isolated from randomly selected samples. genotyping of the virus isolates revealed that the east central south african genotype of chikungunya virus was the etiologic agent of this epidemic. molecular diagnosis is an important tool to identify such new vectorborne viral illnesses. emerging viral infections have become a serious problem in recent years. emergence or reemergence of severe arboviral hemorrhagic fevers caused by mosquitoborne viruses, such as dengue virus and chikungunya (chik) virus, have been frequently reported in the indian subcontinent in the past few years. from the clinical perspective, these infections have similar clinical manifestations and are difficul to distinguish from one another. because the outcomes of these infections vary on the basis of the infecting agent (dengue has a high mortality rate), they pose a diagnostic dilemma for the clinician. therefore, there is a need for a means of definitiv diagnosis and identificatio of the viral agent to prognosticate the outcome. the causative agent chik virus, a single-stranded, positive sense rna, enveloped virus, is a member of the genus alphavirus of the togaviridae family. it is generally transmitted from primates to humans via aedes aegypti and aedes albopictus mosquitoes [ , ] . chik infection produces a self-limiting illness in humans that is often characterized by sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia, and severe and very painful polyarthralgia, which lasts for - days. however, arthralgia may persist for months to years [ , ] . as is the case for most alphaviruses, detection of chik virus depends on isolation of the virus in blood specimens obtained from viremic patients or in infected tissue specimens obtained from blood-feeding arthropods, which are time-consuming. molecular diagnostic tools, such as the conventional rt-pcr, are available for the study of chik virus replication in virus culture supernatants or clinical samples [ , ] . we report clinical observations and laboratory investigations involving virus isolation methods and molecular assays performed for clinically suspected cases of chik fever. of particular interest was the applicability of a novel method of gene amplificatio called real-time loop-mediated isothermal amplifica tion (rt-lamp) as a rapid, sensitive, and specifi real-time method to detect and quantify chik virus in the acute phase of the infection. the study included patients with a history of sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia, and severe and very painful polyarthralgia suggestive of chik infection. patients either reported directly or were referred to nizam's institute of medical sciences (hyderabad, india) for treatment from regions in and around hyderabad, andhra pradesh, south india, during the period march-december . there was no sampling bias or any attempt to specially recruit patients. the study was approved by the institutional ethics committee of nizam's institute of medical sciences (ec/ nims/ / ). written informed consent was obtained from each patient. acute-phase serum samples were obtained during days - after the onset of symptoms. two sets of whole-blood specimens were collected from all patients. one set was used for virus isolation, which was conducted using molecular assays with vacutainer edta tubes (bd biosciences); the other set underwent elisa with sst vacutainer tubes (bd biosciences). plasma and serum specimens were aliquoted in sterile vials and stored at Ϫ Њc at the department of microbiology department at nizam's institute of medical sciences until testing. the samples were transported under cold chain to the virology laboratory at the defense and research establishment (gwalior, india), where all the assays and virus isolations were performed. serologic testing. sixty-fiv of patients reported for follow-up. serum specimens obtained from these patients were tested for the presence of chik virus-specifi igm and igg antibodies using an in-house dipstick elisa kit [ ] . because symptoms of chik fever mimic those of dengue fever, a panel of of serum samples obtained from patients with clinical features similar to those of chik or dengue fever was included in the study. in addition, a panel of serum samples obtained from healthy individuals without any signs and symptoms of chik or dengue fever was included as a negative control. virus isolation. virus isolation was attempted in c / cell lines from rt-pcr-positive plasma samples that were randomly selected during the outbreak. virus isolation was performed using the virus adsorption technique [ ] . in brief, a confluen monolayer of cells grown in a -cm culture flas was adsorbed with . ml of inoculum at Њc for h. after adsorption, the inoculum was replenished with ml of maintenance medium supplemented with % fetal bovine serum. suitable mock-infected cell controls were also incubated for comparison of cytopathic events. cells were incubated at Њc and were observed daily for cytopathic effects. after observation of %- % cytopathic effects, the infected culture supernatant was clarifie by light centrifugation at rpm for min, which was further purifie by sucrose gradient ultracentrifugation. the isolated virus was confi med to be chik virus by rt-pcr. molecular assays. all plasma samples were tested for the presence of chik virus-specifi rna by rt-pcr and rt-lamp. positive and negative controls were included in each run of the assays, and all precautions to prevent cross-contamination were observed. for rt-pcr, rna was extracted using the qiaamp viral rna mini kit (qiagen). one-step rt-pcr was performed using the access quick rt-pcr kit (promega), in accordance with the manufacturer's protocol, employing primer pairs targeting the e gene designed from the nucleotide sequence of the reference s strain (genbank accession number af ; ck , tta cat cac gtg cga ta c; ck- , ctt tc tct cag gg tgc gac ttt). the amplificatio was performed in a -ml total reaction volume with the promega access quick one-step rt-pcr kit, with pmol of each forward and reverse primer and ml of extracted viral rna, in accordance with the manufacturer's instructions. the thermal profil of rt-pcr was Њc for min and Њc for min, followed by cycles of Њc for s, Њc for s, and Њc for s and a fina extension at Њc for min. rt-lamp was performed at a total -ml reaction volume using the loopamp rna amplificatio kit (eiken chemical). the real-time monitoring was accomplished by incubating at Њc for min in a loopamp real-time turbidimeter (la- ; teramecs). real-time monitoring of the rt-lamp amplificatio of chik virus template was observed through spectrophotometric analysis by recording the optical density at nm every s, with the aid of the loopamp real-time turbidimeter (la- ; teramecs). the cutoff value for positivity for the real-time rt-lamp assay was determined by taking into account the time of positivity (in min), at which point the turbidity increases to more than the threshold value (which was fixe at . , which is times more than the mean turbidity value for the negative controls of several replicates). after incubation at Њc for min, -ml aliquots of rt-lamp products were examined by electrophoresis on % nusieve : agarose gel (bma) in tris-borate buffer, followed by staining with ethidium bromide and visualization on a uv transilluminator at nm. to facilitate the fiel application of the rt-lamp assay, the monitoring of rt-lamp amplificatio was also performed with naked-eye inspection. after amplification tubes were inspected for white turbidity by the naked eye after a pulse spin to deposit the precipitate in the bottom of the tube. the inspection for amplificatio was also performed by observing a color change after the addition of ml of sybr green i dye to the tube. positive amplificatio is indicated by green fluo escence, which is permanent and which can be stored for recording purposes [ ] . the rt-pcr-positive amplicons were subjected to doublestranded sequencing with big dye terminator cycle sequencing ready reaction kit on an abi sequencer (applied biosystems). the genotype of the chik virus, based on the partial e gene sequence, was determined by nucleotide sequencing and compared with other globally diverse chik isolates. a dendrogram was constructed by pair-wise comparison of nucleotide sequences of partial e gene (positions - , with respect to the s genome), which classifie all isolates into different genotypes. the phylogenetic tree was constructed with the neighbor-joining method, with a bootstrap analysis of replicates, using mega software, version . [ ] . the chik fever epidemic affected male and female patients at a ratio of : . . the most affected age group was persons aged - years (figu e ). during the acute phase of infection, which lasted for - days, the most common symptoms among patients were fever (temperature, . Њc- Њc) and severe arthralgia and arthritis, which affected the fingers wrists, toes, ankles, and knee joints (table ). the chronic phase of infection was characterized by severe joint pain, which severely limited the patients' ability to walk and perform everyday tasks. almost % of cases reported experiencing prolonged arthralgia (duration, weeks). chik infection has an important economic impact in many tropical countries, and because of the lack of specifi symptoms, the infection cannot be differentiated from dengue or yellow fever [ , ] . a chik fever epidemic is characterized by its sudden disappearance for a considerably long period from a particular geographic area before its resurgence. this has been well documented in the republic of the congo and indonesia. however, in early , a major epidemic of chik fever started in many indian ocean island nations, and in late , it started spreading to several parts of india, after a hiatus of epidemic activity of nearly years. this recent outbreak of chik infection in many parts of southern india is a point of major concern. this was the largest and most severe epidemic, affecting , , persons in andhra pradesh, maharastra, and karnataka states of southern india and spreading to several new areas, with huge public health and administrative concerns to control the epidemic [ ] . although the resurgence of chik fever was anticipated, this epidemic is considered to be unprecedented, owing to the magnitude of morbidity and geographical distribution. in several villages, % of inhabitants were found to be affected [ ] , with an estimated figu e of . million cases to date [ ] . in , outbreaks of chik fever were reported from districts in states across india. the clinical manifestations of cases in the current outbreak match the known description of the disease. analysis of the outbreak suggested that the increased severity of disease may have been associated with a change in the genetic sequence, altering the virus coat protein, which potentially allowed the virus to multiply more easily in mosquito cells [ ] . laboratory diagnosis is critical to establish the diagnosis and to initiate a specifi public health response. the alphavirus species can be characterized by hemagglutination inhibition, elisa, complement fixation and neutralization of viral infectivity using reference serum samples [ , ] . serodiagnosis rests on demonstrating a -fold increase in chik virus igg antibody titer between the acute-and convalescentphase serum samples. because obtainment of paired serum samples is usually not practical, the demonstration of igm antibodies specifi for chik virus in acute-phase serum specimens is done. a chik-positive viral culture, coupled with neutralization by reference serum, is taken to be definitiv proof of the presence of chik virus. pcr results for e and c genome either singly or together constitute a positive result for chik virus [ ] . in the present study, the detection of chik virus rna in . % of samples by rt-pcr and in . % of samples by rt-lamp, as well as the detection of igm antibodies in . % of samples, confi med that the causative agent of this epidemic was chik virus. all patients who had clinically suspected chik virus but whose rt-pcr and rt-lamp results were negative presented days after the onset of fever; this may be the reason for the negative test results. the peak number of positive pcr results occurred on day of illness (figu e ). the rt lamp assay is a novel nucleic acid amplificatio method developed by eiken chemical that has the potential to replace pcr because of its simplicity, rapidity, specificit , and cost-effectiveness [ ] [ ] [ ] . the rt-lamp assay has emerged as a powerful gene amplificatio tool for rapid identificatio of microbial infections and is being increasingly used by various investigators for rapid detection and typing of emerging viruses, such as the west nile, severe acute respiratory syndrome, dengue, and japanese encephalitis viruses [ ] [ ] [ ] . the lamp method is cost-effective, because it requires only type of dna polymerase with strand displacement activity. in the present study, the -step, single-tube, real-time accelerated rt-lamp assay was standardized by targeting the immunodominant e gene for rapid and real-time detection of chik virus. in our study, the rt-lamp assay uncovered additional cases of infection, which were detected by naked eye or with a uv lamp. these samples had negative rt-pcr results, thereby proving the high sensitivity of rt-lamp. there were a few mismatches in the rt-pcr primers (i.e., base in the forward primer and bases in the reverse primer), but they did not affect the test's sensitivity [ ] . all of the rt-pcr-positive samples also yielded positive rt-lamp results. the rt-lamp allows rapid, realtime detection of chik virus in acute-phase serum samples, without requiring sophisticated equipment, and has potential usefulness for clinical diagnosis and surveillance of chik virus in developing countries. real-time quantificatio of the chik virus level can also be performed by rt-lamp with use of a loop turbidimeter. isolation of chik virus from of rt-pcr-positive clinical samples further confi med that the infection was due to chik virus. previous phylogenetic studies showed that strains of chik virus were clustered into distinct groups on the basis of origin: west africa, central/south africa, and asia [ ] . the sequence of chik virus was directly determined from clinical samples without risk of altering the genome by in vitro passaging. mo-lecular phylogenetic analysis revealed that all of these new indian chik virus strains were very closely related to analogous strains from indian ocean island nations and that they formed a distinct clade in ecs african genotype. these new strains are reported to harbor many unique molecular features, making them more virulent and evolutionarily more competent [ ] . the clustering of older indian isolates (including the last outbreak isolates) into the asian genotype indicates that the cause of this outbreak was the sudden introduction of the ecs genotype into the telangana region of andhra pradesh in southern india, rather than in situ evolution of existing strains. because the period of our study was march-december , which is later than the period studied by prasanna et al. [ ] and abu barkar et al. [ ] , to the best of our knowledge (after an extensive search of the pubmed database), we submit that this is the firs report of ecs african genotype of chik virus as the causative agent of an unprecedented epidemic. a clustering of cases among members of the same family ( %) was observed in our study, which is in concordance with the findin that direct human-to-human transmission can occur as a consequence of high viral loads in patients, as was demonstrated in southern france [ ] . although the viremia is transient, the concentration of virus is sufficien to infect feeding vector mosquitoes [ ] . that the rt-pcr and rt-lamp results were positive during the period of peak viral load (i.e., days and of fever) also confi ms this fact. chik infection is a self-limited illness, with joint symptoms and signs usually lasting for months and occasionally for у year [ ] . however, deaths due to chik infection are rare [ ] . indiscriminate use of antibiotics and nonsteroidal anti-inflam matory drugs (especially aspirin) can contribute to thrombocytopenia, gastrointestinal bleeding, and vomiting. this may lead to prerenal acute renal failure and dehydration. these can indirectly contribute to mortality due to chik fever [ ] . a high morbidity rate with no mortality was noted in our study. this is in contrast to the finding from kerala, southern india, where the cause of death for patients with chik fever was probably an underlying illness and was not related to chik infection [ ] . young adults (age, - years) were the most affected persons, and there was a preponderance of female patients in our study ( , by fisher's exact test); these finding are in p ! . concordance with the finding of the study from reunion island [ ] . some patients ( . %) were so disabled that they required hospitalization, but the number of such cases was negligible, and such cases mostly involved elderly patients. the chronic phase of disease resolved over a few months, extending up to months. some patients improved after receiving short-term steroid treatment. because no specifi antiviral therapy exists for chik infection, treatment consists of supportive care, including administration of analgesics and anti-inflammato y medication for joint symptoms. persons with febrile illness that is suspected to be due to chik virus should avoid mosquito exposure for at least days after the onset of illness, to reduce the likelihood of transmitting chik virus to local mosquitoes, which might then transmit the virus to other humans [ ] . although the number of cases being reported has decreased, this epidemic may still be continuing and spreading. therefore, continuous surveillance is warranted to monitor the spread of infection and to track the possible evolution of the virus during the epidemic. the natural history of chik fever is not fully understood. although mortality is rare or infrequent, early diagnosis and vector control will play an important role in preventing the outbreak of epidemics in the future. intraoutbreak studies point toward recent changes in the viral genome that have facilitated the rapid spread and enhanced pathogenecity of infection [ ] . the lack of herd immunity, as evidenced from available studies [ , ] , appears to be the simplest attributable factor. also, the reasons for the current outbreak and the causes behind reemergence of the virus in india have to be further assessed. molecular diagnosis is an important tool to identify new vectorborne viral illnesses, such as chik fever, at an early stage. public health measures need to be improved to prevent such epidemics in the future. there is also an immediate need for an effective vaccine for chik infection. the arboviruses: epidemiology and ecology complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site the alpha viruses combined detection and genotyping of chikungunya virus by specifi reverse transcriptionpolymerase chain reaction detection of west nile and japanese encephalitis viral genome sequences in cerebrospinal flui from acute encephalitis cases in karachi genome microevolution of chikungunya viruses causing the indian ocean outbreak rapid and real-time detection of chikungunya virus by reverse transcription loop-mediated isothermal amplificatio assay molecular evolutionary genetics (mega) software development of monoclonal antibody based antigen capture elisa to detect chikungunya virus antigen in mosquitoes re-emergence of chikungunya virus in india failure to control mosquitoes has led to two fever epidemics in india chikungunya doctor ndtv health information on chikungunya communicable diseases branch. chikungunya fever, laboratory diagnosis of chikungunya fevers. geneva: world health organization detection of loop mediated isothermal amplificatio reaction by turbidity derived from magnesium pyrophosphate formation accelerated reaction by loop mediated isothermal amplificatio using loop primers loop-mediated isothermal amplificatio of dna development and evaluation of a novel loop mediated isothermal amplificatio method for rapid detection of severe acute respiratory syndrome corona virus real-time reverse transcription loop mediated isothermal amplificatio for rapid detection of west nile virus evaluation of a dipstick elisa and a rapid immunochromatographic test for diagnosis of dengue virus infection east central south african genotype as the causative agent in reemergence of chikungunya outbreak in india novel chikungunya virus variant in travelers returning from indian ocean islands genome microevolution of chikungunya viruses causing the indian ocean outbreak chikungunya outbreaks caused by african genotype reemergence of endemic chikungunya update: chikungunya fever diagnosed among international travelers-united states chikungunya fever: clinical manifestation and management infectious diseases surveillance update chikungunya outbreak in reunion: epidemiology and surveillance emergence of chikungunya virus in indian subcontinent after years: a review serological survey in madras city with special reference to chikungunya serosurvey of chikungunya antibody in calcutta metropolis potential conflict of interest. all authors: no conflicts key: cord- - io zsm authors: sidoti, francesca; bergallo, massimiliano; costa, cristina; cavallo, rossana title: alternative molecular tests for virological diagnosis date: - - journal: mol biotechnol doi: . /s - - - sha: doc_id: cord_uid: io zsm several nucleic acid amplification techniques (naats), particularly pcr and real-time pcr, are currently used in the routine clinical laboratories. such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. however, conventional pcr methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. a new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. the main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. in this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional naats. at present, a wide variety of diagnostic techniques are applied for the detection of viral pathogens. traditional diagnostic methods, like virus isolation and serology, have been the mainstay of the clinical laboratory, especially in the past two decades. in recent years, several previously unknown viral pathogens have been discovered for which classical culture is unrealized or even lacks sensitivity. to overcome the shortcomings of the traditional diagnostic methods, molecular techniques have been developed. several nucleic acid amplification techniques (naats), particularly pcr and real-time pcr, are currently used in the routine clinical laboratories. such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. moreover, naats have offered additional advantages over traditional methods by production of easily standardized protocols, thus resulting a potential for automatization with a range of options for real-time detection chemistries. the advent of fully automated systems with faster turnaround times has given clinical laboratories the tools necessary to report out accurate and sensitive results to clinicians. however, all these in vitro nucleic acid amplification methods have several intrinsic disadvantages, such as the requirement for precision thermal cycling between three temperatures during the reaction and an elaborate method for detection of amplified products. moreover, real-time pcr machines are very expensive requiring an instrumentation platform that consists of a thermal cycler, computer, optics for fluorescence excitation, emission collection, data acquisition, and analysis software. in this context, a new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction, and easy detection. these new techniques do not require thermal cycler and can be performed simply by using a heating block and/or water bath with a low-energy consumption. the main isothermal methods reviewed here include loop-mediated isothermal amplification (lamp), nucleic acid sequence-based amplification (nasba), and helicase-dependent amplification (hda). moreover, in this review, design criteria, potential of amplification, and application of these alternative molecular tests in the detection of viral pathogens will be discussed. lamp represents today a better innovative nucleic acid amplification method which exceeds the classical pcr in its reaction simplicity, accuracy, and higher amplification efficiency. the whole procedure is very rapid and the nucleic acid amplification can be completed in less than h under isothermal conditions. the main advantage of the lamp technique is that it does not require thermocyclers and the amplification can be performed simply with a water bath or heating block necessary to maintain the required temperature. moreover, the design of lamp assay is very simple requiring only the dna polymerase along with dntps, reaction buffer, and two sets of specially primers that can be developed using the free software primer explore (lamp primer designing support software program, net laboratory, japan, http://venus.netlaboratory. com). the addition of reverse transcriptase make it possible to amplify cdna from rna sequences (rt-lamp). lamp is a one-step amplification reaction that amplifies target dna from a few copies to - copies and proceeds at isothermal conditions for h or less depending on the efficiency of the designed primers. lamp employs a dna polymerase with strand displacement activity (bst dna polymerase), along with two internal primers (fip, bip), and two outer primers (f , b ) which recognize six different sequences in the dna template, by incubating all the reagents in a single tube at a constant temperature, usually °c which is optimum for the activity of dna polymerase (fig. ) . the chemistry of lamp amplification is based on the principle of strand displacement reaction which has been described thoroughly by notomi et al. [ ] . in particular, the mechanism of the reaction can be explained in three steps, an initial non-cyclic step, a cyclic amplification step, and an elongation step. an animation that is useful for better understanding of the principle is available at the web site http://loopamp.eiken.co.jp/e/ index.html. the addition of a primer set that anneals at the loop structure in lamp amplicons enhances specificity of the reaction and accelerates further the amplification time [ ] . in particular, using these specific primers, named loop-primers (lf, lb), the reaction time is reduced by half, making it a more efficient tool used in the practical applications of lamp. moreover, the employment of reverse transcriptase in addition to dna polymerase allows the synthesis of cdna molecules from rna template. reverse transcriptase is added to the reaction mixture and, after mixing and incubating at a constant temperature between and °c, amplification and detection can be carried out in a single step (rt-lamp). as concerns the visualization of amplified product obtained from lamp reaction, several methods may be used. firstly, product is visualized by agarose gel analysis stained with an intercalating agent such as ethidium bromide or sybr green i using a common uv transilluminator. as the product of the lamp is a mixture of different length dna fragments, the gel will show several bands which will appear as a smear. another method, based on real-time turbidity measurement, allows to quantify the amount of dna template formed by lamp amplification. the increase of turbidity in the reaction mixture is directly proportional to the amount of dna synthesized. precisely, the lamp method yields large amounts of pyrophosphate ions in the course of the amplification reaction leading to a white precipitate of insoluble magnesium pyrophosphate in the reaction mixture. since the production of precipitate correlates with the increase of turbidity, real-time monitoring of the lamp reaction kinetics can be achieved by measurement of turbidity using an inexpensive turbidimeter. gene copy number can also be quantified by using a standard curve obtained from different concentrations of gene copy number plotted against time of positivity. finally, a new detection method of amplified products has been developed [ ] . this method uses fluorescent intercalating dye, like calcein, the fluorescence of which is quenched by the binding of manganese ions bound by pyrophosphate ions produced in the course of the amplification reaction. the presence of fluorescence indicates the presence of dna template and a simple visual detection can be achieved by using an uv lamp. recently, lamp products have also been detected electrochemically in a microchip [ ] . based on these assumptions, it is possible to make a number of considerations. lamp assay is more specific towards the template sequences than classical pcr. this is caused because four primers recognize six separate regions within a target dna and the amplification reaction occurs only when all these six regions are correctly recognized by the primers. furthermore, lamp is more sensitive than conventional dna-based detection systems and its ability to amplify from fewer copies of initial target dna than pcr has been demonstrated [ ] [ ] [ ] [ ] . in particular, the lamp assay was found to be -to -fold more sensitive than pcr with a detection limit of . - pfu of virus [ ] [ ] [ ] . the development of lamp assay is very simple and allows the use of cost-effective reaction equipment. the simplicity of this method comes from the facility of designing primers and from the fact that only the dna polymerase along with dntps, reaction buffer, and a common water bath or heating block are necessary for the development of lamp assay. moreover, lamp has higher amplification efficiency compared with the pcr, with dna being amplified - times. this high amplification efficiency is attributed to no time loss of thermal change because of its isothermal reaction. finally, rt-lamp assay demonstrated faster in comparison to conventional rt-pcr ( min vs - h), because no additional reverse transcriptase step is required. a survey of the literature shows that the lamp has already been applied to detect many kinds of pathogens including viruses and bacteria [ ] [ ] [ ] . in particular, the lamp method has been developed for most emerging human viral pathogens like west nile, dengue, chikungunya, japanese encephalitis, sars, highly pathogenic avian influenza (hpai) h n , and norwalk viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . rt-lamp assays for rapid detection of several respiratory viruses as influenza a and b virus, measles virus, and mumps virus have also been evaluated [ ] [ ] [ ] [ ] [ ] . moreover, the usefulness of lamp for amplification of dna viruses was also reported for cytomegalovirus, herpes simplex virus, varicella zoster virus, human herpes virus - , adenovirus, bk virus, and human papilloma virus type- , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the lamp technology has now been developed into commercially available detection kits and some of them have been adopted as the officially recommended methods for detecting various pathogens. lamp kits for the detection of escherichia coli, mycobacterium, salmonella, legionella, vibrio cholerae, listeria, campylobacter, and criptosporidium have been commercialized [ ] [ ] [ ] [ ] . considering the advantages of rapid amplification, and easy detection, the current focus of lamp methodology is towards a simple diagnostic tool to be routinely employed in resource-limited laboratories in developing countries where many fatal tropical diseases are endemic, without requiring sophisticated equipment or skilled personnel. however, the combination of lamp methodology and innovative microchip technologies may facilitate the realization of novel testing systems to be used by both developed and developing countries in the near future. nucleic acid sequence-based amplification (nasba) nasba technology has provided an alternative method to conventional procedures with a broad application for the detection of several nucleic acid targets. in particular, nasba is an isothermal transcription-based amplification method, first described by guatelli et al. [ ] , particularly suitable for the detection and quantification of genomic, ribosomal, and messenger rna. nasba offers potential advantages compared to conventional rt-pcr. first of all, it is a continuous, isothermal process that does not require a thermocycler and the optimal annealing temperature for primers does not have to be determined empirically. moreover, because nasba is a method based on the isothermal reaction occurring at a temperature of °c, and does not require denaturation, it prevents amplification of dna genome in case of contamination, thus being very selective for rna target amplification. however, the low temperature occurring in the reaction could be representing a risk factor for the specificity of the method. anyhow, the specificity rate is increased by a well-constructed method for detecting amplified products using additional hybridization with target-specific probes. another advantage is that no additional reverse transcriptase step is required, thus saving time and reducing the risk of contamination. the only restriction of nasba method is probably that individual preparation of the chemical reagents mixture is difficult and commercial kits are expensive. the nasba method nasba amplification consists of a repeated process of primer annealing, formation of double-stranded dna molecule containing a t promoter site, and t -rna polymerase mediated transcription of multiple anti-sense copies of rna amplicons (fig. ) . held at °c, the reaction uses two oligonucleotide primers specific to the rna target, p (forward primer), p (reverse primer), and three enzymes: avian myeloblastosis virus reverse transcriptase (amv-rt) which has also polymerase activity, rnase h, and t rna polymerase. during the reaction, a dna intermediate is generated through a process that involves the hybridization of a primer to the rna target. this primer (p ), which contains a t rna polymerase promoter sequence, is then extended by amv-rt to form a rna-dna hybrid. the digestion of the rna component of the hybrid by rnase h permits the binding of a second primer (p ) to the remaining dna strand. the second primer is then extended by amv-rt to form the doublestranded dna intermediate, which contains the t -rna polymerase promoter needed for transcription. finally, the t rna polymerase produces numerous rna copies and once transcription is initiated, the resulting single-stranded rna transcripts, which are anti-sense to the original rna, can serve as a template to start a new amplification process. the amplification product of nasba can be detected by liquid or gel-based probe-hybridization assays, electrochemiluminescence, or microfluidic electrochemical detection [ ] [ ] [ ] [ ] . recently, real-time assays incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. in particular, quantitative real-time nasba assays using molecular beacons have been developed and utilized for the detection and quantification of several rna target in all published real-time procedures whether for commercially available kits or for in-house diagnostic assays [ , ] . these realtime nasba assays appear to be rapid (about . h), specific and sensitive with rna amplification and a targetspecific fluorescent signal achieved simultaneously in one tube with measurements obtained by using a simple fluorometer. real-time nasba methodology seems to be a suitable alternative to other real-time amplification techniques such as rt-pcr without the need for expensive thermocyclers. because nasba amplification involves three separate enzymes with their own kinetic parameters, variability in every measurement is inevitable [ ] . weusten et al. [ ] were the first to describe a mathematical model for rna amplification of both target and internal calibrator rna in a molecular beacon-based nasba reaction to normalize enzyme efficiency differences between reactions. however, the description of this model did not include all of the essential parameters needed to operate the model. consequently, analysis using this model requires software calibrated to each target and is commercially available for only a few specific targets. on the contrary, in our study an alternative method for normalizing nasba data by using a simple time to positivity (tpp) calculation in the presence of an internal control that reduces the variability between replicates has been described [ ] . to date, the role of primers and kcl concentration for nasba optimization has not been considered. nasba is able to specifically amplify target rna by using specific primers in the presence of kcl. initially, the primers' concentration is very high and is not rate limiting; relatively small amounts of primers are consumed in depletion of the initially present pool of rna copies (linear phase of nasba process). at some time point, the primers' concentration do become rate limiting and decline towards zero. at this time point, the dna intermediate levels have reached their peak and rna production proceeds at high speed. from now on the only reaction that can proceed is t rna polymerase-mediated formation of rna from the dna intermediate templates. this time interval represents the second phase of nasba process characterized by an exponential kinetics (fig. ) . in our study, we evidenced for the first time that high concentrations of primers and kcl elongate the linear phase of nasba process by shorting the exponential amplification; whereas, low concentrations of primers and kcl promote the exponential phase [ ] . in particular, in our study we used relatively low concentrations of primers and kcl ( . lm and mm, respectively) to elongate the exponential phase of nasba process, and accordingly, to minimize the reaction-to-reaction variation. nasba has proven to be a useful technique for the highly sensitive detection of several pathogens in clinical, environmental, and food samples including, in particular, different rna viruses (table ) . although nasba methods offer a powerful tools for molecular diagnosis, their sensitivity and specificity are limited by several factors. amplification inhibitors and rna integrity are the main cause of concern when preparing clinical specimens for nasba. efficiency of rna extraction methods is determined by the rna recovery rate and nasba inhibitor reduction during rna extraction. many rna commercial extraction methods have been tested for the reduction or removal of nasba inhibitors. in particular, rna extraction originally performed with phenol-chloroform has been widely replaced by the boom method which is suitable for use in nasba and reagents for this are commercially available [ ] . however, these methods are time consuming, labor intensive, and susceptible to contamination. lately, complete automatization was introduced performing rna extraction within - min on high numbers of samples. several studies showed that robotic automated sample preparation and the performance of the automated magnapure and the nuclisens extraction procedures (easymag and minimag) were more table applications of nasba assay in the detection of several rna viruses enterovirus [ ] [ ] [ ] influenza a virus [ , ] influenza b virus [ , ] influenza a virus (h n v) * [ ] influenza a virus (h n ) [ ] respiratory syncytial virus [ , ] hiv- [ ] [ ] [ ] parainfluenza virus type [ ] parainfluenza virus type [ ] parainfluenza virus type [ ] parainfluenza type [ ] norovirus [ ] metapneumovirus [ ] sars coronavirus (sars-cov) [ ] chikungunya virus [ ] st. louis encephalitis virus [ ] dengue virus [ ] west nile virus [ ] hepatitis a virus [ ] hepatitis c virus [ , ] human rhinovirus [ , ] measles virus [ ] rubella virus [ ] rabies virus [ ] * h n v, h ni variant consistently than manual techniques [ , ] . as concerns the development of in-house real-time nasba assays, a commercial kit is available (''nuclisens easyq Ò basic kit'' ([biomérieux]) [ ] . it contains the necessary reagents for nasba amplification process including amv-rt, rnase h, t rna polymerase enzymes in the form of lyophilized spheres, the enzyme diluent that consists of sorbitol in aqueous solution, the reagent lyophilized spheres containing nucleotides, dithiothreitol, mgcl with their diluent (tris/hcl, % dmso), and kcl solution. the primers and specific probe are to be synthesized for each target. in particular, the design of primers and probe for nasba can be performed using the ''beacon designer tm '' program developed by premier biosoft international (www. premierbiosoft.com), and the stability of predicted structure beacons can be analysed by using the european mfold server (http://frontend.bioinfo.rpi.edu/applications/ mfold/cgi-bin/dna-form .cgi). the amplification conditions for real-time nasba are generally constant, and optimization of conditions for each new assay can be simpler than rt-pcr. the concentration of enzymes is standardized and does not differ from assay to assay. the variable factors that have to be optimized are the kcl, primers and probes concentrations. in conclusion, nasba is a simple and rapid alternative method to conventional procedures, and its isothermal nature and specificity for rna versus dna make it an important technique in rna research and diagnostics. hda is an isothermal amplification reaction inspired by the natural mechanism of the dna replication fork. this new technology mimics dna replication in vivo by using a dna helicase to separate two complementary dna strands (dsdna) into each single-stranded templates for primers hybridization and subsequent extension by a dna polymerase. as the dna helicase unwinds double-stranded dna enzymatically, the initial heat denaturation and subsequent thermocycling are not necessary, and the entire hda reaction can be performed at a single uniform temperature. thus, this alternative technique provides a useful tool to amplify dna in vitro under isothermal conditions with a very simple reaction scheme. the amplification scheme of the hda method is shown in fig. . in this method, double-stranded dna is unwound enzymatically by a dna helicase in the presence of chemical energy. the displaced dna strands are stabilized by single-stranded dna (ssdna)-binding proteins (ssbs). in particular, these ssb proteins bind specifically to the single-stranded part of dna in order to prevent reannealing of the complementary ssdna templates and to protect them from degradation. two sequence-specific primers hybridize to the -end of each ssdna template, and a dna polymerase extends the primers annealed to the templates to produce a dsdna. the two newly synthesized dsdnas are used as substrates by the dna helicase, entering the next round of the reaction. therefore, a simultaneous chain reaction proceeds resulting in exponential amplification of the selected target sequence. it has been reported that rna target as well as dna was also amplified and detected by hda method followed by reverse transcription step [ , ] . initially, the hda systems were developed using escherichia coli uvrd helicase and t bacteriophage gp helicase. these current hda systems will be briefly described in this review with consideration of the processivity and efficiency of dna amplification. hda system using escherichia coli uvrd helicase the first hda system for isothermal dna amplification was developed by using e. coli uvrd dna helicase (* kda) along with a dna polymerase, and two accessory proteins (ssbs): t gene or rb gene proteins [ , ] . initially, e. coli uvrd helicase was chosen due to its ability to unwind blunt-end substrates (dsdna) as well as nicked circular dna [ ] . this hda system mimics the in vivo dna replication and is able to amplify several hundred base pairs of dna with a detection limit ranging from to dna copies in less than h [ , [ ] [ ] [ ] . moreover, to further improve the sensitivity and specificity of dna amplification in the hda reaction a very simple expedient as the use of thermostable uvrd helicase at elevated temperatures ( - °c) was considered. however, the efficient amplification of long target sequences is not possible, probably due to the low processivity and limited speed of dna synthesis by uvrd helicase. it has been reported that uvrd helicase has a limited speed ( bp/s) and processivity (less than bp per binding) [ , ] . the performance of an hda system may be further improved by testing different helicases. a new hda system with high processivity and speed was developed by using the t bacteriophage gp helicase. hda system using t bacteriophage gp helicase (t bacteriophage replisome) the t bacteriophage replisome consists of four proteins necessary for amplification process: t gp helicase-primase, t gp dna polymerase, t gp . (ssb protein), and the processivity factor e. coli thioredoxin (trx) [ , ] . the t gp helicase-primase is an hexameric protein composed by two subunits, the gp a (* kda) with both helicase and primase activities, and the gp b (* kda) with only helicase activity [ , , ] . in the t helicasebased hda system, the helicase t gp unwinds the dsdna at a rate of bp/s with high processivity, whereas the primase domain of t gp produces the primers [ ] . in particular, this hda system has been applied to amplify both long linear and circular ssdna templates, and the primase activity of t gp allows for whole genomes to be amplified without the need for additional dna primers [ ] . as concerns the t gp dna polymerase activity itself is not processive, whereas together with the processivity factor e. coli thioredoxin (t gp dna polymerase-e. coli thioredoxin complex), the speed and processivity are enhanced by up to[ nt/s and [ kb per binding, respectively [ ] . recent progress in understanding the function of helicases has enabled researchers to use a helicase/polymerase pair (helicase/ polymerase fusion complex) which can move in a coordinated way to further improve the speed and the processivity of hda systems, allowing for the amplification of dna fragments up to . kb compared to the original limit of bp [ ] . future experiments will be certainly directed towards improving the performance of hda systems by testing several helicases/polymerases complex, and by optimizing the existing hda systems. applications of hda assay hda assay has been used to detect several viruses in different clinical samples. in particular, tang and colleagues developed an innovative isothermal amplification hda with lateral flow to detect hiv- in human plasma, whereas kim and colleagues developed a qualitative hda method for the detection of herpes simplex virus (hsv) types and from genital lesions [ , ] . moreover, a novel one-tube isothermal reverse transcription-thermophilic hda (rt-thda) system has been developed to detect rna viruses, including enterovirus and ebola virus [ ] . thermophilic hda in combination with enzyme-linked immunosorbent assay was also used by gill et al. [ , ] for the detection of helicobacter pylori. in addition, they also developed a colorimetric method to detect h. pylori by using isothermal ' step step step step fig. amplification scheme of hda method. (step ) dna helicase unwinds doublestranded dna. ( step ) ssb proteins stabilize the displaced dna strands. ( step ) specific primers hybridize to the ssdna template and are extended by dna polymerase. (step ) a double-stranded copy of the dna target is produced hda and gold nanoparticle probes. andresen et al. [ ] incorporated hda on a microarray for quantitative detection of antibiotic-resistant pathogens neisseria gonorrhoeae and staphylococcus aureus. microfluidic chips have also been developed for hda at °c for quantification of sars cdna [ ] . a fully integrated microfluidic device for dna extraction and hda at °c on samples containing live bacteria has been developed by mahalanabis et al. [ ] . this microfluidic device was the first to combine bacterial lysis, nucleic acid extraction, and dna amplification on the same chip. finally, kivlehan et al. [ ] reported for the first time the utilization of a quantitative electrochemical method to monitor in real-time the hda of nucleic acids in less than h at a single constant temperature. the principle of detection consists of monitoring a decrease in the electrochemical current response of a reporter probe during the amplification process. the detection strategy is analogous to that of real-time hda assay. however, this innovative electrochemical method offers some advantages compared to conventional realtime assays being potentially more robust, simpler, and less expensive. isothermal hda kits are currently available and commercially developed at biohelix (beverly, ma, usa). in conclusion, it is expected that more useful and simpler isothermal amplification techniques will be invented to be used for the detection of different pathogens. loop-mediated isothermal amplification of dna accelerated reaction by loop mediated isothermal amplification using loop primers loopmediated isothermal amplification (lamp) of gene sequences and simple visual detection of products simple and accurate determination of cyp d gene copy number by a loop-mediated isothermal amplification method and an electrochemical dna chip detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method a loop mediated isothermal amplification (lamp) method for detection of infectious hematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) development and evaluation of a novel loop mediated isothermal amplification (lamp) method for rapid detection of sars corona virus real-time reverse transcription loop mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loopmediated isothermal amplification assay application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant staphylococcus aureus (mrsa) in blood cultures lamp-an emerging technology for the detection of water-and food-borne protozoan parasites loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific loop-mediated isothermal amplification method rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. microbiology and immunology rapid and real-time detection of chikungunya virus by reverse transcription loop mediated isothermal amplification assay development and evaluation of reverse transcription loop mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus a simple method for the detection of measles virus genome by loop-mediated isothermal amplification (lamp) rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification detection of human influenza a viruses by loop-mediated isothermal amplification rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation mumps virus reinfection is not a rare event confirmed by reverse transcription loop-mediated isothermal amplification rapid diagnosis of human herpesvirus infection by a novel dna amplification method, loopmediated isothermal amplification rapid detection of varicellazoster virus infection by a loop-mediated isothermal amplification method rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (lamp) method detection of human herpesvirus dna by loop-mediated isothermal amplification rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method sensitive and rapid detection of herpes simplex virus and varicella-zoster virus dna by loop-mediated isothermal amplification comparison of loop-mediated isothermal amplification, real-time pcr, and virus isolation for the detection of herpes simplex virus in genital lesions development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus dna development of a loop-mediated isothermal amplification assay for rapid detection of bk virus loop-mediated isothermal amplification method for detection of human papillomavirus type , , , and direct detection of human herpesvirus dna in serum by the loop-mediated isothermal amplification method rapid detection of human herpesvirus dna using loop-mediated isothermal amplification rapid and simple detection of legionella species by lamp, a new dna amplification method loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples sensitive and rapid detection of cholera toxin-producing vibrio cholerae using a loop-mediated isothermal amplification isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication detection of dengue viral rna using a nucleic acid sequence-based amplification assay highly sensitive and specific detection of viable escherichia coli in drinking water human pathogenic cryptosporidium species bioanalytical detection method with single oocyst detection capability. analytical and bioanalytical chemistry pmma biosensor for nucleic acids with integrated mixer and electrochemical detection real-time nucleic acid sequencebased amplification is more convenient than real-time pcr for quantification of plasmodium falciparum development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes increased precision of microbial rna quantification using nasba with an internal control principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons development and implementation of real time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens evaluation of lightcycler as a platform for nucleic acid sequence-based amplification (nasba) in real time detection of enterovirus development of multiplex nucleic acid sequence-based amplification for detection of human respiratory tract viruses development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza a dry cotton or flocked respiratory swabs as simple collection technique for the molecular detection of respiratory viruses using real time nasba detection of novel swine origin influenza a virus (h n ) by real-time nucleic acid sequence-based amplification development and validation of a commercial real time nasba assay for the rapid confirmation of influenza ah n virus in clinical samples clinical evaluation of nuclisens magnetic extraction and nu-clisens analytical specific reagents for the real-time detection of respiratory syncytial virus (rsv) in pediatric respiratory specimens a one-tube quantitative hiv- rna nasba nucleic acid amplification assay using electrochemiluminiscent (ecl) labelled probes quantitation of human immunodeficiency virus type rna in different biological compartments single rapid realtime monitored isothermal rna amplification assay for quantification of human immunodeficiency virus type isolates from groups m, n, and o evaluation of the persistence of infectious human norovirus on food surfaces by using real time nucleic acid sequence-based amplification diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis real time nasba detection of sarsassociated coronavirus and comparison with real-time reverse transcription-pcr evaluation of real time nucleic acid based amplification for detection of chikungunya virus in clinical sample nucleic acid sequencebased amplification assays for rapid detection of west nile and st. louis encephalitis viruses real time nucleic acid sequence-based amplification assay for detection of hepatitis a virus characterization of the quantitative hcv nasba assay evaluation of a new nasba assay for the detection of hepatitis c virus based on the nuclisens basic kit reagents detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season a sensitive and robust method for measles rna detection comparative detection of rabies rna by nasba, real-time pcr and conventional pcr rapid and simple method for the purification of nucleic acids comparison of five methods for extraction of legionella pneumophila from respiratory specimens evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens development and evaluation of nucleic acid sequence based amplification (nasba) for diagnosis of enterovirus infections using the nuclisens Ò basic kit development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid rna detection helicase dependent onchip-amplification and its use in multiplex pathogen detection bacteriophage t gene protein: modulation of protein-nucleic acid and protein-protein association by structural domains snapshot of the genome of the pseudo-t-even bacteriophage rb escherichia coli helicase ii (uvrd) protein can completely unwind fully duplex linear and nicked circular dna characterization of a thermostable uvrd helicase and its participation in helicase-dependent amplification colorimetric detection of helicobacter pylori dna using isothermal helicase-dependent amplification and gold nanoparticle probes improving isothermal dna amplification speed for the rapid detection of mycobacterium tuberculosis an oligomeric form of e. coli uvrd is required for optimal helicase activity helicase-dependent isothermal dna amplification complete nucleotide sequence of bacteriophage t dna and the locations of t genetic elements bacteriophage t : minimal requirements for the replication of a duplex dna molecule characterization of the helicase and primase activities of the -kda component of the bacteriophage t gene protein cloning and expression of gene of bacteriophage t and creation and analysis of t mutants lacking the a primase/ helicase or the b helicase dna replication dna-dependent nucleoside -triphosphatase activity of the gene protein of bacteriophage t escherichia coli thioredoxin confers processivity on the dna polymerase activity of the gene protein of bacteriophage t isothermal dna amplification in vitro: the helicase-dependent amplification system nucleic acid assay system for tier ii labs and moderately complex clinics to detect hiv in low-resource settings a rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types and detection of helicobacter pylori by enzyme-linked immunosorbent assay of thermophilic helicase-dependent isothermal dna amplification helicase-dependent amplification: use in onchip amplification and potential for point-of-care diagnostics real-time pcr array chip with capillary-driven sample loading and reactor sealing for point-ofcare applications an integrated disposable device for dna extraction and helicase dependent amplification real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids key: cord- -gprd di authors: shirato, kazuya title: detecting amplicons of loop‐mediated isothermal amplification date: - - journal: microbiol immunol doi: . / - . sha: doc_id: cord_uid: gprd di loop‐mediated isothermal amplification (lamp) assays are used to detect diverse pathogens. initially, lamp amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental dna. recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. here, methods of detecting lamp amplicons are reviewed. the loop-mediated isothermal amplification (lamp) method was developed by notomi et al. and was patented by eiken chemical co., ltd (patent no. jp , wo a ). in lamp assays, dna is amplified by four specific primers (f , fip, bip, and b ) at - °c within hr, although the speed of amplification can be accelerated by adding two loop primers (lf and lb). thus, lamp is rapid and user-friendly; it can reduce the time required to increase and decrease the incubation temperature, facilitating the choice of incubation device, that is, lamp can be performed using a simple isothermal device, such as heat block and water bath, and the amplification ends faster than general pcrs. the principles of lamp are described on the manufacturer's website (http://loopamp.eiken.co.jp/e/lamp/principle.html) and as an animation (http://loopamp.eiken.co.jp/e/lamp/anim. html). lamp assays can also be used to detect rna by adding a reverse transcription step (rt-lamp). lamp uses four or six primers, which assures high sequence specificity. the primers are also tolerant of point mutation(s) other than mismatches at the ′ end of the primer and in the bip primer. [ ] [ ] [ ] [ ] therefore, lamp enables detection of pathogen genes. lamp assays have been developed to detect gene of bacteria, [ ] [ ] [ ] [ ] [ ] [ ] [ ] parasites, - protozoa, , , and viruses, [ ] [ ] [ ] [ ] including rna viruses such as west nile virus, lamp amplicons can be visualized by electrophoresis in an agarose gel. the four basic lamp primers are constructed based on six specific regions of the target sequence and form a unique dumbbell-like structure, both ends of which have stem-loop structures that each comprise three specific regions. the dumbbell-like structure is extended in a bumper-to-bumper manner; therefore, the amplicons are ladder-like in appearance because they contain various numbers of dumbbell-like structures ( figure ). the specificity of amplification can be confirmed by incorporating restriction enzyme sites. in our rt-lamp for rsv, the subtype a amplicon is cleaved by nlaiii and that of subtype b by xbai. if the amplification succeeds, depending on the number of used enzymes and recognition sites in targeted sequences, several bands can be seen after digestion ( figure ). thus, detecting lamp amplicons by electrophoresis and restriction enzymes can confirm the specificity of amplification. however, electrophoresis is performed under open conditions, leading to contamination of the laboratory with amplified dna. the high sensitivity of genetic diagnostic methods increases the likelihood of false positives. , lamp produces a larger quantity of dna than conventional pcr ( vs. . μg in a μl reaction), suggesting that lamp is more likely to result in contamination of the laboratory. to prevent this, separate reagent mixing and electrophoresis rooms with a one-way workflow are needed. furthermore, training of workers is needed, and the number of tests performed daily by one person should be limited to prevent contamination; however, this is time consuming and costly. the optimum means of avoiding laboratory contamination by amplified dna is performing detection under closed conditions, that is, without opening the reaction tube. mori et al. developed a turbidity-based method for amplicon detection in a closed system, based on precipitation of magnesium pyrophosphate. the pyrophosphate ion is released from deoxynucleotide triphosphate as a by-product of dna polymerization by dna polymerase, and precipitates with magnesium ion in the reaction buffer. , because lamp produces a large quantity of dna, the pyrophosphate ion concentration exceeds the threshold for precipitation, resulting in the formation of a visible magnesium precipitate in an amplicon concentration-dependent manner. by contrast, pcr yields a pyrophosphate concentration insufficient for magnesium pyrophosphate precipitation. the precipitate is visible at the end of amplification (figure a ) and the turbidity can be monitored in real-time using a turbidimeter ( figure c ). a fluorescence detection method using the fluorescent dye calcein has also been developed. calcein, known as fluorexon, is quenched by manganese ions, of which it is deprived by pyrophosphate. therefore, it fluoresces when accompanied by pyrophosphate ion production during the lamp reaction and is visible to the naked eye under ultraviolet light (figure b ). the signal can be monitored using a real-time fluorescence monitor. turbidity monitoring enables the detection of targets under closed conditions without electrophoresis, as well as lamp in the field and at point of care using an isothermal incubator, such as a heat block and water bath. lamp kits for in vitro diagnosis are commercially available in japan for whooping cough, tuberculosis, mycoplasma pneumonia, legionella pneumophila, influenza, and severe acute respiratory syndrome. however, the turbidity signal is derived from a by-product of dna amplification; it is not caused by a primer's specific reaction directly. consequently, it is not possible to completely exclude the possibility of monitoring nonspecific amplification; salt accumulation accompanying the lamp reaction can be induced even by a nonprimer signal or primer dimers. thus, even if host-derived dna fragments or primer dimers form dna amplification nonspecifically, the increased turbidity that accompanies this process can be monitored as a positive signal. although rare, the possibility of nonprimer signals in turbidity monitoring cannot be excluded. hence, validation of the integrity of the primer set is quite important to develop a f i g u r e rsv rt-lamp was performed as described previously. the amplicons were purified using a qiaquick pcr purification kit and treated with nlaiii and xbai at °c for hr. the resulting fragments were separated by electrophoresis on % agarose gels and visualized using ethidium bromide staining and ultraviolet light lamp system with turbidity monitoring. the primers for lamp can be designed using online software (primer-explorer v. or , fujitsu, tokyo, japan; http:// primerexplorer.jp/e/). however, the search results do not guarantee primer specificity. candidates should be screened by strict validation tests to check whether they ever show nonspecific reactions in the presence of host and/or carrier nucleotides and the primer set itself. the primer set can be used for further validations only after it has been shown that it never causes nonspecific dna amplification. labeling primers with a fluorescent dye ensures detection of only primer-derived signals. takayama et al. developed fluorescence rt-lamp using a quenching primer (qprimer) to detect influenza virus and rsv, and nakauchi et al. developed qprimer rt-lamp for rhinoviruses. in this system, a cytosine or guanine residue at the ′ end of the loop primer is labeled with bodipy (boron-dipyrromethene) dye and the labeled and unlabeled loop primers are mixed at a : ratio. upon annealing to its target sequence, qprimer fluorescence is quenched by the paired guanine or cytosine residue (figure ) . therefore, the positive signal can be detected as a reverse sigmoid curve using an isothermal fluorescence reader, such as a real-time pcr instrument (figure ). in addition, use of qprimers reduces the lamp detection time by several minutes relative to turbidity detection, and positive signals are typically detected within min. although qprimer can resolve concerns regarding detection of primer-derived signals directly, nonspecific dna extension at the annealed ′ end and/or primer dimers can result in false positives, so strict screening for primer integrity is still necessary. to overcome this, lamp using a quenching probe (qprobe) was developed. in qprobe lamp, the ′ end of one of the loop primers is labeled with bodipy dye (figure ) ; the qprobe must be designed to be several nucleotides longer than the original loop primer toward the ′ end. table shows the sequences of qprobes used f i g u r e detection of lamp amplicons by turbidity. (a) mers-cov rt-lamp was performed as described previously. a muddy white color indicates precipitation of magnesium pyrophosphate. left two wells, positive; right two wells, negative. (b) amplification was performed using calcein and visualized under ultraviolet light. green fluorescence indicates positivity. (c) rsv rt-lamp was performed as described previously, and turbidity was monitored in real time. the signal is drawn as a power curve f i g u r e schematic diagrams of a qprimer and qprobe. (a) the cytosine or guanine residue is labeled with bodipy. the qprimer and qprobe fluoresce ordinarily. upon annealing to their target sequence, their complementary guanine or cytosine residue quenches the fluorescence. (b) the signal can be detected as a reverse sigmoid curve using a fluorescence meter in qprobe rt-lamp for mers-cov. the sequences in bold are extended relative to the loop primer, and are responsible for the specific reaction, because the sequence exists only in the target sequence and the specific amplicon. although this makes qprobes more difficult to design than qprimers, the labeled dye physically blocks dna extension at the ′ end, preventing nonspecific dna amplification by a labeled primer and enhancing the accuracy of the signal. fluorescence detection requires a fluorometer, which prevents naked-eye endpoint detection. however, portable isothermal fluorometers enable fluorescence detection in the field. for example, the esequant ts (qiagen, https://www.qiagen.com/jp/products/customsolutions/automation/ese-instruments/esequant-ts /) is a well isothermal tube scanner that has a built-in battery pack, and so can be used outdoors. furthermore, a lyophilized rt-lamp reaction mixture in a well tube reduces the time required for mers-cov detection. in other words, the lyophilized reagent contains all necessary components for rt-lamp, and all it takes is just to add extracted rna and water, and it can reduce the time for regent preparation. thus, lyophilized reagents and portable fluorescence lamp devices enable its use in field surveillance as an accurate diagnostic tool. although each detection method has advantages and disadvantages, overall, lamp is a superior assay. its high sensitivity, specificity, and rapidity are advantageous for use in clinical laboratories compared with rapid test kits based on antigen detection. indeed, the sensitivity of lamp is superior to that of rapid test kits based on immunochromatography. , thus, lamp may replace such kits in the future. the author declares that he has no competing interests. http://orcid.org/ - - - the sequences in bold are extended relative to the loop primer. loop-mediated isothermal amplification of dna accelerated reaction by loopmediated isothermal amplification using loop primers real-time reversetranscription loop-mediated isothermal amplification for rapid detection of rift valley fever virus effect of internal primer-template mismatches on loop-mediated isothermal amplification detection of middle east respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (rt-lamp) development of fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) using quenching probes for the detection of the middle east respiratory syndrome coronavirus loop-mediated isothermal amplification (lamp) for the direct detection of human pulmonary infections with environmental (nontuberculosis) mycobacteria the rapid detection of salmonella from food samples by loop-mediated isothermal amplification (lamp) efficacy of loop mediated isothermal amplification (lamp) assay for the laboratory identification of mycobacterium tuberculosis isolates in a resource limited setting detection of mycoplasma pneumoniae by loop-mediated isothermal amplification (lamp) assay and serology in pediatric communityacquired pneumonia development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of bordetella pertussis infection molecular diagnostic tools in mycobacteriology examination of the rapid detection of legionella from bathwater samples by the loopmediated isothermal amplification and polymerase chain reaction methods loop-mediated isothermal amplification (lamp) assay for detection of theileria sergenti infection targeting the p gene rapid detection of opisthorchis viverrini copro-dna using loopmediated isothermal amplification (lamp) development of loop-mediated isothermal amplification (lamp) assays for rapid detection of ehrlichia ruminantium rapid identification of giardia duodenalis by loop-mediated isothermal amplification (lamp) from faecal and environmental samples and comparative findings by pcr and real-time pcr methods loop-mediated isothermal amplification (lamp) detection of babesia orientalis in water buffalo (bubalus babalis, linnaeus, ) in china loopmediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus- rapid diagnosis of human herpesvirus infection by a novel dna amplification method, loop-mediated isothermal amplification rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification development of an improved rt-lamp assay for detection of currently circulating rubella viruses rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification detection of respiratory syncytial virus genome by subgroups-a, b specific reverse transcription loop-mediated isothermal amplification (rt-lamp) development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection multiplex loop-mediated isothermal amplification (m-lamp) assay for the detection of influenza a/h , a/h and influenza b can provide a specimen-to-result diagnosis in min with single genome copy sensitivity development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (h n ) virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) onepot reverse transcriptional loop-mediated isothermal amplification (rt-lamp) for detecting mers-cov false-positive results and the polymerase chain reaction avoidance of pcr false positives detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation avoiding false positives with pcr enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia nucleic acid sequence-based amplification real-time turbidimetry of lamp reaction for quantifying template dna loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products fluorescent quenchingbased quantitative detection of specific dna/rna using a bodipy (r) fl-labeled probe or primer development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assays for rhinovirus detection how to cite this article: shirato k. detecting amplicons of loop-mediated isothermal amplification key: cord- - pjam em authors: stranieri, angelica; lauzi, stefania; giordano, alessia; paltrinieri, saverio title: reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: pjam em the feline coronavirus (fcov) is the etiological agent of feline infectious peritonitis (fip), a lethal disease of felids. the role of molecular methods is controversial for the diagnosis of fip, while essential for the identification of the shedders. thus, a fast and inexpensive method for the detection of fcov could be beneficial, especially in multicat environments. a reverse transcription loop mediated isothermal amplification (rt-lamp) assay was developed. rna extraction and rt-npcr for fcov were performed on thirty-two samples ( faeces, blood, effusions, and lymph nodes) collected from cats. six rt-lamp primers were designed from the same conserved region of rt-npcr, and the assay was run at °c for one hour. results were evaluated through both agarose gel run and hydroxynapthol blue (hnb) dye and then compared with rt-npcr results for the assessment of sensitivity and specificity. the overall specificity was %, but the sensitivity was % and . % for agarose gel and hnb respectively. therefore, rt-lamp seems optimal to confirm the presence of the virus, but not applicable to exclude it. the feline coronavirus (fcov) is the etiological agent of feline infectious peritonitis (fip), a lethal disease of felids. the role of molecular methods is controversial for the diagnosis of fip, while essential for the identification of the shedders. thus, a fast and inexpensive method for the detection of fcov could be beneficial, especially in multicat environments. a reverse transcription loop mediated isothermal amplification (rt-lamp) assay was developed. rna extraction and rt-npcr for fcov were performed on thirty-two samples ( faeces, blood, effusions, and lymph nodes) collected from cats. six rt-lamp primers were designed from the same conserved region of rt-npcr, and the assay was run at • c for one hour. results were evaluated through both agarose gel run and hydroxynapthol blue (hnb) dye and then compared with rt-npcr results for the assessment of sensitivity and specificity. the overall specificity was %, but the sensitivity was % and . % for agarose gel and hnb respectively. therefore, rt-lamp seems optimal to confirm the presence of the virus, but not applicable to exclude it. © elsevier b.v. all rights reserved. feline coronaviruses (family coronaviridae, order nidovirales) are enveloped, single-stranded positive sense rna viruses belonging to the species alphacoronavirus , genus alphacoronavirus of the sub family coronavirinae (gonzalez et al., ) . the feline coronavirus (fcov) possesses a large genome (almost kb) organized in putative open reading frames (orfs) encoding nonstructural proteins involved in virus replication as well as four structural proteins (spike, s; membrane, m; nucleocapsid, n; envelope, e) and five accessory proteins ( a-c, a and b) (kipar and meli, ) . not yet well characterized viral mutations, along with an inadequate immune response of the host, lead to the inevitably deadly disease of felids called feline infectious peritonitis (fip) (pedersen, ; porter et al., ) . the diagnosis of fip is challenging in vivo and must rely on several clinico-pathological tests (e.g. serum protein electrophoresis, agp measurement, effusion analysis) and only the immunohistochemical demonstration of the viral antigen inside the typical pyogranulomatous lesions can be considered as a gold standard (pedersen, ) . the fcov is faecally-orally transmitted, worldwide distributed and highly prevalent in feline populations (addie et al., ) . since most of the molecular and serological tools available up to date are not able to distinguish between the not mutated and the mutated pathogenic form of the fcov, the use of reverse transcriptase polymerase chain reaction (rt-pcr) for the diagnosis of fip is still extensively discussed doenges et al., ; felten et al., ; longstaff et al., ) . on the other hand, pcr is an extremely useful tool for the diagnosis of fcov infection and shedding and consequently for the identification of shedders, when performed on faeces (pedersen, ) . since shedders can spread the virus in the environment for months, rt-pcr should be repeatedly performed to identify both shedders and cats free from the infection (addie and jarrett, ) . loop-mediated isothermal amplification (lamp) is an amplification gene technique developed some years ago (notomi et al., ) which allows to amplify nucleic acids in an hour and under isothermal conditions, and to evaluate the results by observation of the turbidity of the reaction or using different dyes. reverse transcriptase lamp (rt-lamp) has been used to amplify the genome of several coronaviruses of both humans and animals (amer et al., ; bhadra et al., ; cardoso et al., ; hanaki et al., ; nemoto et al., ; pyrc et al., ; thai et al., ; yu et al., ) . to our knowledge, rt-lamp has never been used for the identification of fcovs. the aim of this study was to develop a rt-lamp for the detection of feline coronavirus in the specimens most frequently used in clinical practice for both screening and diagnostic purposes. thirty-two samples from cats ( faeces, blood, effusions, and lymph nodes) submitted to our institution as part of a diagnostic panel for the clinical suspicion of fip or, regarding faeces, for screening purposes, were used. all the specimens were subjected to rna extraction using a nucleospin rna isolation kit (macherey-nagel bethlehem, pa). whole blood and effusion samples were centrifuged ( min at × g) and the obtained pellets were suspended in l of phosphate buffered saline (pbs) by vigorous vortexing and stored at − • c for further rna extraction. faecal samples were suspended at a final concentration of % (wt/vol) in pbs by vigorous vortexing. the supernatant was cleared by centrifugation for min at × g and stored at − • c for further rna extraction. for tissues, approximately mg of sample were thinly shredded on sterile plates using sterile scalpels, followed by vigorous vortexing in rna lysis buffer until complete disruption of the sample. all the further steps were performed according to the manufacturer's instruction. the extracted rna samples were tested for the presence of fcov using a reverse transcription nested pcr (rt-npcr) targeting a bp product of the highly conserved untranslated region ( utr) of the genome of both type i and type ii fcov (herrewegh et al., ) . rt-npcr positive fcov rna from a cat with fip was used as positive control and rnase-free water as negative control. pcr products were visualized under uv transilluminator on a % agarose gel stained with ethidium bromide. the rt-lamp primers targeting the utr of the fcovs were designed using the primer explorer v software (http:// primerexplorer.jp/elamp . . /index.html) based on a nucleotides sequence ( - bp) of the fcov c je strain (accession number: dq ) (table ) . a loopamp rna amplification kit (rt-lamp, new england biolabs, uk) was used to perform rt-pcr lamp and the reaction mixture was set up as follows: × isothermal amplification buffer, mm mgso , . mm dntps, u/ml of warm start dna, u/ml of warm start rtx reverse transcriptase, m of both forward inner primer (fip) and backward inner primer (bip), . m of both f and b primers, m of both loop f and loop b primers, m of hydroxynaphtol blue (hnb) dye (sigma-aldrich ® ) and l of rna template. the reaction mixture was then made up to l with rnase-free water and incubated in a thermal cycler (mycycler, bio-rad laboratories, hercules, ca, usa) for h at • c followed by min at • c for heat inactivation. the same positive control used for traditional rt-npcr, which tested positive also on the first rt-lamp assay, was then used as a positive control in the following rt-lamp assays, while rnase-free water was used as negative control. the products of the reaction were then inspected both by eye, in order to detect the color turning from violet to sky blue in case of positive results with hnb (goto et al., ) , and under uv transilluminator on a % agarose gel stained with ethidium bromide in order to detect a ladder-like pattern in case of positive result (parida et al., ) (fig. ) . results obtained with rt-lamp were then compared with those obtained with the rt-npcr and the sensitivity and specificity of rt-lamp obtained with both hnb and agarose gel were calculated. results are reported in table . all the negative samples using rt-npcr were also negative by rt-lamp, using both gel electrophoresis and hnb for the visualization of the results, leading to a % specificity. on the other hand, a conspicuous number of false negative results was recorded ( / for agarose gel and / for hnb) and the overall sensitivity was % and . % using gel electrophoresis and hnb, respectively. positive samples were characterized by a slight difference in the intensity of coloration, as shown in fig. . the sensitivity of rt-lamp was also different according to specimens: faeces showed a sensitivity of % and %, with gel electrophoresis and hnb respectively. on blood, the sensitivity was % and %, with gel electrophoresis and hnb respectively while on effusions the sensitivity was % with both the visualization methods. only on tissues, the sensitivity resulted to be absolute, but the number of tested samples was too low to be discussed in terms of diagnostic accuracy. based on the results of this pilot study, rt-lamp for fcov, due to its high specificity, appears to be a solid molecular test to confirm the diagnosis of fcov infection, and eventually to support a clinical diagnosis of fip, when performed on specimens from cats with an high pre-test probability of fip (e.g. cats with clinical signs and laboratory findings consistent with fip, like effusions with physico-chemical and cytological features consistent with fip) but its low sensitivity makes this test not reliable in case of negative results (pedersen, ) . the design of this study did not allow us to understand the possible mechanisms responsible for the low sensitivity recorded. technical problems are unlikely since the method described in this study has been developed after testing different primers, working conditions or temperatures (data not included in this short communication). possible explanations of the high rate of false negative results compared with conventional nested rt-pcr include the lower analytical sensitivity of rt-lamp, as previously reported for other coronaviruses when compared with real time rt-pcr (bhadra et al., ) , that allow to obtain positive results only in samples with a high viral burden. in the case the sensitivity of the test might be ameliorated through further studies, the rt-lamp could be extremely useful, due to its low costs and rapidity, in those situations where the table results obtained on the samples tested with rt-npcr (pcr) and lamp and evaluated with agarose gel electrophoresis (lamp gel) and hydroxynaphtol blue dye (lamp hnb detection of fcov must be repeated over time and on a high number of cats (e.g. breeding catteries). an additional future perspective would be the optimization of the test to obtain quantitative results, possibly by establishing a standard curve of color intensity using rna samples with known viral load (e.g. quantification of rna copies by quantitative pcr techniques). moreover, the development of an internal control to assess the integrity of rna in each sample would be advisable before the use of this test in field conditions. the authors declared no conflicts of interest regarding the research, authorship, and/or publication of this article. the authors received no financial support for the research, authorship, and/or publication of this article. use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats feline infectious peritonitis. abcd guidelines on prevention and management a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus 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of severe acute respiratory syndrome coronavirus development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus none. key: cord- - jnfl authors: savan, r; kono, t; itami, t; sakai, m title: loop‐mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens date: - - journal: j fish dis doi: . /j. - . . .x sha: doc_id: cord_uid: jnfl fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. early diagnosis plays a vital role in management of fish and shellfish diseases. traditionally, various biochemical and serological tests have been used for fish disease diagnosis. however, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. polymerase chain reaction and probe‐based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. recently, a novel technique called loop‐mediated isothermal amplification (lamp) has been developed, which is highly sensitive and rapid. lamp has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. in aquaculture, lamp‐based detection of pathogens like edwardsiella tarda, e. ictaluri, nocardia seriolae, tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. in this review, the application of lamp for the detection of aquaculture‐associated pathogens is discussed. fish and shellfish culturists continually manage diseases that threaten the sustainability of aquaculture. the threat of newly emerging pathogens is also a concern for aquaculturists. the rapid diagnosis and prevention of diseases of fish and shellfish in culture systems is important in fish husbandry. efficient management of disease usually begins by preventing the spread of disease. disease diagnosis has been mainly based on clinical signs supported by isolation and identification of the aetiological agent. this requires a rapid and sensitive method to detect pathogens. on-farm laboratories are not usually equipped with diagnostic equipment, thus small-scale aquaculture operations depend on specialist fish disease laboratories for their diagnostic needs. however, laboratories routinely handling diagnostic cases are often very busy and take time to carry out diagnoses. in order for laboratories to handle large number of samples sensitive and rapid diagnostic kits are needed. traditional methods for identifying pathogens are mainly based on phenotypic characters. common techniques used in diagnosis of fish and shellfish diseases include bacteriological analysis, virus isolation, histopathology and enzyme-linked immunosorbent assay (elisa)-based techniques. these are often accurate and sensitive techniques although they require time and trained personnel. nucleic acid-based detection assays have also been used for the detection of pathogens. an excellent review on molecular diagnostics used to detect pathogens associated with fish and shellfish diseases has been published by cunningham ( ) . various techniques, including restriction digestion of nucleic acid, nucleic acid hybridization and dna amplification technology are currently being used. polymerase chain reaction (pcr) and real-time pcr have gained a wide popularity as a diagnostic tool. the advantage of pcr over other nucleic acid techniques is the ease of development of the detection system, its high sensitivity and rapidity. in this paper, we review a novel method of dna amplification known as loop-mediated isothermal amplification (lamp) of a target nucleic acid. this method has already been applied for the detection of several micro-organisms (table ). the advantages and the potential application of this technique in detection of pathogens associated with aquaculture are reviewed. loop-mediated isothermal amplification is a sensitive strand displacement technique (notomi, okayama, masubuchi, yonekawa, watanabe, amino & hase ) . this method amplifies target dna from a few copies to copies in less than an hour under isothermal conditions. it is an offshoot of the basic strand displacement techniques which have been described thoroughly (notomi et al. ) . briefly, four highly specific primers are constructed from the target dna, one set of primers anneal to the target region one after the other on the same strand and the primer which anneals at the later stage displaces the strand formed by the first primer with the help of bst dna polymerase. the bst polymerase has a strand displacement activity. this takes place on both strands and the primers are designed such that loops are formed. the reaction is carried out under isothermal conditions as denaturation of the strand takes place by strand displacement. the reactions produce a series of stem-loop dnas with various lengths. the four primers hybridize against six distinct sequences in the target dna making it highly specific. designing primers for lamp is a complex procedure compared with the normal pcr. a minimum of four primers are required for a lamp reaction. the primers required are one pair of inner-primers, which are usually over mer in length, and shorter outer-primers mer in length. the technical specifications of each primer and the optimized annealing temperatures are described in detail by notomi et al. ( ) . the primers can be developed using primer explorer version software (http://primerexplorer.jp/lamp . . /) specifically designed to develop lamp primers. by using additional sets of loop primers, the reaction time of lamp can be further accelerated (nagamine, hase & notomi ) . using loop primers the time required is reduced by half, making it a more efficient tool in disease diagnostic applications. the lamp reaction is performed by the bst dna polymerase along with dntps and reaction buffer (eiken genome co. ltd, tokyo, japan). the reaction proceeds at isothermal conditions for h or less depending on the efficiency of the developed primers and template dna. the template dna for lamp is denatured for min at °c before setting up the lamp reaction for isothermal amplification (notomi et al. ) . however, studies have shown that non-denatured templates can also be used directly for lamp-mediated detection (nagamine, watanabe, ohtsuka, hase & notomi ) . the lamp reaction is carried out at - °c for - min and the reaction terminated at °c for min (fig. ). the main advantage of the technique is that it does not need thermocyclers. as the amplification in made under isothermal conditions, only a water bath or heating block is needed to maintain the required temperature. several methods may be used to check for the lamp reaction. the products are commonly visualized by agarose gel electrophoresis stained with an intercalating agent such as ethidium bromide or sybr green i. as the lamp reaction produces products of various lengths of stem loop structures, the gel will show products from the minimum length of target dna to the loading well, which appears as a smear and bands at the base of the gel. because amplification of the target dna is so high at the final stage it is vulnerable to contamination in subsequent amplifications. however, an advantage of the large amount of product that is generated is that it can be visualized on a uv-transilluminator by incorporating intercalating agents directly into the lamp-amplified tubes (notomi et al. ) . another method is by assessing the amount of white precipitate formed from magnesium pyrophosphate (mori, nagamine, tomita & notomi ). the dna formed by amplification of prostate-specific antigen by pcr for cycles and lamp for min at °c was . and . lg ll ) , respectively (mori et al. ) . the huge amount of dna formed by lamp allows the detection of turbidity using a desktop centrifuge, as the precipitate accumulates at the bottom of the tube. negative reactions neither show smearing nor turbidity in the reaction mixture. application of lamp in the detection of micro-organisms there are several published reports regarding the use of lamp for the detection of bacterial isolates from humans, fish and the aquatic environment. the first report of the application of lamp to detect bacterial isolates was by maruyama, kenzaka, yamaguchi, tani & nasu ( ) . these authors developed a novel strategy using lamp to detect escherichia coli from the coastal environment, where the stx gene was detected by an in situ method. several reports on lamp-mediated diagnostic methods have been developed for bacterial pathogens associated with fish. the first use of lamp for detection of an aquaculture pathogen was reported for edwardsiellosis (savan, igarashi, matsuoka & sakai ) . edwardsiella tarda was detected from infected japanese flounder, paralichthys olivaceus. lamp primers were designed by targeting the haemolysin gene (fig. ) . the specificity of lamp was tested for five different e. tarda strains isolated from eel (e ), tilapia (e ), flatfish (fpc ), water from eel culture ponds (su ) and sea bream ( ). non-specific amplification was not seen in other enteric bacteria and those which produce enterotoxins. the optimum amplification regime for a clear visible banding pattern was at °c for min. edwardsiella tarda could be figure schematic representation of the procedure used in detecting pathogens by loop-mediated isothermal amplification. the reaction mixture and template is amplified at isothermal temperature and can be detected by staining the agarose gel or by incorporating the intercalating agents in the reaction tube and visualizing. detected from and cfu by lamp and pcr, respectively. thus, in this case, lamp-based detection was much more sensitive than pcr. furthermore, lamp could detect the pathogen from japanese flounder pond water with an e. tarda count of . · cfu. a lamp-based diagnostic system has been recently developed for detection of e. ictaluri from diseased catfish, ictalurus punctatus (yeh, shoemaker & klesius ) . the lamp primers were designed targeting the eip gene. the lamp reaction amplified six different strains of e. ictaluri. non-specific amplification was not observed when tested with other bacterial strains associated with channel catfish. the detection limit by lamp was cfu, more sensitive than real-time pcr and traditional biochemical assays (bilodeau, waldbieser, terhune, wise & wolters ) . edwardsiella ictaluri could be detected using genomic dna from infected brain samples as template. the lamp method has also been applied to the detection of nocardiosis (kawakami, kono & sakai ) . nocardiosis is a serious pathogen affecting yellowtail, seriola quinqueradiata, and amberjack, s. aureovittata. nocardial diagnosis has been mainly achieved by isolation and biochemical identification. recently, pcr detection has been possible for nocardiosis (kono, ooyama, chen & sakai ; miyoshi & suzuki ) . in these studies, the detection limits by lamp and pcr were and cfu, respectively. compared with pcr a -fold higher sensitivity is seen using lamp. furthermore, lamp detection was superior to pcr when spleen dna extracted from infected fish was used as template. detection and diagnosis of viruses is often difficult, and histopathology, virus isolation, western blot and elisa-based detection techniques are often cumbersome, expensive, time consuming and require r & d support to develop viable methods. advent of pcr has not only simplified the development of detection systems, but has also found various applications in virus diagnosis. there are a number of reports regarding the use of lamp recently, detection of white spot syndrome virus (wssv) infecting kuruma shrimp, marsupenaeus japonicus, was reported from our laboratory (kono, savan, sakai & itami ) . the detection limit of the viral dna template was at the fg level, whereas nested pcr-mediated detection was limited to the fg level. detection by lamp was superior to pcr in that it was faster and more sensitive. lamp-mediated detection for koi herpes virus (khv) by targeting the thymidine kinase (tk) gene (aj ) has been developed. the detection limit was found to be similar to pcr primers targeting the tk gene from infected common carp, cyprinus carpio (gunimaladevi, kono, venugopal & sakai ) . screening for khv has become very important as trade of fancy carp is an easy route for geographical spread of the virus and a rapid and efficient system like lamp is very figure loop-mediated isothermal amplification reaction amplifying the haemolysin gene from edwardsiella tarda isolates. the samples denoted as positive are e. tarda isolates. amplification was not seen in the control (-), the no template reaction (nt) and from the non-specific dna template. the products were run on a . % agarose gel stained with ethidium bromide. suitable for this purpose. lamp has also been used to detect red sea bream iridovirus (rsiv) (caipang, haraguchi, ohira, hirono & aoki ) . the rsiv dna was amplified using dna extracts obtained from spleen of infected red sea bream, pagrus major. the protocol developed is times more sensitive than conventional pcr for detecting rsiv. the authors also showed a correlation between the number of dna copies of the virus and the corresponding turbidity, which can be used to quantify virus particles in infected fish. the use of reverse transcription (rt)-pcr as a diagnostic tool for rna viruses has been widely acknowledged; however, rt-pcr suffers from constraints, the cost incurred per sample is high, the time required is high as each sample has to undergo rt and the efficiency is low. with the development of the lamp method, an extended application of reverse transcription (rt)-lamp has been developed (notomi et al. ) . the ability of avian myeloblastosis virus (amv) reverse transcriptase to withstand high temperatures, allows a single tube reaction of both rt and lamp procedures. the first report on the use of rt-lamp was for the detection of the japanese yam mosaic potyvirus from infected leaves, propagules and roots of japanese yam (fukuta, iida, mizukami, ishida, ueda, kanbe & ishimoto a) . recently, protocols for the detection of tomato spotted wilt virus (fukuta, ohishi, yoshida, mizukami, ishida & kanbe ) , severe acute respiratory syndrome (sars) coronavirus (hong, mai, cuong, parida, minekawa, notomi, hasebe & morita ; poon, leung, tashiro, chan, wong, yuen, guan & peiris ) and west nile virus (parida, posadas, inoue, hasebe & morita ) have been developed. of sars samples only % could be detected as positive. lamp was less sensitive than real-time pcr conducted for sars coronavirus (poon et al. ). however, lamp was superior to conventional pcr assay (poon et al. ) . in fish, rt-lamp has been reported for infectious haematopoietic necrosis virus (ihnv) (gunimaladevi, kono, lapatra & sakai ) . this virus affects wild and hatchery-reared salmonids causing widespread mortality. several immunological and molecular methods to detect ihnv have been developed and rt-pcr and real-time pcr are commonly used for ihnv diagnosis. an rt-lamp protocol for detection of ihnv was developed targeting the g-protein of the virus. a comparative analysis of rt-lamp, lamp and nested pcr was conducted. lamp and nested pcr require an additional - min as cdna must be synthesized first. however, rt-lamp can directly use rna as template, where the cdna synthesis and target gene amplification is carried out in a single tube. in this study, lamp was -fold more sensitive than nested pcr. although real-time pcr is a superior method, rt-lamp might be a good alternative as the former can be expensive as a routine diagnostic tool. loop-mediated isothermal amplification has been used for the detection of african trypanosomes, important protozoan parasites causing sleeping sickness in humans (kuboki, inoue, sakurai, di cello, grab, suzuki, sugimoto & igarashi ) . two sets of lamp primers were developed for detecting the trypanosoma brucei group or t. congolense, targeting pfr-a and ribosomal subunit p genes. the authors reported a times higher sensitivity using the lamp protocol compared with normal pcr in detecting trypanosome infection. furthermore, detection of t. brucei from blood samples or cerebrospinal fluid by lamp, shows that the system is not inactivated by tissue and blood-derived inhibitors or genomic dna as seen in pcr. babesia gibsoni is a parasitic infection affecting dogs. detection and analysis of b. gibsoni infection were performed with wholeblood samples by pcr and lamp targeting the s rdna of the parasite (ikadai, tanaka, shibahara, matsuu, uechi, itoh, oshiro, kudo, igarashi & oyamada ) . proliferative kidney disease (pkd) is a serious disease affecting salmonid fish (hedrick, mac-connell & kinkelin ) . a myxozoan parasite, tetracapsuloides bryosalmonae, is the causative agent of pkd. apart from classical may-grunwald-giemsa staining to detect parasites from kidney imprints (klontz & chacko ) , modern techniques like pcr targeting ssu-rdna (kent, khattra, hervio & devlin ) , in situ hybridization (morris & adams ) and mab (adams, richards & de mateo ; saulnier & de kinkelin ) assays are available. recently, el-matbouli & soliman ( ) used lamp for rapid diagnosis of pkdaffected rainbow trout. furthermore, a comparison of pkd-lamp with normal pcr was evaluated. four sets of primers along with loop primers were designed targeting ssu-rdna of t. bryosalmonae. the loop primers were used for the acceleration of the lamp reaction. the pkd-lamp was found to be -fold more sensitive and low amounts of dna as template could be amplified in h. a lamp assay was developed for paracoccidiodes brasiliensis, an endemic fungus causing mycosis in latin america, by targeting the gp gene (endo, komori, ricci, sano, yokoyama, ohori, kamei, franco, miyaji & nishimura ) . dna extracted from paraffin-embedded tissue samples was used as template. the dna extraction, lamp assay and detection required h, while nested pcr required h. no reports exist on the use of lamp for the detection of fungal infections in fish and shellfish. presently, techniques like rapd are used to detect fungi such as aphanomyces (reviewed by cunningham ) . these techniques are technically demanding and have problems with reproducibility and lamp could prove a suitable alternative. the lamp reaction does not progress without the hybridization of six distinct sequences in the target dna by four different highly specific primers, thus it is highly specific. furthermore, the ability of the method to amplify from fewer copies of initial target dna than pcr has been conclusively demonstrated (gunimaladevi et al. (gunimaladevi et al. , kono et al. ; savan et al. ). the efficiency of lamp does not seem to be affected by the presence of non-target genomic dna in the reaction mixture (notomi et al. ) , which is highly desirable in development of a diagnostic system. detection of target dna by lamp compared with detection by two-step nested-pcr was at least equal or more sensitive (gunimaladevi et al. (gunimaladevi et al. , kono et al. ; savan et al. ) . the evidence suggests that lamp is relatively more sensitive than conventional dnabased detection systems. the high sensitivity of the lamp system makes it susceptible to false positives because of carry-over or cross-contamination. amplification and detection should therefore be carried out in separate working areas. as positive reactions are seen as a smear, together with some bands of low molecular weight, and are not seen as a single band as in pcr, the specificity of amplification should be thoroughly validated to ensure that the primers only amplify the target sequence of the specific pathogen. alternatively, the specificity can be determined by cutting the amplified products using restriction enzymes specific to the target sequence. detection of two or more pathogens in a single reaction, as in multiplex pcr or nested pcr, is not possible using lamp. diagnosis of bacterial infections can be cumbersome because isolation and identification takes time and resources. bacterial infection with mixed aetiology makes the diagnosis even more complex. farm managers, together with their previous experience of the clinical signs of diseased fish, rely on diagnostic kits available in the market to detect bacterial agents. the available diagnostic kits are easy to use, do not require expensive equipment but take - h to obtain a result. pcr-mediated detection has been developed for several diseases in aquaculture caused by bacterial or viral agents. the lamp-mediated diagnosis of edwardselliosis, wssv, rsiv, ihnv and khv clearly demonstrates the usefulness of the method as a diagnostic tool. the diagnosis can be based on specific dna markers as target region for the bacteria. lamp can also be used for the detection of bacterial resistance to antibiotics by targeting antimicrobial resistance genes. using lamp less time is required for detection, allowing more time to use management practices to minimize the spread of disease. viral diseases in fish and shellfish can cause mass mortalities and the use of specific pathogen-free stocks is an excellent way to prevent the introduction of disease-causing agents into culture systems. a major problem lies in the introduction of carriers from imported fish stocks, e.g. introduction of infectious pancreatic necrosis virus/ihnv infected fish into japan (yamazaki ) . along with robust legislation, a reliable and sensitive diagnostic technique is needed to detect known and emerging pathogens. the development of diagnostic kits based on nucleic acid detection is simpler and more cost-effective than developing virus specific celllines. nucleic acid-based detection of protozoan and parasitic diseases in fish for efficient management practice in culture systems has been progressing in recent years. many reports and reviews have been published on the detection of parasites using molecular methods in fish and shellfish (gasser & monti ; gasser ; cunningham ) including the use of ribosomal rna genes and spacers by pcr. however, cross-reaction with host tissues because of the presence of complementing conserved rdna sequences has been suggested as a cause of false-positive results (perkins & martin ) . using lamp the specificity could be enhanced as the primers hybridize to six distinct sequences (el-matbouli & soliman ) . development of monoclonal antibodies to pkx, the causative agent of proliferative kidney disease rapid and simple detection of legionella species by lamp, a new dna amplification method a real-time polymerase chain reaction assay of the bacterium edwardsiella ictaluri rapid detection of a fish iridovirus using loop-mediated isothermal amplification (lamp) molecular diagnosis of fish and shellfish diseases: present status and potential use in disease control rapid diagnosis of tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (pkd) in salmonid fish by a novel dna amplification method, loop-mediated isothermal amplification (lamp) detection of gp of paracoccidioides brasiliensis by the loopmediated isothermal amplification (lamp) method rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method use of loop-mediated isothermal amplification of the is sequence for rapid detection of cultured mycobacterium avium subsp. paratuberculosis detection of japanese yam mosaic virus by rt-lamp detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum what's in that band? identification of parasitic nematodes by pcr-sscp of its- rdna detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification a loop mediated isothermal amplification (lamp) method for detection of infectious hematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) proliferative kidney disease in salmonid fishes development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid diagnosis of human herpesvirus infection by a novel dna amplification method, loop-mediated isothermal amplification molecular evidence of infections with babesia gibsoni parasites in japan and evaluation of the diagnostic potential of a loopmediated isothermal amplification method loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples detection of fish nocardiosis by loop-mediated isothermal amplification ribosomal dna sequence analysis of the pkx myxosporean and their relationship to members of the genus sphaerospora methods to detect the organism causing proliferative kidney disease in salmonids sequencing of s- s rrna internal transcribed spacer and its application in the identification of nocardia seriolae by polymerase chain reaction detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification loop-mediated isothermal amplification for detection of african trypanosomes detection of periodontal pathogen porphyromonas gingivalis by loop-mediated isothermal amplification method detection of bacteria carrying the stx gene by in situ loop-mediated isothermal amplification a pcr method to detect nocardia seriolae in fish sample detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation pcr and in situ hybridization of tetracapsula bryosalmonae (pkx), the causative agent of proliferative kidney disease loop-mediated isothermal amplification reaction using a nondenatured template accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus conserved polymerase chain reaction primers fail in diagnosis of parasitic infections loop-mediated isothermal amplification for rapid detection of newcastle disease virus rapid detection of the severe acute respiratory syndrome (sars) coronavirus by a loop-mediated isothermal amplification assay detection of human influenza a viruses by loop-mediated isothermal amplification antigenic and biochemical study of pkx, the myxosporean causative of proliferative kidney disease of salmonid fish sensitive and rapid detection of edwardsiellosis in fish by a loopmediated isothermal amplification method loop-mediated isothermal amplification method targeting the lyta gene for detection of streptococcus pneumoniae sensitive and rapid detection of shigella and enteroinvasive escherichia coli by a loop-mediated isothermal amplification method comparison of loop-mediated isothermal amplification, real-time pcr, and virus isolation for the detection of herpes simplex virus in genital lesions infectious pancreatic necrosis of rainbow trout evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish ictalurus punctatus important bacterial pathogen edwardsiella ictaluri detection of human herpesvirus dna by loop-mediated isothermal amplification key: cord- - pp o authors: lu, renfei; wu, xiuming; wan, zhenzhou; li, yingxue; jin, xia; zhang, chiyu title: a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: pp o covid- has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the covid- virus (sars-cov- ). using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for sars-cov- detection based on its n gene. the assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time pcr machine or visualized via colorimetric change from red to yellow. the limit of detection (lod) of the assay is . copies of sars-cov- rna per μl reaction. the reaction can be completed within min for real-time fluorescence monitoring, or min for visual detection when the template input is more than copies per μl reaction. to evaluate the viability of the assay, a comparison between the rt-lamp and a commercial rt-qpcr assay was made using clinical samples. the sars-cov- rt-lamp assay showed perfect agreement in detection with the rt-qpcr assay. the newly-developed sars-cov- rt-lamp assay is a simple and rapid method for covid- surveillance. since december , an emerging infectious disease , caused by the novel coronavirus sars-cov- , has emerged in wuhan, china [ ] [ ] [ ] . sars-cov- is the seventh coronavirus that causes human infections. like sars-cov and mers-cov, sars-cov- can lead to lethal pneumonia, but it also has a stronger human-to-human transmission capacity than sars-cov and mers-cov [ , ] . as of april , it has caused , infections in countries, including , deaths. the on-going covid- pandemic is a new and huge global public health threat [ , ] . in the absence of suitable antiviral drugs or vaccines, simple, rapid and reliable detection of the sars-cov- infection is critical not only for the prevention and control of the covid- pandemic, but also for clinical treatment [ ] . real-time pcr (qpcr) is the most robust and widely used technology for the qualitative and quantitative diagnosis of viruses, including coronaviruses [ ] [ ] [ ] [ ] . since the outbreak of covid- , of many real-time qpcr (rt-qpcr) assays had been developed, and have played an essential role in laboratory confirmation of sars-cov- infection [ , , ] . however, the rt-qpcr assay requires sophisticated equipment and highly trained personnel, and it is relatively time-consuming (about . - h), which limits its capacity to meet the demand for detecting the virus in a rapidly growing number of patients with covid- infection, suspected infection, or close contact with confirmed cases. therefore, fast, simple, and sensitive point-of-care testing (poct) assays are urgently needed to facilitate the detection of sars-cov- infection. lamp is a simple, fast (within min) and sensitive isothermal amplification technique, that is less dependent on sophisticated equipment, and has been widely used for the development of poct techniques for the detection of viruses and other pathogens in field and/or resource-limited settings [ ] . recently, we developed a mismatch-tolerant version of the lamp method that has higher sensitivity and a faster reaction speed than the conventional ones [ , ] . in this study, we applied the mismatch-tolerant technique to develop novel real-time fluorescent and visual rt-lamp assays for the rapid and sensitive detection of sars-cov- rna, and evaluated the novel assays using clinical samples. sars-cov- viruses are relatively conserved with very low sequence divergence [ ] . to develop rt-lamp assays for sars-cov- detection, we aligned the sars-cov- genomic sequence with those of six other human covs, including sars-cov, mers-cov, hcov-hku- , hcov-nl , hcov-oc and hcov- e. several sets of sars-cov- -specific lamp primers, targeting n, s and rdrp genes, were designed according to the sars-cov- genomic sequence, using the open access primer explorer v. software tool (http://primerexplorer.jp/). the primer specificity was first evaluated via sequence alignment with the other six human coronaviruses and the performance of a homology search using the blast tool in ncbi, and then by performing an rt-lamp reaction with non-template control (ntc). after excluding the primer sets with non-specific amplification in the ntc reaction, one primer set targeting the n gene was found to have higher amplification efficiency, and was thus selected for use in the development of the method (table and figure ). table . primer information of the novel sars-cov- rt-lamp assay. sequence ( '- ') length (nt) f gccaaaaggcttctacgca b ttgctctcaagctggttcaa fip tcccctactgctgcctggagcagtcaagcctcttctcgtt bip tctcctgctagaatggctggcatctgtcaagcagcagcaaag lb tggcggtgatgctgctctt rdrp genes, were designed according to the sars-cov- genomic sequence, using the open access primer explorer v. software tool (http://primerexplorer.jp/). the primer specificity was first evaluated via sequence alignment with the other six human coronaviruses and the performance of a homology search using the blast tool in ncbi, and then by performing an rt-lamp reaction with non-template control (ntc). after excluding the primer sets with non-specific amplification in the ntc reaction, one primer set targeting the n gene was found to have higher amplification efficiency, and was thus selected for use in the development of the method (table and figure ). specificity tests showed that there were no amplification curves or very weak amplification signals observed for all respiratory viruses after min of reaction, indicating the high specificity of the assay (figure ). because corresponding clinical samples or standard strains are unavailable for sars-cov and mers-cov, we did not test the specificity for the two coronaviruses. however, the sequence comparison suggests that the rt-lamp may also amplify sars-cov due to high sequence similarity ( figure b) . specificity tests showed that there were no amplification curves or very weak amplification signals observed for all respiratory viruses after min of reaction, indicating the high specificity of the assay ( figure ). because corresponding clinical samples or standard strains are unavailable for sars-cov and mers-cov, we did not test the specificity for the two coronaviruses. however, the sequence comparison suggests that the rt-lamp may also amplify sars-cov due to high sequence similarity (figure b ). ten-fold serial dilutions of sars-cov- rna standard, from to copies per μl, were used to determine the sensitivity of the rt-lamp assay. rna inputs of × to × copies per μl reaction generated typical s-shaped amplification curves, whereas the rna input of copies did not yield an obvious amplification curve (figure a) , indicating that the rt-lamp can detect as few as copies per reaction. importantly, all amplification curves appeared within min and entered a plateau phase within min when the template input was more than copies (figure a) , indicating that the assay is fast. sensitivity testing showed that when the template input was more than copies of sars-cov- ten-fold serial dilutions of sars-cov- rna standard, from to copies per µl, were used to determine the sensitivity of the rt-lamp assay. rna inputs of × to × copies per µl reaction generated typical s-shaped amplification curves, whereas the rna input of copies did not yield an obvious amplification curve (figure a) , indicating that the rt-lamp can detect as few as int. j. mol. sci. , , of copies per reaction. importantly, all amplification curves appeared within min and entered a plateau phase within min when the template input was more than copies (figure a) , indicating that the assay is fast. standard were inputted. these results indicated that the colorimetric reaction system's sensitivity is similar to that of the fluorescent detection system (figure ). to evaluate the clinical application of the novel rt-lamp assay, a comparison with a commercial rt-qpcr assay was performed, using clinical samples collected from both covid- -suspected patients and control populations. of these samples, and were detected as sars-cov- positive and negative by both assays, respectively ( table ). the concordance rate between both assays was . %. two samples were detected as positive by one assay, but negative by another. the two negative samples detected by the novel rt-lamp assay had high threshold cycle (ct) values (> ) in the rt-qpcr assay, indicating a very low viral copy number. table . comparison of the novel rt-lamp assay with a commercial rt-qpcr assay. sensitivity testing showed that when the template input was more than copies of sars-cov- rna, all reactions ( %) displayed positive amplification. when the template input was and copies, eight and six of the reaction replicates showed positive, respectively ( table ). the limit of detection (lod) of the new rt-lamp assay was calculated to be . copies per reaction. for convenient use, a visual detection version of the sars-cov- rt-lamp assay was developed. the reaction gave clear color change, from red to yellow, for all samples tested for over min ( - min). therefore, the min time period was selected as the optimal protocol (cut-off) for the visual assays (figure b) . a very faint color change was observed when copies of the rna standard were inputted. these results indicated that the colorimetric reaction system's sensitivity is similar to that of the fluorescent detection system (figure ). to evaluate the clinical application of the novel rt-lamp assay, a comparison with a commercial rt-qpcr assay was performed, using clinical samples collected from both covid- -suspected patients and control populations. of these samples, and were detected as sars-cov- positive and negative by both assays, respectively ( table ). the concordance rate between both assays was . %. two samples were detected as positive by one assay, but negative by another. the two negative samples detected by the novel rt-lamp assay had high threshold cycle (ct) values (> ) in the rt-qpcr assay, indicating a very low viral copy number. table . comparison of the novel rt-lamp assay with a commercial rt-qpcr assay. the covid- is a newly emerging, life threatening respiratory disease caused by sars-cov- , a newly identified human coronavirus [ ] [ ] [ ] ] . as the seventh human coronavirus and the third most lethal coronavirus, sars-cov- has a higher affinity to human receptor ace than sars-cov [ , ] , and a greater human-to-human transmission capacity than other human coronaviruses [ , ] . because of its newness, communicability, and rapid spread, sars-cov- has led to a global pandemic and created great international concern. as of april , , people in china and , people in over other countries have been infected with sars-cov- , and a total of , individuals have died from covid- . transmission of sars-cov- appears to mainly occur in the early stage of infection, with higher viral loads in the nasopharyngeal tract, or soon after the symptom-onset of covid- [ , ] . early diagnosis, and the timely implementation of intervention and quarantine measures, are crucial to prevent the spread of the virus and optimize clinical management. therefore, the development of simple, rapid and reliable diagnostic assays is of high priority for the prevention and control of covid- . many rt-qpcr assays have been developed since the outbreak, and contributed to the confirmation of most sars-cov- infections during the pandemic in china [ , , ] . currently, although the covid- pandemic has been contained following the strong public health intervention efforts of the chinese government, the virus is spreading quickly outside china, including into some resource-limited areas. the stringent requirements of facilities and professional workers, and its time-consuming nature, limit the use of rt-qpcr assays in some undeveloped countries. furthermore, the presence of asymptomatic infections and the long incubation period of covid- increase the difficulty of diagnosis, because some sars-cov- carriers may not visit the hospital for diagnosis due to lack of symptoms or signs of infection [ ] . therefore, simple, rapid and sensitive poct detection assays, that can be deployed more widely, will be very helpful for the early diagnosis of sars-cov- infection, and should be developed. one promising poct method, lamp, has been used for the detection of various pathogens because of its high sensitivity, rapid reaction speed, relatively simple operation, and visual determination capability [ , , , ] . we recently upgraded the lamp method to a mismatch-tolerant version [ , ] . compared to the conventional lamp method, the novel version contains an additional . u of high-fidelity dna polymerase, which confers upon it a higher applicability to highly variable viruses, and a - min faster reaction speed. in this study, we established a rapid and sensitive one-step single-tube rt-lamp assay, for the detection of sars-cov- using the mismatch-tolerant technique. the assay can be performed at • c for min in a real-time pcr instrument, for real-time monitoring using fluorescent dye, or for min in a regular pcr machine or heating block (e.g., dry incubator or water bath), for visual detection using ph-sensitive indicator dyes. in the real-time monitoring system, syto is used as a fluorescent dye which has a minimal inhibitory effect on lamp amplification [ , ] . in the visual detection system, cresol red is used as a ph indicator dye because of a clear color contrast between negative (red) and positive (yellow) reactions after min of incubation at • c; a color change from red to orange or yellow is defined as sars-cov- positive [ ] . the novel rt-lamp assay has a high sensitivity, with a lod of . copies per reaction, and shows no cross-reactivity with common human respiratory viruses, including four other human coronaviruses (oc , e, hku- and nl ). in particular, when the template input is more than the lod, the assay has a very fast detection speed of less than min. the robustness of the novel assay was tested using clinical samples from covid- patients and control populations in nantong. the novel rt-lamp assay showed a high consistency ( . %) with a commercial rt-qpcr assay for sars-cov- rna detection. there were four samples showing inconsistent results by both the rt-qpcr assay and the novel rt-lamp assay. the reason for two samples testing positive by the rt-qpcr assay but negative by the rt-lamp assay may be the very low copy numbers (having high ct values of > in the rt-qpcr assay). however, regarding the other two samples tested positive by the rt-lamp assay (with a high time threshold of > min) but negative by the rt-qpcr assay, the reason is speculated to be the presence of viral variants, that have caused mismatches with the primers and/or probe [ , ] . although the sars-cov- detection rate in nasal swabs was reported to be lower than in bronchoalveolar lavage fluid (balf) and sputum, the high viral load in nasal swabs, combined with easy sampling, will facilitate the early diagnosis of covid- , especially for mild and asymptomatic infections [ ] . in view of a mean viral load of . × copies/ml in nasal swabs of covid- patients, the novel assay is sufficiently sensitive for the detection of sars-cov- at an early stage of infection using nasal swab specimens. recently, the nucleic acid extraction-free protocol of the lamp assay was developed by directly adding naoh-treated swabs into the reaction [ ] . the rna extraction-free rt-lamp assay for sars-cov- should be deployed in the future. in brief, the optimal rt-lamp assay for real-time monitoring is a µl reaction mixture, containing: x isothermal amplification buffer; mm mgso ; . viral rna was extracted from µl throat swabs of suspected covid- patients, using an rna extraction kit (liferiver, shanghai, china), and eluted in µl of nuclease-free water for immediate use or storage at − • c. for the specificity tests, total nucleic acids were extracted from µl throat swabs that tested positive for common respiratory viruses and virus culture supernatants (hcov-oc : vr- and hcov- e:vr- ), using the magna pure lc total nucleic acid isolation kit (roche diagnostics gmbh, mannheim, germany) according to the manufacturer's instruction, and then eluted in µl of nuclease-free water. the specificity of the sars-cov- rt-lamp assay was evaluated using common respiratory viruses. two human coronaviruses (hcovs), hcov-oc (vr- ) and hcov- e, were from standard strains of vr- and vr- , respectively, which were purchased from the american type culture collection (atcc). nucleic acids of another viruses (including: influenza a, b, and c viruses; parainfluenza viruses type - ; enterovirus; rsv a and b groups; hcov-hku- ; hcov-nl ; human rhinovirus; human metapneumovirus; adenovirus; and bocavirus) were obtained from positive clinical samples from children with acute respiratory tract infections. to prepare the rna standard, a sars-cov- n gene fragment ( - nt in wuhan-hu- , genbank: mn . ) was amplified from a positive clinical sample with primers containing the t promoter. the rna standard was obtained via in vitro transcription, and quantified by a qubit ® . fluorometer (thermo fisher scientific, usa). the copy number of the rna standard was calculated using the following formula: rna copies/ml = [rna concentration (g/µl)/(nt transcript length × )] × . × . ten-fold serial dilutions of the rna standard, from to copies per µl, were used as the standards to determine the sensitivity of the novel sars-cov- rt-lamp assay. for the lod test, ten-fold serial dilutions of the rna standard, from to copies per µl, were used. each dilution was tested in a set of replicates. to more accurately estimate the lod, additional experiments were performed by adding five-fold dilutions of the rna standard ( , copies, copies, copies, copies and . copies) to the µl reaction volume. the lod was defined as a % probability of obtaining a positive result, using probit regression analysis with spss . software [ ] . to evaluate the performance of the novel rt-lamp assay for sars-cov- detection, throat swabs were collected from suspected covid- patients and individuals who were admitted or quarantined at nantong third hospital. sars-cov- infection was detected with a commercial sars-cov- rt-qpcr kit (liferiver bio, shanghai, china) and the novel rt-lamp assay. because the amounts of rna from clinical samples were very limited, we only performed a comparison between the real-time monitoring rt-lamp assay and the commercial rt-qpcr assay. the concordance rate between both assays was calculated by the formula: (number of consistent results by both methods/total number) × %. for visual detection, a warmstart colorimetric lamp x master mix (new england biolabs, beverly, ma, united states), that uses cresol red as a visual indicator, was used. a µl reaction system was set up, containing: x warmstart colorimetric lamp buffer; . unit of q high-fidelity dna polymerase; . µm each of primers fip and bip; . µm each of primers f and b ; and . µm of loop primer lb. the reactions were performed at • c, and the color change from red to yellow was observed at the , , and min time points. the study was approved by nantong third hospital ethics committee (e : february ). written informed consents were obtained from each of the involved patients. in conclusion, we developed a new, simple and sensitive rt-lamp assay for the fast and accurate detection of sars-cov- . the rt-lamp assay has a high sensitivity, with a lod of . copies of sars-cov- rna per µl reaction, and good specificity regarding common respiratory viruses. validation using clinical samples confirmed that the rt-lamp assay's performance was similar to that of the rt-qpcr assay in the detection of sars-cov- . although their specificity regarding sars-cov and mers-cov was not tested, in view of the lethal nature of these two coronaviruses and sars-cov- , a positive result for any of these three coronaviruses might be of clinical importance. the novel sars-cov- rt-lamp assay has been developed into real-time monitoring and colorimetric versions, both of which will be especially useful in covid- surveillance. author contributions: conceptualization, c.z. and r.l.; methodology, r.l., x.w. and y.l.; validation, c.z. and r.l.; formal analysis, c.z. and x.w.; investigation, r.l., x.w. and z.w.; resources, r.l. and z.w.; data curation, c.z. and x.w.; writing-original draft preparation, c.z.; writing-review and editing, x.j.; visualization, c.z. and x.w.; supervision, c.z.; funding acquisition, c.z. all authors have read and agreed to the published version of the manuscript. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin compensation of ace function for possible clinical management of -ncov-induced acute lung injury cryo-em structure of the -ncov spike in the prefusion conformation the continuing -ncov epidemic threat of novel coronaviruses to global health-the latest novel coronavirus outbreak in wuhan the first disease x is caused by a highly transmissible acute respiratory syndrome coronavirus racing towards the development of diagnostics for a novel coronavirus molecular diagnosis of a novel coronavirus ( -ncov) causing an outbreak of pneumonia a melting curve-based multiplex rt-qpcr assay for simultaneous detection of four human coronaviruses a mismatch-tolerant rt-quantitative pcr: application to broad-spectrum detection of respiratory syncytial virus clinical features of patients infected with novel coronavirus in loop-mediated isothermal amplification of dna a mismatch-tolerant reverse transcription loop-mediated isothermal amplification method and its application on simultaneous detection of all four serotype of dengue viruses a mismatch-tolerant rt-lamp method for molecular diagnosis of highly variable viruses genomic variance of the -ncov coronavirus preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak pattern of early human-to-human transmission of wuhan sars-cov- viral load in upper respiratory specimens of infected patients detection of sars-cov- in different types of clinical specimens clinical characteristics of coronavirus disease in china development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- visual detection of isothermal nucleic acid amplification using ph-sensitive dyes a direct isothermal amplification system adapted for rapid snp genotyping of multifarious sample types methods to determine limit of detection and limit of quantification in quantitative real-time pcr (qpcr) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the biobank of pathogen discovery and big data center, institut pasteur of shanghai, chinese academy of sciences (cas), for storing the standard strains of hcov-oc and hcov- e that were previously purchased from the american type culture collection (atcc). we also thank lulu zuo at pathogen discovery and evolution unit, institut pasteur of shanghai, cas for her technical support. ace angiotensin converting enzyme ii poct point-of-care testing balf bronchoalveolar lavage fluid ncbi national centre for biotechnology information the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. coronavirus key: cord- -sduz j authors: bokelmann, l.; nickel, o.; maricic, t.; paabo, s.; meyer, m.; borte, s.; riesenberg, s. title: rapid, reliable, and cheap point-of-care bulk testing for sars-cov- by combining hybridization capture with improved colorimetric lamp (cap-ilamp) date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: sduz j efforts to contain the spread of sars-cov- have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can easily be applied to large numbers of people. however, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation, sophisticated laboratory equipment and trained personnel to achieve throughputs that allow whole communities to be tested on a regular basis. here we present cap-ilamp (capture and improved loop-mediated isothermal amplification). this method combines a hybridization capture-based rna extraction of non-invasive gargle lavage samples to concentrate samples and remove inhibitors with an improved colorimetric rt-lamp assay and smartphone-based color scoring. cap-ilamp is compatible with point-of-care testing and enables the detection of sars-cov- positive samples in less than one hour. in contrast to direct addition of the sample to improved lamp (ilamp), cap-ilamp does not result in false positives and single infected samples can be detected in a pool among uninfected samples, thus reducing the technical cost per test to ~ euro per individual. the recent global outbreak of coronavirus disease has led governments to take drastic measures to contain the spread of the severe acute respiratory syndrome coronavirus (sars-cov- ). most affected countries resorted to more or less stringent lockdown measures including restrictions on travel, public gatherings and the closing of institutions such as schools, kindergartens and universities. these restrictions and closings have usually been implemented broadly, affecting the lives of infected and uninfected people alike, as no reliable and economical mass testing approach for decentralized point-of-care identification of infected individuals exists. reverse transcription followed by quantitative pcr (rt-qpcr) is the most widely used method to detect rna viruses such as sars-cov- . however, its need for trained personnel and expensive instrumentation and global shortages of resources for rna purification has spurred search for viable alternatives. loop-mediated isothermal amplification (lamp) can rapidly amplify small target nucleic acid sequences under isothermal conditions (notomi et al. ) and has been applied for molecular diagnostics (wong et al. ) . the reaction requires four to six primers and produces concatemers of double-stranded amplification products. these can be detected directly, using intercalating dyes (e.g. sybr green, syto-dyes), its reaction with triphenylmethane dye precursors and acid hydrolysis (miyamoto et al. ; trinh and lee ), or using cleavage with crispr enzymes coupled with lateral flow color detection of the cleavage product (broughton et al. ). however, these methods require opening of the tube after the reaction, thus posing the threat of cross-contaminating future reactions with the amplified product. amplification can also be detected indirectly by hydroxynaptholblue or phenol red based detection of the release of protons and/or pyrophosphate generated during dna synthesis (tanner et al. ) . recently, a number of studies explored ways to detect sars-cov- rna using rt-lamp (lamb et al. ; yang et al. ; zhang et al. ) but they required time consuming rna isolation steps before the reaction. there have also been attempts to add sample directly into the reaction without prior purification (buck et al. ; dao thi et al. ). however, it was noted that the ph of nasopharyngeal swab samples often varies and can adversely affect readouts. here we describe a method to detect sars-cov- rna of a single infected individual within a bulk sample comprised of up to individual patient samples by combining a hybridizationcapture-based rna extraction approach with smartphone app-assisted colorimetric detection of rt-lamp products, a procedure that can be performed in less than one hour ( figure a) . we compared the sensitivity of the method to standard extraction rt-qpcr protocols in a diagnostic lab and validate its performance on gargle lavage samples from a hospital, a nursing home previously affected by covid- , and round robin samples from a reference institution of the german medical association. we evaluated three published rt-lamp primer combinations targeting either the orf a gene or the n gene of the sars-cov- genome (lamb et al. ; zhang et al. ) using a dilution series of synthetic viral rna (twist biosciences, san francisco, ca, usa) and chose the two most sensitive primer sets (cv - and cv - , supp. table ) table ). using both primer combinations, we detected synthetic viral rna copies after - minutes incubation at °c as measured by fluorescence real time rt-lamp (supp. figure a and c). combining the primers for the orf a gene and the n gene did not increase sensitivity (supp. figure d ). amplification in lamp reactions is often detected colorimetrically by a ph sensitive dye that changes color when extensive dna synthesis lowers the ph of the reaction (tanner et al. ) . as noted in a previous study (dao thi et al. ) , biological samples such as nasopharyngeal swab eluates may change the ph when added to the lamp reaction directly, leading to false positive results. we found that nasopharyngeal swab eluates tend to be more acidic than gargle lavage samples and that adding gargle lavage directly to a lamp reaction at a final concentration of % leads to false positive results in . % of cases even before the isothermal incubation (supp. figure a and b). it would therefore be preferable to detect the amplification product directly rather than indirectly by a ph change. to achieve this, we deposited a drop of . microliters of a , x concentrated solution of the dye sybr green i to the cap of the tube before the reaction is initiated. shaking the tube after the isothermal amplification dissolves sybr green i, stops the reaction and allows visual detection of sars-cov- via a color change from orange/red to intense yellow ( figure c ). the color of the reaction can be objectively quantified as a single numerical hue value, that is insensitive to light intensity changes and can be derived from the red, green, blue (rgb) color model (cappi et al. ; yin et al. ) , by using freely available 'camera color picker' smartphone apps. we used the 'palette cam' app (alexander mathers, app store) for extracting rgb values before conversion to hue. an additional advantage is that this detection strategy allows us to include acidifying enhancing enzymes in the lamp reaction, namely tte uvrd helicase, which prevents unspecific late amplification of artefacts all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint induced by primer interactions (supp. figure a and b) and thermostable inorganic pyrophosphatase which increases reaction speed (miyamoto et al. ) . however, addition of patient gargle lavage directly to this improved lamp (ilamp) reaction promoted false positives ( . %) (supp. figure a ). presumably, this unspecific amplification is due to dna from the oral microbiome, food or host cells as it can be prevented by prior λ exonuclease treatment that preferentially digests '-phosphorylated dna leaving nonphosphorylated primers and ilamp product intact (supp. figure b ). because, gargle lavages from known infected patients did result in false negatives for all seven samples we employed an initial quick extract (lucigen, middleton, wi, usa) lysis (ladha et al. ) to release more viral rna from the capsid. even when these optimized reaction conditions were used, reactions resulted in one false negative out of the seven gargle lavages from infected patients and the single sars-cov- negative gargle lavage sample was false positive (supp. figure b ). we therefore employed a rapid ( min) bead-capture enrichment purification akin to mrna isolation, using two oligonucleotides flanking the rt-lamp target sites ( figure b , supp. table ) immobilized on paramagnetic beads. this step eliminates non-target nucleic acids and other unwanted components in the biological samples but also concentrates the viral rna. comparing the ct-values of rt-qpcr targeting the e gene (corman et al. ) after silica-based rna-extraction with hybridization capture targeting the e gene for the same volume of gargle lavage suggests a capture efficiency of roughly % ( figure d ). to allow comparison to rt-qpcr we captured viral rna with a biotinylated probe for the e gene and not for the orf a and n gene as used for rt-ilamp. when rna is concentrated from µl gargle lavage to µl final volume and µl input volume is used in ilamp, this results in a detection limit of - viral genome copies per µl of sample before capture. the reagents required for the ilamp reaction can be pre-mixed and freeze-thawed at least twice. cap-ilamp could be performed at point-of-care as no bulky equipment and only pipettes, a thermoblock, and a magnetic rack are needed ( figure e ). we used cap-ilamp to test gargle lavage samples from hospital patients and elderly nursing home inhabitants and employees either individually or in pools. these samples had previously been tested by a rt-qpcr assay targeting the sars-cov- e gene (maricic et al. ) and showed either no amplification or ct values ranging from . to . ( figure a ). cap-ilamp targeting the sars-cov- orf a gene or the n gene results in an orange/red and intense yellow color for sars-cov- rna negative and positive samples, respectively ( figure b ). of the samples which we tested individually ( figure c) , six had been previously tested all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint positive in the rt-qpcr assay. scoring the color of the ilamp reactions using a smartphone 'camera color picker' app shows that hues for sars-cov- negative and positive samples are clearly separated, below ° for the former and above ° for the latter, respectively. all six negative samples were correctly identified as negative in the cap-ilamp assays targeting the orf a and the n gene. of the six samples that were sars-cov- positive in the rt-qpcr assay, four were positive in the cap-ilamp assay while the remaining two were false negative. when one twentieth of input volume were used they were positive, suggesting that some residual inhibition originating either from the biological sample or from lysis/binding buffer carryover exists in these extracts. to investigate whether it is possible to detect single infected individuals in pools of gargle lavage samples, we created eleven pools of patient samples each, all of which had been tested negative in rt-qpcr assay and in the cap-ilamp assays for the orf a and the n gene ( figure d ). in order to determine if components of the pooled gargle lavage still inhibit the rt-lamp reaction after capture, we took subsamples of the negative pools and added copies/µl of artificial viral rna before cap-ilamp. all pools were positive in both cap-ilamp assays, showing that there was no substantial inhibition in these extracts after capture. to investigate whether it is possible to detect a single infectious individual within a pool, we added different single positive patient samples (ct < ) to three pools of healthy individuals so that / ( . %) of the final volume was composed of the infected sample. in all three cases, the cap-ilamp assays targeting the orf a and the n gene were positive, demonstrating that a single infectious individual can be detected in a pool of samples. the two sars-cov- positive samples previously found to be inhibited when tested individually ( figure c ) were correctly identified as positive when pooled ( figure d ), potentially because inhibition is rare and is diluted out in the pool. an additional wash step after hybridization capture should thus be applied for individual sample diagnostic testing. all tested gargle lavages from single healthy individuals (n= ) and pools of healthy individuals (n= ) correctly tested negative for both the orf a gene and n gene in the cap-ilamp assay ( figure c and e), indicating that false positive results which were sometimes observed when samples are added directly into the ilamp reaction (supp. figure a perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint amounts of sars-cov- (ring , , and ), different coronaviruses (ring hcov oc and ring hcov e) or no virus (ring ) and had previously been tested via rt-qpcr (maricic et al. ) ( figure a ). to prevent sporadic inhibition as observed for some gargle lavage samples, the first wash step after capture hybridization was performed twice. as shown in figure b , all four sars-cov- -containing samples were correctly and consistently identified by both the orf a and n gene assays while all samples devoid of virus or containing a different coronavirus were correctly classified as being negative. we have established cap-ilamp as a reliable and rapid diagnostic test for sars-cov- . our method overcomes problems associated with standard rt-lamp ph-dependent colorimetric detection which is prone to false positives due to ph-variability in gargle lavages and off-target amplification. gargle lavage cause no discomfort and is thus more suitable than pharyngeal and nasal swabs for frequent and repeated testing of apparently healthy individuals (saito et al. ) . they have the additional advantage that they do not expose the person collecting samples to any risk of infection. in contrast to rt-qpcr approaches, no complicated equipment such as qpcr machines is required. removal of inhibitors and enrichment of target nucleic acids by on-bead capture, combined with a double assay of two sars-cov- -specific target sites and control reactions to detect inhibitor carryover and contaminated reagents ensure accuracy. as quality controls we recommend dedicated water negative controls and sample-specific inhibition/positive controls containing sample and synthetic viral rna for each the orf a and the n gene assay. a scheme for evaluating the results is shown in supp. table . due to the removal of non-target nucleic acids and the inclusion of lamp enhancing enzymes in cap-ilamp, we did not detect false positive results, which were often reported in previous lamp-based detection methods (teng et al. ; abbasi et al. ; hardinge and murray ) . the inclusion of dye in the cap of the tube allows to keep the tube closed for detection after amplification thus preventing the cross-contamination of future assays. we quantified color as a single numeric hue value using a freely available smartphone app, which allows objective point-of-care detection without the ambiguity of an individual's perception of color. yin et al. (yin et al. ) already described application of hue for hydroxynaptholblue color scoring that is in need of a custom-made 'smart cup' reaction tube holder as well as an app that is not freely available. consequently, cap-ilamp outperforms other published sars-all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint cov- detection methods (broughton et al. ; buck et al. ; corman et al. ; dao thi et al. ) in some key aspects as shown in table . the cap-ilamp assay passed an official round robin test used to evaluate testing accuracy across diagnostic laboratories. we show that it is possible to detect a single infected individual in a pool of samples, opening the prospect of rapid screening of large numbers of people in public decentralized settings such as in schools, hospitals, airports, prisons, retirement homes or border crossings. this also allows daily testing of people at risk or working in critical infrastructure. the cap-ilamp method can be readily applied to numerous human respiratory pathogens, plant pathogens, animal pathogens, food-borne diseases, viruses, protozoan parasites, and fungi, for which primer combinations have been developed (wong et al. ). these include the eight diseases prioritized by the who (as of july ) that pose the greatest public health risk due to their epidemic potential and/or whether there is no or insufficient countermeasures (kurosaki et al. ; fukuma et al. ; osman et al. ; oloniniyi et al. ; huang et al. ; ma et al. ) . for testing in remote areas or developing countries master mixes for ilamp could potentially be lyophilized for transportation or long term storage, as shown previously (chen et al. ; carter et al. ) . sampling of gargle lavages was done as described previously by our group (maricic et al. ). all individuals included in this study were asked for their voluntary assistance to participate and each individual gave written informed consent before entry into the study. the study was approved by the ethics committee of the saxonian medical chamber (ek-allg- / - ). all procedures utilized in this study are in agreement with the declaration of helsinki. ml of sterile water was filled into sterile urine cups. the full water volume was gargled for seconds before spitting it back into the cup. analyzed anonymized gargle lavages from hospitalized covid- patients and round robin samples (instand) were kept frozen below - °c for - months. in solution capture purification of sars-cov- nucleic acids per reaction µl dynabeads myone streptavidin c bead suspension (thermo fisher scientific, # . ) are pelleted using a magnetic rack and washed twice with µl combined x lysis/binding buffer (lysbb: mm tris-hcl, ph . , mm licl, . % lids, mm edta, mm dtt). washed beads are then resuspended in µl x lysbb before addition of pmol cv _btn and pmol cv _btn (see supp. table all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . for the rt-qpcr assay) and rotation for min at room temperature. beads are pelleted and supernatant is discarded before resuspension in µl alkaline wash solution ( mm naoh, and min incubation at room temperature. beads are pelleted with a magnetic rack and supernatant is discarded. beads are then washed once with µl x lysbb before resuspension in µl x lysbb. prepared bead suspension can be stored at °c or used directly. to µl ready-made bead suspension an equal volume of patient gargle lavage (sample), water (negative control) or sample with k copies of artificial sars-cov- rna (twist biosciences, # ) (positive and inhibition control) is added and mixed by pipetting. the reaction is incubated at °c for min and then placed on a magnetic rack. the supernatant is discarded and the beads are thoroughly resuspended in µl wash buffer ( mm tris-hcl, ph . , mm licl, mm edta). beads are pelleted and supernatant is discarded, repeat this step once for individual or inhibited samples. wash beads once with µl low salt buffer ( mm tris-hcl, ph . , mm licl, mm edta) and discard supernatant. beads are resuspended in µl elution buffer ( mm tris-hcl, ph . , mm edta) and rna is eluted by heating to °c for min. transfer the supernatant to a fresh . ml strip tube or use directly as input for rt-ilamp or rt-qpcr. for each sample two assays targeting either the orf a or n gene are performed. primer mixes for both assays are prepared in bulk for quick reaction assembly (supp table ). µl of rt-ilamp master mix containing either the orf a (cv - ) or n gene (cv - ) primer sets (supp. tables ) and µl of capture eluate are combinded to obtain a final reaction volume of µl ( x warmstart® colorimetric lamp master mix (neb, ipswich, ma, usa, #m l), mm atp, µm syto , x primer mix, . ng/µl tte uvrd helicase (neb, #m s), . u/µl thermostable inorganic pyrophosphatase (neb, #m l), . u/µl protector rnase inhibitor (sigma aldrich, st. louis, mi, usa, # )). for color reactions, . µl sybr green i is applied to the lid of the tube without getting into contact with the reaction liquid. for transport or preparation of large numbers of reactions it is also possible to immobilize the dye by drying for min at °c in an oven. if amplification curves should be recorded on a qpcr machine, no sybr green i is needed. the reaction is incubated at °c for min. if sybr green i was applied to the cap of the tube, the reaction is then stopped by shaking and can be evaluated by eye or with any 'camera color-picker' app on a smartphone within a minute. we used the 'palette cam' app to score rgb values and extracted the hue. this can be easily done using a web tool (e.g. https://www.rapidtables.com/convert/color/rgb-to-hsv.html). hue can also be scored directly when using e.g. 'color grab (color detection)' app for android or 'aurora' app for ios. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . (d) capture efficiency was estimated relative to automated silica-based rna extraction based on copy number estimates for rt-qpcr assay targeting the sars-cov- e gene. (e) equipment necessary for cap-ilamp: pipettes and pipette tips, pre-mixed reagents (including ilamp master mix and capture bead suspension), stable buffers (lysis/binding buffer,wash buffer, low salt buffer, elution buffer), a magnetic rack and a thermoblock. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint table : comparison of sars-cov- detection methods. ten important aspects of different published sars-cov- detection methods and cap-ilamp are compared and ranked optimal (green), sub-optimal (light green), and critical (orange). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint supplementary table : evaluation of cap-ilamp results. for each of the two assays (targeting either the sars-cov- orf a or n gene), a water negative control as well as a sample-specific positive/inhibition control comprised of sample and artificial viral rna should be included. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint supplementary fig. . evaluation of different amounts of tte uvrd helicase in lamp reactions. amplification plots of negative controls (left panel) and positive controls containing copies of artificial sars-cov- rna (right panel). amount of tte uvrd helicase in µl reaction are indicated on the right. the primer set used targeted the n gene (cv - ), the assay proceeded at °c. fig. direct lamp of patient gargle lavage samples can lead to false positives and false negatives. (a) hues measured for negative (n= ) and positive (n= ) controls after rt-ilamp reactions (left side) and after rt-ilamp of sars-cov- -negative (n= ) gargle lavage samples directly added to the reaction mix. (b) hues measured after rt-ilamp reactions preceeded by a λ exonuclease (neb) digestion step ( u per reaction for - min at °c) for negative (n= ) and positive (n= ) controls (left side), sars-cov- -negative (n= ) and sars-cov- -positive (n= ) gargle lavage samples directly added to the reaction mix (middle) and sars-cov- negative (n= ) and sars-cov- -positive (n= ) gargle lavage samples directly added to the reaction mix digested with quickextract (qe) and heat inactivated for min at °c before lamp (right). schematic drawing of λ exonuclease digestion shown on the right. primers and lamp reaction products do not possess a '-phosphate and are thus not a target for λ exonuclease digestion. all reactions contained µg t gene protein (neb) as it could alleviate inhibition of some gargle lavages in initial experiments. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint optimization of loop-mediated isothermal amplification (lamp) assays for the detection of leishmania dna in human blood samples crispr-cas -based detection of sars-cov- standard operating procedures for sars-cov- detection by a clinical diagnostic rt-lamp assay label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance lyophilized visually readable loopmediated isothermal reverse transcriptase nucleic acid amplification test for detection ebola zaire rna development of lyophilized loop-mediated isothermal amplification reagents for the detection of leptospira detection of novel coronavirus ( -ncov) by real-time rt-pcr screening for sars-cov- infections with colorimetric rt-lamp and lamp sequencing rapid detection of lassa virus by reverse transcription-loop-mediated isothermal amplification reduced false positives and improved reporting of loop-mediated isothermal amplification using quenched fluorescent primers a rapid and specific assay for the detection of mers-cov development and evaluation of a simple assay for marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method a -min rna preparation method for covid- detection with rt-qpcr rapid detection of novel coronavirus (covid- ) by reverse transcription-loop-mediated isothermal amplification rapid and specific detection of all known nipah virus strains' sequences with reverse transcription-loop-mediated isothermal amplification a direct rt-qpcr approach to test large numbers of individuals for sars-cov- method for colorimetric detection of doublestranded nucleic acid using leuco triphenylmethane dyes loopmediated isothermal amplification of dna rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (rt-lamp) development and evaluation of loop-mediated isothermal amplification assay for detection of crimean congo hemorrhagic fever virus in sudan gargle lavage as a safe and sensitive alternative to swab samples to diagnose covid- : a case report in japan visual detection of isothermal nucleic acid amplification using ph-sensitive dyes specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for taura syndrome virus by colorimetric dot-blot hybridization a foldable isothermal amplification microdevice for fuchsin-based colorimetric detection of multiple foodborne pathogens loop-mediated isothermal amplification (lamp): a versatile technique for detection of micro-organisms rapid detection of sars-cov- using reverse transcription rt-lamp method synergistically enhanced colorimetric molecular detection using smart cup: a case for instrument-free hpv-associated cancer screening rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp supplementary fig. . sensitivity of different primer sets targeting sars-cov- rna. (a) amplification curves we thank the management of diakonische dienste leipzig, the personnel and residents of altenpflegeheim emmaus in leipzig, and the personnel of the department of laboratory medicine at hospital st georg leipzig for their cooperation. funding was provided by the max planck society, the nomis foundation, and the immunodeficiencycenter leipzig. the authors declare no conflict of interest. key: cord- -u ohm p authors: liu, xiaonan; zhang, chao; zhao, mengye; liu, kewu; li, hang; li, ningning; gao, linlin; yang, xuemin; ma, ting; zhu, juanli; hui, wenli; hua, kai; cui, yali title: a direct isothermal amplification system adapted for rapid snp genotyping of multifarious sample types date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: u ohm p genotyping of single nucleotide polymorphisms (snps) in point-of-care (poc) settings could be further improved through simplifying the treatment of samples. in this study, we devised an accurate, rapid and easy-to-use snp detection system based on direct loop-mediated isothermal amplification (lamp) without dna extraction, known as direct-lamp. samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with naoh, can be used directly in amplification. the turnaround time was about min from sample collection to provision of results. the accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (mthfr) c t and aldehyde dehydrogenase- (aldh ) glu lys, which are better known for their critical role in folate and ethanol metabolism, respectively. completely consistent genotyping results reveal that direct-lamp is generally concordant with sequencing. this system can serve as a very promising platform in the fields of disease predisposition, drug metabolism and personalized medicine. the most abundant source of genetic variation (approximately %) in the human genome is represented by single nucleotide polymorphisms (snps), which can account for heritable inter-individual differences in complex phenotypes (mhlanga and malmberg, ; yu et al., ) . hence, snp detection has great potential for direct clinical application by providing highly accurate diagnostic information for facilitating early diagnosis, prevention, and treatment of genetic diseases (everitt et al., ; moore et al., ) . because of its accuracy, dna sequencing is considered to be the gold standard for snp detection analysis. however, given the subsequent massive data analysis required by the method, sequencing is more suitable for discovery of novel snps rather than detection of specific known snps (grant et al., ; hendre et al., ; muhammad et al., ) . other technologies rely on sample amplification, including dna microarrays michal et al., ; zhang et al., ) , real-time polymerase chain reaction (rt-pcr) (martinez-serra et al., ; psifidi et al., ) , selfsustained sequence replication ( sr) (ren et al., ) , strand displacement amplification (sda) (toley et al., ) , high resolution melting (hrm) (norambuena and copeland, ) , mass array (kriegsmann et al., ; suthandiram et al., ) , and multiplex pcr-rflp method (loo et al., ) also offer sensitive approaches for snp detection. however, the tremendous potential of snp detection in clinical practice has so far often been limited by the lack of efficient screening methods which cannot meet the requirements of point-of-care (poc) testing strategies due to their complex experimental procedures and long testing times (tost and gut, ) . based on the status quo, development of a new detection platform that shortens assay times and simplifies procedures would be highly desirable. saving time from dna purification, a conventional step in snp detection that not only complicates the procedure, but also enhances the risk of invalid results and cross-contamination, is a promising approach. it has been reported that new snp detection methods have been developed based on pcr that do not require dna extraction from whole blood, hair root and saliva (cascella et al., ; hallie and reena, ; hayashida et al., ) . by treating them with specific chemicals, samples can be used directly in pcr without an initial dna purification step, which would save approximately min compared with conventional snp detection systems. however, pcr-based detection methods still require long processing times due to thermal cycling. in order to shorten the testing time further, it would be interested to adopt another simple and fast detection method, instead of pcr. loop-mediated isothermal amplification (lamp) is a highly specific, sensitive, and rapid gene amplification method that was established by notomi et al. (notomi et al., ) . this method amplifies nucleic acids under isothermal conditions and employs self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers (duan et al., ; tomita et al., ) . lamp has been used for detection of pathogens via the amplification of gene segments, including japanese encephalitis virus infection (mori et al., ) , rapid genotyping of carcinogenic human papillomavirus and herpesvirus (mori et al., ) , and the detection of middle east respiratory syndrome coronavirus (bhadra et al., ; mori et al., ; notomi et al., ) , demonstrating the method's great potential in molecular diagnostics. based on the advantages of this technology, lamp has been used for genotyping with buccal swab samples, combining gold nanoparticles functionalized with ssdna and colorimetric turbidimetry detection (carlos et al., ) . although it no longer requires dna purification, this method still takes h to perform due to its complex experimental procedure. methylenetetrahydrofolate reductase (mthfr) is the key enzyme in folate metabolism. mutations in the mthfr gene would influence the activity of this enzyme and increase the risk of various diseases, such as cancer, neurological disorders, cardiovascular diseases, and pregnancy complications (nazik et al., ) . aldehyde dehydrogenase- (aldh ) is responsible for the oxidation of aldehydes in the liver. differences in aldh expression contribute to a wide variety of human diseases, including cardiovascular disease, diabetes, and cancer. in addition, genetic polymorphisms of aldh alter susceptibility to ethanol intake, as well as the risk of alcoholism and alcoholic complications, and aldh may possess important therapeutic potential against alcoholism and other forms of myocardial damage (chen et al., ) . in this study, we devised a direct-lamp procedure, amplifying nucleic acids with various samples (including whole blood, dried blood spot, buccal swab and saliva) without dna purification, which is essential for conventional nucleic acid detection methods. the turnaround time was about min from sample collection to provision of results. mthfr c t, and aldh glu lys were utilized as models for snp detection with the direct-lamp method. accuracy was evaluated against clinical samples, and complete consistency between genotyping results reveals that this direct lamp procedure generally concords with sequencing. the proof of concept scheme described in this paper uses direct-lamp for snp detection. as exhibited in fig. a , samples are treated with naoh solution, followed by amplification under isothermal conditions using specifically designed primers. for convenience, we refer to the wild type as "wt" and the mutation type as "m". for each sample to be detected, two separate reactions (m tube and wt tube) are run simultaneously using the same sample. two sets of allele-specific primers (wt sequence and m sequence) are added to two different tubes for amplification, respectively. the result can be obtained automatically through a real-time fluorometer. the operating procedures of the direct-lamp only take min from sample collection to result. based on point mutations in the mthfr c t and aldh glu lys genes, four specific primers were designed to discriminate each snp. as shown in fig. b , the direct-lamp procedure relies on the specific primers, with forward inner primer (fip) and backward inner primer (bip) that are designed to contain a snp nucleotide at the ′ terminus. each reaction includes two common primers (f and b ) and two special primers (fip-wt and bip-wt for wild type allele or fip-m and bip-m for mutation type allele). the fluorescence signal coming along with target fragment amplification would be read in real-time only when the ′-end of the specific primer is complementary with the template. the structures of the specific primers used in this study are presented in fig. b . the target snp was characterized by using four different primer regions specifically designed to recognize six distinct regions on the target gene, which were selected to ensure that the primers would specifically amplify the c t and glu lys substitutions. the result depicted in fig. c illustrates that the direct-lamp could accurately detect all possible homozygotes and heterozygotes of mthfr c t with whole blood sample. for mthfr c t and aldh glu lys polymorphism genotyping, respectively, two sets of primers for direct-lamp were designed using the primer . software program (primer-e ltd., plymouth, uk), including two outer primers (f and b ) and four inner primers (fip-wt, fip-m, bip-wt and bip-m) that recognize six distinct regions of each target gene. two primers were also designed for sequencing. the sequences of the primers are shown in supplementary table s . all primers were synthesized by invitrogen biotechnology ltd. (shanghai, china). matched fresh human whole blood and dried blood spot samples were collected from unrelated chinese volunteers at the shaanxi provincial people's hospital (xi'an, china) with informed consent. whole blood sample was collected using edta-coated tubes. dried blood spot sample was prepared by dropping the peripheral blood on the filter paper and drying in the air. matched saliva and buccal swab samples were obtained from chinese volunteers at northwest university (xi'an, china) with informed consent. saliva samples were collected in eppendorf tubes. buccal swab sample was collected by swabbing the insides of cheeks times. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). peripheral blood and saliva samples were collected in edta-coated tubes and eppendorf tubes, respectively, followed by mixing of the samples with mm naoh in a : ratio, with a final volume of μl, and incubation at room temperature for min. in total, μl of the mixture was taken for subsequent amplification. the buccal swab head was cut off (about mm under the head) and placed into μl of mm naoh, followed by incubation at room temperature for min, and μl of the mixture was taken for subsequent amplification. dried blood spot of mm diameter was put into an eppendorf tube and mixed with μl of mm naoh, followed by incubation at room temperature for min, and μl of the mixture was taken for subsequent amplification. the mixture must be used immediately after preparation. samples treated with naoh were observed using an optical microscope (ix , olympus optical co., ltd., tokyo, japan) with w halogen light source (u-lh l- , olympus); cells to be observed were stained with rapid wright-giemsa staining solution kit (sangon biotech co., ltd., shanghai, china). the initial conditions of the direct-lamp procedure were adopted as described by zhang et al. ( ) . the direct-lamp was performed with different combinations of primer concentration ( . μm fip/bip with . μm f /b , . μm fip/bip with . μm f /b , . μm fip/ bip with . μm f /b and . μm fip/bip with . μm f /b ) to determine the best primer concentration. the best reaction temperature was also investigated by running the direct-lamp at , , , , or °c for min. the four sample types were used to establish the direct-lamp, and the genotypes of the used samples had been sequenced. the reaction mixture contained μl of isothermal master mix (optigene ltd., uk) (including geobacillus dna polymerase, thermostable inorganic pyrophosphatase, optimized buffer including mgcl , dntps and ds-dna dye), μl of primer mix consisting of four primers each for f and b primers at . μm, fip and bip primers at . μm, μl sample treated solution, and ddh o up to μl. the reaction was performed in -well . -ml tubes, with incubation at °c for min. the fluorescence intensity of the ds-dna dye was simultaneously monitored in a realtime fluorometer (genie ii from optigene ltd. uk). the detection limit of optimized direct-lamp was evaluated by a serial dilution of the target concentrations ( %, %, %, . %, . %, . %, . %) of whole blood sample and saliva sample with two different genotypes (wild type and homozygous mutation, confirmed by sequencing) of mthfr c t and aldh glu lys. the four sample types with two different genotypes (wild type and homozygous mutation which confirmed by sequencing) of mthfr c t and aldh glu lys, respectively, were used to evaluate the specificity of the detection system. the accuracy of the optimized direct-lamp was further verified using clinical samples, which had been obtained with informed consent. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). the genotype of each sample was analyzed by direct-lamp and compared with the results obtained via sequencing by bgi (beijing genomic institute, beijing, china). based on the statistical data, the coincidence rate and total agreements of three genotypes of mthfr c t and aldh glu gly were calculated to evaluate the accuracy of our method. in the current study, samples were treated with naoh to induce cells lysis and dna release, saving time ordinarily spent on extraction of dna from sample. to evaluate the effect of naoh on the performance of direct-lamp, the morphologies of leukocytes and oral epithelial cells in whole blood and saliva were observed, and the ph value at each step of direct-lamp process was assessed. in fig. a , a great number of cellular debris were found with the whole blood treated with naoh, while integrated cells were observed when blood was incubated with physiological saline. similar results were found for saliva treated with naoh and physiological saline, respectively. the results indicated that naoh treatment lead to cell lysis, followed by release of dna for use in subsequent amplification. in fig. b , it is shown that the ph value of naoh solution was . , and by adding the naoh solution to blood sample and saliva sample, the ph value of the mixture was . - . . however, the ph value drops dramatically to . - . by mixing the naoh treated sample with reaction buffer, and no significant difference in ph value was observed between the reaction buffer with and without naoh treated sample. the reason can be explained as the buffering effect of tris-hcl buffer, which provided a benign working environment for dna polymerase, according to verma et al. ( ) . to determine the best working condition, the performance of the direct-lamp was evaluated under different concentrations of primers and reaction temperatures by using whole blood sample with two different genotypes of mthfr c t (wild type and homozygous mutation, which were confirmed by sequencing). firstly, the best combination of primer concentrations was found to be . μm of the fip/bip and . μm of the f /b (fig. a) . the reaction temperature of direct-lamp was also optimized, as shown in fig. b ; the best amplification efficiency and specificity was obtained when the reaction temperature was °c. to evaluate the performance of the direct-lamp, a serial dilution of the target concentrations of whole blood sample and saliva sample with two different genotypes (wild type and homozygous mutation which confirmed by sequencing) of mthfr c t and aldh glu lys, respectively, were used to determine the detection limit. the x. liu et al. biosensors and bioelectronics ( ) - amplification efficiency reflected by the threshold time is inversely proportional to the dilution ratio of biological sample (fig. ) . accurate genotyping results were still obtained with the direct-lamp when biological sample was diluted to times. the specificity of the detection system for every target and sample type was evaluated, accurate genotyping results were still obtained under interference of background in all cases (supplementary fig. s and fig. s ), demonstrating great potential for direct clinical application. the accuracy of the direct-lamp was further verified with clinical samples (matched whole blood and dried blood spot samples, matched buccal swab and saliva samples) with informed consent. the study was approved by the ethics committee of the college of life sciences, northwest university (xi'an, china). mthfr c t of whole blood samples and aldh glu lys of saliva sample were also sequenced by bgi. in fig. , the threshold time of wild type were defined as a negative value, the threshold time of mutation type were defined as a positive value. for mthfr c t genotyping, samples of homozygous mutation, samples of wild type and samples of heterozygous mutation were observed with whole blood in fig. a , which was same as those found with dried blood spot in fig. b . for aldh glu lys genotyping, sample of homozygous mutation, samples of wild type and samples of heterozygous mutation were observed with saliva in fig. c , which was same as those found with buccal swab in fig. d . the genotyping results provided by the direct-lamp showed no discrepancies with those found with sequencing. as shown in supplementary table s , the derived allele frequencies of mthfr c t based on the direct-lamp detection were % and % for c and t, respectively (calculated from: c: f +f / , t: f / +f ), which is in agreement with those reported by hui et al. . as shown in supplementary table s , the observed allele frequencies of aldh glu lys were % and % for g and a respectively, which is also in agreement with those reported by eng et al. ( ) in the chinese population. loop-mediated isothermal amplification (lamp) was established by notomi et al. and this method has been used for detection of pathogens via the amplification of gene segments, demonstrating the method's great potential in molecular diagnostics. based on the advantages of this technology, some researchers are working on this technology can be applied for snps detection. a few reports indicated that this method has been applied for snps detection successfully (carlos et al., ; iwasaki et al., ; kwong et al., kwong et al., , nakamura et al., ; nakamura and ito, ; zhang et al., ) , which shorten the testing time further than pcr. however, dna purification or complex clinical sample pre-treatment are required for snps detection in these methods. the sample pre-treatment procedures including repeating incubation, elution and centrifugation consume a lot of time and energy during the detection process. although commercial kits simplify the procedure of dna purification and clinical sample pre-treatment, there are restrictions on the clinical sample types, such as colorless clinical samples are required (fta™ indicated micro card, whatman, uk) . the need for rapid snps detection to provide highly accurate diagnostic information is realized by all major health organizations, including the centers for disease control and prevention and the world health organization. however, no method combining lamp exists for snps detection that can be performed within a single patient visit ( min) directly from various clinical samples. here, we have established a rapid, easy-to-use and accurate snp detection platform using direct-lamp, which enables us to use whole blood, dried blood spot, buccal swab or saliva as samples for genotyping without dna purification. the whole testing procedure required only min from sample collection to result, which is much faster than with conventional snp detection systems. this is the first report to introduce the pretreatment of various kinds of samples with a simple naoh solution before lamp for genotyping. designed as a universal genotyping platform, this direct-lamp protocol has been successfully applied not only to the four sample types previously mentioned, but also verified with clinical sample, indicating that this genotyping platform could be applied to more sample types in further study. the treatment of sample with naoh plays an important role in this direct-lamp, making the whole testing procedure convenience and useable. first of all, compared with other snp detection methods, the dna purification step is eliminated in our assay with the help of naoh treatment, reducing the sample preparation time. secondly, using naoh-treated sample simplifies the complex procedures of conventional genotyping methods and eliminates the problem of cross-contamination, which happened in traditional dna purification processes. furthermore, various natural dna polymerase inhibitors in whole blood, such as hemoglobin, igg, lactoferrin, and proteases (adams, ; connelly et al., ; monroe et al., ) , can be inactivated by treatment with naoh solution. by using direct-lamp, qualitative analysis can be carried out by reading the fluorescence signal automatically in real time. the major advantage of this assay system is rapid qualitative answer in ''yes'' or ''no'' according to the fluorescence signal. compared with conventional snp detection methods based on pcr or dna sequencing, direct-lamp shows significant advantages (jung et al., ; ngo et al., ) . for conventional genotyping methods that require purified dna as template, such as pcr-microarray, qpcr and dna sequencing, the process of dna purification required a fair amount of time during the whole testing, whereas the present assay system requires less than min for sample preparation. traditional genotyping techniques based on pcr always require more than . h for dna amplification, whereas the present methods based on lamp shorten the testing time further. the process of nucleic acid amplification under isothermal conditions, employing self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers, enable this special identification system, which is faster than pcr-based methods, which use only two primers to recognize two regions (duan et al., ; tomita et al., ) . thus, compared with conventional snp detection techniques usually requiring a complex operation procedure that limits their application, the present methods provide an easy-to-operate onsite technique for genotyping with high efficiency. therefore, the direct-lamp will greatly benefit molecular diagnostics by dramatically reducing turnaround time, which makes this snp detection method very promising for poc testing in clinical diagnostics. with further development for additional snps and sample types, direct-lamp could enable rapid clinical decision-making, improve management of disease prevention and facilitate personalized medicine in each level of medical institutions. in this study, we presented a direct-lamp for snp detection by using whole blood, dried blood spot, buccal swab or saliva as samples without dna purification. this is the first report that a snps detection platform combined the direct amplification and lamp for genotyping with various clinical sample validation. our data have demonstrated that this system is highly applicable for detection of snps in clinical samples due to its unparalleled advantages, such as short processing time, simple procedure and accuracy. since detection of snps has significant clinical value, in our laboratory this method has already been extended to detection of snps in other genes related to folic acid metabolism. it is envisaged that our newly established direct-lamp could be quickly adapted for the detection of other snps of genes that are associated with disease risk, drug metabolism, or drug reaction. analytical sciences the international plos one , e this study was supported by the national natural science foundation of china ( ), national natural science foundation of china ( ), shaanxi provincial nano-biomedical detection innovation team founds ( kct- ) and northwest university graduate innovation and creativity funds (yzz ). supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.bios. . . . key: cord- -aw rc authors: Österdahl, marc f.; lee, karla a.; lochlainn, mary ni; wilson, stuart; douthwaite, sam; horsfall, rachel; sheedy, alyce; goldenberg, simon d.; stanley, christopher j.; spector, tim d.; steves, claire j. title: detecting sars-cov- at point of care: preliminary data comparing loop-mediated isothermal amplification (lamp) to polymerase chain reaction (pcr) date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: aw rc background: a cost effective and efficient diagnostic tool for covid- as near to the point of care (poc) as possible would be a game changer in the current pandemic. we tested reverse transcription loop mediated isothermal amplification (rt-lamp), a method which can produce results in under min, alongside standard methods in a real-life clinical setting. methods: this prospective service improvement project piloted an rt-lamp method on nasal and pharyngeal swabs on residents of a high dependency care home, with two index covid- cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (rt-pcr). we recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of a single swab using rt-lamp compared with the current standard, rt-pcr, as per standards for reporting diagnostic accuracy studies (stard) guidelines. results: the novel method accurately detected / rt-pcr positive cases and identified a further positive cases. eight further cases were negative using both methods. using repeated rt-pcr as a “gold standard”, the sensitivity and specificity of a single novel test were and % respectively. ppv was % and npv was %. incorporating retesting of low signal rt-lamp positives improved the specificity to %. we also speculate that hypothermia may be a significant early clinical sign of covid- . conclusions: rt-lamp testing for sars-cov- was found to be promising, fast and to work equivalently to rt-pcr methods. rt-lamp has the potential to transform covid- detection, bringing rapid and accurate testing to the poc. rt-lamp could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread. current diagnosis of covid- relies on centralised laboratory-based rt-pcr (reverse transcription polymerase chain reaction) testing. although pcr provides a relatively rapid result, it is limited by the bottlenecks of transportation to the laboratory and the requirement to batch samples in a large run. moreover, alternative technologies to rt-pcr requiring different reagents, and dry swabs would reduce the strain on laboratory and clinical supplies, allowing greater numbers of tests to be performed [ ] . it is abundantly clear that urgent research is needed to enable health services globally to plan resources and this research must both move rapidly from bench to bedside and be scalable and rapidly available. in light of this urgency, we present a preliminary evaluation of a novel, quick test for covid- that can be implemented at the point of need. point-of-care (poc) testing may be critical to enable rapid detection of disease when an outbreak is suspected. this is particularly important in community settings like care homes, where multiple vulnerable patients reside together, and covid- can spread quickly if not identified early [ ] . older residents are at higher risk of mortality from covid- [ ] , and care homes have reported significant outbreaks both in the united kingdom (uk) and internationally [ ] . however, they have limited access to laboratory diagnostic services. a rapid, poc test would allow early case identification, and implementation of increased infection control measures to prevent further spread to residents and staff, as recommended by the world health organization (who) [ ] and british geriatric society [ ] . between th february and th april , six independent groups have posted preprints of submitted manuscripts evaluating novel rt-lamp testing methods against rt-pcr as gold standard (table ) . since then, a number [ ] of other groups have published high-quality studies demonstrating that rt-lamp has the potential to replace rt-pcr as a means for detecting sars-cov- (severe acute respiratory syndrome coronavirus ) within rna extracted from nose -throat swabs and endotracheal secretions/bronchoalveolar lavage fluid [ , , ] . to this end, we used a combination of magnetic bead viral genome capture and optimised rt-lamp (reverse transcriptase loop-mediated isothermal amplification) for amplification and detection of the sars-cov- genome; targeting the orf ab gene, to show proof of principle. the assay runs at °c allowing simpler and cheaper instrumentation to be used with rapid results (< min from swab to result). it can be used without a hospital laboratory and is suitable for a mobile testing unit model. compared to rt-pcr, the method has a high sensitivity and specificity in laboratory evaluation [ ] but is yet to be proven in clinical settings. the setting was a national health service (nhs) high dependency care home (category continuing care), where an outbreak was suspected. all residents were eligible for inclusion. on day (monday th march) two patients experienced fever and had other classical symptoms of covid- , arousing clinical suspicion. rt-pcr testing was performed on day and reported as positive to determine the extent of spread in the home, and protect patients and staff, on days & nasal and pharyngeal swabs were performed in all patients in the care home and analysed using multiplex tandem rt-pcr. on day a single rt-lamp swab was used to sample the throat, followed immediately by the nose. patients' vital signs (including temperature, heart rate, blood pressure, respiratory rate and oxygen saturations) were noted in the weeks before the known outbreak to determine whether the start of the outbreak may have occurred prior to the presumed day . standards for reporting diagnostic accuracy studies (stard) guidelines were used; stard guidelines aim to improve the completeness and transparency of reporting studies of diagnostic accuracy, to allow readers to assess the potential for bias and to evaluate its generalisability [ ] . in order to protect staff and patients, isolation and barrier nursing with full personal protective equipment were instituted for all patients. all patients were sampled on day and day using pharyngeal (day ) and deep nasal (day ) specimens (swabs) collected which were immediately placed into viral transport media (vtm) for rt-pcr or dry for the rt-lamp assay. staff taking the swabs were also swabbed and were negative for sars-cov- using rt-lamp. samples were urgently couriered to the hospital and microsensdx laboratory. the hospital performed multiplex tandem rt-pcr according to standard protocols with the rt-pcr test targeting the orf ab gene only; the limit of detection of the rt-pcr was not determined by the lab or manufacturer, but for this technology it is typically < copies per μl nucleic extract input [ ] . input volume for rt-pcr was μl of sample eluted to μl, with just μl of this used in the assay. if patients were positive on day , day samples were not analysed, but have been stored for later analysis. the rt-lamp method employed was the microsensdx rapiprep® sars-cov- research use test (see fig. ). this method used magnetic bead capture to maximise the yield of target nucleic acid during sample preparation from the dry swab, which is followed by rt-lamp to amplify and detect the sars-cov- genome, targeting the orf ab gene alone. the assay runs at °c allowing simpler and cheaper instrumentation which can yield results in min on average, often giving identification of positives in < min. results from this assay were compared to multiplex tandem pcr performed twice in the case of negative patients. input volume for rt-lamp was μl of the rna extract, which was the entire eluate from the magnetic bead extraction. the sensitivity, specificity, positive predictive value and negative predictive value were calculated using clopper-pearson confidence intervals (ci) by comparing our day rt-lamp result to rt-pcr. a patient was considered positive by rt-pcr if either a day / result or a day test result was positive. in view of the urgency of the covid- pandemic and the need to act quickly in the outbreak, formal ppi consultation for this clinical improvement study was not possible. the study was discussed extensively with the care team and virology department and senior management. this project was a clinical service improvement and the requirement for research ethics committee (rec) approval was therefore waived in line with nhs health research authority guidance (http://www.hradecisiontools.org.uk/research/). in the spirit of participant involvement, the study was discussed with all capacitous patients in the care home. all were enthusiastic to be involved and could see the value of rapid testing. in addition, relatives of all the patients who lacked capacity were appraised of the study and given a chance to comment, and for their relative to not take part. one family felt an additional swab might be intrusive, but all others were keen to be involved, gave some suggestions about swab technique (nasal vs. pharyngeal) and for the results to be shared for the benefit of others. twenty four residents were present in the care home on day . two patients lacked capacity and had no contactable next of kin to inform of the project. in one patient their informant did not agree to repeated testing as a service improvement. twenty one patients were included in the study (fig. ) . study participants were aged between and years (median years) and were predominantly female ( %). / died due to covid- , and / died from unrelated causes (for one patient, a progressive end-stage malignancy) within days of their positive test (table ) . testing results are shown in table . we defined cases as being rt-pcr positive on one of two tests at day or , and negative if negative on both tests. using this definition, / patients in the facility were covid- positive (rt-pcr ). of these cases, were identified with a single swab for rt-lamp, giving a sensitivity of % ( % ci - %) and positive predictive value of % ( %ci - %) ( table ) . this represented an improved rate of detection compared to single swab rt-pcr both in our sample and previous estimates. the specificity of the rt-lamp test was % on a single test and % on retesting lamp positives with low signal. three cases were initially identified as low positive using rt-lamp which were negative on rt-pcr, giving a total of patients testing positive on either rt-pcr or rt-lamp (table ). of these patients, patient had a high grade temperature of . °c on d of testing, patient had a temperature < . °c in the days prior to testing and patient had a temperature of . °c in the days prior to testing (table ). all three remained well at day with no other explanation for symptoms, such as upper respiratory or urinary tract infections. the routine test protocol now recommended by microsensdx includes retesting of low positive samples. repeat rt-lamp tests on samples from the three low positive patients were negative on repeat at day . it is possible that the rt-pcr results for one or more of these patients represent false negatives on day . of the two patients positive for rt-pcr and negative using rt-lamp one was contemporaneously symptomatic, and the other was well at the time of testing but had suffered a significant flu-like illness for the weeks prior to day . many patients in the home had altered vital signs in the week leading up to testing, with / negative cases, and / positive cases showing signs, e.g. fevers or reduced oxygen saturations. low temperatures (< °c were detected in a minority of covid pcr positive patients ( table ). the development of cases in the home and testing results are illustrated in fig. . no adverse events related to testing were reported. in a time of global crisis, it is critical that data are quickly shared on new testing methods, so that they can be scaled up more rapidly. to this end, we present data from patients in a care home tested within days of an outbreak in the home. in this patient group, a single rt-lamp test had a sensitivity of % and a specificity of % on single test compared to a "better than gold standard" of two consecutive rt-pcr swabs. the specificity of the rt-lamp improved to % when the new protocol of retesting low positive lamp tests is performed. we feel that this level of sensitivity is "clinically workable" in a time of crisis, particularly if repeated testing is utilised, and safeguards are put in place to guard against overconfidence in negative individuals, but this is somewhat subjective as no defined threshold of acceptability exists. it is comparable to other estimates of a single-swab rt-pcr test in our clinical experience and in posted pre-prints [ , ] . combined with the rapid result time, rt-lamp may have additional clinical utility to standard rt-pcr. the rt-pcr negative, rt-lamp initial low positive samples may indicate a lack of specificity of the low-level rt-lamp signals. given that some infected patients are assumed to be have been clinically asymptomatic and given that the rt-lamp assay used here tests more of the swab eluate than the pcr, these may be real positive results at day , that have missed by the rt-pcr. further testing and further studies will resolve this issue. in addition, we found fever > . °c, as expected was a common symptom, but hypothermia (t < . °c) and desaturation were also noted. the finding of hypothermia is important. it is a recognised symptom of sepsis and the systemic inflammatory response syndrome, particularly in older people [ ] . however, current phe and who covid- guidelines do not include hypothermia as a symptom. larger scale studies on prevalence of hypothermia, as well as other nonclassical symptoms, would shed more light on the presentation of covid- in institutionalised patients. loop-mediated isothermal amplification (lamp) was developed as a rapid and reliable, cheaper method to amplify from a small amount target sequence at a single reaction temperature, obviating the need for sophisticated thermal cycling equipment [ ] . two of these used only proven pcr-positive throat and nasal swabs and demonstrated sensitivity > % for rt-lamp methods targeting the orf ab gene when compared with gold standard rt-pcr [ , ] . only the studies by yang [ ] and yan [ ] included samples from both sars-cov- positive and negative patients and was thus able to produce both a sensitivity and a specificity. the remaining two groups, both based in the united states, lacked access to, or clearance to work with, sars-cov- samples and used either inactivated hiv with synthesised lamp sequences [ ] or other synthesised rt-lamp sequences [ ] . the majority of studies focused on the highlyconserved orf ab gene primer, also targeted by the rt-lamp method used by the microsensdx rapiprep® sars-cov- method. our study is the first "real world" study comparing the effectiveness of rt-pcr and rt-lamp testing in a group of patients at high risk for covid- and represents an important progression to clinical use for this novel sars-cov- testing method. we planned to perform rt-lamp testing just once due to a high degree of confidence that a single test would have satisfactory accuracy, allowing clinical decisions to be made immediately. however, the discrepant samples were fully concordant on re-test. as such, our standard for comparison was not a single rt-pcr, but two separate swabs for rt-pcr sent on consecutive days, thus representing what could be considered a "better-than gold standard" for comparison. however, as the pandemic has progressed, it has become apparent that there is no true "gold standard" for covid- testing with highly-anticipated antibody testing not always proving helpful; even in mild disease, antibodies in pcr positive patients may not be detected [ ] . we have been able to perform these tests quickly in a group at high risk for severe disease, and a setting where early identification of infected patients is key to preventing further spread. many other studies so far have used laboratory samples to estimate efficacy but have been unable to estimate the clinical utility. swabs were taken by the same clinician, minimising the risk of technical error or observer biases. all rt-lamp samples were tested in the microsensdx laboratory, and rt-pcr in the hospital laboratory, and there was no viral transport medium on the rt-lamp swabs. our samples were shipped to microsensdx because a level biosafety cabinet was available in the company's laboratory for initial sample handling and, due to the urgency of the study, there was not time to install a suitable cabinet in the care home. in the future a poc facility may still require a level cabinet, however recent developments in sample collection devices that inactivate the virus immediately after swabbing are expected to eliminate the need for operator protection and so a biosafety cabinet will not be required. actual poc testing, and or viral medium could be used to optimise performance further but use of dry swabs could ease issues with supply of viral transport media. we are limited by a small sample size, so our estimates have wide confidence intervals. however, they appear to be concordant with other (pre-print) studies on rt-lamp performed purely on laboratory samples. cost is a significant issue when large-scale testing within the setting of a pandemic is considered. the combined sample preparation and lamp assay kit at list price from microsensdx is equivalent in cost to a separate sample extraction kit and pcr test kit used in the reference laboratory. however, the lamp instrument is significantly cheaper than a pcr machine (by a factor of - x) providing a cost saving. subsequent technology developments in the lamp assay since this study was performed early in the pandemic include conversion to a colorimetric signal that can be read by eye, potentially negating the need for instrumentation altogether. additionally, lamp assays are currently being trialled with saliva. use of this rapid test could facilitate early identification of cases and enactment of infection control measures as required. we speculate that this could significantly reduce spread and subsequent mortality in care home residents, a speculation which could easily be tested if the method was more widely available. the test may also be suitable for use in other community settings such as pharmacies and care agencies, as well as emergency departments, and prisons or residential settings for homeless people where rapid diagnosis would be most useful. an area of global concern is covid- spread in developing countries, where reported cases are increasing. inexpensive poc testing that is not dependent on skilled and centralised technicians will be vital for less well-resourced countries and economies. however, evaluation in these settings would be advised to replicate its effectiveness. there is an urgent need for a rapid, robust and costefficient poc test that can be used in care homes, community settings and away from centralised large-scale laboratories, without the need for skilled technicians. magnetic bead capture and rt-lamp amplification and testing for sars-cov- was found to be promising, rapid, easy to use and to work equivalently to standard multiplex tandem pcr methods. definitive studies to evaluate this method in larger cohorts are underway. rt-lamp has the potential to transform covid- detection, bringing rapid and accurate testing to the poc. covid- : testing times a single and two-stage, closed-tube, molecular test for the novel coronavirus (covid- ) at home, clinic, and points of entry rapid detection of novel coronavirus/ severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp rapid detection of covid- coronavirus using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid detection of sars-cov- using reverse transcription rt-lamp method rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay fermented barley and soybean (bs) mixture enhances intestinal barrier function in dextran sulfate sodium (dss)-induced colitis mouse model characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention phenolic compounds from red wine and coffee are associated with specific intestinal microorganisms in allergic subjects prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study probiotics, prebiotics and synbiotics-a review development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (rt-lamp) system potentially to be used for reliable and high-throughput screening of covid- standard operating procedures for sars-cov- detection by a clinical diagnostic rt-lamp assay development of reverse transcription loopmediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus (sars-cov- ) stard guidelines for reporting diagnostic accuracy studies: explanation and elaboration impact of fermented foods on human cognitive function-a review of outcome of clinical trials correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of -ncov infections the impact of body temperature abnormalities on the disease severity and outcome in patients with severe sepsis: an analysis from a multicenter, prospective survey of severe sepsis loop-mediated isothermal amplification of dna covid- : two antibody tests are "highly specific" but vary in sensitivity, evaluations find publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank participants, their families and staff at the care home for their help. authors' contributions mfo, kal, mnl and cjs collected clinical data. mfo and kal contributed equally to this paper as joint first authors, and with cjs wrote the manuscript. cjs took all swabs. mfo performed the data analysis. rh, as, tds, cjs and sw were involved in project set-up and planning. sdg and sd provided lab and virology expertise. the authors reviewed and approved the final manuscript. this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. testing was provided free of charge by microsensdx, who processed the rt-lamp tests. cw and sw both provided comments on the interpretation of the results. pcr tests were performed as part of routine clinical care. no other funding was sought for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. in line with procedures relating to a clinical service improvement, all capacitous participants, and relatives in case of non-capacitous participants, were verbally appraised of the project and given the opportunity to not take part. written appraisal was not practical due to visiting restrictions. this approach was approved by the institutional review bodies. competing interests cjs is supported by hefce funding. cs and sw are employees of microsensdx ltd. testing was provided free of charge by microsensdx. other authors report no conflict of interest. key: cord- - qx svs authors: buck, m. d.; poirier, e. z.; cardoso, a.; frederico, b.; canton, j.; barrell, s.; beale, r.; byrne, r.; caidan, s.; crawford, m.; cubitt, l.; gamblin, s.; gandhi, s.; goldstone, r.; grant, p. r.; gulati, k.; hindmarsh, s.; howell, m.; hubank, m.; instrell, r.; jiang, m.; kassiotis, g.; lu, w.-t.; macrae, j. i.; martini, i.; miller, d.; moore, d.; nastouli, e.; nicod, j.; nightingale, l.; olsen, j.; oomatia, a.; o'reilly, n.; rideg, a.; song, o.-r.; strange, a.; swanton, c.; turajlic, s.; walker, p. a.; wu, m.; reis e sousa, c.; consortium, crick covid- title: standard operating procedures for sars-cov- detection by a clinical diagnostic rt-lamp assay date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: qx svs the ongoing pandemic of sars-cov- calls for rapid and cost-effective methods to accurately identify infected individuals. the vast majority of patient samples is assessed for viral rna presence by rt-qpcr. our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than , rt-qpcr results since its commencement at the beginning of april . however, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. in this work, we present a clinically-validated standard operating procedure (sop) for high-throughput sars- cov- detection by rt-lamp in minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive. the current pandemic caused by novel coronavirus sars-cov- , first detected in late in the province of wuhan, china, has rapidly spread worldwide, infecting more than million individuals as of june [ ] [ ] [ ] . infection with sars-cov- can lead to development of covid- , a disease associated with severe acute respiratory syndrome, responsible for hundreds of thousands of deaths globally. controlling the spread of sars-cov- relies on the ability of healthcare systems to quickly identify infected individuals, which has mainly relied on rt-qpcr for viral rna detection . international competition for commercial kits and reagents has negatively impacted the ability of many countries to scale up testing capacity to deal with the increased demand caused by rampant infection. implementing rt-qpcr testing programs requires specialised laboratory equipment and reagents, presenting additional challenges. in an effort to increase the diagnostic capacity for sars-cov- infection in the uk, the francis crick institute, a biomedical research institute based in london, rapidly repurposed its staff and facilities in late march to serve as a clinical diagnostic testing facility through a partnership between a major local healthcare provider (university college london hospitals national health services trust) and an accredited clinical diagnostic laboratory (health services laboratories, hsl), termed the crick covid- consortium (ccc) . the pipeline utilises a series of in-house buffers to first inactivate patient samples received from care homes and hospitals, and to then extract rna before using a ce marked commercial kit to detect sars-cov- by rt-qpcr. patient results are reported through a custom online web portal that interfaces with the reference laboratory . in order to avoid dependence on any singular testing methodology, to continue increasing testing capacity, and to provide a potential means to deliver diagnostics at the point-of-care, the ccc was also tasked with developing and validating alternative sars-cov- testing strategies. herein, we describe the use of loop mediated isothermal amplification pcr coupled with reverse transcription (rt-lamp) as a robust method for sars-cov- detection in clinical specimens . the entire strand displacement and amplification procedure is carried out at a single temperature in less than minutes, alleviating the need for a thermocycler qpcr system. diagnostic tests utilising this technique have been developed for rna viruses and other pathogens [ ] [ ] [ ] , and there are now several preprints focusing on the use of rt-lamp to detect sars-cov- - . we set up a sars-cov- rt-lamp assay using the warmstart colorimetric lamp x mastermix commercialised by new england biolabs (neb), which allows for visual assessment of dna amplification. alternatively, dna amplification can be quantified on a real-time pcr machine by complexing the reaction with the dna dye syto . we made use of primers developed by zhang et al. targeting the nucleocapsid all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint phosphoprotein (n gene) of sars-cov- and, in a parallel reaction, primers that detect human s rrna to control for specimen quality . our results demonstrate that within the ccc pipeline, rt-lamp can readily replace rt-qpcr as a means for detecting sars-cov- transcripts within rna extracted from nosethroat swabs and endotracheal secretions/bronchoalveolar lavage fluid. rt-lamp for the n gene shows absence of non-specific amplification and cross-reactivity with other human coronaviruses or respiratory viruses, and displays a sensitivity threshold almost equivalent to the gold standard rt-qpcr. switching to rt-lamp translates into a ten-fold decrease in total reagent cost and a potential four-fold increase in pipeline output. additionally, we provide preliminary data suggesting that rt-lamp can be performed without prior rna extraction, allowing rapid and cost-effective testing that could potentially be extended to point-of-care. the entire workflow was validated under extended governance by public health authorities during the pandemic and inspected by a qualified ukas assessor against genqa guidelines to verify compliance to iso : equivalent standard (us equiv. cap/clia). as such, the standard operating procedure (sop) developed here is ready to be deployed for diagnostic testing of sars-cov- . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint all the samples processing through the ccc pipeline was done in accordance with sops described recently . a redefined reaction, running, and reporting sop can be found in the supplemental methods. rt-lamp reaction was performed in a total volume of µl, mixing . µl warmstart colorimetric lamp x master mix (new england biolabs, m ), . µl of x primer mix, . µl syto green fluorescent nucleic acid stain (thermofisher scientific, s ), . µl of nuclease-free water (ambion, am ) and µl of sample unless stated otherwise. x syto solution at µm in nuclease free water was prepared for a final concentration of . µm in the final rt-lamp reaction. x primer mix was prepared from µm desalted dna primers obtained from sigma custom oligos service. x primer mixes for n gene and s contained fip and bip primers at µm, f and b at µm, lf and lb at µm. rt-lamp was ran following a standard operating procedure (cf. appendix) on a , fast, quantstudio , , or real-time pcr system (applied biosystems). a negative no template control (ntc) and a positive control (supplied by hsl, sars-cov- clinical sample) were included on every run. experiments utilising laboratory grown sars-cov- were performed in containment level at the francis crick institute (fci) by trained personnel according to health and safety guidelines. we found that the assembled reaction mix was unstable when kept at o c for more than a few hours, and sensitive to freeze-thaw when kept at - o c for more than a day. the rt-lamp should be performed using freshly prepared reaction mix. the individual components should be stored at - o c until use and avoid repeated freeze/thaw cycles. as outlined recently , the ccc was formed in partnership with hsl, a uclh ukas accredited lab, who already had a covid- rt-qpcr test in scope. all samples were received and communicated by hsl under their accreditation and the ccc rt-lamp assay was validated against their existing rt-qpcr test and the ccc's validated rt-qpcr test, which uses a ce marked commercial kit (bgi). given the urgent timeframe required to implement testing, it was all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint not possible to secure official clinical laboratory accreditation for the francis crick institute. however, full measures were taken to ensure that the ccc rt-lamp test was evaluated, verified and performed for diagnostic use in an environment that adhered to equivalent international standards (iso : , us equiv. cap/clia), overseen and audited by hsl. these measures were implemented under the advice and oversight of registered professionals from existing nearby iso accredited medical laboratories, and included writing and following clinical diagnostic sops for every stage of the pipeline from sample reception, processing to result reporting by qualified clinical scientists prior to results being communicated to patients by hsl. additional sops were followed for sample storage, disposal of materials, batch certification of reagents and incident reporting. appropriate risk assessments, training and competency assessment procedures were established and documented. record sheets were created to document the receipt, batch acceptance testing, and start/end of use dates for key reagents and consumables. an inventory of all key equipment was compiled which, where appropriate, included details of service and calibration records. systems were also established for the control of all key documents (version implementation, distribution and acknowledgement), audit trailing (what samples were tested when, by whom, with what equipment and using which consumable/reagent batches), and the recording of all untoward incidents/issues (thus facilitating appropriate investigation, rectification and recurrence prevention). samples were barcoded and tracked using the crick clarity library information management system (lims). all key documents are available at https://www.crick.ac.uk/research/covid- /covid -consortium. nhs governance was extended by a specific memorandum of understanding for diagnostic testing between uclh and the francis crick institute enabled by nhs england. assurance of the pipeline was performed in collaboration with genqa, following their checklist for non-accredited laboratories and the lab and ccc workflow were inspected by a qualified ukas assessor against the genqa guidelines to verify compliance to is : equivalent standard. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint rt-lamp was performed with µl of rna in a total reaction volume of µl unless stated otherwise. each sample was tested in two separate reactions, one with primers targeting the n gene of sars-cov- , and the second targeting human s rrna to control for specimen integrity and quality ( figure a ). the rt-lamp mastermix contains a colorimetric ph indicator that turns from pink to yellow upon dna amplification ( figure b ). in addition, we benchmarked the rt-lamp method by measuring dna amplification using a sybr based dye in a real-time pcr machine. when measuring fluorescence every minute ('cycle'), double stranded dna accumulation follows a characteristic exponential amplification phase that eventually plateaus ( figure c and d ). an initial characterization of the technique was performed using rna samples purified from patient nasopharyngeal swabs, of which were positive for sars-cov- as assessed by the ccc pipeline via rt-qpcr . rt-lamp could detect sars-cov- in the positive samples with no amplification detected in all negative samples, displaying % concordance to our current clinical diagnostic platform ( figures c and e ). internal control signal was detected in all samples ( figure d ). the background signal of the sars-cov- rt-lamp assay was assessed by running rna elution buffer ( samples) or nuclease free water ( samples) no template controls (ntcs) (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . figure b ). with the n gene rt-lamp, no signal was observed with any of the distinct virus containing specimens tested, confirming that the assay is highly specific for sars-cov- ( figure b ). in order to ascertain the sensitivity of the n gene rt-lamp assay, rna from a sars-cov- quantified gene copy number standard obtained from the uk national institute for biological standards and control (nibsc) was extracted and assessed by limiting dilution. the results indicate that the limit of reliable detection of the n gene rt-lamp assay is between - copies of n gene ( figure a -b). this was confirmed using -fold serial dilutions of rna extracted from laboratory-grown sars-cov- quantified using the nibsc standard ( figure c ). notably, a highly linear response was observed, even in the presence of % triton-x , which has been proposed as a means of inactivating sars-cov- . the rt-lamp assay reproducibility and precision were determined by extracting rna times from a confirmed covid- positive patient sample through the ccc pipeline and assessing by n gene and s rt-lamp in independent experiments, performed by two different operators ( figure d ). the coefficient of variation was . for the n gene rt-lamp analysis and . for the s rrna internal control. we also verified that rt-lamp can be performed in a -well plate format using a quantstudio real-time pcr machine with equivalent results ( figure s ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the rt-lamp assay was benchmarked against the rt-qpcr methods used by the reference clinical diagnostic laboratory, hsl, and that used by the ccc validated clinical diagnostic platform by assessing clinical samples processed in parallel by both laboratories with duplicate rt-qpcr analyses. rna extracted by the ccc pipeline was then tested by rt-lamp in duplicate runs. rt-lamp detected positives in both experiments, which was % concordant with results obtained by the ccc's rt-qpcr assay using a ce marked kit from bgi and hsl's n gene rt-qpcr assay. however, positives with ct > near the limit of detection (ct = ) of the bgi rt-qpcr assay, which were termed 'borderline positives', lastly, we asked if rt-lamp could be performed on dry swabs without the need for rna extraction. recent work shows that . % triton x- inactivates sars-cov- , and we observed that performing rt-lamp with samples diluted in % triton x- does not all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint affect the sensitivity of the assay ( figure c ). we reverse-engineered swab specimens by coating hospital-grade swabs in serial dilutions with hek t cell supernatant of sars-cov- crude suspension generated in the laboratory. swabs were left to dry overnight, and placed in . % triton x- for - minutes at room temperature before assessment. the rt-lamp test used . µl of the solubilised material to maximise the sensitivity of the assay ( figure ). in parallel, samples were processed by the ccc pipeline and interrogated by rt-qpcr with a calculated equivalent amount of rna. 'rt-lamp pre' (without rna purification) results were % concordant to those generated by the ccc pipeline using rt-qpcr, suggesting that rt-lamp may potentially be performed without traditional rna extraction. however, the use of rt-lamp on material from swabs without rna extraction requires clinical validation using bona fide nasopharyngeal specimens to ascertain reliability and compatibility with various transport media. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint rapid and reliable detection of sars-cov- is required to efficiently diagnose infected individuals and to provide governments and health systems with guidance for treatment and quarantine strategies to reduce the risk of transmission. the ccc was formed to address a critical testing shortage in the london area, especially for frontline healthcare workers, with the additional goal of rapidly validating and disseminating sops for others to scale up their own diagnostic programs. the majority of testing is currently performed by rt-qpcr amplification of viral rna obtained from nasopharyngeal samples. in the face of continued global demand and competition for reagents and resources and to minimise reliance on any singular testing strategy, we have outlined here procedures to utilise rt-lamp as a costeffective and high throughput alternative to rt-qpcr for detecting sars-cov- in clinical specimens. implementing this testing method would reduce the ccc's operational costs ten-fold when accounting for all consumables (data not shown), provide the ability to scale up our output further ( figure s ), and decrease reporting turnaround time in comparison to our current method. we confirmed that the assay is highly specific for sars-cov- and displays lack of cross-reactivity with other respiratory viruses, including seasonal coronaviruses. assay accuracy and robustness is denoted by an absence of false positives and the further introduction of a melt curve stage allows increased confidence in the identification and reporting of genuine positives. when performed with extracted rna, the n gene rt-lamp assay displays a sensitivity comparable to clinically approved rt-qpcr methods. additionally, preliminary experiments demonstrate that rt-lamp can be performed directly without rna extraction, by inactivating virus-containing dry swabs in a detergent-based solution. however, these experiments did not make use of true nasopharyngeal swabs that are likely to contain material that can cause rna degradation or inhibit the assay (unpublished observations). our methodology makes use of a real-time qpcr machine that measures dna amplification using a sybr based dye, allowing for real-time detection and standardised reporting. we did not test extensively if rt-lamp could be assessed by the colorimetric indicator alone, although some of our data suggests that the colour change readout is concordant with the syto dye results (figures , and ) . if rt-lamp were coupled with a colorimetric read-out and the need for rna extraction could be obviated, the result would be a testing modality that tremendously reduces the cost and time for sars-cov- diagnostics and allow its application at point-of-care and in remote areas where sophisticated testing infrastructures currently do not exist. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . the clinical call from the reference laboratory is indicated above the graph. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . in-house generated samples tested by rt-lamp assay directly from dry swabs without traditional rna extraction procedures (red squares) compared to ct values derived from the ccc rt-qpcr bgi assay (dark and light blue dots) and the rt-lamp assay following rna extraction (yellow squares). the clinical call from the reference laboratory is indicated above the boxes. ct values of 'undetermined' are plotted as "ct = / " for illustrative purposes. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . reference left y-axis reference right y-axis all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . [ ] summary table all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin laboratory testing of sars-cov, mers-cov, and sars-cov- ( -ncov): current status, challenges, and countermeasures scalable and resilient sars-cov- testing in an academic centre loop-mediated isothermal amplification of dna loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid detection of zika virus in urine samples and infected mosquitos by reverse transcription-loop-mediated isothermal amplification sars-cov- detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification shotgun transcriptome and isothermal profiling of sars-cov- infection reveals unique host responses, viral diversification, and drug interactions development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus (sars-cov- ) methods of inactivation of sars-cov- for downstream biological assays enhancing colorimetric lamp amplification speed and sensitivity with guanidine chloride no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity key: cord- -q neat x authors: zhang, haoqing; xu, ying; fohlerova, zdenka; chang, honglong; iliescu, ciprian; neuzil, pavel title: lamp-on-a-chip: revising microfluidic platforms for loop-mediated dna amplification date: - - journal: trends analyt chem doi: . /j.trac. . . sha: doc_id: cord_uid: q neat x nucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (lamp), exhibit characteristics ideal for point-of-care (poc) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. other key advantages of lamp are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making lamp suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. in this review, we discuss the trends in miniaturized lamp techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for poc applications alongside our opinion of the future development of miniaturized lamp. polymerase chain reaction (pcr) [ ] is currently the most widely used nucleic acid amplification test (naat) method. the integration of heating, and, especially cooling control modules used for thermal cycling to perform pcr increases the challenges and cost of the point-of-care (poc) system's design and operation. this demands careful design for the pcr system to optimize thermal properties, which directly influences its performance, as extensive transition times between temperature segments of each cycle could result in non-specific amplification results. with an advent of microfluidics methods over the past years, the pcr technique was one of the first utilizing this technology [ ] . since then, the microfluidics were extensively exploited for pcr applications in their conventional [ ] , quantitative [ ] , and digital form [ , ] , including different poc assays [ , ] . integration of a conventional pcr with pre-or post-processing samples is difficult, as it requires specially developed hybridization probes, which can pass the nucleic acid (na) denaturation steps with a temperature of z c [ ] . isothermal amplification (ia) methods became promising alternatives [ ] to pcr, especially for poc applications [ , ] . ia conducts deoxyribonucleic acid (dna) amplification at elevated constant temperatures, simplifying the system design and operation. the temperature range required to perform ia is typically from z c to z c, lower than the one used for pcr denaturation, making the ia system simpler in comparison with pcr and suitable for integration with sample preparation [ e ] . the most widely used ia method is loop-mediated isothermal amplification (lamp) as it provides excellent specificity in comparison with other ia methods [ ] . lamp utilizes six specific oligonucleotides sequences in the initial (material-producing) step and then four sequences during amplification, elongation, and recycling steps. the process is based on primer annealing followed by auto-cycling strand displacement. it was originally performed at elevated temperatures of z c [ e ] (fig. ) . the lamp is a rapid, sensitive technique for na amplification, with applications in clinical diagnostics and pathogen detection [ e ] , food safety testing [ ] , and others. here, we present the trends in the lamp-on-a-chip systems for naat including its microfluidic, paper-based and digital variants. for each specific system, we reviewed their impacts and potentials. we also briefly described the detection methods used for lamp techniques as well as lamp applications. the potential of lamp-on-a-chip technology relies on simplified hardware, easy options to integrate it with sample preparation steps, and lamp detection capability. the insensitivity of the lamp technique to the inhibitors presented in the clinical sample makes lamp an exciting method for the detection of infectious diseases or genetic maladies. nucleic acids amplification methods are primarily required to be performed, as the original number of either dna or ribonucleic acid (rna) copies in the clinical sample is insufficient for their direct detection. they can be categorized according to the operating procedures, as either temperature cycling or isothermal. polymerase chain reaction is the most common technique used for na amplification [ , ] . the reagents are dna polymerase, two sets of primers (oligonucleotides), and deoxynucleoside triphosphates. the pcr protocol requires a temperature-cycling process in three sub-steps: denaturation of a double-stranded dna (dsdna) molecule to two single-stranded dna (ssdna) molecules at z c, annealing of the target sequence of specific primers to ssdnas at z c, and enzymatic elongation to finish the dsdna at z c. the cycling is typically repeated e times to achieve a detectable number of dna copies. the speed of the pcr process is defined mainly by the activity of dna polymerase and the heat transfer rate defined by the hardware setup. the microfluidic pcr systems have rapid heat transfer due to a small sample volume and high surface-to-volume ratio. they can be divided into two major groups according to the temperature cycling method: -stationary chamber pcr [ ] is also called "time domain pcr" [ ] . a defined volume of sample positioned in a chamber [ ] or sealed in a droplet [ ] is heated and cooled, either actively using an external element, or passively by simply switching the heater off with a stationary sample. different heating methods are developed for a fast process, including infrared-mediated [ ] , plasmonassisted heating [ ] , and ultrafast with active heating and passive cooling, achieving cycles pcr in < min [ ] . -continuous flow pcr method can be called, more generally, the "space domain pcr". the sample moves between heaters with fig. . schematic of lamp. reproduced with permission from ref. [ ] , open access. different temperatures. the chip in early reported work [ ] had a serpentine channel build over three heating blocks. overall, the continuous flow pcr system does not need the temperature cycling from the heaters but does need an external pumping unit and an accurate flow control. recently, a pcr system with an ultimate reaction time, was demonstrated achieving a cycle time of between z . s and z . s, resulting in the total time required for cycles of pcr within an incredible range of z s to z s [ ] . the pcr system's utilization for poc is limited by the system hardware and the sample-to-answer protocol complexity. numerous steps are required to remove polymerase inhibitors from clinical samples. isothermal amplification techniques require simple hardware and they are rather insensitive to polymerase inhibitors, making the amplification process robust. they typically require a processing time within a range from z min to z min, significantly longer than microfluidic-based pcr running time. the most promising ia techniques suitable for conduction in microfluidic devices are nucleic acid sequence-based amplification (nasba) [ ] , recombinase polymerase amplification (rpa) [ ] , helicase dependent amplification (hda) [ ] , and lamp [ ] . nasba [ ] is an ia method operating at a temperature of z c, designed to amplify target rna. nasba can achieve up to z fold target amplification in z min [ ] . this technique requires the initial removal of secondary structures from target rnas to achieve specific amplification products conducted at z c. this method, integrated with a sample preparation, was demonstrated to detect the norovirus in oysters [ ] . rpa [ ] is an ia operating entirely at a relatively low temperature range from z c to z c. it can amplify a target sequence using a recombinase, single-strand binding proteins, and a stranddisplacement polymerase. rpa has the advantage of a fast reaction with relaxed requirement for temperature control. this method has been demonstrated by the lab-on-a-foil system, based on a centrifugal microfluidic cartridge. the system was capable of detecting < dna copies in z min [ ] . another rpa-performing device, constructed of a substrate comprising of a paper made from a combination of glass fiber and cellulose could detect human immune-deficiency virus (hiv). results were detected by lateral flow strips achieving a limit of detection (lod) of copies in z min [ ] . hda [ ] is another type of ia working in a temperature range from z c to z c using a dna helicase for denaturation of dsdna into ssdna. unfortunately, its speed of long-target sequence amplification is a limiting factor. the strength of this method is its combination with paper-based microfluidics, as demonstrated by the detection of mycobacterium tuberculosis [ ] . lamp [ ] is one of the ia methods for dna amplification, operating at temperatures of z c. its low running cost and the simplicity of the hardware makes lamp one of the most promising techniques for genetic analysis in low-resource settings. the development of lamp-on-a-chip techniques will be further elaborated in the next sections. microfluidics-based lamp chips with different structures were proposed for poc diagnostics. here, we summarized the development of those chips based on single gene detection, multi-gene detection, and reverse transcription lamp (rt-lamp) (fig. ) . a magnetic bead-based lamp system, integrating na extraction and temperature control, was proposed for the rapid detection of methicillin-resistant staphylococcus aureus (mrsa) ( fig. a ) [ ] . the clinical samples, along with the specific nucleotide-coated magnetic beads, the washing buffer, and the reaction mixture were first loaded into the chambers of three different chips. the dna was then extracted from the bacteria (cell lysis), bound to magnetic particles, and concentrated, using a magnetic field followed by lamp. the fluids transportation and their mixing were assisted by a vacuum pump and a micro valve. the temperatures for cell lysis and lamp were controlled by a built-in micro temperature control module. the results were analyzed using a spectrophotometer and the lod was z fg ml À . this system was significantly faster in comparison with standard mrsa detection, as timeconsuming cell culturing was eliminated. also, the risk of contamination during cell culture was greatly suppressed as the system keeps the bacteria enclosed. nevertheless, the solution movement in the chip and the optical detection were achieved by external components, such as the pump and spectrophotometer. a microfluidic disk-based lamp chip, integrating sample preparation and detection, was developed [ ] (fig. b ). the disk consisted of a pressure-sensitive adhesive material film sandwiched between two poly(methyl methacrylate) (pmma) layers. the lamp reagents and the solution containing the dna template were preloaded into respective chambers and sealed within the chip, prior to the experiment. the solution mixing was achieved by modulating the rotation rate of the disk, alternating the centrifugal force. the chip was kept at an elevated temperature using a hot air gun with a chip temperature monitored by an infrared camera. the results were optically detected as fluorescence change using sybr green i intercalating dye with achieved lod of z pg ml À . the large size of the hardware, due to bulky temperature and disk rotation control systems, made it challenging for poc applications. single gene detection using a compact integrated microfluidic lamp chip is rather costly. therefore, chip structures were modified to meet the requirements of multiplexed gene detection with high throughput. an octopus-like multi-channel chip was fabricated using the soft lithography method (fig. c ) [ ] . the microchannels were connected to the related wells via gradient bridges. the designed microchannels present a low-mass-transfer coefficient to avoid the "cross-talk". the different sample solutions were loaded into the chamber by capillary force and sealed. then the lamp was performed at z c. the results were detected using an optical sensor, obtaining lod < copies$ml À . nevertheless, the sample pre-treatment, heating, and optical detection was performed off chip. compared with the previous design, the lamp disk system was proposed, integrated with dna extraction and amplicon detection using a colorimetric lateral flow strip (fig. d ) [ ] . it consisted of three major microfluidic layers from top to bottom for solution loading and transportation, dna extraction and amplification, and lateral flow strip detection, respectively. the reagents for lamp were loaded and sealed in the chamber. the chip was mounted on a centrifugal system, and the solution mixing and transportation via microchannels were controlled by operating different revolutions per min. three identical units on the chip allowed simultaneous multi-gene detection with lod of colony-forming units. nevertheless, the off-chip temperature control module limited its application for poc diagnostics. an rt-lamp system (fig. e ) [ ] , made from polydimethylsiloxane (pdms), was designed to integrate rna extraction and one-step rt-lamp with an external temperature control. the sample was transported by operating the integrated pneumatic pump and valve. the rna was extracted using specific probeconjugated magnetic beads after sample loading. the rt and lamp steps were performed using integrated micro-heater systems. this research presented a hands-off integrated system for virus detection from the sample containing rna using the one-step rt-lamp process. however, off-chip electrophoresis was performed for amplicon detection. table . an integrated rt-lamp system for rapid detection of the zika virus ( fig. f ) was proposed [ ] . the disposable chip consisted of four independent multifunctional reactors. the rna was extracted and transferred into the amplification chamber. the chip was then placed inside a thermally insulated portable chamber, heated by an exothermic chemical reaction to perform lamp. the results were recorded using a mobile phone camera, but it was also directly visible using the naked eye. this low-cost, simple, integrated sample-to-answer-based rt-lamp system underlines the potential of this technology for poc applications. a combination of multiplexing and rt-lamp has also been demonstrated detecting two rnas, both from hiv, thus increasing the specificity of its diagnostics [ ] . classical microfluidic lamp devices require pumping systems and valves for fluid manipulation, increasing the cost and operational complexity of the system. the researchers explored the potential of much simpler paper-based devices, well-known for their robustness, cost-effectiveness, and user-friendliness [ , ] . in this section, we summarize their characteristics, showing their potential applications (fig. ) . a critical aspect for paper-based na amplification is the paper's material selection. the nonspecific binding of dna molecules to the paper's fibers as well as paper selffluorescence, negatively influence the noise level and thus the lod. the most commonly used paper types are either nitrocellulosebased fta [ e ] or glass-based [ ] . a disposable cassette for the detection of hiv was proposed [ ] (fig. a) . the cassette, made from pmma, consisted of a single amplification chamber connected to the inlet and outlet ports. the fta membrane was incorporated into the chamber for rna isolation, concentration, and purification. as a result, the captured rna was directly used as a template for rt-lamp, without the elution or removal of the amplification inhibitors. a thin film heater, together with a conventional thermocouple, was mounted to the cassette holder to provide key components of a closed-feedback loopthermal control system. the fluorescence of the sample was monitored in real-time using a portable and compact optical detector, achieving an lod of < hiv virions. the system could be improved to detect nucleic acids related to other pathogens in saliva and urine, as well as in water. a magnetic-based sliding-strip device [ ] was fabricated for gene detection (fig. b) . a magnetic strip with an fta-based disc was sandwiched between two magnetic layers, containing serial ports for sampling, washing, amplification, and detection. the linear motion of the sliding strip acted as a valve to control all processing steps. the probe's fluorescence was detected using a hand-held ultraviolet source, achieving an lod of z cells. roll-to-roll thermal imprinting (molding) technology was proposed for the fabrication of integrated pdmsepaper microfluidic device for molecular diagnostics [ ] . this technology enabled the scaling up of production to thousands of devices in an hour (fig. c) . the mold was made by photolithography on a nickel plate. the pdms coating and pattern replication were performed on alcoated paper. the metal coating effectively suppressed the paper's auto-fluorescence and enhanced fluorescence intensity, due to its high reflectivity. a rolling cylinder was used to transfer microfluidic structures in pdms by thermal imprinting. the performance of this integrated device was validated by the detection of viral rna by lamp. the method created a bridge between academic research and industrialization in poc diagnostics, upscaling fabrication processes. an integrated sample-to-answer microcapillary-based lamp system [ ] was presented for single nucleotide polymorphism genotyping (fig. d) . the fta was placed inside this capillary as well as all reagents for dna extraction, purification, and amplification. the sample flow was controlled by capillary forces as well as an external pressure. once the sample pretreatment was completed, the glass capillary was kept at a temperature of z c for an hour, using an external heating source, to complete the lamp and the results were subsequently observed by the naked eye. a pdms-paper hybrid sample-to-answer device incorporating lamp and lfa was proposed (fig. e) to enhance lamp sensitivity [ ] using the shunt and pdms barrier. it consisted of following four layers from top to bottom: a polyvinyl chloride for later flow, a glass fiber for performing lamp, an fta for sample loading and extraction, and a cellulose for waste absorption. the reagents for lamp were pipetted onto the strip and then heated up using a hand-held heating system [ ] . this device was capable of performing a simple colorimetric readout, achieving a lod of z pm, -fold signal enhancement compared with the lfa strip without the shunt and pdms barrier. this chip-based system is a good candidate for poc, due to its portability and simplicity. these molecular diagnostic platforms generated a wide range of applications in medicine, healthcare, agriculture, environment, and water monitoring due to their low cost, rapidity, and accuracy. digital pcr (dpcr) was reported almost two decades ago [ ] and sparked the interest of numerous applications primarily for early cancer detection and non-invasive, prenatal diagnostics. naturally, the dpcr system has lamp alternatives (fig. ) . based on the sample compartmentalization methods, digital lamp (dlamp) systems can be grouped into two categories: droplet-based and well-plate-based systems. the droplet-based dlamp devices utilizes the sample droplets suspended in oil, thus preventing dropletto-droplet cross-contamination. the chip-based dlamp devices are of two types, both containing micro-reaction chambers. the sample is loaded into wells, either via a microfluidic network and then sealed by oil, or by pipetting it into wells and sealed by a lid. results from both the droplet-based and chip-based dlamp devices are analyzed in the same way as dpcr results, using either flow cytometry or digital image processing. the first chip-based dlamp system was proposed in [ ] . this self-digitalized chip was fabricated based on the pdms having microwells. the sample and the oil were sequentially loaded using force originated by air pressure (fig. a ). the chip with the loaded sample was placed into a water reservoir, keeping the temperature at z c to perform the lamp process. during the same year, a self-priming system for chip-based dlamp was presented [ ] . its major advantage was the absence of external pumping or valves for the manipulation of the sample as table characteristics of lamp-on-a-chip. fta stands for flinders technology associates, producing the fta membranes, and lfa stands for lateral flow assays. well as the oil. the pdms chip was kept in a vacuum environment to remove air from the pdms. the sample was loaded into the chip and pulled into the chambers due to the force caused by pressure difference between the external ambient and low internal pressure with degaussed pdms working as a sorption pump (fig. b) . however, the system independence on fluidic elements, such as pumps and valves, was compromised by a low "digitalization" level having only wells. a droplet-based dlamp device was presented in [ ] (fig. c) . the droplets were generated using a classical cross between two microfluidic channels and then transferred into a heated region to perform amplification. the droplet volume was z pl with a processing time of z min required for a volume of z ml. that is equivalent to amplifying dna in z droplets in h. the elevated temperature of z c was achieved by placing the dlamp device on a thermoelectric element. an optical setup was used for fluorescence detection, using a dual excitation wavelength and a dual detection band. results monitoring was performed by two methods: dna binding to evagreen and calcein dye-based indicators. another method was introduced using disposable polymeric dropchip, generating the droplets by centrifugal force [ ] . once the droplet generation was completed, the chip was transferred to a thermal cycler to perform lamp in z h and the amplification results were monitored by a microarray scanner. the chip consisted of two loosely connected plates, enabling them to slip over each other, thus named "slipchip" technology [ ] (fig. d) . the sample droplets were generated and then mixed by layer slipping. the slipchip contained wells and was used to test antimicrobial susceptibility, measuring the phenotypic response of escherichia coli (e. coli) present in a clinical sample of urine. the results of slipchip technology showed a fast processing time of < min. monitoring methods used for lamp are commonly adopted procedures developed earlier for pcr and other bio-molecular techniques. their utilization for lamp has been extensively studied and was summarized [ ] . mainstream detection methods of lamp results often rely on naked-eye monitoring to observe precipitates [ ] , dna-binding dyes [ ] , and colorimetric indicators [ ] . off-chip end-point detections are commonly conducted by performing gel electrophoresis [ ] . there are also real-time monitoring optical methods, such as turbidity measurement monitoring optical attenuation of the fluid [ ] and fluorescence with a suitable dye [ ] . electrochemical methods can also be either real-time or endpoint detected, and they are also commonly used for lamp monitoring, either in form of a sensor [ ] or biosensor [ ] . finally, there are antibody-based methods such as immunochromatic technique (lateral flow dipstick) [ ] and the gold standard for protein detection such as antibody enzyme-linked immunosorbent assay [ ] . besides these major techniques, researchers have also tested numerous other methods based on a nano-au probe [ ] , field effect transistors [ ] , surface plasmon resonance [ ] , bioluminescence [ , ] , or the giant magnetoresistive effect [ ] . nevertheless, compared with mainstream detection methods, these techniques are not commonly used as they require chemical preparation or additional instruments to perform product detection. the lamp method is an innovative technique for gene amplification, due to the simplicity of its hardware [ ] . it has been utilized in a wide range of applications, including infectious disease diagnostics [ , ] and food safety tests [ ] . in particular, lamp is an effective gene amplification method for poc devices [ ] . with the progression of globalization, the spread of infectious diseases is threatening people's health and lives around the world, regardless of regions' wealth or development status. it is essential to diagnose and cure infected patients to prevent the spread of diseases. rapid, accurate, and sensitive diagnostic tools to identify pathogens, ideally in poc format, are vital to tackle the spread of infectious diseases [ ] . lamp was first recognized as a potent alternative to pcr in , when it was shown to detect the rna of a severe acute respiratory syndrome virus [ ] . since then, it has attracted a lot of attention and has become an increasingly popular method for clinical diagnosis. so far, lamp has been employed for rapid detection of both dna and rna possessing human-and nonhuman-carried viruses, such as mrsa [ ] , pseudorabies [ ] , tuberculosis [ ] , influenza a [ ] , zika [ ] , nervous necrosis [ ] , and aqua pathogens in fish [ ] . food safety and food quality control are, inevitably, an important part of the public-health system. lamp has been used in food safety detection [ , e ] including staphylococcus aureus [ ] , salmonella [ e ], e. coli o [ ] , and vibrio parahaemolyticus [ ] , as well as food allergens [ ] . lamp is an isothermal amplification technique that can detect target sequences with high specificity and sensitivity. its technology has been further developed recently, having advantages over classic pcr techniques, such as isothermal operation. the specificity of the pcr achieved by annealing at an optimized temperature is substituted by lamp technology with more complex primer designs. however, a processing time of a single sample lamp cannot compete with ultimate pcr systems, capable of performing the amplification in < min [ ] . the dlamp technique seems different, as its conduction was reported in < min, a difficult time to achieve for dpcr [ ] . therefore, lamp has its merits, as the heating system is simpler, and there is no temperature cycling and cooling as required for pcr. also, the sample pretreatment module is easier to incorporate due to a more robust lamp process compared to pcr. the results analysis of lamp is also much easier, as the resulting fluorescence after amplification can be observed by the naked eye. a lower lod can be better achieved by lamp in food-borne pathogen detection than pcr [ ] . moreover, the simple structure of lamp chips provides opportunities for mass production and industrialization. as a result, lamp-based integrated systems [ ] are more suitable for application in poc diagnostics [ ] than pcrs. lamp-based diagnostics are not likely to replace conventional techniques such as pcr; they will coexist in parallel, as each one has its strengths and weaknesses. since its discovery, lamp has been further developed, often involving the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification. lamp is easy to handle, and is compatible with multiple detection methods, including turbidity detection, real-time fluorescence detection, and end-point visualization. in previous sections, we discussed lamp's potential based on its specificity, efficiency, speed (more related to the dlamp), and practical implementations. the utilization of lamp was demonstrated for the diagnosis of viruses, bacteria, and allergens in food. nevertheless, there is still much room for improvement in detection methods for the integration of lamp in poc applications. ideally, the detection modules have to be miniaturized and integrated with the on-chip lamp system. thus, developing a mobile biosensor with a lamp technology application may result in significant advancement towards the detection of pathogens directly from clinical samples, which is especially important in cashstrapped countries or when working with highly contagious clinical samples. due to its high specificity, lamp can also be used for the early detection of genetic disorders, an important aspect of prevention diseases associated with them and their treatment. in summary, lamp could be the ideal method for diagnostics in an everyday poc setting. specific enzymatic amplification of dna in vitro: the polymerase chain reaction, cold spring harbor symp chemical amplification: continuous-flow pcr on a chip pcr microfluidic devices for dna amplification polymerase chain reaction in microfluidic devices digital polymerase chain reaction technology e recent advances and future perspectives advances in microfluidic pcr for point-of-care infectious disease diagnostics point-of-care diagnostics for global 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a spiral chip for absolute quantification of nucleic acids this work was supported by the "foreign experts program" of p.r. china (w ). it was also partially supported by the grant agency of the czech republic under the contract ga - s and from the ministry of education, youth and sports of the czech republic under the project op vvv ceitec nanoþ (cz. . . / . / . / _ / ). key: cord- -yzhsdz c authors: soares, r. r. g.; akhtar, a. s.; pinto, i. f.; lapins, n.; barrett, d.; sandh, g.; yin, x.; pelechano, v.; russom, a. title: point-of-care detection of sars-cov- in nasopharyngeal swab samples using an integrated smartphone-based centrifugal microfluidic platform date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: yzhsdz c with its origin estimated around december in wuhan, china, the ongoing sars-cov- pandemic is a major global health challenge, resulting in more than million infections and . million deaths. the demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. while high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. aiming at developing cost-effective viral load detection systems for point-of-care covid- diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (lamp) of viral rna directly from heat-inactivated nasopharyngeal swab samples. the discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used as a versatile post-nucleic acid amplification signal enhancement strategy, allowing fluorescence detection via a smartphone camera and simple optics. the platform provided sample-to-answer analysis within hour from sample collection and a detection limit between and rna copies in l reaction volume. furthermore, direct detection of non-extracted sars-cov- rna in nasopharyngeal swab samples from patients with ct values below (n= plus pcr negative samples) was achieved with ~ % sensitivity and % specificity, thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. these results show significant promise towards bringing routine point-of-care covid- diagnostics closer to resource-limited settings. , with a peak in the order of copies per throat swab on day after onset of symptoms . however, in the context of viral viability and risk of transmission, it has been observed that the probability of isolating infectious sars-cov- is less than % when the viral load in the respiratory tract is below ~ . x rna copies/ml . thus, it has been recently advocated that more frequent and cost effective testing with lower sensitivity, instead of lengthy and sparse pcr testing in centralized labs, can provide a more efficient strategy for containment by ( ) rapidly detecting the viral load peak early upon infection and ( ) avoiding unnecessary isolation during the long viral load decrease period detectable only with high analytical sensitivity and with minimal to zero risk of crosshuman infection . in this context, it is expected that nucleic acid amplification tests can still provide higher sensitivity and potentially specificity than antigen rapid tests , thus being the ideal solution if costs and equipment complexity can be kept low. within the landscape of the ongoing pandemic, it has become clear that testing is paramount to reduce the spread of infection and ease the impact on health systems combined with a smart and data-driven management of social distancing policies. however, while developed countries can economically and logistically support an exponential increase in lab-based testing and social distancing measures, such a course of action is hardly feasible in rls . thus, to support routine sars-cov- diagnostics on a global scale but particularly in rls, a cost-effective, rapid, sensitive, portable and simple to use device to detect rna directly from biological specimens would have a significant impact. concerning the development of portable analytical devices, the use of rt-pcr is suboptimal considering the intrinsic technical complexity of performing several precise heating-cooling cycles up to > °c . thus, in the context of pathogen detection, several groups have been actively exploring the use of loop-mediated isothermal amplification (lamp) as a potential alternative, requiring only a constant and relatively lower temperature of ~ °c . concerning the miniaturization of lamp towards integrated analytical platforms, its combination with centrifugal microfluidics - and/or smartphone-based signal readout - has been showing significant promise to achieve a true sample-to-answer operation. a few remarkable examples of lamp-based miniaturized modules using either of these approaches are: ( ) capture of lamp amplification products with anti-dig antibodies followed by generation of a tmb precipitate measured using a standard light source and a smartphone camera ; ( ) digital microfluidic platform with temperature monitoring/control provided by a thermal . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint imaging camera and sybr green i derived fluorescence transduction by naked eye or smartphone camera ; ( ) microfluidic cartridge combining immune-capture, lysis and lamp to detect viable bacteria using a reader platform comprising two light sources for fluorometric and/or turbidimetric analysis resorting to a smartphone camera ; ( ) a hermetic container providing power-free chemical-based heating for lamp amplification followed by detection using a smartphone flashlight and camera for fluorometric detection ; ( ) centrifugal platform combining silica-based dna extraction and integrated lfa strips to multiplex the detection of multiple lamp products using anti-dig antibodies and colorimetric detection ; ( ) centrifugal platform with automated bead-beating lysis followed by direct rt-lamp by continuous measurement of fluorescence with uvc illumination and a standard camera ; and ( ) centrifugal platform incorporating non-contact heating of the disc and colorimetric detection of lamp products using a white led for illumination and filtered photodiodes for signal acquisition . here, lamp, centrifugal microfluidics, smartphone-based detection and recent developments in rt-lamp applied to the detection of sars-cov- rna - are combined to develop a novel cost-effective and fully integrated platform for covid- diagnostics directly from heat-inactivated nasopharyngeal samples. the direct detection from heat-inactivated samples was achieved using ( ) a one-pot combination of reverse transcriptase and polymerase enzymes for robust isothermal amplification and ( ) a novel agarose bead-based signal enhancement strategy for improved fluorometric detection, thus avoiding the impact of collection media on weakly-buffered ph responsive colorimetric amplification mixtures . considerable advances in lamp-based detection of sars-cov- rna have been achieved since the beginning of the pandemic, with several primer sets and potential detection strategies being reported in the past months. in this context, particular attention has been given to ph-based colorimetric signal transduction considering the simple visual interpretation of the results . however, mildly buffered systems are highly prone to interference from different biological samples and collection media, typically . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint requiring a previous silica-based extraction and elution with di-water for improved robustness . fluorescence detection using dna intercalator dyes is an equally simple and more interference-forgiving approach but requires the tackling of two key limitations, namely ( ) the relatively more complex signal transduction, particularly in miniaturized systems concerning low fluorescence signal intensities and filtering requirements and ( ) the high numbers (typically different sequences) and design complexity of lamp primers results very often in a variable degree of intra-(hairpins) and cross-primer hybridization (primer dimers), which result in significant non-specific fluorescence signal . this signal is particularly relevant at room temperature due to the lower hybridization stringency, hindering a simple, non-real-time, post-lamp measurement. here, the developed device incorporates an agarose bead-based strategy combined with centrifugal microfluidics to fundamentally tackle both these limitations by significantly enhancing the negative-to-positive signal ratio. using a set of primers that we have previously developed and resorting to a combination of ssiv and bst . for lamp and sybr green i for fluorescence generation, the obtained rt-lamp results are shown in figure -a. performing a dilution series of an rna fragment with a bp sequence matching the orf ab of sars-cov- in water, copies per μl reaction could be detected within min of amplification time, in agreement with the typical lod requirements of fda-approved covid- diagnostic technologies . strikingly, while the increase in fluorescence signal magnitude from negative to positive measured at °c was about fold, the difference was reduced to ~ . -fold at room temperature (~ °c) due to an increase in signal in the non-amplified samples. this background, arising from the increased hybridization of primer dimers/hairpins at lower temperatures, was addressed using the novel bead-based sample processing strategy described in figures -b and -c. this strategy dramatically improved the signal-to-noise ratio at room temperature, thus facilitating signal acquisition when portability and cost reduction are a priority. multimodal ligands as is the case of n-benzyl-n-methylethanolamine (nbnm) combining anion exchange and other interactions, i.e. hydrophobic and hydrogen bonding, are typically used in chromatography to capture host cell dna as an impurity or plasmid dna as target product directly from complex matrices. here used for sample processing, this ligand, when modified on agarose beads with an average porosity of ~ nm, was found to have a remarkable selectivity for short oligonucleotides with lengths of - bp and a cutoff region between - bp (figure -b) . according to the results in figure is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint μl of lamp mixture (negative input vs negative output), while the titers of lamp products amplified from copies of template rna remained constant above bp (positive input vs positive output). these observations were confirmed using fluorescence microscopy in the presence of sybr green i as dsdna selective intercalator. according to the results in figure -c, the primers in a lamp mixture without rna template (negative) result in a very high fluorescence measured at bead level, which is ~ . -fold higher compared to a positive lamp mixture ( copies of rna template). this observation is in agreement with the electropherograms in is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure . characterization of lamp assay and bead-based signal enhancement. a-dilution series of sars-cov- rna fragment spiked in water. the rna copies were added to a μl reaction mix and measured in triplicate. b (top)-capillary electropherograms of the lamp master mixes incubated at °c for min in the presence (positive) or absence (negative) of sars-cov- rna fragment copies before (input) and after (output) processing using nbnm beads packed on a disc. b (bottom)-capillary electropherogram of a dna ladder ( - , bp) before (input) and after (output) processing using nbnm beads packed on a disc. peaks below and above and bp, respectively, correspond to the boundaries of the electropherogram. c-fluorescence microscopy of nbnm beads packed on a pdms microcolumn after flowing a lamp mixture pre-incubated at °c for min in the presence (positive) or absence (negative) of sars-cov- rna fragment copies. the signals were measured as average grayscale intensities on the highlighted regions. Δsignal refers to the difference in signal magnitude between the positive and negative sample measured on the solution upstream of the beads ( . rfu) and directly on the beads ( rfu). aiming at bringing cost-effective, rapid and sensitive covid- diagnostics to rls, the main goal of this work was to develop a robust but minimally complex and portable platform for sars-cov- rna detection directly from heat inactivated is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint two components via the narrow μm deep section. to process the sample added to the disc, the sequence of steps schematized in figure -b was followed. the heat-inactivated swab sample containing template rna was mixed with the lamp reagents and a total of μl of the mixture was added to the channel, the access holes were sealed and the whole disc incubated at °c. after amplification, the disc was subjected to four cycles of ramping the rotation linearly from to , rpm and vice-versa ( cycle meaning - , - rpm). during the ramp-up, the liquid is forced through the beads due to the centrifugal force and held back by an increase in pressure inside the sealed oc. during ramp-down, the decrease in centrifugal force allows the oc to decompress pushing the liquid backwards, while the denser agarose beads are still held in place by the centrifugal force. this back and forth motion with cycles allows a complete capture of the primers in solution according to the previous results in figure schematics and working principle of the on-disc lamp fluorescence signal readout with bead-based signal enhancement. a-pmma disc comprising parallel channels packed with dried nbnm agarose beads. the disc is fabricated with layers, a bottom (b) mm pmma layer with embedded μm deep channels, a middle (m) layer comprising patterned double-sided psa defining a μm deep sieving channel preventing the flow of beads into the outlet chamber (oc), and a top (t) mm pmma layer with inlet and outlet access holes. b-sequential operation of the disc after adding μl of sample. the sequence comprises the ( ) sealing of the inlet and outlet holes, ( ) lamp by heating the disc at °c for minutes, ( ) ramping up the rotation speed to , rpm to force the solution through the packed beads, followed by a ramp down to rpm, resulting in a backflow of the liquid due to the pressure difference between the oc (positive pressure) and the lamp region (negative pressure). the final two steps can be repeated multiple times to ensure complete capture of the target molecules in solution. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint the portable platform developed to combine the centrifugal and heating modules required to perform the lamp followed by the bead-based signal enhancement is shown in figure . the top centrifugal module shown in figure -a uses a dc motor controlled by a microcontroller board and a hall-effect sensor to complete the rotation protocol. after the rotation protocol, the measurement is performed through a pmma lens in the cover lid covered on the backside with a μm polyimide film which is aligned with the smartphone camera with a custom-made adapter. the polyimide film serves as emission absorption filter to block the excitation light from the nm mw laser embedded on the platform . the laser diode is aligned at an angle of ~ . ° relative to the disc ( figure s ) to take advantage of total internal reflection of the pmma-air interface between the disc and the lens to minimize residual leakage of blue light into the camera sensor. the heating module assembled below the centrifugal platform is shown in figure -b. this module comprises a pmma housing with a removable front piece to insert the disc. the disc enclosed by the housing is sandwiched in contact with two copper plates which are actively heated by two resistive silicone mats. the temperature control is achieved with a feedback loop measuring the temperature between the copper plates and the heating mats on both sides of the stack. the pmma housing and the insulating foam on each side of the heating mats serves to minimize convective and conductive heat dissipation. to minimize non-specific amplification of hybridized primer dimer and primer-template pairs at lower temperatures, rapid heating is achieved by initially setting the temperature of the copper plates at °c according to the plot in is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . platform combining centrifugal and heating modules for lamp-based diagnostics coupled with fluorometric readout using a standard smartphone camera. a-photographs of the key components of the centrifugal module. the bottom side of the pmma lens is covered with a kapton (polyimide) film working as emission filter. b-photographs and schematics of the key components of the heating module. the disc is enclosed in a pmma housing and in between two copper plates to maximize the rate of heat transfer and maintain a constant temperature. the starting temperature of the copper plates (continuously measured between the plates and the heating mats) is set above °c to strike a balance between maximum heating rate inside the disc channels ( s to reach °c) while preventing overshooting. the developed platform was subsequently characterized by testing a dilution series of orf ab rna fragment spiked in di-water (figure ) . a dilution series using the complete platform was tested and the results in is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint copies performing independent measurements for each concentration). the analysis of the signal in each channel was performed by measuring the grayscale intensity profile of the green channel (average of pixels) pixels along the interface of the beads and the solution. centering the x pixels line profile at the bead-solution interface (negative pixel values - to in solution and positive values to on the beads), the response was described as a -pixel moving average relative to the intensity of the solution. a negative/positive decision threshold was defined according to . times the standard deviation of the relative signal obtained at the end of the line profile (pixel ) for independent negative controls. the obtained sensitivity for the on-disc lamp was lower than that obtained in the rt-lamp tests (figure -a) , where % of the samples with /copies per reaction showed a positive signal. the relatively lower sensitivity is hypothesized to arise from non-specific adsorption of enzymes and/or template to the pmma channel, having a significantly higher surface area in contact with the μl of solution compared to a standard reaction tube. efforts to improve channel passivation strategies or application of alternative materials are envisioned to maximize performance. the solution, to allow the maximization of the signal-to-noise ratio. the bead-based primer depletion was also validated as a simultaneous means of inactivating the reaction post-lamp, thus avoiding higher temperatures and longer assay times for enzyme inactivation (figure -c) . it was observed that when the primer depletion cycles were performed before amplification ( °c for min), no lamp products were obtained with initial rna template titers as high as copies/reaction. overall these results confirm the triple functionality of the agarose beads for ( ) signal intensity enhancement, ( ) sample preparation to remove non-specific background and ( ) simple reaction inactivation at room temperature. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure . characterization of on-disc signal generation after on-disc lamp. a-relative grayscale intensity (green channel) measured at the interface between the solution and beads ( pixels each side) performing the lamp in the presence of increasing copy numbers of sars-cov- rna fragment. the threshold value was calculated as the average difference between signal magnitude of the beads and solution minus . times the standard deviation of independent negative measurements. differences in signal magnitude at pixel above and below the threshold are considered negative and positive, respectively. b-enhancement of signal-to-noise ratio in solution provided by the nbnm beads. all images (green channel only) were acquired using a smartphone camera combined with illumination provided by the nm laser diode and polyimide film as emission filter. c-bead-based lamp inactivation. the lamp was performed on the disc after first flowing the mix through the nbnm beads according with the same rotation protocol used for the measurements. table were tested and preanalyzed by rt-pcr targeting the e and n genes. all samples were first tested in duplicate using rt-lamp and the results are compiled in figure is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint table were grouped in categories, namely ( ) very high -average ct < (red), ( ) high -ct between and (yellow), ( ) medium -ct between and (green), ( ) low -ct > (blue) and ( ) negative (dark blue). color coding applies to a, b and c. a-rt-lamp analysis of clinical samples using a benchtop real-time thermocycler (micpcr). all samples were measured in duplicate. the inset plot shows the correlation between average pcr ct value of each sample and time to positivity (ttp) measured for rt-lamp. ttp was determined as the required incubation time at °c to increase the fluorescence intensity above the threshold. the threshold was fixed as the highest background fluorescence (before amplification) measured among all tested samples. b-analysis a set of samples in each ct range using the integrated on-chip platform with an incubation time of or min at °c. scale bars: mm. c-relative signal measured at pixel for each nasopharyngeal swab sample. relative values below the threshold are considered positive for sars-cov- rna. the threshold and relative signal values in b and c were determined as previously described in section . . we introduce a novel portable bead-based centrifugal microfluidic platform and demonstrate lamp based viral rna detection directly from heat-inactivated nasopharyngeal swab samples. the platform achieves two major breakthroughs in the scope of point-of-care viral diagnostics. firstly, a versatile agarose bead-based strategy was developed to significantly improve signal transduction after lamp by removing the intrinsic background of primer dimer interactions when using intercalating fluorescent dyes. secondly, the developed centrifugal and heating modules serve as a complete is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint package for sample-to-answer analysis directly from heat-inactivated nasopharyngeal samples in less than hour of total processing time, combined with simple smartphonebased signal acquisition. the limit of detection of ~ ct (approximately x - x copies/ml ) and inexpensive analysis are a suitable combination for frequent screening to allow detection of the spike in viral load early upon infection and minimize the risk of transmission . in the context of the current sars-cov- pandemic, the full centrifugal platform is adaptable to any smartphone, costs less than usd and is compatible with cost-effective and scalable lamp reagents under investigation . these features can potentially pave the way to bring routine and scalable diagnostics to rls, as well as expanding the current diagnostic capacities in high-income countries by bringing viral rna detection directly to the field. both these avenues are paramount to improve the containment of viral spread and simultaneously minimize the need for extreme lockdown policies. the pdms microchannels used for fluorescence microscopy characterization were fabricated as described in detail elsewhere . briefly, microchannels comprising a x μm cross section converging into a x μm cross-section aimed at trapping agarose beads with an average diameter of μm, were fabricated using standard su- mold replication techniques. -gauge inlet and outlet access holes were punched using a blunt syringe and the channels were sealed against corning glass slides after an oxygen plasma treatment ( sec, femto science cute, w, pa o ). the discs were designed using autodesk® fusion (education license) and cut into mm thick pmma sheets using computer-numerical-control milling machine (roland modela mdx- a). the disc layers were bonded together using clear medical grade pressure-sensitive adhesive (arcare® ). the microchannels on pressuresensitive adhesives were cut using a cutter plotter (graphtec ce - ). after aligning the pmma layers with the pressure-sensitive adhesive layer, the disc was placed in a manual press machine overnight to ensure uniform bonding. detailed dimensions of each microchannel are shown in figure s . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint the heating module consisted of two silicone heater mats ( w, x mm, v dc; rs pro article # - ) which were used to heat up mm thick copper plates ( mm diameter.). the heater mats are self-adhesive and the copper plates were attached to the mats. a mm thick pmma sheet served as a chamber for the heating module, having a slot, made with a cnc milling machine, to insert the disc. heater mats with the attached copper plates in the center were attached on either side of the pmma sheet, thus forming a heating chamber for the disc with an open slit on the side for inserting disc. the thickness of the copper plates and pmma sheets was chosen to ensure direct contact with the two copper plates upon insertion of the disc into the chamber. the centrifugal module ( x x mm) was fabricated using mm thick pmma sheets which were cut using cnc milling machine and assembled using m bolts and nuts. it consisted of two chambers; the bottom chamber served as the housing for the electronic components and the top chamber is where the rotation of the disc and the signal acquisition was done. the control of the whole platform is achieved using an arduino is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint provides information to the user regarding ongoing assay steps. further technical details and exploded view is shown in figure s . the nbnm agarose beads were packed in the discs by first diluting the bead resin stock (capto adhere, cytiva) in di-water at % (v/v). μl of the diluted bead stock were then added to each channel on the disc and subsequently centrifuged at rpm for s. the excess solution was manually removed from the outlet and the disc was subsequently placed in a vacuum chamber at pa for min to dehydrate the beads. after dehydration, the disc was centrifuged a second time at rpm for s to ensure a homogeneous packing. the bp-cutoff of the beads packed inside the discs was characterized by microfluidic capillary electrophoresis in a bioanalyzer system with a dna kit (agilent technologies, usa). the packing in the pdms microchannels was performed by first preparing a suspension of beads in a % peg (w/w) solution by adding μl bead stock to μl peg solution. the suspension was flowed into the microchannels at μl/min using a ne- syringe pump (new era pump systems, usa). the channels were then rinsed with di-water flowed at μl/min for min to ensure removal of any residual peg and salts. to dehydrate the beads, the pdms device was placed in a vacuum chamber at pa for min and subsequently stored at room temperature until further usage. the beads inside the pdms devices were characterized by fluorescence microscopy in a nikon ti-eclipse inverted microscope equipped with a lumencor sola light engine and a fitc filter cube. the acquired fluorescence microscopy images were analyzed using imagej software (nih, usa). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint we obtained nasopharyngeal samples from karolinska university hospital, huddinge (stockholm) collected between may th and june st . samples were collected either using sigma-transwab or sigma-virocult kits (mwe, uk) as described in detail elsewhere . we used pseudo-anonymized surplus material previously collected for clinical diagnostics of sars-cov- . this is in accordance with the swedish act concerning the ethical review of research involving humans, which allows development and improvement of diagnostic assays using patient samples which were collected to perform the testing in question. additional ethical approval for rt-lamp diagnosis was obtained by the appropriate swedish authority (dnr - , etikproevningsnaemnden). all samples were pre-analyzed on the genexpert xpress sars-cov- system (cepheid, usa) and ct values for e and n genes were obtained. for the lamp experiments, μl of each sample was heat-inactivated at °c for minutes and stored at - °c until further processing. the sample containing the rna template (in vitro synthetized rna fragment or inactivated sars-cov- ) was first combined with the lamp master mix at a ratio of % (v/v) and μl were subsequently added to each of the channels in the disc. both inlet and outlet access holes of all channels were sealed with a sheet of psa (arseal™ ) covering the entire disc surface. the sealed disc was subsequently inserted into . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint the heater module and incubated at °c for - min. the disc was then transferred to the centrifugal platform and subjected to acceleration/deceleration cycles from to , rpm taking min each. after centrifugation, the laser diode was turned on and each channel was sequentially imaged using a mid-range smartphone camera (oneplus t, china). the acquired rgb images were processed using imagej software (nih, usa) measuring the grayscale profile of the green channel along the interface between the solution and the packed beads ( pixels long and average of pixels perpendicular to the line). the grayscale intensity on the beads was normalized relative to the solution for each channel and was smoothed with a -pixel moving average before further processing. , , - . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint viral copy titer was measured in quadruplicate. b-fluorescence intensity measured on capto adhere beads, or solution upstream of the beads after flowing μl of the preamplified mixture (after the amplification cycles in a) through the bead-packed microchannel. both fluorescence intensity on the beads and in solution were measured using fluorescence microscopy and imagej software (nih, usa) by measuring the grey scale intensity in both regions ( -bit images). the beads provide an increase of ~ fold in fluorescence signal intensity. error bars correspond to the standard deviation of independent pcr amplification and bead-capture experiments. the inset plot shows the correlation between cycle threshold values of the tested viral loads and fluorescence intensity measured on the beads. c-microscopy images of the beads with increasing copy numbers of sars-cov- genomic rna (initial copy numbers before pcr). . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure s . schematics of the bead capture selectivity in the presence of positive or negative samples amplified with lamp or pcr. in the case of lamp, the fluorescence is generated by a dsdna fluorescent intercalator (e.g. sybr green i). for pcr, the fluorescence is generated using a molecular beacon (e.g. fam-bhq- pair). in the tested setup, the pcr amplicon has a length of bp and is able to effectively penetrate the pores of the agarose beads. green droplets indicate strongly fluorescent solutions, while white droplets indicate non-fluorescent or weakly fluorescent solutions. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure s . exploded view of the centrifugal platform. the smartphone adapter design depends on the specific position of the camera being used and can be adapted to ensure an optimal focal distance when measuring the fluorescence signal on the microchannels. a kapton™ μm thick polyimide film is attached between the lens and the disc to block the excitation light from the laser diode. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure s . position and incidence angle of the laser light relative to the disc. the light from the laser has a perpendicular beam divergence of ° (fwhm) and a parallel beam divergence of °. the higher perpendicular divergence ensures illumination of the entire solution and bead region along the microchannels. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint viral hemorrhagic fever diagnostics diagnostics in ebola virus disease in resource-rich and resource-limited settings advancing rapid point-of-care viral diagnostics to a clinical setting viral load of sars-cov- in clinical samples substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov- ) sars-cov- detection, viral load and infectivity over the course of an infection rouphael, n. triplex real-time rt-pcr for severe acute respiratory syndrome coronavirus limits of detection of approved rt-pcr kits for the novel sars-coronavirus- (sars-cov- ) simpler, faster, and sensitive zika virus assay using smartphone detection of loop-mediated isothermal amplification on paper microfluidic chips smart cup: a minimally-instrumented, smartphone-based point-of-care molecular diagnostic device rapid detection of covid- coronavirus using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay development of a reverse transcription-loopmediated isothermal amplification as a rapid early-detection method for novel sars-cov- development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus (sars-cov- ) rt-lamp for rapid diagnosis of coronavirus sars-cov- rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification a colorimetric rt-lamp assay and lamp-sequencing for detecting sars-cov- rna in clinical samples integrated fluorescence detection of labeled biomolecules using a prism-like pdms microfluidic chip and lateral light excitation impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral rna multimodal chromatography: debottlenecking the downstream processing of monoclonal antibodies plasmid dna purification using a multimodal chromatography resin formation of silver nanoclusters in transparent polyimides by ag-k ion-exchange process multi-center evaluation of cepheid xpert(r) xpress sars-cov- point-of-care test during the sars-cov- pandemic detection of sars-cov- using non-commercial rt-lamp reagents and raw samples the application of microbeads to microfluidic systems for enhanced detection and purification of biomolecules key: cord- - o jo uk authors: chen, hao-tai; zhang, jie; sun, de-hui; chu, yue-feng; cai, xue-peng; liu, xiang-tao; luo, xue-nong; liu, qing; liu, yong-sheng title: rapid detection of porcine circovirus type by loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o jo uk a method of loop-mediated isothermal amplification (lamp) was employed to develop a rapid and simple detection system for porcine circovirus type (pcv ). the amplification could be finished in min under isothermal condition at °c by employing a set of four primers targeting the cap gene of pcv . the lamp assay showed higher sensitivity than the conventional pcr, with a detection limit of five copies per tube of purified pcv genomic dna. no cross-reactivity was observed from the samples of other related viruses including porcine circovirus type (pcv ), porcine parvovirus (ppv), porcine pseudorabies virus (prv) and porcine reproductive and respiratory syndrome virus (prrsv). the detection rate of pcv lamp for clinical samples was . % and appeared greater than that of the pcr method. the lamp assay reported can provide a rapid yet simple test of pcv suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction. porcine circovirus (pcv) is a small, non-enveloped, spherical single-stranded dna virus, and can be classified as a member of the genus circovirus of the family circoviridae . two distinct genotypes of pcv, designated pcv type (pcv ) and pcv type (pcv ) have been identified. pcv shares an approximate % nucleotide sequence homology with pcv . pcv was identified as a contaminant of porcine kidney cell cultures and considered nonpathogenic for swine . however, pcv is now generally accepted as the major infectious agent involved in postweaning multisystemic wasting syndrome (pmws) (bolin et al., ) . clinical signs of pcv infection in pigs include progres-sive weight loss, paleness, dyspnoea and, occasionally, diarrhoea and icterus. histopathological findings include histocytic infiltration and lymphocyte depletion of lymphoid tissues, interstitial pneumonia and, less frequently, hepatitis and nephritis (kim and chae, b; segalés and domingo, ) . two major open reading frames (orfs) have been recognized for the genome of pcv ; orf , called the rep gene, which encodes a protein of . kda involved in virus replication (mankertz et al., ) , and orf , named the cap gene, which encodes the major immunogenic capsid protein of . kda (cheung, ; nawagitgul et al., ) . the capsid protein has the type-specific epitopes (mahe et al., ) , which suggests that orf contributes to the development of pmws and thus has potential for protective immunization in a vaccine (liu et al., ) , and for type-specific diagnosis (blanchard et al., ) . epidemiologic data suggest that the virulence of pcv is strongly related to the presence of the capsid protein (cho et al., ) . accumulated evidence indicates that pcv replicates in the lymph nodes, lung, liver, spleen, heart and kidney of infected pigs. this results in impairment of the immune system through degradation of lymphoid tissues (kennedy et al., ) and through changes in the proportions of lymphocyte subsets present in peripheral blood (darwich et al., ) . the presence of pcv in tissues of pigs with pmws had been proven by virus isolation, polymerase chain reaction (pcr), in situ hybridization and immunohistochemistry kim and chae, a) . real-time pcr is a sensitive assay for the detection of pcv (brunborg et al., ; chung et al., ; olvera et al., ) . although specialized equipment such as a thermal cycler is needed, pcr-based detection methods are commonly accepted because of their high sensitivity and specificity. loop-mediated isothermal amplification (lamp) is a novel amplification method which was developed originally by notomi et al. ( ) . the most significant advantages of lamp are the ability to amplify specific dna sequences under isothermal conditions between and • c and a visible result within - min. the method has been applied successfully to the detection of human influenza a virus, severe acute respiratory syndrome coronavirus and newcastle disease virus (hong et al., ; pham et al., ; poon et al., ) . however, the use of lamp for detecting pcv has not been reported to date. in this study, we evaluated the potential of lamp for the development of a simple and rapid detection system for pcv . the pcv -bj vaccine strain was used to develop a lamp method (beijing bio-pharmaceuticals corporation). monolayers of pk- cells (atcc ccl- ) grown in -cm culture flasks were infected with an inoculum of pcv and cytopathic effects (cpe) were monitored daily. upon observation of between and % cpe, the supernatant from the infected culture was collected, centrifuged at × g for min and stored in aliquots at − • c until use. field isolates of pcv , porcine parvovirus (ppv), pseudorabies virus (prv), and porcine reproductive and respiratory syndrome virus (prrsv) were identified by conventional pcr (or rt-pcr) and sequencing. a total of clinical samples that were diagnosed as pcv positive are shown in table , including peripheral blood and tissues of lymph nodes, lung, liver, kidney, heart and spleen. these clinical samples were taken from pcv -antibody-positive pigs tested by elisa. among them, samples were identified as positive by pcr and sequencing and the other eight samples were identified by virus isolation. dna was extracted from blood, lymph nodes, lung, liver, kidney, heart and spleen samples taken from pcv -infected and healthy pigs, using a dneasy tissue kit (qiagen) according to the manufacturer's instructions. after extraction, dna was eluted in l of elution buffer and stored at − • c. rna was extracted directly from prrsv samples by using trizol reagent (invitrogen). complementary dna (cdna) was synthesized using l of the eluted rna with oligo(dt) primers and the reverse transcriptase kit (takara corp., japan) according to the manufacturer's instructions. the highly conserved sequences in the capsid protein-coding region of pcv were selected as the target for lamp and pcr. a set of four primers for the cap gene was designed for lamp by alignment of six pcv genomic sequences (accession nos. ay , ay , dq , dq , ef and ef ). primers f, b, fip and bip for lamp are shown in table , and the f and b primers were also used in pcr. ; lanes - , different pcv copy numbers subjected to pcr ( , , , , and copies/tube, respectively); lanes - , different pcv copy numbers subjected to lamp assay ( , , , , and copies/tube, respectively). pcr products showed a specific amplification for the cap gene of pcv -bj with a detection limit of copies, whereas detection limit for lamp was five copies per reaction. the lamp reaction was carried out in a conventional water bath by mixing . m each of fip and bip primer, . m each of f and b primer, . mm each deoxynucleoside triphosphate, u of bst dna polymerase (new england biolabs) using the manufacturer's supplied × buffer (containing mm of mgso , . m betaine) and l of extracted template dna or cdna in a . ml eppendorf tube. the amplification reaction was performed at • c for min and then terminated by heating at • c for min. lamp products were analyzed by . % agarose gel electrophoresis. pcr was carried out in a l reaction volume containing . mm of each deoxynucleoside triphosphate, and l of × buffer, u of taq polymerase (nippon gene), m each of primers f and b, and . l of extracted dna or cdna. the amplification regime was min at • c, followed by cycles of • c for min, • c for s and • c for min, with a final elongation for min at • c. the pcr was carried out in the gene amp pcr system (applied biosystems). pcr products were subjected to electrophoresis on a . % agarose gel. the detection limit of lamp was tested and compared with pcr by using the same templates at identical concentrations. serial dilutions of , , , , and copies of dna from pcv -bj strain were used in this assay. in addition, clinical samples were analyzed with the lamp reaction and the sensitivity of detection was compared between lamp and pcr. to assess the specificity of lamp, potential cross-reactions with dna of pcv , ppv, prv and cdna of prrsv were examined. pcv -bj strain genomic dna was used as the positive control and dna extracted from healthy swine tissues was used as the negative control. pcr products were sequenced with an automated abi model a stretch dna sequencer. dnastar software was applied to align the sequences and blast searching of genbank was used to assess homology with the known capsid protein gene sequences of pcv . a successful lamp reaction with pcv -specific primers at • c for min produced many bands of different sizes upon agarose electrophoresis, since the lamp products consisted of several inverted-repeat structures. the amplification by lamp showed a ladder-like pattern, whereas the pcr product was a specific dna band. the result indicated that the detection limit of the pcv lamp was five copies per reaction whereas that of pcr was copies (fig. ) . the detection sensitivity of lamp was therefore fivefold better than for conventional pcr. in order to evaluate the optimal tissues for viral detection and to compare the sensitivity of pcv detection by lamp and pcr, dnas from tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from pcv -infected pigs were extracted and subjected to lamp and pcr. there was a % positive detection rate for both pcr and lamp on extracts of lung, kidney and heart tissue. however, lamp showed higher sensitivity than pcr for the detection of pvc dna in blood, lymph nodes, liver and spleen tissue samples (table ) . overall, the detection rate of pcv lamp for clinical tissue samples was . % and appeared better than that for the pcr method. dna extracted from tissues of healthy animals, pigs infected with pcv , ppv and prv, and cdna from prrsv were used as templates for pcv lamp. agarose gel electrophoresis analysis indicated that the pcv lamp reaction did not detect pcv , ppv, prv, or prrsv, and gave a negative reaction with tissues of healthy swine. only with the pcv -bj dna did the pcv primer set give a positive reaction (fig. ) . pmws was first observed in piglets of a high-health herd in canada in (harding and clark, ) , and appeared to be an emerging disease that affected swine herds in many countries of north america, europe and asia choi et al., ) . pcv (which differs markedly from pcv ) was commonly found in pigs with pmws . several researchers reported that ppv, prv and prrsv could also reproduce symptoms typical of pmws rodriguez et al., ; rovira et al., ) . thus, the development of a simple and rapid diagnostic tool that could detect pcv and differentiate it from pcv , ppv, prv and prrsv in the same samples would be of significance for epidemiological surveillance and prediction of the severity of pmws outbreaks in swine herds. lamp is a new diagnostic method which is quite simple, requiring only a conventional water bath or heat block for incubation under isothermal conditions. another useful feature of lamp is that its products can be observed directly, by naked eye, because a white precipitate of magnesium pyrophosphate forms in the reaction tube (mori et al., ) . adding sybr green i to lamp reactions can increase the ease and sensitivity of detection by the naked eye (iwamoto et al., ) . some samples from blood, lymph nodes, liver and spleen that were positive by lamp were not detected as positive by pcr. the greater sensitivity of lamp (as compared to pcr) for detecting pcv detection accords with the sensitivity reported for lamp methods used to detect newcastle disease virus, japanese encephalitis virus, mumps virus and west nile virus (okafuji et al., ; parida et al., ; parida et al., ; pham et al., ) . the lack of cross-reaction observed with pcv , ppv, prv and prrsv suggest that the pcv lamp system possesses reliable specificity in addition to high sensitivity. the presence of pcv in blood and many tissues following natural infection (darwich et al., ; kennedy et al., ) was confirmed by the pcv lamp method using, in the present study, clinical samples of blood, lymph nodes, lung, liver, kidney, heart, and spleen. the optimal tissues for pcv lamp are probably the blood, lymph nodes, lung, kidney and heart because these gave a % detection rate in the lamp assay. lamp is a simple and timesaving procedure, allowing results to be obtained within h, whereas the pcr method typically requires - h. compared with pcr, the lamp method appears to be a fast and sensitive tool for the clinical diagnosis of pcv infection. nonetheless, the reliability of this assay should be further evaluated by large-scale investigation. in conclusion, a pcv lamp assay was developed successfully and shown to be a simple, highly sensitive, rapid and reliable method for the clinical diagnosis of pcv infection. porcine circoviruses: a review isolation of porcine circovirus-like viruses from pigs with a wasting disease in the usa and europe an orf protein-based elisa for porcine circovirus type antibodies in post-weaning multisystemic wasting syndrome postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type porcine circovirus quantitation of porcine circovirus type isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a taqman-based real-time pcr transcriptional analysis of porcine circovirus type . virology serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to porcine circovirus type porcine postweaning multisystemic wasting syndrome in korean pig: detection of porcine circovirus infection by immunohistochemistry and polymerase chain reaction real-time pcr for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type in naturally infected and challenged pigs pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus : an immune riddle coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome recognizing and diagnosing postweaning multisystemic wasting syndrome (pmws). swine health prod development and evaluation of a novel loopmediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type alone or in combination with porcine parvovirus multiplex nested pcr compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome a comparison of the lymphocyte subpopulations of pigs experimentally infected with porcine circovirus and/or parvovirus quantitative, competitive pcr analysis of porcine circovirus dna in serum from pigs with postweaning multisystemic wasting syndrome differential recognition of orf protein from type and type porcine circoviruses and identification of immunorelevant epitopes identification of a protein essential for replication of porcine circovirus characterization of novel circovirus dna associated with wasting syndromes in pigs detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation open reading frame of porcine circovirus type encodes a major capsid protein loop-mediated isothermal amplification of dna rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification comparison of porcine circovirus type load in serum quantified by a real time pcr in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus loop-mediated isothermal amplification for rapid detection of newcastle disease virus detection of human influenza a viruses by loopmediated isothermal amplification aujeszky's disease virus infection concurrent with postweaning multisystemic wasting syndrome in pigs experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus postweaning multisystemic wasting syndrome (pmws) in pigs: a review this work was supported in part by grants from the national key technologies r&d program of china (no. bad a ). this study was also supported by the national high-tech r&d program (no. aa a - - ) and the national natural science foundation of china (no. and no. ). key: cord- -gb vgs authors: mekata, tohru; kono, tomoya; savan, ram; sakai, masahiro; kasornchandra, jiraporn; yoshida, terutoyo; itami, toshiaki title: detection of yellow head virus in shrimp by loop-mediated isothermal amplification (lamp) date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: gb vgs reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for detecting the structural glycoprotein gene of yellow head virus (yhv). the rt-lamp assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. the whole procedure is very simple and rapid, and reaction time and temperatures were optimized for min at °c, respectively. detection of gene amplification could be accomplished by agarose gel electrophoresis. the standardized rt-lamp procedure was used to detect yhv in the heart and gill from infected shrimp. thus, the rt-lamp assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for yhv detection in shrimp. yellow head virus (yhv) is an enveloped, rod-shaped particle (approximately nm × nm) with prominent surface projections (approximately nm) and an inner helical nucleocapsid (chantanachookin et al., ; wang and chang, ; loh et al., ) . based on the virion morphology and the presence of a single-stranded rna genome (wongteerasupaya et al., ) , yhv was previously reported as a rhabdovirus . however, it was subsequently demonstrated that the yhv genome is positive-sense rna (tang and lightner, ) . sequence identity, genome organization and gene expression have indicated that gill associated virus (gav) and yhv are related to coronaviruses, toroviruses and arteriviruses and are classified in new taxa (family roniviridae, genus okavirus) within the order nidovirales (cowley et al., (cowley et al., , sittidilokratna et al., ; cowley and walker, ) . * corresponding author. tel.: + ; fax: + . e-mail address: itamit@cc.miyazaki-u.ac.jp (t. itami). the development of a loop-mediated isothermal amplification (lamp) assay for detection of white spot disease virus (wsdv) dna was described by kono et al. ( ) . the lamp assay is a novel approach to nucleic acid amplification that amplifies dna with high specificity, selectivity and rapidity under isothermal conditions. therefore, a thermal cycler is not needed. the lamp assay originally described by notomi et al. ( ) is based on the principle of the reaction performed by a dna polymerase with strand displacement activity and a set of two specially designed inner primers and two outer primers. lamp is highly specific for the target sequence because of the recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences in the later stages of the lamp reaction. the amplification efficiency of the lamp method is extremely high because of its no time loss for thermal change, based on its isothermal reaction. therefore, the lamp assay has the advantage in specificity, selectivity and rapidity over other nucleic acid amplification methods (mori et al., ) . the lamp assay is also useful for rna template detection upon the use of reverse transcriptase (rtase) together with dna polymerase. in this paper, the rt-lamp assay for detection of yhv rna in shrimp is described. black tiger shrimp (penaeus monodon) with or without yhv clinical signs were collected from shrimp farms in songkhla, thailand. shrimp samples were kept on ice for rna extraction. rna extraction from the gills was carried out using an rna extraction kit (high pure rna tissue kit (roche diagnostics, germany) according to the manufacturer's instructions. briefly, gill tissues ( - mg) were homogenized with the lysis buffer. rna was then eluted from spin columns in a final volume of l of elution buffer and stored at − • c until use. yhv-specific rt-lamp primers were designed according to published sequence of yhv structural glycoprotein gene (gen-bank accession number: af ; jitrapakdee et al., ) using primer explorer version (https://primerexplorer.jp/ lamp . . /index.html). a set of four primers composed of two outer and two inner primers was designed. the two outer primers are known as the forward outer primer (f ) and the backward outer primer (b ), which helps in strand displacement. the inner primers are known as the forward inner primer (fip) and the backward inner primer (bip). each primer has two distinct sequences corresponding to the sense and anti-sense sequences of the target, one for priming in the first stage and the other for self-priming in later stages. fip contains f c region (complementary to f ), a tttt spacer and the f region. bip contains the b c region (complementary to b ), a tttt spacer and the b region. fip and bip were hplc-purified. the location of the primers within the rna fragment is shown in fig. . the rt-lamp was carried out in a total volume of l reaction mixture with a loopamp rna amplification kit (eiken chemical co. ltd., japan) according to the manufacturer's instructions. briefly, l of target rna was mixed with l ( pmol) of each yhv-fip and yhv-bip, . l ( pmol) of yhv-f and yhv-b , . l of × reaction mix, . l of distilled water and . l enzyme mix containing bst dna polymerase and amv reverse transcriptase. after incubation at • c for , , or min, the reaction was terminated by heating at • c for min. the reaction temperature ( , and • c) was also optimized. the rt-lamp products were electrophoresed in a % agarose gel to determine the optimal condition. ten-fold serial dilutions ( − to − diluted) of rna extracted from yhv-infected shrimp was used as a template for rt-lamp according to determined conditions. after the reaction, rt-lamp products were electrophoresed on a % agarose gel and visualized using a gel document system (ultra-violet products, japan). nested rt-pcr was carried out using a commercial kit, iq (farming intelligene technology corporation, taiwan) for detecting yhv and gav. the first-step rt-pcr was carried out in l reaction volume containing . l of rt-pcr premix (reaction buffer, dntps and yhv/gav-specific primers), . l of iqzyme dna polymerase ( u/l), . l of rt enzyme mix, . l of rna extracted from yhv. ten-fold serial dilutions ( − to − ) of template rna were used to determine the sensitivity of the detection. the amplification regime was min at • c, min at • c followed by cycles of • c for s, • c for s and • c for s, then final elongation for s at • c and s at • c. after rt-pcr reaction was completed, . l of nested pcr premix and . l of iqzyme dna polymerase were added. two-step pcr reaction profile was followed by cycles of • c for s, • c for s and • c for s, then final elongation for s at • c and s at • c. two-step pcr products were electrophoresed in a % agarose gel to visualize the specific products. to determine specificity of rt-lamp method, rt-lamp was carried out with the different sources of rna template, i.e. rnas or dnas of wsdv-infected shrimp, taura syn-drome virus (tsv)-infected shrimp or healthy shrimp using commercial rna extraction kit, high pure rna tissue kit. instead of using the commercial kit for extraction of rna from shrimp, . m naoh (wang et al., ) was used. rna was extracted from gill tissues of shrimp samples showing yellow head disease clinical signs. twenty-five micrograms of sample was homogenized in l . m naoh on ice. then, l homogenization was diluted with l tris-hcl buffer. the same weight of shrimp gill sample was used for the extraction of rna using high pure rna tissue kit following the manufacturer's instruction. a series of -fold dilutions ( − to − diluted) of extracted rna were used as template for rt-lamp. rt-lamp was performed using determined condition. rt-lamp products were electrophoresed and analyzed in a % agarose gel. the rt-lamp was carried out using rna as template in order to determine the optimal temperature and reaction time. rt-lamp products were detected at both and • c. however, the product at • c showed a clearer reaction bands and this temperature was used as an optimal temperature. no amplification of template was found in the reaction time of and min. for the reaction time of and min at • c, lamp products were detected. however, for the complete amplification, the reaction time of min was selected as an optimal reaction time. the results are shown in fig. . lane , molecular size marker (/x /hinc ii digest); lanes - , rt-lamp and nested rt-pcr carried out using concentrations of rna ( − , − , − , − , − , − and − ), respectively. all products were electrophoresed on a % agarose gel and stained with ethidium bromide. in order to determine the sensitivity of detection limit, rt-lamp and nested rt-pcr were carried out using various concentrations ( − to − dilution) of rna extracted from yhv-infected shrimp as template. rt-lamp detected at a concentration of − dilution as a template, while the nested rt-pcr detected − diluted. the sensitivity of detection limit by rt-lamp is times lower than that of nested rt-pcr (fig. ) . the cross-reaction with other shrimp disease viruses, i.e. wsdv, tsv and healthy shrimp rna was also carried out to determine the specificity of rt-lamp method. positive result for rt-lamp was found none of them (fig. ) . this indicates that this rt-lamp method is a high specificity for yhv. the template rna extracted by the commercial rna extraction kit (high pure rna tissue kit; roche diagnostics) provided higher sensitivity for rt-lamp ( − dilution) than that by quick method ( − dilution) using . m naoh (fig. ) . in this study, the rt-lamp diagnostic protocol was carried out for the detection of yhv in shrimp. two sets of primer (outer and inner) used were able to amplify a bp sequence of structural glycoprotein gene. the optimal condition of rt-lamp reaction for the detection of yhv-rna was shown as • c and min. however, it was also found that the reaction could be terminated within min. this suggests rt-lamp is a more rapid method for the detection of shrimp virus, compared to the rt-pcr method which takes - min for rt reaction and at least - h for conventional pcr method. in addition, it was shown that rt-lamp method used for yhv detection specifically reacts only to yhv-infected shrimp. no cross-reaction with wsdv-infected shrimp was found. fig. . rt-lamp products with different source of rna/dna template, i.e. from heart of yhv-infected shrimp (positive), tsv-infected shrimp (negative), wsdv-infected shrimp (negative) and healthy shrimp (negative). rt-lamp products: lane , molecular size marker (/x /hinc ii digest); lanes and , yhv-infected shrimps; lanes and , tsv-infected shrimp; lanes and , wsdv-infected shrimp; lane , healthy shrimp. all the products were electrophoresed on a % agarose gel and stained with ethidium bromide. fig. . rt-lamp products with various concentrations of template ( − , − , − , − , − and − ) extracted using commercial kit and quick method. (a) commercial rna extraction kit: lane , molecular size marker (/x /hinc ii digest); lanes - , − , − , − , − , − and − , respectively. (b) . m naoh method: lane , molecular size marker (/x /hinc ii digest); lanes - , − , − , − and − . rt-lamp was carried out using series of concentrations of rna. all products were electrophoresed on % agarose gels and stained with ethidium bromide. the sensitivity of rt-lamp was found to be times lower than that of nested rt-pcr using an iq kit for the detection of yhv/gav. however, nested rt-pcr detection needs at least - h, comparing rt-lamp within one hour. as an efficient diagnostic method, the rapidness for detection should be also considered. the sensitivity of rt-lamp also depends on the quality of rna template, as the results showed quick method for rna extraction using . m naoh. this rapid technique resulted in lower sensitivity for the detection of virus, compared to using a commercial kit, although this method may take only min for rna extraction. therefore, for the confirmatory diagnosis shrimp showing the typical signs of yellow head disease that are heavily infected with the virus can be diagnosed with this method in short period of time for rna preparation. this study proposed the first rt-lamp protocol as an alternative method for the rapid detection of yhv with high sensitivity and specificity. this protocol is useful for the detection of low concentration of yhv from several tissues of cultured shrimp during early stages of infection. additionally, this method could be used as both screening and confirmatory diagnosis for suspected shrimp, even though the virus titer is relatively low. this technique is recommended as an applied protocol for health management program and disease surveillance of shrimp in hatcheries as well as in grow-out pond, in order to prevent the disease outbreak. histology and ultrastructure reveal a new granulosis-like virus in penaeus monodon affected by yellow-head disease yellow head virus from thailand and gill-associated virus from australia are closely related but distinct prawn viruses gillassociated virus of penaeus monodon prawns: an invertebrate nidovirus with orf a and orf b genes related to arteri-and coronaviruses the complete sequence of gill-associated virus of penaeus monodon prawns indicates a gene organisation unique among nidoviruses identification and analysis of gp and gp structural glycoproteins of yellow head nidovirus of penaeus monodon shrimp detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification viral pathogens of the penaeid shrimp detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp loop-mediated isothermal amplification of dna the complete orf b-gene sequence indicates yellow head virus is an invertebrate nidovirus a yellow head virus probe: application to in situ hybridization and determination of its nucleotide sequence a simple method of preparing plant samples for pcr yellow head virus infection in the giant tiger prawn penaeus monodon cultured in taiwan yellow-head virus of penaeus monodon is an rna virus this study was supported partly by a grant-in-aid for science research from the ministry of education, science, culture, sports, science and technology of japan. key: cord- -rn pkk authors: michiwaki, yuhei; tanaka, tatsuya; wakamiya, tomihiro; tabei, yusuke; samura, kazuhiro; suehiro, eiichi; kawashima, masatou title: emergent carotid artery stenting following intravenous alteplase infusion after rapid negative diagnosis for covid- by loop-mediated isothermal amplification assay: a case report date: - - journal: world neurosurg doi: . /j.wneu. . . sha: doc_id: cord_uid: rn pkk background during the coronavirus disease (covid- ) pandemic, a rapid screening method for covid- detection is needed to decide the appropriate strategy to treat stroke patients. in acute ischemic stroke treatment, the efficacy and safety of emergent carotid artery stenting (ecas) for hyperacute ischemic stroke (hais) due to internal carotid artery stenosis (ics) have not been sufficiently established. case description a -year-old man with hais caused by severe ics was treated via intravenous alteplase infusion. the patient underwent screening for covid- by the loop-mediated isothermal amplification (lamp) assay shortly after arrival at our institution. the lamp result was obtained within minutes, during intravenous alteplase infusion, and turned out to be negative. the symptom of hemiplegia worsened during alteplase infusion, and he, therefore, underwent ecas after administration of aspirin ( mg). recanalization was achieved successfully by ecas, and dual antiplatelet therapy and argatroban were administrated following ecas. hemorrhagic complications or re-stenosis/occlusion of the carotid artery were not observed. he was discharged without neurological deficits days following ecas. because of the rapid negative diagnosis for covid- using the lamp method, ecas could be performed following standard procedures, along with infectious defense, without delay. conclusions this case report suggests that ecas for hais due to ics following intravenous alteplase can be an effective treatment, along with appropriate antiplatelet medication and management in select patients. during the covid- pandemic, the lamp assay for covid- detection might be a suitable diagnostic strategy preceding stroke treatment because of the rapid turnaround time. during the coronavirus disease (covid- ) pandemic, a rapid screening method for covid- detection is needed to decide the appropriate strategy to treat stroke patients. in acute ischemic stroke treatment, the efficacy and safety of emergent carotid artery stenting (ecas) for hyperacute ischemic stroke (hais) due to internal carotid artery stenosis (ics) have not been sufficiently established. a -year-old man with hais caused by severe ics was treated via intravenous alteplase infusion. the patient underwent screening for covid- by the loop-mediated isothermal amplification (lamp) assay shortly after arrival at our institution. the lamp result was obtained within minutes, during intravenous alteplase infusion, and turned out to be negative. the symptom of hemiplegia worsened during alteplase infusion, and he, therefore, underwent ecas after administration of aspirin ( mg). recanalization was achieved successfully by ecas, and dual antiplatelet therapy and argatroban were administrated following ecas. hemorrhagic complications or re-stenosis/occlusion of the carotid artery were not observed. he was discharged without neurological deficits days following ecas. because of the rapid negative diagnosis for covid- using the lamp method, ecas could be performed following standard procedures, along with infectious defense, without delay. this case report suggests that ecas for hais due to ics following intravenous alteplase can be an effective treatment, along with appropriate antiplatelet medication and management in select patients. during the covid- pandemic, the lamp assay for covid- detection might be a suitable diagnostic strategy preceding stroke treatment because of the rapid turnaround time. j o u r n a l p r e -p r o o f the coronavirus disease (covid- ) pandemic has had a significant impact on treatment paradigms of all diseases. [ ] [ ] [ ] [ ] for instance, patients with acute ischemic stroke (ais), who must be treated appropriately and promptly, should be screened for covid- simultaneously. - therefore, a rapid screening method for covid- is needed to help decide the proper treatment strategy for stroke patients. carotid artery stenting (cas) is a standard treatment procedure for internal carotid artery stenosis (ics) ; however, the efficacy and safety of emergent cas (ecas) for hyperacute ischemic stroke (hais) due to ics have not been sufficiently established. herein, we aimed to report a case of hais due to severe ics, which was successfully treated with ecas following intravenous alteplase infusion. moreover, the patient underwent screening for covid- by the loop-mediated isothermal amplification (lamp) method shortly after admission, and ecas was performed using standard procedures without any further delay because the lamp assay revealed a negative diagnosis for covid- immediately before ecas. this case report not only demonstrates the effectiveness of ecas for treatment of hais, but also shows the value of using the lamp assay for covid- screening to enable prompt treatment of hais. j o u r n a l p r e -p r o o f a -year-old man was admitted to our institution due to sudden dysarthria and slight left hemiplegia in may . he had a history of atrial fibrillation, hypertension, and diabetes mellitus, and had been receiving oral administration of dabigatran. due to the covid- pandemic, a nasopharyngeal swab specimen collected from the patient was examined using the lamp assay immediately upon arrival according to the protocol of our institution. the duration from onset to admission was minutes, and the score on the national institutes of health stroke scale (nihss) was / . computed tomography (ct) performed minutes following admission showed a slight ischemic change at the right frontal lobe (alberta stroke program early computed tomography score: ). ct angiography demonstrated severe ics and poor blood flow in the right intracranial internal carotid artery (ica) ( figure a , b). the patient was diagnosed with hais due to ics and was then treated with intravenous alteplase (time from onset to infusion: minutes; time from admission to infusion: minutes). magnetic resonance imaging (mri) performed during intravenous alteplase infusion revealed sporadic infarction in the right cerebrum ( figure c ). the symptoms of hemiplegia improved upon the initiation of intravenous alteplase infusion, but got worse again during the alteplase infusion. thereafter, we decided to perform an ecas following administration of aspirin ( mg). as the lamp assay revealed a negative diagnosis for covid- during intravenous this case report highlights two important findings. first, ecas following intravenous alteplase infusion for hais due to ics was effective and safe in this patient. second, the lamp assay was a suitable screening tool for covid- preceding stroke treatment because the results could be obtained rapidly. intravenous alteplase has been proven to improve patient outcomes following hais, , and endovascular thrombectomy has become the standard treatment for hais due to large vessel occlusion in the anterior circulation. [ ] [ ] [ ] [ ] [ ] [ ] however, the efficacy of ecas for hais due to ics has not been established. since alteplase is a thrombolytic drug, it cannot be used to treat plaque-based atherosclerotic stenosis. moreover, chronic atherosclerotic stenosis could not be removed easily by mechanical thrombectomy. therefore, it seems reasonable to assume that ecas can contribute to recanalization success in patients with hais due to ics. in the present case, we decided to perform ecas because the symptoms of hemiplegia worsened, despite intravenous alteplase infusion. while some studies have reported the efficacy and safety of ecas, , most of them performed cas within a few days to two weeks following the onset of ais, and studies demonstrating successful ecas for hais are rare. one of the reasons that the efficacy of ecas has not been sufficiently ascertained is thought to be the risk of ica occlusion caused by in-stent thrombosis due to the insufficient efficacy of antiplatelet agents administered j o u r n a l p r e -p r o o f before ecas. although administration of antiplatelet medication before elective cas has been recommended, the ideal regimen of ecas for hais has not been established. high dose antiplatelet medication could decrease the risk of in-stent thrombosis; however, it may increase the risk of intracranial hemorrhage, particularly in patients who have undergone intravenous alteplase treatment. deguchi et al. reported three cases of ecas for hais immediately after intravenous alteplase treatment. they administrated antiplatelet agents at the loading dose (cases and : mg clopidogrel + mg aspirin; case : mg clopidogrel) before ecas; case presented with an asymptomatic intracranial hemorrhage, and an ica occlusion due to in-stent thrombosis occurred in case . in the present study, the patient was administrated mg aspirin before ecas, which was less than the dose used in a previous study. the patient presented neither in-stent thrombosis nor hemorrhagic complications. therefore, the dose of antiplatelet agents used or the postoperative management performed in this case might have been appropriate. additionally, we used carotid guardwire and a carotid wallstent under pfc. one study has indicated that ecas for ais under pfc is effective and safe. this case report provides evidence that could improve the management of ecas cases following intravenous alteplase infusion. with the recent development of endovascular treatment, effective and safe therapeutic or management strategies for hais can be established. the outbreak of covid- has had a major impact on the treatment of stroke. [ ] [ ] [ ] [ ] although patients with hais should be treated rapidly, physicians have to simultaneously evaluate the patients for covid- infection. [ ] [ ] [ ] real-time polymerase chain reaction (rt-pcr) is a standard method for covid- detection; however, it takes a long time to obtain the results. therefore, the optimal time window for the treatment of stroke may be exceeded if the physicians wait until a rt-pcr result is known; conversely, stroke treatments performed without waiting for the diagnostic test results for covid- involve risks of nosocomial infections. practically, stroke treatment might have to be performed under personal protective equipment (ppe), including n respirators. , , , these clinical problems can be overcome if a rapid and simple diagnostic method for covid- screening can be developed. in the case presented here, we used the lamp assay for covid- screening. the lamp assay is a rapid, sensitive, and effective visual nucleic acid amplification method that has been widely applied for the detection of certain viruses. [ ] [ ] [ ] the results of the lamp assay can be obtained in minutes, with high sensitivity and specificity. , moreover, the lamp assay does not require expensive reagents or instruments. in this case, because the result of the lamp assay revealed a negative diagnosis for covid- during intravenous alteplase infusion, ecas could be achieved with standard equipment and procedures according to the protocol of our institute without delay. thus, the lamp assay might be suitable for covid- detection during stroke treatment because the diagnosis can be rapid. however, some uncertainty remains about whether a negative result on the lamp assay can confirm whether the patient is truly covid- -free; therefore, it is advisable to use ppe, even for patients with a negative diagnosis of covid- . , , , future clinical cohort studies evaluating the lamp assay for use in covid- screening in clinical practice will be necessary. in addition, in cases that require extremely prompt treatment for stroke when a team cannot wait even for the results of a lamp assay, it seems unavoidable that one must prioritize the stroke treatment without knowing the patient's covid- infection status while following the appropriate procedures for preventing infection. , , , rapid and accurate diagnostic methods for detecting covid- should, therefore, be developed or improved upon urgently. this case report demonstrates that ecas for ais due to ics following intravenous alteplase infusion can be an effective treatment option along with appropriate antiplatelet medication and management in select patients. during the covid- pandemic, the lamp assay for covid- detection might be a suitable diagnostic method preceding stroke treatment because the diagnosis can be made rapidly. a novel coronavirus from patients with pneumonia in china world health organization challenges and potential solutions of stroke care during the coronavirus disease (covid- ) outbreak protected code stroke: hyperacute stroke man-agement during the coronavirus disease (covid- ) pandemic letter: academic neurosurgery department response to covid- pandemic: the university of miami/ jackson memorial hospital model preparing a neurology department for sars-cov- (covid- ): early experiences at columbia university irving medical center and the new york presbyterian hospital neurosurgical impact of coronavirus disease (covid- ): practical considerations for the neuroscience community long-term results of carotid artery stenting versus endarterectomy in high-risk patients national institute of neurological disorders and stroke rt-pa stroke study group. tissue plasminogen activator for acute ischemic stroke thrombolysis with alteplase to . hours after acute ischemic stroke a randomized trial of intraarterial treatment for acute ischemic stroke randomized assessment of rapid endovascular treatment of ischemic stroke thrombectomy within hours after symptom onset in ischemic stroke stent-retriever thrombectomy after intravenous t-pa vs. t-pa alone in stroke endovascular thrombectomy after large-vessel ischaemic stroke: a meta-analysis of individual patient data from five randomised trials endovascular therapy for ischemic stroke with perfusion-imaging selection safety and effectiveness of emergency carotid artery stenting for a high-grade carotid stenosis with intraluminal thrombus under proximal flow control in hyperacute and acute stroke emergency carotid artery stent placement in patients with acute ischemic stroke carotid artery stenting in acute stroke carotid artery stenting for acute ischemic stroke patients after intravenous recombinant tissue plasminogen activator treatment society of neurointerventional surgery recommendations for the care of emergent neurointerventional patients in the setting of covid- rt-lamp for rapid diagnosis of coronavirus sars-cov- loop mediated isothermal amplification (lamp) assays as a rapid diagnostic for covid- rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay computed tomography; ecas, emergent carotid artery stenting hyperacute ischemic stroke; ica, internal carotid artery; ics, internal carotid artery stenosis mri, magnetic resonance imaging; nihss, national institutes of health stroke scale; pfc, proximal flow control; ppe, personal protective equipment we would like to thank editage (www.editage.com) for english language editing. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. j o u r n a l p r e -p r o o f acknowledgements we would like to thank editage (www.editage.com) for english language editing. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord- - sqovwb authors: li, hao; li, kai; bi, zhen; gu, jun; song, deping; lei, dan; luo, suoxian; huang, dongyan; wu, qiong; ding, zhen; wang, leyi; ye, yu; tang, yuxin title: development of a reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for the detection of porcine pegivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: sqovwb a simple and accurate reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay was developed and evaluated for the detection of porcine pegivirus (ppgv). the specific rt-lamp primers targeting the conserved regions of ns a genes were designed and used to detect ppgv. the optimal reaction parameter for rt-lamp assay was ℃ for min. the detection limit of the rt-lamp assay was copies of ppgv genome, which was times more sensitive than that of the conventional rt-pcr and comparable to nested rt-pcr and quantitative rt-pcr (qrt-pcr). there was no cross amplification with other related rna viruses. in the clinical evaluation, the rt-lamp assay exhibited a similar sensitivity with nested rt-pcr and qrt-pcr. the results indicated that rt-lamp assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of ppgv in field settings. porcine pegiviruses (ppgv) are small enveloped viruses with a single-stranded, positive-sense rna genome that have recently been assigned as the pegivirus genus in the flaviviridae family (berg et al., ) . ppgv genome contains a single large open reading frame (orf) encoding a polyprotein, which is cleaved into individual proteins including e , e , x, ns , ns , ns a, ns b, ns a, and ns b. pegiviruses are detected in diverse mammalian hosts including pigs, humans, horses, chimpanzees, bats, monkeys, and rodents (yang et al., ) . all known pegiviruses have been classified into species (a-k), and ppgv is a member of pegivirus k. ppgv was first discovered in germany in (baechlein et al., ) , and then reported in the united states in (yang et al., ) . in china, ppgv was first identified in guangdong province (lei et al., ) , where vesicular diseases broke out in a swine farm in . the serum samples were submitted to the veterinary diagnostic laboratory of jiangxi agriculture university, china for diagnosis. the tested results indicated that foot-and-mouth disease virus (fmdv) and seneca valley virus (svv) were negative but positive for ppgv by a rt-pcr assay. although it remained yet unknown about the association between ppgv infection and the disease, more epidemiological studies are needed to address the clinical effect of ppgv on swine health. currently, there are some methods available for ppgv diagnosis, including conventional rt-pcr, nested rt-pcr, and semi-quantitative rt-pcr (yang et al., ; lei et al., ; chen et al., ) . therefore, a rapid and accurate method to detect ppgv is urgently needed for monitoring its presence. in the past decade, loop-mediated isothermal amplification (lamp) has become an effective technique, which exhibits high sensitivity and specificity for diagnosing important pathogens in medicine and veterinary medicine (notomi et al., ) . lamp is a nucleic acid amplification-based method that generally requires a group of four specific external and internal primers (notomi et al., ) . besides, additional loop primers can be used to further accelerate the reaction (nagamine et al., ) . in this study, a reverse transcription lamp (rt-lamp) assay was established and evaluated for surveying the prevalence of ppgv in porcine serum samples. a set of three specific primers pairs was designed to amplify the ns a gene, which is suitable for rt-lamp and relatively conserved in ppgv genome. the developed rt-lamp assay showed high sensitivity and specificity for detection of the ppgv. the results tested by rt-lamp assay on clinical samples were % correlated to that of nested rt-pcr and qrt-pcr, indicating the assay could be used for the surveillances of ppgv infection. in this study, serum samples (n = ) of -week-old pigs with a vesicular disease from guangdong province, china in were collected. extraction kit (takara, china), and then stored at - ℃ until use. the extracted rna was converted to the first strand of cdna using primescript rt reagent kit (takara, china) following the manufacturer's instructions. for the construction of plasmid standards, an -bp fragment of the ns a gene of ppgv was initially amplified using a pair of primers (table ) with the st strand of cdna as template. afterwards, the purified fragment was cloned into the pmd -t vector (takara, china) according to the manufacturer's protocol. to assess the successful construction of the plasmid, designated as pmd-ppgv, dna sequencing was performed by sangon biotech company (shanghai, china). the concentration of pmd-ppgv plasmid was measured by a nanodrop spectrophotometer (thermo fisher scientific, usa). the copy number of the cloned gene was quantified as follows: [copy/ μl = plasmid concentration (g/μl) / [(plasmid length) × ) × ( . × )] (park et al., ) . serial dilutions of plasmid standards ( × - × copies) were used as templates for the determination of detection limit of pcr, which would be used as a positive control in the rt-lamp assay system. the three pairs of rt-lamp primers targeting the conserved regions of ns a genes were designed based on the sequences of the reference strain (ppgv_gd/ch/ , genbank: mg . ; ppgv_s / /ger/ , genbank: ku . ; ppgv_ /ger/ , genbank: nc_ . ; ppgv_ f/ger/ , genbank: ku . ; ppgv_ / nd/ , genbank: mg ; ppgv_ /mo/ , genbank: mg ; ppgv_ /ia/ , genbank: mg ) using the online software primer explorer v (http://primerexplorer.jp/e/), which included an outer pair (f and b ), an inner pair (fip and bip), and a loop pair (lf and lb). the inner primers fip and bip contained f and b sequences with the complementary sequences of f and b (f c and b c) in their ′-terminal, respectively (fig. ) . a pair of primers (f and r ) was used for amplifying the ns a gene in conventional rt-pcr and meanwhile served as outer primers for the nested rt-pcr (table ). the primer pair of f and b was used as inner primers for nested pcr. a pair of primers (f and r ) was used for qrt-pcr targeting the ns a gene which was designed using the online software primer (http://bioinfo.ut.ee/primer - . . /). the specificity of the primers was then examined by a basic local alignment search tool (blast) search (http://www.ncbi.nlm.nih.gov/blast) against the ncbi database. the results demonstrated that there was no hits, that is, no nonspecific primer binding site was observed. the final volume of μl reaction mixtures for rt-lamp was prepared, which contained μl of bst dna polymerase (neb, usa) ( u/ml), . μl of × isothermal amplification buffer, μl of betaine ( m), μl of mgso ( mm), μl of dntp ( . mm), μl of each inner primers fip and bip ( μmol), . μl of each outer primers f and b ( μmol), . μl of each loop primers lf and lb ( μmol), . μl of amv reverse transcriptase (takara, china) ( u/μl), μl of rna template, and the sterile distilled water was set as a negative control template. to determine the optimal reaction conditions, the rt-lamp reactions were incubated at different temperatures of , , , , , and ℃ for , , , , , and min, respectively. finally, the reaction was terminated by heat inactivation at ℃ for min. the amplified dna products from the rt-lamp were analyzed by . % agarose gel electrophoresis with the addition of . % ethidium bromide using a gel electrophoresis system (liuyi, china). the results were also observed directly by color change from orange to green with staining due to the presence of sybr green Ⅰ (thermo scientific, usa). after the amplification was completed, μl of coloring agent was added to each reaction tube, and the results were then examined visually. in rt-lamp assay with ppgv rna as template, the amplified dna products showed characteristic ladders of multiple bands on an agarose gel, indicating that the final products were a mixture of stem-loop dna with different stem lengths (khan et al., ) . the results of the rt-lamp reaction were also determined by direct visualization of the color change under daylight and uv light as shown in fig. . in contrast, lamp-specific dna bands and color changes were not observed in the negative control. meanwhile, the dna products of rt-lamp showed the highest intensity when the reaction was carried out for min (fig. ) . in regard to temperature, the optimal reaction temperature of rt-lamp was ℃. therefore, the optimized parameter for the established rt-lamp was ℃ for min. to determine the detection limit of the rt-lamp, -fold serially diluted plasmid standards of pmd-ppgv ( × - × copies) were used as templates in μl rt-lamp reaction system under optimized conditions. the sterile distilled water was set as a negative control template. nested rt-pcr was performed based on the protocol established in our laboratory (lei et al., ) . for the pcr step of the nested rt-pcr, the primer sets of f /r and f /b respectively served as outer ( st set) and inner primers ( nd set) ( table ). the reaction system contained μl of plasmid standards of pmd-ppgv ( × - × copies) or μl of the first pcr product, . μl of × pcr buffer (ta-kara, china), . μl of rtaq ( u/μl), μl of mgcl ( mm), μl of dntp ( . mm), and μl of each primer ( μmol). two rounds of nested pcrs were performed under the following conditions: ℃ for min; cycles at ℃ for s, ℃ for min, and ℃ for min; and a final extension of ℃ for min (wang et al., ) . the conventional rt-pcr was carried out under the same reaction condition as above in nested rt-pcr using the primer pair of f and r based on the procedure described previously (lei et al., ) . the products of nested rt-pcr and conventional rt-pcr were analyzed by . % agarose gel electrophoresis with . % ethidium bromide. for qrt-pcr, the primer set of f and r , targeting conserved region of the ns a gene of ppgv, was used ( table ). the qrt-pcr assay was performed by using a commercial qpcr kit (takara, china) based on the manufacturer's procedure. each μl reaction mixture consists of μl of tb green fast qpcr mix, μl of plasmid standards of pmd-ppgv ( × - × copies), . μl of ( μmol) each forward and reverse primers and . μl of rox. the amplification parameters included an initial denaturation at ℃ for min followed by cycles of ℃ for s and ℃ for min. the melting curve analysis was measured using the software supplied with abi fast real-time pcr system (applied biosystems, usa). the results demonstrated that the detection limit of the developed rt-lamp was copies/μl, which was much higher than conventional rt-pcr ( copies/μl), while it was comparable to that of the nested rt-pcr ( copies/μl) and qrt-pcr ( copies/μl), respectively (fig. ) . to assess the specificity of rt-lamp for ppgv, cdnas of seneca valley virus (svv), food and mouth disease virus (fmdv), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), and swine acute diarrhea syndrome coronavirus (sads-cov) were used, and the plasmid standards of pmd-ppgv was used as the positive control. the amplified products were analyzed by . % agarose gel electrophoresis with . % ethidium bromide and addition of sybr green Ⅰ staining solution. the results indicated that there was no cross amplification of the rt-lamp assay for these control viruses. using ppgv rna as a template, positive dna bands were amplified as expected (fig. ) , indicating that the rt-lamp assay developed was specific for ppgv. to further evaluate the developed rt-lamp assay, serum samples (n = ) from finishing pigs and sows collected in guangdong and jiangxi province in china during october to january were tested (lei et al., ) . total rna was extracted and used as a template for conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp according to aforementioned methods. as shown in table , the positive sample rates of conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp were . % ( / ), . % ( / ), . % ( / ), . % ( / ), respectively, suggesting these data were consistent with our previous findings (lei et al., ) . the detailed information is listed in table s . the results showed the rt-lamp had a similar sensitivity to nested rt-pcr and qrt-pcr, which was superior to conventional rt-pcr. pegiviruses have a broad host range and infect diverse animal species. ppgv is recently proposed as a novel species in the pegivirus genus. ppgv epidemics have been confirmed in germany in , the united states in , and china in . although several pigs positive for ppgv exhibited vesicle and lameness (yang et al., ) , there is limited evidence that infections were associated with a clinical disease. therefore, the clinical significance and epidemiological investigation of ppgv infection should be further studied. nevertheless, the establishment of a sensitive, cost-effective, and rapid assay for detecting ppgv is urgently needed. in this study, a rt-lamp assay was developed to monitor ppgv in pig populations. compared to other diagnostic methods, such as conventional rt-pcr, nested rt-pcr, and qrt-pcr (baechlein et al., ) , rt-lamp assay may eliminate a need for complex procedures and expensive instruments, which makes it suitable for poorly equipped laboratories and clinics (lopez-jimena et al., ) . rt-lamp is considered to be an alternative diagnostic tool in terms of its ability to rapidly amplify target nucleotide sequence(s) under isothermal conditions. lamp has been widely used to detect a variety of important swine pathogens, including pdcov (zhang et al., ) and pedv (mai et al., ) . in the present study, the rt-lamp reaction conditions were optimized in terms of reaction time and temperature. the detection limit of rt-lamp assay established is times higher than that of conventional rt-pcr, and had a similar sensitivity to nested rt-pcr and qrt-pcr. rt-lamp developed in the study showed a high specificity as it did not cross react with other related porcine viruses. in the clinical evaluation, the positive rate of rt-lamp were consistent with that of nested rt-pcr and qrt-pcr, indicating the ppgv-specific rt-lamp assay was a valuable tool for the rapid detection in field settings. in summary, we developed a rapid, simple, accurate, and cost-effective rt-lamp assay for detection of ppgv, and the assay could be used as an alternative for the clinical diagnosis of ppgv infection and epidemiological investigation. none. pegivirus infection in domestic pigs discovery of a novel human pegivirus in blood associated with hepatitis c virus co-infection semiquantitative duplex rt-pcr reveals the low occurrence of porcine pegivirus and atypical porcine pestivirus in diagnostic samples from the united states diagnostic accuracy of loop-mediated isothermal amplification (lamp) for detection of leishmania dna in buffy coat from visceral leishmaniasis patients detection and genetic characterization of porcine pegivirus from pigs in china development of a single-tube one-step rt-lamp assay to detect the chikungunya virus genome development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay loop-mediated isothermal amplification of dna loop-mediated isothermal amplification (lamp): principle, features, and future prospects loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus integrating nested pcr with high-throughput sequencing to characterize mutations of hbv genome in low viral load samples detection and genetic characterization of porcine pegivirus in pigs in the united states a simple and rapid identification method for newly emerged porcine deltacoronavirus with loop-mediated isothermal amplification rapid specific and visible detection of porcine circovirus type using loop-mediated isothermal amplification (lamp) this work was supported by the national key research and development program (grant number yfd ), the natural science foundation of jiangxi province (grant number acb and bab ), the graduate research & innovation projects in jiangxi (grant number yc -b ), and science and technology project of education department of jiangxi province (grant number gjj and gjj ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- -viz uam authors: dong, qing; liu, quanyi; guo, lulu; li, dan; shang, xudong; li, bingling; du, yan title: a signal-flexible gene diagnostic strategy coupling loop-mediated isothermal amplification with hybridization chain reaction date: - - journal: analytica chimica acta doi: . /j.aca. . . sha: doc_id: cord_uid: viz uam abstract recent study proves that the combination of loop mediated isothermal nucleic acid amplification (lamp) with one-step strand displacement (osd) is of great help to improve the sequence specificity during genetic detection. however, because osd is incapable of signal amplification, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. with the purpose to overcome this challenge, herein a more advanced and practical sensing principle is developed with the osd replaced by an amplifiable nucleic acid circuit, hybridization chain reaction (hcr). the very contagious norovirus (nov) was employed as the model target. compared with lamp-osd, the lamp-hcr can detect as few as copies of nov gene in % fecal samples with significantly enlarged signal change and signal-to-background ratio. therefore, more reliable detection is achieved. moreover, due to the high compatibility of hcr, the final lamp-hcr products can be flexibly transduced into different types of readouts, including fluorescence, flow cytometer (fcm) and even a personal glucose meter (pgm). this further enlarges the operating environments for the detection from hospital labs, bedsides, to potential off-the-shelf devices in local pharmacies. especially when using fcm or pgm, with the assistance of magnetic beads (mbs), the detection shows even higher tolerance capability to complicated biological matrices. molecular diagnostic is a series of techniques which are used for sensitively analysing biomarkers in the genome and the proteome-genetic code of individuals in vitro [ ] . in the past several decades, it was rapidly developed to guide patient care management in the field of infectious diseases, cancers and so on. the increasing demand for the information of genetic and genomic leads to the rapid advances of molecular techniques [ ] . among most molecular diagnostic methods for nucleic acid biomarkers detection, polymerase chain reaction (pcr) has been the most widely used method for vastly increasing the number of nucleic acid molecules [ e ] . to make the molecular diagnostic test simpler and more portable, a suite of isothermal amplification techniques have been invented, such as rolling circle amplification (rca) [ ] , helicase-dependent amplification (hda) [ ] , recombinase polymerase amplification (rpa) [ ] , and loop-mediated isothermal amplification (lamp) [ , ] . these methods can realize the exponential amplification at constant temperature which could facilitate the point-of-care (poc) gene detection [ e ] . however, these assays are generally still impractical for real applications, because of the resource poor settings as well as the high frequency to generate false positive results [ e ] . with the purpose of improving the detection specificity and achieving more simple and reliable readout, we recently developed an innovation in which one-step nucleic strand displacement reaction (usually shortened as osd) was adapted, instead of traditional fluorescent intercalating dyes, to probe the products of isothermal amplifications (e.g., lamp). due to the high sequence specificity, the possibility of false positive signals has been reduced at the maximum degree. even though, unlike the intercalating dyes that can provide signal amplification via interacting with all the base pairs of the lamp amplicons [ e ], one osd probe can be activated by a single lamp amplicon [ e ] . therefore, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. in order to overcome the above potential shortage and further enable the detection practical in more possible operation environments and settings, herein we import another innovation through updating the osd probe with an enzyme-free nucleic acid circuit, the hybridized chain reaction (hcr). as an integration of multiple osds, the hcr reaction was firstly invented by pierce group [ ] and immediately became a starring signal amplifier due to high amplification efficiency and compatibility to different readouts [ e ]. in assistance of hcr, a variety of proof-ofconcept targets, including cancer cells, microrna, metal ions, and many aptamer ligands have been detected with improved sensitivity [ e ]. in this paper, through designing partial of lamp amplicon sequence as the input sequence to trigger hcr reaction, we for the first time couple the lamp and hcr together to achieve both ultra-sensitivity and significant signal change for general gene targets. and different transduction strategies were developed to make sure the products of lamp-hcr could be flexibly monitored via many kinds of available settings (readouts), e.g. gel electrophoresis, flow cytometer (fcm) and even a commercial personal glucometer (pgm). the practicability of the method has thus been largely improved. recently, the very contagious noroviruses (novs) have become the leading cause of sporadic gastroenteritis across all age groups, especially pediatric populations. it was reported that % of fecal samples from chinese pediatric out patients were novpositive with more than % belonging to gii [ , ] . as a response to the urgent demand for more accurate and reliable detection, here the gene segments of nov gii was specifically employed as the model target [ ] . in spite of their high sensitivity, earlier reports like electron microscopy [ , ] , immunology-based detection (radioimmunoassay (ria) [ , ] , enzyme-linked immunosorbent assays (elisa) [ , ] ) and nucleic acids biomarkers detection based on gene amplification techniques (pcr [ , ] , nucleic acid sequence-based amplification (nasba) [ , ] , lamp [ , ] ) always lack of specificity and false positive results can easily arise. herein, the powerful exponential amplification of lamp results in ultrasensitive nov gene detection with a detection limit of copies, while the hcr gives rise to very good specificity and reliable signal changes in all kinds of readouts. and all of the three readouts can be compatible with fecal samples. compared with fluorescence (gel electrophoresis and fluorescent reader), the fcm and pgm signals were more stable and interference-resistant because of the importance of magnetic beads (mbs) separation. all of the chemicals used in this work are of analytical grade unless otherwise indicated. betanie was purchased from sigma-aldrich (st. louis, mo, u.s.a.). bst . dna polymerase, dntp, mgso ,  isothermal buffer (  iso buffer) were purchased from new england biolabs (ipswich, ma, u.s.a.). all the oligonucleotides used in this work were ordered from sangon biotech (shanghai, china), and the sequences were listed in table s (in supporting information (si)). all modified dna sequences were purified by high performance liquid chromatography (hplc). all unmodified dna sequences were purified with polyacrylamide gel electrophoresis. all oligonucleotides were stored in  te (ph . ) at À c. buffers used here were:  iso buffer ( mm a ml lamp reaction mixtures containing different copies of templates, . mm each bip and fip primers, . mm each b and f primers, m betaine, and . mm dntps in a total volume of ml  iso buffer were incubated at c for min, followed by chilling on ice for min. and then ml of units/ml bst . dna polymerase were added to initiate the lamp reaction solution, followed by a constant c reaction for h. then, the samples were analysed by electrophoresis on a % agarose gel. each well was loaded with ml of sample and an additional ml of  orange loading dye ( % glycerol, . % orange g). the electrophoresis was developed at v for min, then agarose gels were stained with gelred. the standard procedures of lamp reaction were optimized by real-time fluorescence with the osd reaction. annealed osd reporter (at a final concentration of nm reporter-f, nm reporter-q) was added to the lamp reagents with different ratios of primers and dntps, or with different concentrations of mgso . ml of units/ml bst . dna polymerase were added to initiate the lamp reaction. and then, ml of the lamp-osd solutions were transferred into a -well plate, which was maintained at different constant temperatures ( , , or c). fluorescence signals were recorded with a lightcycler . lamp reactions in different concentration of fecal samples were performed using the similar protocol to the real-time lamp reaction. here, the human fecal was diluted to a concentration of wt% with  pbs, and then centrifuged min at , rpm. the supernatant was collected and filtered with . mm millipore filter, and then stored at À c until further use. for the standard hcr reaction, the fluorescein modified h and h (i.e., fam-h and fam-h ) were annealed in  iso buffer, respectively. mixtures in  iso buffer in a total volume of ml containing nm fam-h , nm fam-h , . m nacl and certain amount of dna mimic (i.e., mimic-t) were incubated h at c. to analyze the hcr product, ml of the hcr products were mixed with ml  orange loading dye and loaded into the % native page gel. after the electrophoresis, the gel was then stained with gelred. lamp products were heated to c for min, followed by chilling on ice for min. the hcr reaction mixture containing ml aliquot of the ml lamp products, nm annealed fam-h (or h ), nm annealed fam-h (or p eh , a well-designed dna sequence with -base extension at the ' end of h ), . m nacl in a total volume of ml  iso buffer were incubated at c for h. the reactions were evaluated by electrophoresis in a % agarose gel. each well was loaded with ml of sample and an additional ml of  orange loading dye. the electrophoresis was developed at v for min, and then agarose gels was stained with gelred. firstly, ml of mg/ml mbs were washed times with  iso buffer. the mbs were isolated by using an external magnetic rack and re-suspended in ml  iso buffer. then, ml of . mm annealed biotinylated h (bio-h ) was incubated with the mbs on a rotator for h. the unbound bio-h was removed by washing the mbs/bio-h in  iso buffer at least times. then, ml certain amount of mimic-t, . m nacl, nm annealed fam-h and nm annealed fam-h were added into the mbs/bio-h , rotating at c for h to conduct the hcr reaction. after at least times of washing with ml  iso buffer, each sample was diluted to ml buffer before subjected to the flow cytometer. for analysis, , events were collected for each sample. fluorescence signals of the hcr products-anchored mbs were detected by fl channel with nm laser excitation. the mean fluorescence intensities (mfi) of the fluorescent histograms were used for the quantitative analysis of mimic-t. this protocol was also suitable to lamp products, except that mimic-t was replaced by ml aliquot of the ml lamp products. ml of mg/ml mbs were washed times with  iso buffer. then ml of different samples in  iso buffer were added into the mbs, respectively, for h incubation and rotation at c. after at least washes, the fcm detections were performed. mbs/bio-h were prepared almost in the same manner as hcr-fcm measurement. then ml superblock™ (tbs) blocking buffer was added to mbs/bio-h for h rotation on a rotator at c. the magnetically separated mbs/bio-h was then incubated with ml . m nacl, nm annealed h , nm annealed p eh and certain amount of mimic-t in  iso buffer, rotating at c for h. the unbound dna were removed by washing for at least times, and then the mbs were suspended in ml buffer and incubated with ml of the mg/ml p -inv (thermostable invertaselabelled p -sh) conjugates for . h on a rotator at c. after at least washes to remove the excess p -inv, the mbs were resuspended in ml buffer and transferred into an equal volume of mm sucrose. the mixture was incubated for min at c to allow invertase-mediated catalytic conversion of sucrose to glucose. subsequently, . ml of the reaction mixture was transferred to a glucometer strip and the amount of glucose was measured by using personal glucometer. the above protocols for detecting mimic-t were also suitable for lamp products, except that mimic-t was replaced by ml aliquot of the ml lamp products. ml of mg/ml mbs were washed times and re-suspended in ml buffer. then, ml mixture of . mm annealed bio-reporter-f and reporter-q was incubated with mbs on a rotator for h, and unbound dna were removed by washing mbs with buffer for at least times. after that, the mbs were re-suspended in ml buffer and ml lamp products to a total volume of ml, performing fcm detection. our strategy was built as the following steps (scheme ): the standard lamp reaction was carried out for producing flower-like amplicons which contain four different single strand loops (i.e., floop, fc-loop; b-loop; bc-loop). a standard hcr reaction uses one single stranded oligonucleotide as trigger, and two hairpin oligonucleotides, h and h , as substrates. the trigger successively opens h and h via two osd reactions, and then starts an elongation cascade reaction continuously to form long, nicked duplex products. herein the sequence of h and h were designed by using the bc-loop as the trigger. h , h , and the lamp amplicons were mixed with bio-h -labelled mbs to trigger the hcr at the same time. in detail, the hairpin structure of bio-h could be opened by the hybridization with bc-loop of the amplicons, leading to the subsequent cascade of hybridization reaction between h and h . the nicked double stranded dna (dsdna) could be finally formed on the surface of mbs. on one hand, if the probe h and h were modified with fluorochrome, the hcr triggered by lamp could result in the accumulation of large amount of fluorophores on the mbs. by reading the fcm signals of the fluorescent mbs, the nov gene can be directly analysed. on the other hand, the well-designed p eh serves as a tail probe and the bases extension could not disturb the lamp to trigger hcr. in this case, after the hcr, the numerous tailed sequences, which were extended from the nicked dsdna on the mbs' surface, could hybridize with its complementary dna labelled with invertase (i.e., p -inv). followed by the washing and separation steps, the re-suspended mbs with invertase could hydrolyze sucrose into glucose which can be detected by using the commercial pgm. the steps were initially verified separately. here the four primers set of lamp for nov gii gene was self-designed, using copies to  copies as the template. the standard procedures of lamp reaction were optimized by real-time fluorescence with the osd reaction (fig. s , si) . the osd reporter was designed for bc-loop sequence (fig. a) . after the optimization, . mm each bip and fip primers, . mm each b and f primers, m betaine, . mm dntps, mm mgso , and ml of units/ml bst . dna polymerase in  iso buffer were employed as the lamp reagents, followed by a constant c reaction. under the optimal conditions, as few as copies nucleic acid templates of nov could be detected within h in . % fecal samples ( fig. b and c) . it should be noted actually the lamp-osd is already a ready-to-use method for efficient nov detection. however, the concern is even with plenty of optimizations, the signal magnitude of the fluorescence signal (nov positive) over background (nov negative) is still not satisfied. sometimes the signals may even be too small to be distinguished from the non-specific fluorescence increase generated by unknown interferences, especially when fecal sample concentrations are increased ( fig. d and e) or non-specific amplifications (fig. s , in si) are induced. actually, this problem may commonly exist in the detection for many other gene targets. that is the reason why amplifiable circuit (hcr) has to be imported as an enhancement to the detection reliability. the efficiency of hcr was assessed by using mimic-t with the same sequence as bc-loop (fig. ) . the % native page gel (without or with gelred stain) results show that hcr reaction took place with the mimic-t introduced ( fig. a and b, lane e ) . however, the h and h could exist steadily without mimic-t ( fig. a and b, lane e ). fcm analysis also shows the same results. in fig. c and d, the fluorescence increased significantly with the increased concentration of mimic-t. while there is no obvious signal increase in the absence of the mimic-t compared to the bare mbs. to testify the cascade hcr amplification on mbs, some control experiments were carried out. fig. e and f show about orders of scheme. . schematic illustration on coupling lamp with hcr for molecular diagnostic via flow cytometer (fcm) and personal glucometer (pgm). magnitude the mfi increase than that of one-step reaction where the pre-hybridized bio-h and mimic-t were immobilized on the mbs, with nm fam-h serving as the target to form a : binding. and there is about orders of magnitude signal increase than that of the assay only using fam-h where no additional fam-h was introduced during the hcr process together with nm mimic-t. when fam-h and nm mimic-t were mixed with bio-h -labelling mbs, no signal increase was observed compared with bare mbs. these results clearly demonstrate the signal amplification in hcr approach. note that, the non-specific adsorption of dnas on mbs (fig. s , in si) and the optimization of the concentration of hcr reagents (fig. s , in si) were also well investigated. following these steps, we integrated lamp and hcr for nov templates detection. the agarose gel electrophoretic characterization of the amplification products reveals the success of this lamp to hcr signal transduction (fig. s , in si) . in the fcm assay, the lamp amplicons ( ml), nm each of fam-h and fam-h were mixed with bio-h -modified mbs and incubated for h prior to magnetic washing and separation. the completely washed mbs with hcr products were used for fcm measurement to the definitive detection of the synthetic template of nov. fig. a shows the fluorescence signal of the mbs detected in fl channel. the mfi of the tested mbs of~ represents the negative controls (  iso buffer with no template or noncognate rotavirus lamp products) (fig. b ). while as low as copies of the nov template could be detected (mfi is ) without the dose-discrimination, because the primers have been consumed in the given lamp reaction time ( h). to detect the nov templates in fecal samples, rather than in buffer, the nov templates were spiked into different percentages of fecal samples (from . % to %). the agarose gel electrophoretic result shows that it makes no difference of lamp reactions in fecal samples compared with the lamp reaction in buffer (fig. e) . following the optimization of the reaction, the nov template spiked into % human fecal also gave consistent fcm results without any loss in signal intensity and detection sensitivity ( fig. c and d) . these results exhibited the mbs based lamp-hcr strategy realized robust nov detection and was more resistant to interference than the real-time fluorescence assay, where the fluorescence signal could be decreased with the increased concentrations of fecal samples ( fig. d and fig. s , in si). the reproducibility test performed in % human fecal consists of three parallel assays, detecting both nov positive samples (  copies of nov dna) and buffer negative controls on three days, respectively ( fig. e and f) . as expected, only small standard deviations were yielded by all nine sets of measurements (i.e., . mfi and mfi for negative response ( mfi) and positive response ( mfi), respectively). besides sensitivity, selectivity and reproducibility, the re-usability of the bio-h -labelled mbs after detection is easy to regenerate by heating the mbs at c for min, immediately followed by a quick separation of the bio-h -labelled mbs from other reagents (i.e., fam-h , fam-h and lamp products), leading to the regenerated modified mbs. the successful regeneration could be verified by the subsequent detection with the same result as that of the first one (fig. s , in si) . note that, the bio-h -labelled mbs could be reused at least but may be not limited to twice. the aggregation of mbs could occur under too much repeated regeneration, resulting in unreliable detection results. encouragingly, the cost of per analysis could be controlled within dollar, which makes it suitable for poc gene biomarker analysis. as we know, the most commonly used commercially available poc device is the pgm because of its portable size, low-cost and easy operation. lu group has firstly reported the aptasenors by using pgm to detect a series of targets [ ] . previously, we developed the sweet spot for molecular diagnostics by coupling lamp with osd, which could directly transduce middle east respiratory syndrome coronavirus and zaire ebola virus templates into glucose signals [ ] . however, it shows that the signal amplitude (dglucose meter signal, dgms ¼ positive signal e negative signal) is not high (~ mg/dl). in our lamp-hcr to pgm assay, the hcr reaction on mbs could be triggered by lamp amplicons in the presence of h and p eh . here, p eh with a tail sequence can hybridize with its complementary dna labelled with invertase (p -inv). after washing and separation steps, the invertase attached on mbs hydrolyzed sucrose into glucose, which could be detected by pgm. firstly, mimic-t was chosen for investigating the hcr process (fig. a) . with the increasing concentration of mimic-t, the signal amplitude (dgms) of pgm gradually increased. then, instead of mimic-t, lamp amplicons could also trigger the hcr on the surface of mbs, following the hybridization of p -inv with the hcr products on the mbs. fig. b shows a positive signal compared with negative control (non-cognate rotavirus lamp products). remarkably, as few as copies of nov in % human fecal sample can produce an obvious amplitude, reaching up to mg/dl. the agarose gel electrophoretic result also shows that hcr reaction occurred with nov lamp amplicons introduced (fig. c) . the reproducibility test of this pgm assay was also carried out under the same experimental condition as the fcm assay. the dgms values for detecting  copies of nov in % human fecal on three days were comparable (fig. d) , showing a very low standard deviation of mg/dl. the amplification effect of the hcr function was further confirmed using a lamp to osd detection by fcm. as shown in fig. a , an osd duplex (hybridized between sequence bio-reporter-f and sequence reporter-q) was immobilized on mbs via the biotin labelled on bio-reporter-f. and for signal reporting, fluorophore fam and its quencher was, in respective, tagged onto bio-reporter-f and reporter-q. in presence of lamp amplicons, the bc-loop could initiate a strand displacement reaction and kick the reporter-q away from bio-reporter-f, finally leaving bio-reporter-f/bc-loop (in amplicons) duplex on the mbs. therefore, the mbs were lightened up with higher fluorescence emission. when such a process was transferred onto fcm assay, the sample with novs lamp amplicons showed a tiny right-migration in the fl channel, indicating the success of the lamp-osd detection. however, the absolute (fig. b ) and relative mfi (fig. c ) values were all much smaller than those obtained from the lamp-hcr detection. in summary, we have successfully developed a highly sensitive and selective detection method for novs both in buffers and fecal samples via the combination of lamp with either osd or hcr. in general, compared with various novs gene detection methods (table s , in si), the present work proved much more reliable and reproducible. the lamp reactions and transducers (i.e., osd or hcr) we have developed are comparably sensitive but also sequence-specific to ensure low false positives. the sensitivity down to gene copies is mainly contributed by the lamp reaction. when the osd signal suffers unsatisfied signal change, hcr reaction can further provide the ultra-selectivity and higher signal-tobackground ratio which has usually and seriously affected the accuracy for lamp and other enzymatic amplifications. the detection is very practical, low-cost and almost enough to be ready-to-use. and the readouts should be very flexible, exhibiting great potential to be used in the diagnosis of gastroenteritis in the hospital or even at home with the read out by the portable pgm. molecular diagnostic techniques a unified approach to utilize phenotypic, full pedigree, and genomic information for genetic evaluation of holstein final score analysis of enzymatically amplified globin and hla-dq dna with allele-specific oligonucleotide probes identification of group a rotavirus gene types by polymerase chain reaction digital pcr analysis of circulating nucleic acids rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids isothermal recombinase polymerase amplification (rpa) of schistosoma haematobium dna and oligochromatographic lateral flow detection phosphorothioated primers lead to loop-mediated isothermal amplification at low temperatures rapid screening method for male dna by using the loop-mediated isothermal amplification assay double-enzymesmediated bioluminescent sensor for quantitative and ultrasensitive point-ofcare testing coupling sensitive nucleic acid amplification with commercial pregnancy test strips a magneto-dna nanoparticle system for rapid detection and phenotyping of bacteria multiple snps detection based on lateral flow assay for phenylketonuria diagnostic sample-to-answer droplet magnetofluidic platform for point-of-care hepatitis c viral load quantitation integrated magneto-chemical sensor for on-site food allergen detection integrated biosensor for rapid and point-ofcare sepsis diagnosis rapid detection of norovirus from fecal specimens by real-time reverse transcription-loopmediated isothermal amplification assay loop-mediated isothermal amplification (lamp): a new generation of innovative gene 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detection of nucleic acids in complex biological fluids probing spatial organization of dna strands using enzyme-free hairpin assembly circuits luorescent cascade amplification method for sensitive detection of salmonella based on magnetic fe o nanoparticles and hybridization chain reaction cytometric bead assay for sensitive dna detection based on enzyme-free signal amplification of hybridization chain reaction an efficient nonlinear hybridization chain reaction-based sensitive fluorescent assay for in situ estimation of calcium channel protein expression on bone marrow cells multivalent capture and detection of cancer cells with dna nanostructured biosensors and multibranched hybridization chain reaction amplification label-free and enzyme-free homogeneous electrochemical biosensing strategy based on hybridization chain reaction: a facile, sensitive, and highly specific microrna assay a simple colorimetric dna detection by target-induced hybridization chain reaction for isothermal 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that exhibits wide spectrum binding activities a luciferase immunoprecipitation system (lips) assay for profiling human norovirus antibodies surveillance of norovirus contamination in commercial fresh/frozen berries from heilongjiang province, china, using a taqman real-time rt-pcr assay real-time rt-pcr for norovirus screening in shellfish the microfluidic chip module for the detection of murine norovirus in oysters using charge switchable micro-bead beating rapid detection of noroviruses in fecal samples and shellfish by nucleic acid sequence-based amplification detection of murine norovirus by reverse transcription loop-mediated isothermal amplification development of one-step reverse transcription loop-mediated isothermal amplification for norovirus detection in oysters using personal glucose meters and functional dna sensors to quantify a variety of analytical targets a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to glucometers the authors declare no conflict of interests. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.aca. . . . key: cord- - iwljdoi authors: chen, qin; li, jian; fang, xue-en; xiong, wei title: detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: iwljdoi a conserved nucleic acid fragment of the nucleocapsid gene of swine transmissible gastroenteritis coronavirus (tgev) was chosen as the target, six special primers were designed successfully. loop-mediated isothermal amplification (lamp) was developed to detect the tgev by incubation at °c for h and the product specificity was confirmed by hphi digestion. standard curves with high accuracy for tgev quantization was constructed by adding × sybr greeni in the lamp reaction. the assay established in this study was found to detect only the tgev and no cross-reaction with other viruses, demonstrating its high specificity. by using serial sample dilutions as templates, the detection limit of lamp was about pg rna, times more sensitive than that of pcr and could be comparable to the nest-pcr. swine transmissible gastroenteritis coronavirus (tgev), as a member of the coronaviridae, is a kind of single-stranded rna virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry. the traditional detection methods, including virus isolation, virus immunodiagnostic assays and pcr tests have the shortcomings, such as precise instruments requirement, elaborate result analysis demand, high cost, long detection time and so forth, which prevent these methods from being widely used [ ] [ ] [ ] [ ] . loop-mediated isothermal amplification (lamp) is a novel nucleic acid amplification method, which amplifies dna/rna with high specificity, sensitivity and rapidity under isothermal condition [ ] . it has already found wide application in rna virus detection, such as foot-and-mouth disease virus [ ] , swine vesicular disease virus [ ] , taura syndrome virus [ ] , severe acute respiratory syndrome coronavirus and h n avian influenza virus [ , ] . in this study, lamp method was applied in developing qualitative and quantitative detection system of tgev, while its specificity and sensitivity were assessed. swine transmissible gastroenteritis coronavirus (tgev, strain h), porcine reproductive and respiratory syndrome virus (prrsv), pesudorabies (prv), porcine parvovirus (ppv) derived from their passages in cell culture were provided by shanghai entry-exit inspection and quarantine bureau (shciq); nucleic acids of footand-mouth disease virus (fmdv) and classical swine fever virus (csfv) were obtained from chinese academy of inspection and quarantine (caiq). total genomic rna was extracted using trizol kit (invitrogen, usa). dna was extracted by dna blood mini kit (qiagen, germany). after elution in μl nuclease-free water, rna/dna samples were stored at - °c before use. the original concentration of rna/ dna sample was about ng/μl. complete genome sequences of fifteen different tgev strains/isolates and nine other similar viruses were obtained from genbank, and the homology was analyzed using the vector nti. the conserved fragment with high homology was chosen as the target region which and used to design the tgev lamp primers by the primer explorer v software http://primerexplorer. jp/e/. the target rna of tgev was first reverse transcripted using superscript™ ii (invitrogen, usa) and then amplified by pfu dna polymerase using forward primer: ggaagagaactgcaggtaa and reverse primer: ccatcttcctttgaagtcca. the amplified product was purified from agarose gels and then cloned into e. coli jm using the pmd -t vector. the target plasmid with the original concentration of . × copies/μl was extracted by the plasmid mini preparation kit and identified by the nm absorption spectroscopy, which was then used as the standard for the quantitative analysis. the amplified products were digested with hphi to confirm its specificity. the result of tgev-lamp was analyzed by agarose gel electrophoresis and fluorescence by adding × sybr greeni in the lamp reaction. the specificity of tgev-lamp was examined by the use of rna (or dna) extracted from five other pig disease viruses. the sensitivity of tgev-lamp was evaluated by comparing with pcr [ ] and nest-pcr[ ], using -serial tgev rna dilutions ( - to - ) as templates. lamp primers were designed using the primer explorer v software based on a conserved fragment of the nucleocapsid gene (fig. ) . the primers including outer primers (f and b ), inner primers (fip and bip) and loop primer (lf and lb) were shown in table . tgev deprived from the cell culture was first qualitatively analyzed by lamp. amplification products were analyzed by agarose gel electrophoresis. as shown in fig. (lane ), amplification could be carried out at °c and showed a ladder-like pattern on the gel while the negative control gave no bands (lane ). the specificity of the lmap product was confirmed by hphi digestion. predictable product of the -bp motif was resolved on the gel as theoretical expected (fig. , lane ) . standard control was used to develop real-time fluorescence lamp for quantitatively analyzing tgev. dynamic curves for tgev quantification was generated by serially diluting the standard control from . × to . × copies/μl (fig. ) . the log linear regression plot (standard curves) was obtained by plotting the time-to-positive (ttp) values against genome copies. the correlation coefficients were . (fig. ) five other pig viruses were used to confirm the specificity of the lamp for tgev detection. the results showed that only tgev detected gave amplification products while no amplification available to other viruses (fig. ) . the sensitivity of lamp was demonstrated by comparing with pcr tests using serial dilutions ( - to - ) of tgev rna samples as template. as shown in fig. , lamp and nest-pcr were able to detect - dilution (about pg rna), whereas pcr could only amplify the - dilution. therefore, the sensitivity of tgev-lamp could be comparable to nest-pcr, -fold higher than pcr. it is very important to find out a conserved nucleic acid fragment for designing specific lamp primers and developing efficient, accurate lamp assay [ , ] . in this study, the nucleic acid sequence homology of tgev strains/isolates and other similar viruses available from genbank were analyzed by vector nti software. the most conserved fragment of bp was found in the nucleocapsid protein gene which showed highly homology among different tgev strains/isolates (more than %) and low homology among other similar viruses (less than . %). the tgev lamp primers targeting the conserved sequence were designed successfully by the primer designer v software. the target region was amplified successfully by the lamp with a characteristic ladder-like pattern of bands from bp on the gel. this is because the final products of lamp are a mixture of stem-loop dnas with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand [ , ] . after digestion with hphi, -bp motif was resolved on the gel as expected, demonstrating the specific structure of amplification products, which could also be validated by nucleic acid sequencing. as a kind of nucleic acid amplification method, lamp could not only qualitatively detect the tgev, but also quantitatively analyze the virus. in this study, real time fluorescence lamp for quantitatively detection of tgev was established by adding × sybr greeniin the lamp reaction. three standard curves established by tgev standards displayed the good correlation between the ttp and virus copies, implicating the great potential in quanlitatively detecting tgev. five other viruses were used in this study to confirm the specificity of lamp. the results showed no amplification in all viruses tested, which makes the lamp more accurate and reliable for tgev detection. the high specificity of lamp is most probably attributable to recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences during the second reaction stage. the sensitivity of lamp was evaluated using various tgev-rna dilutions as templates. the assay exhibited almost equivalent sensitivity to the nest-pcr and -fold higher than the pcr, indicating that the lamp is a more powerful diagnosis tool to detect tgev in lower copy conditions [ ] . in addition, the tgev-lamp developed has advantages in its rapid detection and simple operation. the only equipment required for the reaction is a water bath or heat block. the assay developed is a faster detection method for the tgev detection, only taking about h, which means the whole diagnosis including rna extraction, amplification and product detection could be completed within one and a half hour after receiving of the samples. it is anticipated that with the advantages of specificity, sensitivity, reliability, rapidity and easy manipulation, lamp will turn out be a powerful molecular tool for the tgev detection in practice. in conclusion, this study demonstrates that the lamp method established could detect only the tgev and no cross-reaction with other viruses, the detection limit was about pg rna, which was times more sensitive than that of pcr and could be comparable to the nest-pcr. virus isolation and serum antibody-responses after infection of cats with transmissible gastroenteritis virus -brief report use of monoclonal antibodies in blocking elisa detection of transmissible gastroenteritis virus in faeces of piglets an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus loop-mediated isothermal amplification of dna novel reverse transcription loopmediated isothermal amplification for rapid detection of foot-andmouth disease virus a: one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus rapid and sensitive detection of taura syndrome virus by reverse transcription loop-mediated isothermal amplification development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific figure standard curves of real-time fluorescence tgev-lamp negative control. figure sensitivity of lamp for tgev detection. a, lamp; b, pcr; c, nest-pcr the authors declare that they have no competing interests. key: cord- -yjbcvf o authors: cardoso, tereza c.; ferrari, heitor f.; bregano, lívia c.; silva-frade, camila; rosa, ana carolina g.; andrade, alexandre l. title: visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye date: - - journal: mol cell probes doi: . /j.mcp. . . sha: doc_id: cord_uid: yjbcvf o a sensitive reverse-transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for the rapid visual detection of turkey coronavirus (tcov) infection. the reaction is performed in one step in a single tube at °c for min, with hydroxynaphthol blue (hnb) dye added prior to amplification. the detection limit of the rt-lamp assay was approximately ( ) eid( / μl) tcov genome, and no cross-reaction with other avian viruses was observed. the assay was evaluated further in tissue suspensions prepared from the ileum and ileum–caecal junctions of infected turkey embryos; % of these samples were positive in the rt-lamp assay. all individual feces samples collected in the field were considered positive by both conventional rt-pcr and rt-lamp. in conclusion, rt-lamp with hnb dye was shown to be a sensitive, simple assay for the rapid diagnosis of tcov infection, either directly from feces or in association with virus isolation methods. the brazilian turkey industry is the second most productive in the world, and last year, more than million carcasses were produced. in , an outbreak of poult enteritis mortality syndrome (pems) was detected for the first time and found to be caused by a group coronavirus [ ] . cases of tcov enteritis are still reported in the u.s, canada and several european countries [ e ] . prior to more extensive sequence analysis, tcov had been identified as a member of group of the coronavirus genus, but it has since been definitively classified as group with other avian coronaviruses. it is a pleomorphic enveloped virus with a size of approximately e nm in diameter and nm-long clubshaped projections around virions. its genome is linear, positive sense, single stranded rna. the major structural proteins of tcov include the glycoprotein spike (s and s ), a membrane (m) protein and a nucleocapsid (n) protein. the s protein is highly variable and the spike gene structure remains unclear, despite several genetic studies of tcov having been conducted [ ] . the amino terminal s region of several coronaviruses contains receptor binding domains, whereas the s region consists of a transmembrane domain that induces cell fusion and pathogenesis [ ] . in addition, the s gene is more conserved and shares only % of similarity with other coronaviruses such as infectious bronchitis virus (ibv) [ , ] . conventional virological methods for detecting tcov include virus isolation (vi), immunofluorescent antibody assay (ifa), reverse transcriptase polymerase chain reaction (rt-pcr) and realtime rt-pcr (rrt-pcr) [ , , , ] . although rt-pcr and rrt-pcr methods are widely applied [ , ] , rt-loop-mediated isothermal amplification (lamp) is simpler, using only a water bath or heat block, and is highly efficient because the reaction is isothermal and requires no time for thermal changes [ ] . moreover, tcov isolates can be propagated in turkey embryos, but the virus cannot yet be propagated in cell culture [ ] . the objective of this study was to develop and validate a reversetranscription loop-mediated isothermal amplification (lamp) method for the direct detection of viral rna from tissues and feces collected from experimentally and naturally infected birds. the results were directly compared with those obtained from conventional ifa and rt-pcr. turkey coronavirus (tcov/br/ ) was obtained from a brazilian turkey farm suffering from severe cases of acute enteritis, described in [ ] . this tcov strain was successfully propagated in embryonated turkey eggs supplied for a commercial hatchery [ ] . viral purification, rna isolation and subsequent rt-pcr amplification were carried out using a pooled intestinal tissue suspension of infected intestines harvested from inoculated embryos, as in our previous study [ ] . the virus titration was performed with -fold dilutions into five groups of five -day-old embryonated turkey eggs. the % embryos infectious dose (eid ) was calculated according to reed and muench ( ) by analyzing macroscopic lesions in the intestinal tract [ , ] . intestinal tissues infected at a .  eid titer were homogenized in a -fold volume of minimal essential medium (gibco-brl, invitrogen, carlsbad, ca), clarified by centrifugation at  g for min and filtered consecutively through . -mm and . -mm membrane filters (millipore, bedford, ma). the presence of tcov in the filtrate was confirmed using rt-pcr of a portion of the utr (untranslated) region [ ] . extensive testing of the inoculums excluded the presence of pathogens other than tcov. embryo tissues (ileum and ileumecaecal junction portions) were prepared by infecting -day-old embryonated turkey eggs with . ml . eid tcov via the amniotic sac, as described previously [ , ] . a control group of turkey embryos at the same age were inoculated with sterile phosphate buffered solution (pbs). three days after infection, embryos were submitted to an external exam, and samples of ileum and ileumecaecal junction tissues were collected. the tissues were stored at À c, fixed in % neutral buffered formalin and embedded in paraffin blocks. ifa was performed in tissue sections, as described previously [ ] in order to detect viral antigens along the cell surface. intestinal suspensions (is) were prepared by cutting slices from the ileum and ileumecaecal junctions from a total of samples collected from infected and from uninfected embryos. the specimens were homogenized in volumes of minimal essential medium (mem) ph . and clarified by centrifugation at  g for min. the supernatant was first filtered through a . -mm paper filter (millipore) and then twice through a . -mm syringe filter (corning Ò ). these suspensions were heated at c for min in a water bath before total rna extraction was performed. fecal samples (n ¼ ) were obtained from a commercial flock that had experienced an outbreak of pems this year. the samples were collected directly from the cloaca and individually stored at À c until use. total rna was extracted from is and feces with a standard trizol protocol, based on guanidinium isothiocyanate and acidephenol, with some modifications [ , ] . each -ml aliquot of clinical suspension was mixed with -ml trizol reagent and incubated for min at room temperature. after the addition of -ml chloroform, the solution was mixed vigorously for s and centrifuged at ,  g for min. the upper aqueous phase was mixed with an equal volume of cold isopropanol and incubated on ice for min. the total rna precipitate was then pelleted by centrifugation at ,  g for min and washed with ethanol. the rna was dissolved in ml diethylpyrocarbonate(depc)-treated sterile double-distilled water and stored at À c. the primers were designed based on s sequence ( e position) information obtained from genbank and assigned the following accession numbers: tcov- /br/ (fj ); tcov- /br/ (fj ), tcov- /br/ (fj ); tcov- (eu ); tcov-atcc (eu ); tcov-gh (ay ); tcov-gi (ay ); tcov (canadian isolate, nc_ ). all primers were designed with primerexplorer v. , a software program for lamp primer design (http://primerexplorer.jp/e/). the names and sequences of each primer are shown in table . to test the specificity of the established rt-lamp, other viruses, including newcastle disease virus (ndv, la sota vaccine strain), turkey astrovirus (tastv- , genbank accession number fj ) and infectious bronchitis virus (ibv-m strain) were tested. the median embryo infectious dose (eid ) of the tcov used in the sensitivity test was table sequences of primers used in this study. sequence ( all samples were also tested by conventional rt-pcr, as described previously [ ] . each assay was conducted in triplicate. a positive amplification was indicated by a color change from violet to sky blue, as shown in fig. a , and verified by agarose gel electrophoresis (fig. c) . the detection limit of rt-lamp and conventional rt-pcr is illustrated in fig. b and c. ten-fold serial dilution of tcov demonstrated that rt-lamp is able to detect eid / ml , whereas the rt-pcr is able to detect eid / ml titrated virus. ifa analysis indicated that out of tested samples were positive, whereas % of samples tested positive by rt-lamp. a number of ileumecaecal junction samples from the infected embryos were scored as negative for tcov infection with ifa (table ). these false negative results were likely observed because viral antigens are not normally expressed on the cell surface until after viral rna transcription [ ] . when conventional rt-pcr and rt-lamp performed in the same samples were directly compared, ileumecaecal junction samples negative by rt-pcr were negative by ifa and samples were considered negative with conventional rt-pcr (table ) . this phenomenon could be explained by properties of dna polymerization offered by the use of four primers in lamp assay which can amplify few amounts of viral rna into cdna [ ] . probably those negative samples generated in conventional rt-pcr were collected from low viral infection samples with titers lower than eid / ml and not visualized on agarose gel electrophoresis system [ ] . all negative samples collected from non-infected embryos were negative in all tests (data not shown). a perfect correlation was found between conventional rt-pcr and rt-lamp results for feces collected in the field (table ) . no false positives (i.e., cross-reaction) of tcov rt-lamp were detected in samples of other avian viruses (fig. b) . rt-lamp employs an initial reverse transcriptase step followed by dna polymerization with a set of four primers specially designed for a specific target, representing a novel nucleic acid amplification method [ ] . one of the most attractive features of this rt-lamp assay is that the results can be observed and determined by hydroxynaphthol blue (hnb) dye-mediated visualization using the naked eye; furthermore, tubes do not have to be opened after amplification. the results reported here indicate that this diagnostic technique could reliably be used to detect tcov in field samples and may be used to effectively monitor infections, as demonstrated previously for realtime rt-pcr for the same target gene [ ] . although no quantitative data was generated in this study, rt-lamp associated with spectrophotometric evaluation can yield absolute values, as demonstrated previously [ e ]. the major advantage of rt-lamp over conventional molecular methods is time; only min are required for the reaction and more than h for rt-pcr. in addition, the reaction is conducted in a water bath rather than a thermocycler, and its results can be interpreted by direct visualization rather than gel electrophoresis, which also save time. in summary, the rt-lamp assay is novel, simple, visual and provides a sensitive and specific tool for the rapid detection and diagnosis of tcov in simply equipped laboratories and under field conditions in developing countries. its ease of use will support epidemiological programs in the turkey industry. detection of turkey coronavirus in commercial turkey poults in brazil poult enteritis and mortality syndrome in turkeys in great britain enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the united states between specific real-time reverse transcription polymerase chain reaction for detection and quantification of turkey coronavirus rna in tissues and feces from turkeys infected with turkey coronavirus coronaviruses in poultry and other birds the molecular biology of coronavirus isolation and propagation of coronaviruses in embryonated eggs validation of an immunohistochemistry assay to detect turkey coronavirus: a rapid and simple screening tool for limited resource settings pathology and virus tissue distribution of turkey coronavirus (tcov) in experimentally infected chicks and turkey poults emergence of a group coronavirus through recombination loop-mediated isothermal amplification of dna reverse transcription loop-mediated isothermal amplification for rapid detection of infectious bronchitis virus in infected chicken tissues rapid detection of infectious laryngotracheitis virus isolates by loop-mediated isothermal amplification loop-mediated isothermal amplification for rapid detection of newcastle disease virus this work was supported by fapesp (fundação amparo à pesquisa do estado de são paulo) and cnpq. we are indebted to the technical team from sadia-unidade uberlândia, mg brazil. tereza cristina cardoso is recipient of cnpq council grants. key: cord- -vi rth o authors: zhang, chao; yao, yao; zhu, juan-li; zhang, si-nong; zhang, shan-shan; wei, hua; hui, wen-li; cui, ya-li title: establishment and application of a real-time loop-mediated isothermal amplification system for the detection of cyp c polymorphisms date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: vi rth o single-nucleotide polymorphisms (snps) represent the most widespread type of genetic variation (approximately %) in the human genome, and the demand to overcome such variation has received more attention now than ever before. the capacity to rapidly assess snps that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. in this work, a rapid one-step snp detection method, real-time loop-mediated isothermal amplification (rt-lamp), was first applied for cyp c polymorphisms testing. the optimized method was established with specifically designed primers for target amplification by real-time detection in approximately min under isothermal conditions. rt-lamp amplified few copies of template to produce significant amounts of product and quantitatively detected human dna with compatible specificity and sensitivity. the success in the establishment of this rt-lamp protocol for cyp c polymorphism testing is significant for the extension of this technique for the detection of other snps, which will further facilitate the development of personalized medicine. clinical observations beginning in the s have suggested that individuals exhibit differences in their responses to drugs and that these variations could be inherited . the detection of dna sequence variations provides valuable insight into the diagnosis of genetic-related diseases and conditions, especially for early-stage treatment and response monitoring . thus, it is critically important to select a method with high sensitivity and specificity to detect single or small numbers of nucleotide polymorphisms , . current detection methods rely on sample amplification combined with meticulous control, including polymerase chain reaction (pcr), nucleic acid sequence-based amplification (nasba) , self-sustained sequence replication ( sr) , strand displacement amplification (sda) and direct sequencing. although these technologies are currently considered the gold standard for laboratory-based dna detection and diagnostics, these methods cannot meet the requirements of point-of-care testing (poct) strategies due to the high set up and operating expenses and the requirements for high-precision equipment. loop-mediated isothermal amplification (lamp) is an outstanding gene amplification procedure with high specificity, sensitivity and rapidity that was established by notomi et al. . the process amplifies nucleic acids under isothermal conditions and employs self-recurring strand-displacement synthesis primed by a specially designed set of target-specific primers , thus clearly distinguishing this technique from existing genetic tests . lamp is characterized by the use of six specifically designed primer regions to recognize eight regions on the target dna; thus, the specificity is extremely high . amplification and detection of a gene can be completed in a one step by incubating the mixture of sample, primers, dna polymerase with strand displacement activity and substrates under isothermal conditions between and °c. lamp has been used for the diagnosis of pathogens via the detection of gene segments, e.g., the diagnosis of infectious diseases, such as japanese encephalitis virus infection , rapid genotyping of carcinogenic human papillomavirus and herpesvirus , and the detection of middle east respiratory syndrome coronavirus [ ] [ ] [ ] . numerous investigations have demonstrated that this special identification system is more accurate than pcr-based methods, which use only two primers to recognize two regions. although detection of human dna polymorphisms using lamp is challenging, especially for single-nucleotide polymorphisms (snps) due to the complex nature of dna compared with microbes and viruses , snps represent the most widespread type of genetic variation (approximately %) in the human genome , and the capacity to rapidly test patients for snps that are correlated with disease predisposition, drug metabolism and disease development is a key step for the development of personalized medicine . thus, the wide application of this simple, rapid and low-cost genotyping lamp method in snp detection is imperative. numerous lines of evidence have strongly suggested that genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors and other drug targets are associated with inter-individual differences in drug treatment response . sequence variations in drug target proteins, drug-metabolizing enzymes, and drug transporters can alter drug efficacy, drug side effects, or both to cause variable drug responses in individual patients . for example, on march , , the us food and drug administration approved a black box warning regarding the diminished effectiveness of clopidogrel in patients who carry two loss-of-function alleles (poor metabolizers) , i.e., cyp c * (g a) and cyp c * (g a) alleles, which account for % and % of the nonfunctional alleles in whites and asians, respectively , . the warning addressed the need for polymorphism genotyping to identify altered clopidogrel metabolism in patients . we developed a rapid, one-step snp detection method (rt-lamp) that enables the detection of the cyp c allele in approximately min under isothermal conditions. the optimized rt-lamp technique is more suitable for point-of-care testing and will further facilitate on-site screening. the successful establishment of an inexpensive, rapid and real-time lamp protocol for cyp c * and cyp c * detection is significant for the extension of this technique for genotyping other snps. our results suggest applications for this rt-lamp assay system for both basic research and clinical diagnosis in pharmacogenomics. plasmid construction and identification. in this study, four plasmids were constructed by recombining the specific sequences of cyp c * g g, cyp c * a a, cyp c * g g and cyp c * a a. using the primer pairs * -seq-f/* -seq-r and * -seq-f/* -seq-r, listed in table , -bp cyp c * and bp cyp c * fragments were amplified and sequenced by beijing genomics institute (bgi; beijing, china), indicating the successful incorporation of the four plasmids (data not shown). based on the point mutations of the cyp c * (g a) and cyp c * (g a) genes, two sets of rt-lamp primers were designed to discriminate the snps. as shown in fig. , the basic principle of rt-lamp involves the use of specific primers, with a forward inner primer (fip) and backward inner primer (bip) that are designed to contain a snp nucleotide at the ′ terminus, each reaction including two common primers (f and b ) and two specific primers (fip-g and bip-g for g allele and fip-a and bip-a for a allele). the structures of the lamp primers and products based on this study are presented in fig. , and the information regarding the primer names and sequences are provided in table . the target snp was characterized using six different regions (f /f c-f /f c and b /b c-b /b c) specifically designed to recognize distinct regions on the target gene, which were designed to ensure that the primers would specifically amplify the g a and g a substitutions. the results depicted in fig. illustrate that the rt-lamp method could accurately detect and discriminate all possible homozygotes and heterozygotes of cyp c * (g a) and cyp c * (g a) snps. sensitivity of the rt-lamp assay. the sensitivities of the rt-lamp assay were tested using -fold serial dilutions of the four constructed plasmids. the detection limit of the rt-lamp assay was × copies of plasmid (the result for the cyp c g g plasmid is presented in fig. a ; the results for the other three plasmids are presented in supplementary fig. s ), indicating that the rt-lamp method was efficient and specific in snp detection under a constant temperature with greater than -fold increased sensitivity compared with conventional pcr , . moreover, as shown in fig. , a standard curve was generated using -fold dilutions of plasmids and calculated by regression analysis comparing the t peek with the copy number. the high correlation coefficient (r = . ) indicated that the rt-lamp assay could be applied in dna quantification. to test the reliability of the rt-lamp system optimized in this study, the accuracy of the rt-lamp assay was further verified using clinical samples. in addition, all of these samples were also assessed via conventional pcr (as-pcr) and sequenced by bgi. the detected genotypes together with their frequencies are presented in table . the observed allele frequencies were . %, . %, % and %, for * g, * a, * g and * a, respectively (calculated from: * g: f + f + f / + f + f / ; * a: f + f / + f / ; * g: f + f + f + f / + f / ; * a: f + f / + f / ). the frequencies of the cyp c * and cyp c * alleles were similar to those reported by chen et al. in a chinese population. the comparison of rt-lamp with as-pcr and direct sequencing revealed no discrepancies. in recent years, dna testing technology has been extensively used in the areas of diagnosis and disease detection. the lamp technique is a unique assay with high efficiency and high accuracy that was developed rapidly. as described in some reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the lamp assay has been widely used in the detection of pathogenic microorganisms. however, to the best of our knowledge, reports regarding the detection of mutations in human genomic dna, especially snps, using the lamp method are lacking. in this study, the successful establishment of an inexpensive, rapid and real-time lamp protocol for cyp c snp genotyping expanded the scope of application of this technique to human gene mutation detection. rt-lamp is a one-step method wherein the amplification itself is the signal for snp detection, whereas the difficulty in developing this technology involves the suppression of non-specific amplification . to help overcome this difficulty, the target snp is characterized using six different primer regions specifically designed to recognize eight distinct regions on the target gene , . in this work, by adding or subtracting a few nucleotides in the primer regions, the t m value and gc content were calculated until the six different primer regions were suitable for the lamp reaction. as a result, the primer regions were selected as noted in fig. (f -f and b -b ) . furthermore, as the arrangement and composition of human genomic dna is very complex, sequence alignment was necessary to avoid false recognition of the specific site. thus, primer-blast software (http://www.ncbi.nlm. nih.gov/tools/primer-blast) was used to ensure that the chosen primer regions were specific to the target snp, which helped avoid mismatches and locate the target snp as accurately as possible. the secondary structures of these primers may cause non-specific results in the lamp reaction given that the selectable sequence area for primer design is limited to less than four hundred nucleotides surrounding the target snp site . hence, the inner primers were designed (fig. ) to minimize the impact of the secondary structure given that the inner primers are the main component for dna strand extension. moreover, according to tomita et al., the formation of a starting structure is the key initiating step of lamp . specific nucleotides were added to the ′ termini ( ′ -term) of the fip and bip primers to establish a complete and effective starting structure, as shown in fig. . consequently the starting structure would be successfully established only when the ′ -term nucleotide was exactly matched with the target snp (fig. a) . otherwise, the starting structure was blocked, as shown in fig. c , and the amplification could not proceed. in conclusion, non-specific amplification was effectively suppressed in this work through special primer design and accurate target location. in addition, using real-time fluorescence detection equipment, amplification and detection can be performed in a closed tube, which could reduce the risk of contamination. thus, the rt-lamp method that was developed has an excellent sensitivity for detection of cyp c polymorphisms (as shown in fig. a ). similar results were observed by singh et al. and lee et al. with the same sensitivity of × copies. moreover, a standard curve with a high correlation coefficient was obtained in this study, as shown in fig. b , indicating that except for microorganism quantification [ ] [ ] [ ] , the rt-lamp assay can also be applied in human dna quantification. therefore, the rt-lamp method can be used for the determination of trace amounts of dna of interest among copious background dna, such as specific mutation detection in circulating tumour dna , , and can be applied for complex gene quantification, which is clinically meaningful , . in summary, as a rapid, feasible and cost-efficient point-of-care (poc) snp detection method, we demonstrated that rt-lamp could quantitatively detect human genomic dna with high specificity and sensitivity in a single step. moreover, the lamp method can amplify few copies of template to significant levels in min and can be used for both dna and rna targets . thus, this poc detection method should be helpful in basic research in a variety of fields, including medicine, pharmaceuticals, environmental hygiene, food security, and pharmacogenomics testing. peripheral blood and genomic dna extraction. peripheral blood samples were collected from unrelated chinese volunteers using edta-coated tubes at the shaanxi provincial people's hospital (xi'an, china) with informed consent. the study was approved by the ethics committee of the national engineering research center for miniaturized detection systems, xi'an, china. all methods were performed in accordance with these approved guidelines. the genomic dna from the volunteer was isolated from μ l of blood using a whole blood genomic dna isolation kit (xi'an goldmag nanobiotech co., ltd., xi'an, shaanxi, china), according to the manufacturer's instructions. the final dna quality and concentrations were measured using a nanodrop c/ uv-vis spectrophotometer (thermo fisher scientific, wilmington, de, usa), according to the manufacturer's instructions. primer design and synthesis. for each snp, six primers for rt-lamp were designed, including two outer primers (common primers, f and b ) and four inner primers (specific primers, fip-g, fip-a, bip-g and bip-a) that recognize distinct regions of the cyp c * and cyp c * alleles (rs and rs ). conventional pcr primers were designed based on the principle of as-pcr using the primer . software program (primer-e ltd., plymouth, uk). all oligonucleotide primers were synthesized by invitrogen biotechnology ltd. (shanghai, china). plasmids for cyp c * and cyp c * . plasmids - gg, - aa, - gg and - aa, which contain the cyp c * g g, cyp c * a a, cyp c * g g, and cyp c * a a genes, respectively, were constructed using the pmd tm optimization of rt-lamp reaction. the initial condition of the rt-lamp reaction was adopted from zhang et al. . the lamp reaction mixtures were incubated for min at , , , , , or °c to determine rt-lamp conventional pcr (n = ) total sequencing (n = ) total agreement (%) frequency (%) * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* * /* table . gene test results and frequency of clinical samples of cyp c alleles (type-specific concordance among rt-lamp, conventional pcr and direct sequencing). * /* : cyp c * g g type, cyp c * g g type; * /* : cyp c * a a type, cyp c * g g type; * /* : cyp c * g g type, cyp c * a a type, * /* : cyp c * g a type, cyp c * g g type; * /* : cyp c * g g type, cyp c * g a type; * /* : cyp c * g a type, cyp c * g a type. the optimal reaction temperature. then, the lamp reaction was performed at the optimal reaction temperature for , , , , and min to determine the optimal reaction time ) with the following parameters: one step of min at °c; cycles of s at °c, s at °c, s at °c; and one step of min at °c. all pcr products were detected by electrophoresis on a . % (w/v) agarose gel containing goldview nucleic acid stain (an alternative to ethidium bromide, xi'an heart biological technology co., ltd for the samples to be sequenced, a -bp fragment for cyp c * and a -bp fragment for cyp c * were amplified using sequencing primers (table ). the pcr products were sequenced by the beijing genomic institute sensitivity of rt-lamp assay. the sensitivities were assessed using the optimized rt-lamp assay with pharmacogenomics-drug disposition, drug targets, and side effects dna diagnostics: nanotechnology-enhanced electrochemical detection of nucleic acids array-based dna diagnostics: let the revolution begin a nanoliter fluidic 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dna reference material innate reverse transcriptase activity of dna polymerase for isothermal rna direct detection determination of abo blood group genotypes using the real-time loop-mediated isothermal amplification method this work was funded by the national science and technology major projects for "major new drugs innovation and development" of china (nos. zx - ) and northwest university graduate innovation and creativity funds (nos. yzz ). key: cord- -t y w ef authors: luo, zichao; ang, melgious jin yan; chan, siew yin; yi, zhigao; goh, yi yiing; yan, shuangqian; tao, jun; liu, kai; li, xiaosong; zhang, hongjie; huang, wei; liu, xiaogang title: combating the coronavirus pandemic: early detection, medical treatment, and a concerted effort by the global community date: - - journal: research (wash d c) doi: . / / sha: doc_id: cord_uid: t y w ef the world health organization (who) has declared the outbreak of novel coronavirus, known as -ncov, a pandemic, as the coronavirus has now infected over . million people globally and caused more than , fatalities as of april , . coronavirus disease (covid- ) causes a respiratory illness with symptoms such as dry cough, fever, sudden loss of smell, and, in more severe cases, difficulty breathing. to date, there is no specific vaccine or treatment proven effective against this viral disease. early and accurate diagnosis of covid- is thus critical to curbing its spread and improving health outcomes. reverse transcription-polymerase chain reaction (rt-pcr) is commonly used to detect the presence of covid- . other techniques, such as recombinase polymerase amplification (rpa), loop-mediated isothermal amplification (lamp), clustered regularly interspaced short palindromic repeats (crispr), and microfluidics, have allowed better disease diagnosis. here, as part of the effort to expand screening capacity, we review advances and challenges in the rapid detection of covid- by targeting nucleic acids, antigens, or antibodies. we also summarize potential treatments and vaccines against covid- and discuss ongoing clinical trials of interventions to reduce viral progression. the recent global outbreak of covid- has led to a public health emergency. as of april , , over . million confirmed cases were reported to who from countries and territories [ ] . on january , , who declared the covid- outbreak as the sixth public health emergency of international concern, following h n ( ), polio ( ), ebola in west africa ( ), zika ( ), and ebola ( ) [ ] . the rapid global expansion and rising fatalities have raised grave concerns on the viral spread across the globe. with the rapid increase in the number of confirmed cases, who classified the global covid- outbreak as a pandemic on march , [ ] . covid- can spread from person-to-person and animal, and transmission of infection may occur with exposure to symptomatic patients or asymptomatic individuals. coronaviruses (covs) (corona: crown-like shape) are enveloped, single-stranded rna viruses that belong to the order nidovirales in the subfamily coronaviridae. covs are divided into four genera: alpha (α), beta (β), gamma (γ), and delta (δ) (figure (a) ) [ ] . alpha-and beta-covs infect mammals, while gamma-and delta-covs primarily infect birds [ ] . before december , six types of covs had infected humans, including two α-covs (hcov- e and hcov-nl ) and four β-covs (hcov-oc , hcov-hku , sars-cov, and mers-cov). the first two β-covs (hcov-oc and hcov-hku ) mainly cause self-limiting upper respiratory infections, while the other two β-covs (sars-cov and mers-cov) are mostly associated with severe respiratory illness [ , ] . full-genome sequence analysis of -ncov confirms that it is a β-cov, distinct from sars-cov and mers-cov [ ] . investigations reveal that -ncov shares~ % sequence identity with sars-cov while maintaining~ % nucleotide identity to the sars-like covs (zc and zxc ) from bats [ ] . a recent report suggests that a bat cov (ratg ) is % identical to -ncov [ ] . a typical cov genome is a single-stranded, positivesense rna (+ssrna) (~ kb) enclosed by a ′-cap and ′ -poly-a tail [ ] . the genome size of -ncov is , nucleotides, encoding amino acids, with a g+c content of % [ ] . the -ncov genome contains two flanking untranslated regions (utrs) on ′ -and ′ -terminals, one single long open reading frame ab (orf ab) encoding a polyprotein and at least five other orfs encoding structural proteins, and eight accessory proteins (figure (c) ). the first orf (orf a/b) is about two-thirds of the whole-genome length and encodes the nonstructural proteins (nsp - ) . the other one-third of the genome contains four orfs encoding the spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, whereas other orfs encode accessory proteins (figure (b) and (c)). most of the nonstructural proteins are essential for -ncov replication, while structural proteins are responsible for virion assembly and viral infection [ , ] . the m and e proteins are required in viral assembly, while the n protein involves rna genome assembly. the s protein, a surface-located trimeric glycoprotein of covs, is the primary determinant of cov tropism, as it binds to the membrane receptor on host cells, mediating viral and cellular membrane fusion [ ] . the s protein of -ncov reportedly binds to angiotensin-converting enzyme (ace ), a homolog of ace on host cell membranes, contributing to -ncov cell invasion [ ] . moreover, this particular s protein shows a higher binding affinity to ace than the s protein of sars-cov, enabling -ncov to invade host cells more effectively [ , ] . recently, a transmembrane glycoprotein, cd , also known as basigin or emmprin, has been confirmed as another receptor for binding of the -ncov s protein, thereby mediating viral invasion [ ] . the e protein is an integral membrane protein that regulates viral life cycles, including pathogenesis, envelope formation, assembly, and budding [ ] [ ] [ ] . among the four structural proteins, protein e appears to have the highest antigenicity and the most significant potential as an immunogenic target, highlighting the possibility of developing protein e-derived peptides as a -ncov vaccine [ ] . systemic studies of proteins s and e have inspired scientists to take creative approaches to design anti-covid- drugs. although some covid- patients show no symptoms, most patients have some common symptoms such as fever, cough, fatigue, sputum production, shortness of breath, sore throat, and headache. in some severe cases, infections can cause pneumonia, severe acute respiratory syndrome, kidney failure, and death. according to the who-china joint report [ ] , on average, people infected with -ncov develop mild respiratory symptoms and fever, - days after infection (mean incubation period, - days; range, - days). people over years of age and those with hypertension, diabetes, or cardiovascular diseases are at high risk for severe illness and death. in comparison, children under years appear to be infected minimally by -ncov (around . % of all reported cases). based on the chinese center for disease control and prevention (china cdc) report (from , patient records, dated february ), among the confirmed cases, . % of patients are - years of age, . % of patients have mild-to-moderate disease, . % have a severe illness, and . % are critically ill [ ] . notably, the mortality rate of children under years is . %, while people aged over years have the highest mortality rate of . %. currently, there are no effective antiviral drugs or specific vaccines against covid- . thus, there is an urgent need for rapid detection to prevent further spread, to reduce the intensity of the pandemic, and to slow the increase in cases. recently, several new technologies, including lamp-lfa, rpa-lfa, rpa-crispr, and other nanomaterial-based igg/igm kits, have been adopted for -ncov detection. a significant number of drug candidates, including chemical drugs, biological drugs, nutritional interventions, and traditional chinese medicine (tcm), have been proposed for clinical trials after the -ncov outbreak. in this review, we concentrate on the most significant developments in -ncov detection and provide an overview of medical treatments and vaccines currently in development to combat and contain the disease. properties of the virus and biomarkers that hosts exhibit after infection. these biomarkers include viral proteins and nucleic acids, as well as antibodies induced in response to viral infection. the most common -ncov detection methods include viral nucleic acid detection and serum antibody (igg or igm) detection. a confirmed case should have at least one of the following criteria: (i) a positive result for -ncov nucleic acid, using real-time pcr tests from respiratory or blood samples; (ii) a high homogeneity between viral gene sequencing from respiratory or blood samples and known -ncov; and (iii) serum samples positive for igm or igg to -ncov, or seroconversion in igg, or a fourfold or more significant increase in igg antibody titer to -ncov in the recovery phase than in the acute phase [ ] . . . . high-throughput sequencing ( nd -generation sequencing). high-throughput sequencing (hts) technology contains various strategies that depend on a combination of library preparation, sequencing and mapping, genome alignment, and data analysis [ ] (figure (a) ). unlike the sanger sequencing method ( st -generation sequencing) [ ] , nd -generation sequencing has been widely applied in genome sequencing, transcriptional profiling (rna-seq) disease mapping, and population genetic studies. the wholegenome nucleotide sequence of -ncov was identified and compared with the full-length genome sequence of coronavirus from bats [ ] through hts. hts-based technology is also applied to detect -ncov. for example, wang et al. developed a hts method based on nanopore target sequencing (nts) by harnessing the benefits of target amplification and long-reads for real-time nanopore sequencing [ ] . this nts strategy detects -ncov with higher sensitivity ( -fold) than standard qpcr, simultaneously with other respiratory viruses within - h. moreover, all targeted regions can be identified by nts in higher copies of samples ( - copies/ml) within min, indicating the potential for rapid detection of an outbreak in the clinic. for h sequencing data, reads mapped to -ncov differed remarkably from those of negative controls in all targeted regions at concentrations ranging from to copies/ml. importantly, nts can identify mutated nucleic acids. however, the nts platform cannot readily detect highly degraded nucleic acid fragments that are less than base pairs in length [ ] . moreover, the strategy requires tedious sample preparation and lengthy turnaround time. although hts technology provides fast, low-cost dna sequencing, it is not suitable for detection in clinics. on the other hand, the hts strategy may be suitable for amplicon sequencing or de novo sequencing of a whole genome [ ] . reaction (rt-pcr). rt-pcr is considered the gold standard to detect nucleic acids extracted from -ncov specimens qualitatively. positive results indicate infection with -ncov. rt-pcr is an advanced technique for coronavirus detection because of its optimized sensitivity, specificity, and simplicity for quantitative assay [ , ] . it provides accurate and reliable identification for confirmed and suspected cases. there are many commercial -ncov detecting kits with oligonucleotide primers and probes (sybr green or taqman chemistries) for detecting double genes of -ncov (nucleocapsid n gene and orf ab/e/orf b/s gene). this strategy usually requires four steps: sample collection (respiratory swabs), sample preparation (rna isolation), one-step qrt-pcr, and data analysis ( figure (b)) [ ] . real-time rt-pcr has been adopted as the gold standard diagnostic approach for -ncov worldwide. however, rt-pcr is time-consuming ( - h) and requires wellequipped laboratories and skilled technicians, thereby limiting full deployment in developing countries. . . . reverse transcription-isothermal amplification (rt-iamp)-based detection. isothermal amplification technology has been developed to eliminate the need for a high-precision instrument in rt-pcr assays. this approach can amplify dna at isothermal conditions without a thermocycler [ ] . there are mainly four isothermal amplification technologies for nucleic acid detection: lamp, rpa, nucleic acid sequence-based amplification (nasba), and transcriptionmediated amplification (tma) [ ] . in nasba and tma assays, input rna is converted to a double-stranded dna intermediate with a promoter region. detection of rna using dna polymerase-based amplification requires a reverse transcriptase step. lamp and rpa do not require thermal or chemical melting with the aid of enzymes. combined with a visual detection platform, such as a lateral flow assay (lfa) or organic dyes, lamp and rpa have been widely employed in viral detection kits. lamp is a rapid, one-step amplification technique that amplifies nucleic acids with high sensitivity and specificity at an optimal temperature of °c [ ] . lamp processing comprises three steps: an initial step, a cycling amplification step, and an elongation step (figure (a) ). lamp employs six primers to amplify targeted genes by creating stem-loop structures that promote new dna synthesis using a dna polymerase with strand displacement activity. the two inner primers (fip, bip) and two outer primers (f , b ), along with loop structures (lf, lb), create multiple initiating sites in the growing dna products, enabling rapid amplification. lamp is also highly specific since the amplification reaction occurs only when the primers correctly recognize all six regions. a reverse transcriptase step is included in the lamp reaction to allow rna targets to be detected [ ] . rt-lamp offers improved sensitivity and specificity in screening sars-cov, hcov-nl , and mers-cov compared to conventional real-time rt-pcr [ ] [ ] [ ] . recently, yu et al. used a commercial lamp kit to amplify fragmented orf ab genes of -ncov (figure (b) and (c)) [ ] . they optimized the lamp system through incubation at °c for different periods using a -ncov-positive rna sample as the template. results require a min reaction time at °c, and detection sensitivity is comparable to that of the taqman-based qpcr approach ( copies). rt-lamp employs two additional protocols for -ncov rna detection. park et al. performed rt-lamp at °c for min to identify the nsp , s, and n genes of -ncov using colorimetric detection [ ] . the sensitivity of this rt-lamp assay was copies of -ncov rna. the other rt-lamp protocol was conducted at °c for min to detect the orf ab, e, and n genes simultaneously [ ] . the results confirmed the specific nature of orf ab and the high sensitivity of the n gene. based on an analysis of clinical specimens, the sensitivity of this rt-lamp was similar to conventional rt-pcr, and the specificity was %. interestingly, ei-tholoth et al. designed a two-stage isothermal amplification procedure by combining rpa ( °c) with lamp ( °c) to detect synthesized dna fragments of -ncov [ ] . the test was performed in closed tubes within h using either fluorescence or colorimetric detection. this method has a sensitivity of times better than conventional lamp and rt-pcr, suggesting a rapid, sensitive, point-of-care test for use at home. rpa is an isothermal dna amplification method that utilizes a specific combination of enzymes and proteins (recombinase, single-strand binding (ssb) protein, and strand-displacing dna polymerase) to amplify target genes step : cycling amplification step : elongation step : initiation step : ssb binds to displaced strand step : bsu initiates polymerization step : parental strand is displaced and elongation continues step : recombinase binds primers step : ssb ds to displaced strand step : bsu tiates polymerization step : tal strand is displaced elongation continues step : recombinase binds primers rapidly at a constant low temperature between and °c in as little as min [ ] . rpa usually requires four steps to achieve dna amplification: formation of a recombinaseprimer complex, strand invasion, d-loop formation (stabilized by ssb, dna polymerization through the use of strand-displacing dna polymerase), and dna amplification ( figure (d)) [ ] . results of rpa can be detected by agarose gel electrophoresis, quantitatively measured using twis-tamp ™ probes, or simply applied in lateral flow assays. apart from dna target amplification, rpa formats have been developed for the detection of rna targets (rt-rpa) by adding a reverse transcriptase enzyme to reaction mixtures [ ] . because rpa-(rt-rpa-) based detection achieves more rapid and sensitive results and operates efficiently, it has been widely adopted to detect animal and human pathogens, such as hand, foot, and mouth disease (hfmd) virus, human immunodeficiency virus (hiv), bovine coronavirus, or mers-cov [ ] [ ] [ ] [ ] . currently, rpa has been applied to detect -ncov, in combination with other technologies, such as crispr or microfluidic technology. repeat-(crispr-) based detection. the crispr-associated protein (cas ) system (crispr/cas ) is a revolutionary gene-editing toolbox that can modify target genes with high precision and that can control various types of genetic diseases in preclinical studies [ ] [ ] [ ] [ ] . due to the collateral nucleic acid cleavage activity of cas effectors, crispr/cas systems have also been widely used in nucleic acid detection with fluorescent and colorimetric signals [ ] . there are mainly two kinds of crispr/cas systems for diagnostics, based on the cutting activity of cas protein on nucleic acids outside of the grna target site: the crispr/cas a and crispr/cas a systems. the crispr/cas a system (specific high-sensitivity enzymatic reporter unlocking (sherlock)) was developed by zhang's group, based on the collateral effect of an rnaguided and rna-targeting crispr effector, cas a (figure (a)) [ , ] . the detection system is highly sensitive and specific because it is capable of single-molecule nucleic acid detection. subsequently, they developed an enhanced sherlock version (sherlockv ) detection system with a . -fold improvement in detection sensitivity and lateral flow readout. sherlockv has been used to detect dengue and zika virus single-stranded rna or mutations in clinical samples, showing great potential for multiplexable, portable, rapid detection of nucleic acids [ ] . recently, they combined rt-rpa technology with the sherlock system (namely crispr diagnostics) to detect the s and orf ab genes of -ncov (figure (b) and (c)) [ diagnostics-based test can be conducted in hour and can be read using a dipstick. the analysis is performed at °c and °c, and its detection sensitivity is ten copies per microliter of input, exhibiting unique advantages, such as high sensitivity, specificity, speed, and suitability for point-of-care testing. however, this approach needs to be validated using real patient samples. unlike the crispr/cas a system, the crispr/cas a system is based on the collateral effect of cas a on singlestranded dna (ssdna). chen and colleagues combined cas a ssdnase activation with rpa technology to create a new approach, named dna endonuclease-targeted crispr trans reporter (detectr), with attomolar sensitivity for dna detection (figure (a)) [ ] . detectr was also validated with clinical samples, showing the capacity for rapid, specific detection of human papillomavirus (hpv) [ ] . recently, detectr was investigated to identify the nucleic acid of -ncov. lucia et al. applied the detectr (crispr-cas a and rt-rpa) to detect the rnadependent rna polymerase (rdrp), orf b, and orf ab genes of -ncov using synthetic rna fragments as samples [ ] . remarkably, all steps of the test were completed in h, and results were visible to the unaided eye. the limit of detection for orf ab was ten copies/μl. the advantages of this method are its portability and low cost (~us$ per reaction). but this proposed approach also needs to be validated with clinical samples before commercialization. another detectr-based -ncov detection strategy was developed by chiu's lab [ ] . they employed lamp, crispr/-cas , and lateral flow assay to detect the e and n genes of -ncov in clinical samples. this protocol supplied rapid (~ min), low-cost, and accurate ( % specific vs. % specific for qrt-pcr) detection of -ncov in respiratory swab samples. realistically, crispr/cas-based -ncov detection technology is highly specific, rapid, and low cost, but the detection strategy also needs to be validated using clinical samples. microfluidic-based detection. the abovementioned methods are based on relative quantification, because they require external calibration with genetic standards or inner reference dna templates, resulting in unavoidable errors and other uncertainties. on the contrary, methods that do not need standard curves can provide a quantitative analysis of nucleic acids using absolute quantification of genetic copies. recently, digital pcr and digital lamp have been achieved with microelectromechanical and microfluidic technologies [ , ] . microfluidic or lab-on-a-chip techniques use microsized channels to process or manipulate fluids. microfluidics has been widely utilized in various fields, including drug screening, tissue engineering, disease diagnostics, and nucleic acid detection [ , ] . based on its portability and ultralow sample consumption, microfluidics shows significant promises in clinical applications [ ] . regarding nucleic acid analyses, microfluidic devices aliquot diluted nucleic acid samples into hundreds to millions of discrete nanoliter chambers. the isolated chambers contain only one or zero target molecule according to a poisson distribution. consequently, the abso-lute copy number of target nucleic acid can be calculated from the number of positive and negative reactions, based on the poisson distribution formulas [ , ] . both digital pcr and digital lamp have employed microfluidics for pathogen analysis, which is suitable for covid- detection. for instance, ottesen and colleagues used digital pcr to amplify and analyze multiple genes on a microfluidic chip [ ] . this chip consisted of parallel chambers and micromechanical valves. the micromechanical valves segmented chambers into independent pcr reactors after the sample flowed into chambers through connection channels. the chip was able to detect several kinds of genes with parallel sample panels. digital pcr can also be conducted with droplets generated by the microfluidic chip. however, the detection of fluorescent signals in droplets requires special instruments, such as flow cytometers, which may limit its application in point-of-care testing. additionally, the high temperature in pcr amplification tends to evaporate the reaction liquid (nanoliter or even femtoliter), leading to unacceptable errors. using airtight devices or high pressure delays liquid evaporation but complicates the devices and increases testing costs. digital lamp is more compatible with microfluidics than digital pcr because it is executed at a moderate temperature. this simplifies microfluidic devices and reduces testing costs. many microfluidic devices have been reported for nucleic acid detection using digital lamp, such as self-digitization chips, self-priming compartmentalization chips, and dropletgeneration chips [ ] [ ] [ ] . as an example, xia et al. designed a mathematical model using the monte carlo method according to the theories of poisson statistics and chemometrics [ ] . the mathematical model illustrated influential factors of the digital lamp assay, guiding the design and analysis of digital lamp devices. based on the established mathematical model, they fabricated a spiral chip with chambers ( . nl) for pathogen detection ( figure (a)-(c)). this spiral chip operated at °c without visible liquid evaporation and achieved a quantitative analysis of nucleic acids over four orders of magnitude in concentration with a detection limit of copies per ml. this portable gadget shows significant promise in future point-of-care testing. microfluidics, combined with enzyme-dna nanostructures, is also applied to detecting -ncov. ho et al. developed a modular detection platform (termed envision) consisting of an integrated circuit of enzyme-dna nanostructures for direct and versatile detection of pathogen nucleic acids from infected cells [ ] . built-in enzymatic cascades in the envision microfluidic system supply a rapid color readout for detecting hpv. the assay is fast (< h), sensitive (limit of detection < attmol), and readily quantified with smartphones. recently, they adopted the envision microfluidic system to detect -ncov [ ] . preliminary results showed that the envision platform is sensitive, accurate, fast (within . - h), and inexpensive (less than $ per test kit). this novel platform works at room temperature and does not require a heater or special pumps, and it uses a minimal amount of samples, making it highly portable. however, this platform needs to be further validated with real clinical samples. antigen and antibody. as mentioned above, the primary diagnostic methods are virological detection involving viral nucleic acids. another approach to detection is with serological assays that measure antigens or antibodies present in the host. such testing provides vital information about host exposure to -ncov and is useful for detection and surveillance purposes. for instance, this method greatly helps medical professionals to determine whether some recovered patients have a higher risk of reinfection. however, the disadvantage is that one should be cautioned that in the early stages of covid- infection, the host's antibodies are often not within the detectable range of serological test kits. besides, there was no proven evidence on the duration of igm or igg antibodies circulating in the host after recovery. it could be merely a short time frame for detection. as such, serological tests should not be solely used for covid- diagnosis. early diagnosis of -ncov infection is of utmost importance both for medical teams to manage patients effectively and for policymakers to curb the viral spread. presently, elisa in cell culture extracts has proven to be the working "gold standard" for laboratory diagnosis of -ncov [ ] . elisa is a plate-based assessment method for detecting and quantifying biomolecules, including peptides, proteins, antibodies, and hormones. elisa techniques depend on specific antibodies to bind target antigens and a detection system to indicate the presence of antigen binding. in an elisa, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. detection is accomplished by assessing the conjugated enzyme activity after incubation with a substrate to produce a measurable product [ ] . recently, coronavirus proteins have been widely used in elisa to diagnose sars-cov or other viruses within the coronavirus family [ ] . in a bold, novel approach, a team of infectious disease experts in singapore utilized an elisa against -ncov to ascertain that suspected subjects were infected with covid- and discovered the connection between two covid- clusters in the local community [ ] . using blood samples taken from alleged covid- patients, the researchers detected antibodies targeting the spike protein that prevented the virus from killing cells in laboratory tests. they verified that a couple allegedly infected with covid- had the disease because they had exceedingly elevated levels of virus-specific antibodies in their blood. interestingly, pcr tests on the couple yielded negative results. because the couple had recovered from the -ncov infection, they had no viral genetic materials in their bodies, but the antibodies persisted. there were also other reports of using elisa to diagnose -ncov infection [ , ] . each study confirmed the high reproducibility and specificity of elisa in diagnosing covid- patients accurately in clinics. research has established that the presence of immunoglobulin m (igm) indicates a primary defense against viral infections. this igm defense occurs before the production of high-affinity and adaptive immunoglobulin g (igg) that is critical for prolonged immunity and immunological memory [ ] . from a previous study on sars infections, both igm and igg antibodies could be detected in the patient blood after - days and beyond days, respectively [ ] . given that -ncov belongs to the same family of coronaviruses including mers and sars, -ncov should also generate igm and igg antibodies in infected humans. therefore, the detection of igm and igg antibodies may provide epidemiologists with crucial information on viral infection of test subjects, allowing them to adjust policies to combat the pandemic more effectively. point-of-care lateral flow immunoassays are performed qualitatively to quickly determine the presence of -ncov by detecting anti- -ncov igm and anti- -ncov igg antibodies in human plasma, serum, or whole blood. a typical device is shown in figure (a). reddishpurple lines in the readout indicate the presence of -ncov igm and igg antibodies in the sample. lfa is based on the lateral chromatographic flow of reagents that bind and interact with the sample. as the sample flows through the test device, starting at the sample pad region, the anti- -ncov igm and igg antibodies, if present, bind tightly to -ncov antigen-labeled gold nanoparticles, located on the conjugated pad. when conjugated products in the sample continue to move up the strip, anti- -ncov igm antibodies and anti- -ncov igg antibodies bind to anti-human igm (m line) or anti-human igg (g line), respectively. no visible lines can be seen when the specimen does not contain anti- -ncov antibodies because no labeled complexes bind at the test zone. igg-labeled gold colorimetric nanoparticles serve as the control when they bind to anti-rabbit igg antibodies at the control line (c) (figure (b) ). lfa has proven useful in detecting -nco-vigm/igg antibodies in clinical studies, demonstrating . % test sensitivity and . % specificity in human blood, serum, and plasma samples. results from six patients are shown in figure (c). common -ncov detection methods are summarized in table . in clinics, medical imaging tools are indispensable and form an essential component of viral diagnosis, as well as for monitoring viral progression [ ] . they have also been used for follow-up in outpatient settings for coronavirus-related pulmonary disorders. just like both sars and mers, pulmonary complications in covid- patients have been observed. learning from the well-documented sars and mers studies, ct imaging results in the acute and chronic periods of covid- are invariant, but not always present [ ] [ ] [ ] [ ] . evidence is found in previous studies on sars and mers. the glass opacities observed are not always found in covid- patients. crucially, preliminary imaging discoveries indicate that covid- yields nonspecific results as well [ ] [ ] [ ] . radiologists are presently striving to any characteristics specific to covid- , although present medical information remains limited. given the precarious situation, there is a pressing need for alternative, complementary diagnostics. ct is one example. covid- patients often develop "ground glass" lung opacities [ ] . as such, a ct imaging scan can readily identify lung abnormalities in human subjects, thereby enabling early treatment against covid- . ct has demonstrated some common imaging characteristics in covid- patients. these features include bilateral, multifocal, ground glass opacities, with a peripheral distribution (figure (a)) [ ] . crucially, more than half of patients under study presented multilobar involvement and lesions more prominently in the lower lobes of their lungs. given its feasibility and ease of use, ct has become an essential tool for the -ncov infection diagnosis. from a radiological perspective, the advantages of using ct imaging could expedite the rate of diagnosis. it also supports the current shortage and heavily reliant on technical knowhow during rt-pcr testings. nonetheless, one limitation is that it should be cautiously utilized as a diagnostic approach because there are no proven, evidence-based clinical benefits of using ct. it could also cause false securities if results are negative. other limitations include requirements of relatively high-dose ct scans and long-term, continuous usage, which can altogether be logistically challenging and deplete additional medical resources. transmission electron microscopy (tem) has been used for many years and has had a profound impact on our understanding of illnesses, including viral infections. the thousandfold enhanced resolution provided by tem enables investigators to visualize viral morphology and to classify viruses into families [ ] . mechanistically, tem operates based on interactions between electrons emitted from a source and materials under examination. in the present context, it is usually -ncov in a cellular sample [ ] . the detector collects a multitude of signals from transmitted electrons, before processing them to reveal viral morphology and location within cells [ ] . typical specimen preparation for tem includes sample fixation, embedding, sectioning, staining, and loading onto the tem copper grids [ , , ] . -ncov sampling typically uses supernatants from patient airway epithelial cells. infected cells are fixed and dehydrated before embedding in resin. a negatively stained, film-coated grid for examination is similarly prepared for contrast enhancement. -ncov virus particles seen with tem are shown [ ] (figure (b) ). tem enables microbiologists to rapidly diagnose patients with a single examination of a single tissue sample. there are three general approaches to develop potential antiviral treatments of the human coronavirus. firstly, standard assays may be used to evaluate existing broadspectrum antiviral drugs. secondly, chemical libraries containing existing compounds or databases may be screened. thirdly, specific, new medications based on the genome and biophysical understanding of -ncov can be designed and optimized. therefore, this section will discuss some of the potential -ncov therapeutics obtained through these general approaches. besides chemical and biologic drugs commonly used in antiviral therapies, we further elaborate on how nanomaterials, nutritional interventions, traditional chinese medicine, and stem cell therapy can be potentially used for treatment or as an adjuvant to reduce the mortality and morbidity rate of -ncov patients. finally, to end this section, we highlight vaccines as a key therapeutic option to eradicate covid- through herd immunity without getting the disease. . . chemical drugs. there are currently no approved antiviral drugs to treat covid- , and patients must depend upon their immune systems to combat the infection. a full-fledged treatment plan has yet to emerge, and both academia and pharmaceutical companies are racing to develop new treatments and vaccines to address covid- . research into the cellular and molecular pathogenesis of -ncov has provided essential insights with the hope of developing viable therapies. while researchers are working on cures or preventive measures for covid- [ ] , a more robust, efficient, and economical way to tackle the disease is to repurpose existing drugs into a viable therapeutic strategy. drug repurposing, also termed drug repositioning, refers to the process of discovering new therapeutic applications for existing drugs. it offers various advantages over traditional de novo drug discovery, i.e., reduced cost and drug development time, established drug characteristics, and, most importantly, established safe dosages for human use [ ] . repurposed drugs often negate the need for phase clinical trials and can be used immediately [ , ] . at present, repurposed drugs are the only option available at treatment centers for covid- patients. as covid- is a viral infection, the most obvious choices for repurposed drugs come from known antiviral drugs [ ] . antiviral creation strategies focus on two approaches: targeting viruses or targeting host cell factors. in this section, we will review antiviral drugs prescribed or proposed against covid- based on the antiviral drug creation strategies. when an infection occurs, the virus gains entry into a host cell by attaching itself to the host cell surface (figure ) [ ] . this relies on numerous interactions between the virion surface and the specific proteins on the cell membrane. in general, these surface proteins have other functions but are serendipitously recognized by the virus as entry receptors [ ] . molecules that prevent such recognition, either by competitive binding or by downregulating the receptors, are known as viral entry inhibitors (figure (a) ). these inhibitors are valuable as therapeutics since blocking infection early in the life cycle reduces cellular and tissue damage associated with viral replication and production of viral progeny. as mentioned above, coronavirus particles comprise four structural proteins: the s, e, m, and n proteins [ ] [ ] [ ] [ ] . the s protein is the most crucial in viral attachment, fusion, and entry [ ] . it comprises two subunits. s facilitates attachment to the host cell receptor, while s mediates membrane fusion of the virion and the host cell. as mentioned above, viruses have specific attachment sites. sars-cov recognizes ace as its host receptor, while mers-cov recognizes dipeptidyl peptidase [ , ] . like sars-cov, -ncov also targets host ace [ ] [ ] [ ] . biophysical and structural analysis indicates that the -ncov s protein binds ace with higher affinity than the sars-cov s protein [ ] . therefore, it is vital to target ace for the development of viral entry inhibitors. to the best of our knowledge, not much is known about ace -specific inhibitors that are commercially available or under commercial development [ ] . however, ace stimulators have been used in the treatment of hypertension, cardiac diseases, and diabetes mellitus to regulate the reninangiotensin system [ , ] . there are also ace inhibitors known for treating the diseases mentioned, but these lack inhibitory activity toward ace due to their distinct substrate-binding pockets [ ] [ ] [ ] [ ] . in brief, there are concerns that both ace stimulators and ace inhibitors can increase the expression of ace , which in turn may increase susceptibility to viral host cell entry [ , ] . much work needs to be done on ace -targeting drugs, and controversial issues that lie beyond the treatment pathway need to be addressed soon. a small antiviral molecule, umifenovir, has entry inhibitory effects on the influenza virus. umifenovir targets hemagglutinin for its anti-influenza virus effect [ ] [ ] [ ] . hemagglutinin, a viral cell surface protein, facilitates infection by undergoing a conformational change when the virus binds to host cells [ ] . umifenovir interacts with hemagglutinin to stabilize it against low ph-induced conformational change via the formation of an extensive network of noncovalent interactions that prevent hemagglutininmediated membrane fusion [ , ] . it also interacts with phospholipids by altering membrane fluidity [ ] , which is vital for the fusion process. this is most likely due to umifenovir's molecular interactions (bearing both the h donor and acceptor groups) with the interfacial region of the lipid bilayer by competing for the hydrogen bonding of phospholipid c=o groups with water molecules [ ] . this renders lipid bilayers of host cells less prone to viral fusion [ ] . no studies have shown that umifenovir is effective in inhibiting sars-cov or -ncov. wang et al. reported that patients with mild/severe covid- recovered after prescription of combined lopinavir/ritonavir, arbidol (umifenovir), and shufeng jiedu capsule (a traditional chinese medicine) [ ] . on the other hand, dong et al. found in an in vitro study that arbidol may effectively inhibit -ncov infection at a concentration of - μm [ ] . chloroquine, also a small molecule, is a quinine analog used to prevent and treat malaria. similar to umifenovir, chloroquine exhibits its inhibitory effect on influenza by ph stabilization. chloroquine is a weak base and becomes protonated intracellularly in a manner described by the henderson-hasselbalch law [ ] . it can raise lysosomal ph to facilitate autophagy intracellularly [ ] [ ] [ ] . chloroquine also alters the signaling pathway of enzymes, causing enzyme glycosylation, ultimately inhibiting viral replication in host cells [ , ] . liu et al. claimed that chloroquine could inhibit sars-cov entry by changing glycosylation of the ace receptor and s protein [ ] . chloroquine's effective inhibition of sars-cov was demonstrated in vitro on primate cells and human rectal cells [ , ] . hydroxychloroquine is a derivative of chloroquine with an additional hydroxyl group. these two chloroquines share similar struc-tures and mechanisms. both have shown in vitro antiviral activities toward -ncov [ ] [ ] [ ] . hydroxychloroquine was more effective than chloroquine in inhibiting -ncov in vitro on primate cells [ ] . until now, chloroquine has shown apparent efficiency and safety against -ncov in clinical trials conducted in china [ ] . currently, chloroquine or hydroxychloroquine has been administered to hospitalized -ncov patients on an uncontrolled basis in various countries, including china and the usa [ ] . however, it must be noted that chloroquine and hydroxychloroquine cause ocular toxicity [ ] . hydroxychloroquine is reportedly less toxic than chloroquine, making it more attractive as a prescription drug [ , ] . nevertheless, more investigation and clinical trials are needed to evaluate further their efficacy and safety in treating -ncov. proteases are essential enzymes that regulate cell life processes such as cell growth and death, blood clotting, inflammation, fertilization, and infection [ ] . viral entry into host cells requires s protein priming by host proteases, which subsequently enables the fusion of viral and cellular membranes [ ] . membrane fusion enables the release of the viral genome into the host cytoplasm, initiating rna translation into protein. most viruses also encode their proteases to protect viral proteins by modulating host cell responses. while proteases are vital for cell life processes, they have become promising targets for antiviral therapeutic agents. protease inhibitors prevent viral replication by binding selectively to viral proteases or blocking proteolytic cleavage of protein precursors necessary for the production of infectious particles [ ] . it is noteworthy that protease inhibitors were a major therapeutic breakthrough of antiviral drug design in the mid- s for the treatment of hiv. most hiv protease inhibitors have found prominent clinical use (figure (b) ). coronavirus s proteins can be primed by a multitude of proteases [ ] . hoffmann et al. demonstrated that the s protein of -ncov could be primed by serine protease tmrpss [ ] . similarly, both sars-cov and mers-cov can be activated by other tmprss family members [ ] . tmprss family proteases are widely expressed in the respiratory tract [ , ] , which is likely the reason that coronaviruses cause acute respiratory distress syndrome. upon successful priming, the viral genome encoding rna and several nonstructural proteins, including coronavirus main protease ( clpro), papain-like protease (plpro), and rdrp, are released [ ] [ ] [ ] . the single-stranded positive rna is translated into viral polyproteins by ribosomes in the host cell cytoplasm. the polyproteins are then cleaved into effector proteins by viral proteases: clpro and plpro. plpro also acts as a deubiquitinase that may remove specific host cell proteins (e.g., interferon regulatory factor (irf ) and nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb)), thus weakening the immune system [ , , ] . both host and viral proteases are essential therapeutic targets in the case of covid- . camostat mesylate is a small molecule that has shown an excellent therapeutic effect for chronic pancreatitis treatment by targeting proteases [ , , ] . camostat mesylate primarily inhibits enzymatic autodigestion of the pancreas [ ] . in vivo studies on rats with pancreatic fibrosis showed that camostat mesylate inhibits inflammation, cytokine expression, and fibrosis in the pancreas [ ] . it has an additional clinical benefit for pancreatic pain by preventing enzyme-evoked activation of pain receptors [ ] . as mentioned above, the tmprss family, especially tmrpss , is most likely the protease targeted by a coronavirus. camostat mesylate inhibits tmprss activity on primate cells in vitro, completely blocking membrane fusion between the host cell and the viral mers-cov particle [ ] . zhou et al. claimed that camostat mesylate displays an inhibitory effect in mice for sars-cov infection [ ] . recent research by hoffmann et al. showed a promising in vitro inhibitory effect of this serine protease inhibitor in sars-cov and -ncov on human lung cells, showing potential as a viable option for covid- treatment [ ] . unfortunately, in vitro and in vivo data for camostat mesylate against coronaviruses are limited. more investigation is required to evaluate camostat mesylate as a potential therapeutic against covid- . lopinavir-ritonavir is a coformulated antiretroviral drug with excellent efficacy against hiv- . the lopinavir has a core molecular structure identical to ritonavir. the thiazolyl end group and -isopropylthiazolyl group in ritonavir are replaced by the phenoxyacetyl group and a modified valine, respectively, in which the amino terminus has six-membered cyclic urea attached. in brief, lopinavir is a potent protease inhibitor developed from ritonavir with high specificity for hiv- protease [ ] . it represents a higher proportion of the coformulation. lopinavir contains a hydroxyethylene scaffold mimicking a standard peptide bond cleavable by hiv- protease [ ] . this results in the production of noncontagious viral particles. on the other hand, ritonavir binds to hiv- protease, interrupting the maturation and production of viral particles [ ] . a clinical study from hong kong has shown that the combination of lopinavir-ritonavir and ribavirin treatment for patients against sars-cov had an overall favourable clinical response [ ] . it has been demonstrated that lopinavirritonavir targets clpro of -ncov and further indicated that clpro might also be the targets of protease inhibitors for other coronaviruses [ ] . regrettably, a recent clinical trial using lopinavir-ritonavir in wuhan, china, reported that hospitalized adult patients infected with -ncov did not benefit from the treatment [ ] . given such conflicting clinical data, physicians must carefully weigh lopinavir-ritonavir as a covid- treatment. darunavir is another antiretroviral protease inhibitor drug effective against hiv- . darunavir is designed for multidrug-resistant hiv- protease variants, due to its molecular structure, which introduces more hydrogen bonds compared to conventional antiretroviral medicines. in general, changes in van der waals and hydrogen bonding interactions between inhibitors and proteases affect the potency of antiretroviral drugs [ ] . aside from enzymatic inhibition, darunavir inhibits protease dimerization [ ] . the dimerization of hiv protease is essential for the acquisition of its proteolytic activity for the maturation of viral particles [ ] . lin et al. claimed that darunavir inhibits -ncov. the group has used molecular modeling to evaluate darunavir binding to clpro and plpro proteases and found targeted activity against the latter [ ] . nevertheless, the therapeutic effect of darunavir in covid- clinical cases remains untested [ ] . this may be in part due to potential side effects, such as liver damage and severe skin rashes [ , ] . these contraindications must be carefully evaluated if darunavir is to be considered a potential therapeutic agent for covid- . polymerases are enzymes essential for viral replication to produce viral progeny. viral dna and rna polymerases are responsible for duplicating the viral genome and facilitating transcription and replication [ ] . replication inhibitors (figure (c)) interfere with the production of viral particles by blocking enzymatic activity, ultimately causing chain termination during viral dna or rna replication [ ] . there are four types of viral polymerases in viruses: rna-dependent rna polymerases, rdrp, dna-dependent rna polymerases, and dna-dependent dna polymerases. in the section protease inhibitors, we mentioned that rdrp is released upon successful priming. rdrp is a necessary polymerase that catalyzes the replication of rna from an rna template for coronaviruses [ ] . release of rdrp from the virus initiates the synthesis of a full negativestrand rna template to be used by rdrp to replicate more viral genomic rna, which eventually turns host cells into virus factories [ ] . therefore, rdrp is an attractive therapeutic target to prevent host cells from producing viruses. ribavirin is a synthetic guanosine nucleoside analog that mimics purines, including inosine and adenosine, and ribavirin has been used in the treatment of respiratory syncytial virus [ ] . it has only one ring at the heterocyclic base, compared with guanine's two rings. notably, ribavirin has a ribose sugar moiety with a hydroxyl group at the ′ -carbon position, enabling preferential activity in rna-related metabolism [ , ] . ribavirin inhibits cellular enzyme and inosine monophosphate dehydrogenase involved in purine nucleotide biosynthesis [ , ] . ribavirin is also known for its inhibitory effect on viruses by forcing viral genome replication to become catastrophically error-prone. it is likely that as a nucleoside analog, ribavirin is incorporated by rdrp into the newly synthesized viral genome, where it induces mutagenesis [ , ] . although ribavirin has proven effective against viral infections, its mechanism of action has not been firmly established, and there are several proposed mechanisms of action that require further validation [ , ] . ribavirin was initially used in treating sars; however, ribavirin treatment lacked an in vitro antiviral effect and caused adverse side effects including anemia, hypoxemia, and decreased hemoglobin levels [ ] . however, ribavirin was used as the primary treatment during the mers outbreak [ ] . in general, clinical studies of ribavirin treatment for sars and mers did not show strong evidence of efficacy against these coronaviruses [ ] [ ] [ ] . there have been no studies of ribavirin's efficacy against covid- . therefore, the use of ribavirin remains controversial and requires more investigation for a better understanding of its mechanism of action, efficacy, and toxicity, even though it is a widely available drug. favipiravir is a synthetic guanine analog frequently used for influenza treatment [ ] . structurally, favipiravir is closely related to ribavirin, in which it shares the same carboxamide moiety [ ] . while ribavirin interacts with the viral polymerase directly, favipiravir must be phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl- ′ -triphosphate (rtp) [ , ] . the viral polymerase mistakenly recognizes favipiravir-rtp for a purine nucleotide, thereby disrupting viral genome replication [ , ] . favipiravir has not been used against sars and mers previously, but interestingly, it has been shown to reduce viral infection of -ncov [ , ] . in a clinical study involving patients infected with -ncov, conducted in shenzhen, china, favipiravir showed better efficacy than lopinavir-ritonavir in terms of disease progression and viral clearance [ ] . another clinical study involving patients with covid- conducted in hubei province, china, also demonstrated that those treated with favipiravir had a higher recovery rate compared to those treated with umifenovir (preprint) [ ] . more clinical data are needed to validate favipiravir's efficacy and safety in -ncov treatment. remdesivir is a trial synthetic adenosine analog that has not yet been clinically approved [ ] . it was synthesized and developed by gilead science in for ebola virus infection [ ] . remdesivir needs to be metabolized into its active form, gs- , to initiate its activity. the active form of remdesivir inhibits viral rna polymerase and evades proofreading by viral exonuclease, causing an interruption in viral rna production [ , , ] . it has been demonstrated that remdesivir is effective against mers-cov infection in vivo and -ncov in vitro [ , ] , showing great potential as a therapeutic agent for -ncov. the drug is currently being validated in clinical trials [ ] . given that antiviral drugs have previously demonstrated reasonable inhibition of coronaviruses and therapeutic efficacy against coronavirus outbreaks, umifenovir, chloroquine, hydroxychloroquine, lopinavir-ritonavir, and ribavirin have been recommended in the latest guidelines for diagnosis and treatment of covid- , updated on february [ ] . recent studies also demonstrated that some antibiotics potentially inhibit -ncov replication. anderson et al. (preprint) recently developed the first bat genome-wide rna interference (rnai) and crispr libraries and identified mthfdi as the critical host factor for viral infections [ ] . mthfdi is a trifunctional enzyme involved in the one-carbon (c ) metabolic pathway, participating in the cellular production of purine, dtmp, and methyl groups [ ] . anderson et al. demonstrated that purine synthesis activity of mthfdi is an essential activity for viral replication, making mthfdi a potential target for developing antiviral drugs [ ] . they further explained that an mthfd inhibitor, carolacton, restricts replication of influenza virus, mumps virus, melaka virus, zika virus, and, most importantly, -ncov [ ] . carolacton is a secondary metabolite derived from the mycobacterium sorangium cellulosum. it is a macrolide ketocarbonic acid. carolacton has been studied as an antibacterial compound against biofilms of pathogenic streptococcus mutans and growth of pathogen streptococcus pneumoniae [ , ] . it has no toxic effect against eukaryotic cells [ ] . it has recently been identified as a potent inhibitor of mthfdi, and its mechanism of action is presumably due to the ability of carolacton to bind with mthfdi [ ] . more research is needed to validate the mechanism of action, efficacy, and safety of carolacton as a possible treatment for covid- . on the other hand, ivermectin is originally a medication used to treat parasite infestation. it comprises different analogs of avermectin: , -dihydroavermectin b a and , -dihydroavermectin b b, at a ratio of : [ ] . they are macrolide antibiotics isolated from the fungus streptomyces avermitilis. it has reportedly stopped hiv-i proliferation by inhibiting interaction of the retroviral integrase protein with adapter protein (importin), responsible for the nuclear protein import cycle [ ] . caly et al. reported that ivermectin successfully inhibited -ncov in vitro but the mechanism of action is unclear [ ] . since ivermectin is an approved drug, it shows great potential as a therapeutic agent for covid- . in vivo work or clinical trials need to be done to confirm its efficacy and safety for treatment against covid- . potential drugs for covid- are summarized in table . . . nanodrug delivery system. nanomaterials have recently been utilized for the treatment of diseases such as cancer [ ] [ ] [ ] and various types of infections [ , ] . the ease of modification of surface properties, large surface area [ ] , and multivalent interactions with targets [ ] imbues nanomaterials with massive potential as highly efficacious covid- therapeutic options. however, to the best of our knowledge, no nanoparticle treatment option has been applied to covid- . nonetheless, results obtained from nanoparticle research against other viruses have shown promising potential. for example, fujimori et al. utilized a cui nanoparticle to treat h n influenza through the generation of reactive oxygen species (ros) that inactivate the virus [ ] . silver nanoparticles also show much promise in treating covid- with their broad antiviral properties against a multitude of viruses, including hiv, hepatitis b virus, herpes simplex virus, respiratory syncytial virus, and monkeypox virus [ ] . the broad antiviral properties of silver nanoparticles and the generality of ros inactivation suggest that these nanoparticles can be utilized therapeutically without any modifications. nanoparticles could also be used for drug delivery. recently, herold and sander demonstrated the use of pulmonary surfactant-biomimetic nanoparticles to encapsulate a stimulator of interferon gene (sting) agonist, ′ , ′ -cyclic guanosine monophosphate-adenosine monophosphate, as an adjuvant in a variety of influenza vaccines [ ] . using nanoparticles as a delivery agent, immune cells were activated without excessive inflammation in the lung. this could provide a considerable benefit for use in covid- vaccines in the future, but as the field is still relatively new, especially in medicinal applications, safety should remain a key consideration in the adoption of nanoparticles in humans. in addition to chemical medicines, another vital form of therapy for covid- may be the use of biologics. currently, interferon-α b nebulization of , to , iu/kg twice a day for to days is one of the main treatments for covid- in children, and it has demonstrated efficacy in reducing the viral load during early stages of infection [ , ] . another promising biologic drug is convalescent plasma, the plasma of patients who have recovered from covid- [ , ] . antibodies in the donated plasma could confer temporary, passive immunity against covid- , allowing patients time to develop active immunity. clinical trials are currently ongoing [ , ] , and preliminary results announced from the chinese hospitals have been promising. on the other hand, human monoclonal antibodies or their fragments developed in the lab have shown encouraging results as well. tian et al. confirmed the binding of a human monoclonal antibody cr to the receptor-binding domain (rbd) of -ncov with high affinity [ ] , highlighting the therapeutic potential of cr toward covid- , though further in vitro and in vivo studies are required before it could be used clinically. another supportive treatment for covid- involves dietary interventions. various research studies have shown supplementation of multiple vitamins and minerals such as vitamins a, c, and d and zinc can reduce the severity of respiratory infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, most of these studies targeted children below the age of who were suffering from malnourishment or preexisting diseases. therefore, vitamin and mineral supplementation may offer more significant benefits to covid- patients in developing countries. moreover, aggressive supplementation of calories and protein in nutritionally at-risk patients has shown significant benefits in reducing mortality [ ] . using a modified nutrition risk in critically ill (mnutric) score, kalaiselvan and coworkers demonstrated that . % of mechanically ventilated patients have high nutritional risks (mnutric score ≥ ), accompanied by long intensive care unit (icu) stays and high mortality rates [ ] . an estimated % of covid- patients require icu care, and of these critically ill patients, most need mechanical ventilation [ , ] . therefore, nutritional intervention using aggressive calorie and protein supplementation may provide substantial benefits to a significant number of critically ill patients. evidence of such benefits may be provided by the clinical trial (nct ) that is expected to end in july . . . traditional chinese medicine. traditional chinese medicine (tcm) is considered a prospective supplementary treatment of covid- , due to its impressive performance in treating sars in [ ] . first, tcm shows a generalized antiviral effect through direct inhibition of viruses and control of inflammation. for example, weng et al. reported that the smabucus formosana nakai (a traditional medicinal herb) ethanol stem extract displayed strong anti-hcov-nl activity [ ] . moreover, tcm can alleviate damage induced by inflammatory reactions and immune responses initiated by viral infections. single and combined chinese medicines could mitigate the cytokine storm by clearing the heat and toxicity in the body. for instance, tcm approaches were adopted to prevent and treat sars in and h n influenza in [ ] . as of february , , over . % of total confirmed cases (over , cases) had been treated with tcm, showing that tcm yields excellent outcomes. notably, in a trial of cases with mild symptoms, tcm achieved remarkable therapeutic effects, demonstrated [ , ] in vitro antiviral activities against bat kidney cells [ ] not known none ivermectin genome replication antiparasitic drug (broad-spectrum). in vitro antiviral activities against -ncov on primate cells [ ] not known none n/a: not available. note: "coronaviruses" only target sars-cov and mers-cov. by faster clinical symptom disappearance and reduction of fever, shorter disease course, higher cure rate (by %), and lower rate of moderate-to-severe cases [ ] . to date, the national health commission (nhc) of the people's republic of china has published seven editions of the guidelines for diagnosis and treatment of covid- [ ] . since the fourth version, a list of tcm prescriptions (including tcm soup and tcm capsules) has been recommended for patients based on the stage of the disease and their symptoms [ ] . according to the th edition of the guidelines, there are three kinds of tcm prescriptions recommended for different stages of patients: the medical observation period, the clinical treatment period, and critical condition (details in table ) [ ] . among tcm recipes, the qing fei pai du decoction is strongly recommended for treatment of covid- by the nhc of the people's republic of china, because it gave a cure rate of over % of covid- patients in a clinical trial involving confirmed cases [ ] . another tcm recipe, xue bi jing injection is specifically recommended for treating critically ill covid- patients, because it suppresses severe sepsis, according to the china food and drug administration. it also promoted significant improvement in cases of severe community-acquired pneumonia (cap) [ ] . therefore, tcm could be an alternative prophylactic approach to covid- and a supplementary treatment in combination with western medicine to cure covid- . . . stem cell therapy. stem cell therapy is a promising treatment strategy for degenerative diseases, including huntington's disease, parkinson's and alzheimer's diseases, and chronic diseases such as cardiac failure and diabetes [ ] . a clinical study showed that transplantation of mesenchymal stem cells (mscs) significantly lowered the mortality of patients with h n -induced acute respiratory distress syndrome (ards), with no harmful effects [ ] . as h n and -ncov share similar genome structures and corresponding infection mechanisms, as well as related clinical symptoms (lung failure), msc-based therapy could be a possible alternative for treating covid- . currently, stem cellbased therapy for covid- is being conducted by different hospitals in china. doctors from baoshan hospital (yunnan province, china) used human umbilical cord mesenchymal stem cells (hucmscs) to treat a -year-old critically ill woman with covid- . two days after the rd injections of stem cells, the woman recovered, and the throat swab test for covid- turned negative [ ] . another clinical trial involving stem cell therapy was conducted in seven confirmed covid- patients in different clinical stages in beijing youan hospital (beijing, china). two to four days after intravenous transplantation of ce -mscs, all symptoms such as high fever, weakness, and shortness of breath disappeared in all seven patients without observed adverse effects, indicating that mscs can cure or significantly improve functional outcomes [ ] . there are at least other trials using stem cells to treat covid- in china, according to the who report. vaccines are another promising treatment to prevent or cure specific viruses. currently, there is no effective vaccine against -ncov. fortunately, two covid- vaccines are undergoing clinical trials. the first is moderna's mrna- , an mrna vaccine, which started at kpwhri in seattle, usa, on march , [ ] . it targets the spike protein of -ncov. the other vaccine, ad -ncov (a recombinant novel coronavirus disease vaccine (adenovirus type vector)), was conducted at tongji hospital in wuhan, china, on march , [ ] . the trial was jointly developed and administered by cansino biologics inc. and the academy of military medical sciences. ad -ncov, a genetically engineered vaccine, expresses the -ncov s protein using replication-defective adenovirus type as an expression vector, thereby inducing a virus-specific immune response to prevent covid- . there are also several other types of covid- vaccines, including deoptimized live attenuated vaccines, protein vaccine, dna vaccine, rna vaccine, and subunit vaccine, all of which are in the preclinical stage (table ) [ ] . notably, there is a new microneedle array (mna) approach based on delivering coronavirus s subunit vaccines against covid- . expedited by prior experience in developing vaccines against mers, this approach was developed within weeks and enabled long-term induction of potent virus-specific antibody responses. significantly, the mna work can be extended to other emerging infectious diseases. however, these will require further clinical studies for efficacy and safety, which requires more time. according to the th edition of the diagnostic criteria [ ] , patients severely or critically ill with covid- should receive comprehensive antiviral treatment, including lopinavir/ritonavir, arbidol, or shufeng jiedu capsule. meanwhile, they also need additional treatments, according to their symptoms, including respiratory support (oxygen therapy, high-flow nasal cannulas, or noninvasive ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation-(ecmo-) based therapy), circulatory support, or continuous renal replacement therapy. the main therapeutic approaches proposed for covid- are summarized in table . as the most recent pandemic, covid- induces much fear. it is highly infectious and is transmitted asymptomatically. as such, our best options to slow and prevent transmission are to understand the origin of -ncov, its transmission route, and associated disease pathways and systems. generally, a pathogen must remain viable outside the host to allow for environmental spread [ ] . collective effects of many biotic and abiotic factors determine the period that the pathogen can survive [ ] . as of now, covid- is thought to be transmitted directly from person-to-person through liquid (droplets) and, more importantly, transmitted indirectly via contact with contaminated surfaces. -ncov remains viable for a fairly long period outside the human body (up to hours) and is more stable on plastic and stainless steel than on copper and cardboard [ ] . therefore, aerosol and fomite transmission of -ncov is possible, as the virus lingers among particles or fibers, in airborne liquid droplets, and on surfaces, in some cases for days [ ] . although there are currently insufficient data on the inactivation of environmental -ncov, data from other coronaviruses can be used as a reference. however, it should be noted that biocidal agents may only limit the survival of coronavirus in critical environments and have no efficacy for infected patients. given the high transmissibility of covid- , its propensity for asymptomatic transmission, and its persistent nature, confirmed patients could only be quarantined and treated in adequately equipped facilities. this also applies to anyone who has come into contact with these patients. as such, contact tracing is still a mainstay for disease control. confirmation can be achieved only when specific diagnostic methods have been employed. chest ct imaging is useful as an initial evaluation for covid- , as ct confirmation is often possible even before symptoms appear; therefore, it is recommended for suspected covid- cases [ ] . once the primary diagnosis reveals abnormal chest ct findings, a nucleic acid test should be performed to confirm whether a patient is infected. once a person is confirmed positive, tests such as c-reactive protein (crp), complete blood count, urinalysis, biochemical indicators (i.e., liver enzymes, myocardial enzymes, and renal function), blood coagulation function, arterial blood gas analysis, and cytokine levels should be performed to monitor the patient's condition [ ] . chest ct should be performed as a follow-up to treatment as well [ ] . the currently adopted procedure in identifying potential covid- cases in china is summarized in figure . on a community scale and beyond, strict controls over human traffic are essential to limiting disease transmission. by establishing lockdowns, china has been able to bring the crisis under control. other nations are now following the chinese's approach in restricting movement of residents within their borders. as evidenced globally, social distancing is essential to halt the spread of covid- . on the other hand, individuals have the responsibilities to follow the guidelines given by the authorities, to practice good hygiene, and to behave responsibly. covid- , like the past epidemics, does not recognize political boundaries, ethnicity, or gender. the disease has challenged the economic and medical infrastructure of the entire globe. as evidenced by events of the past few months, the impact of the outbreak depends upon how well we are prepared to face such a challenge. only with time will we be able to fully evaluate the measures that are being taken against covid- today. previous coronavirus epidemics like sars and mers have expedited the process of finding useful diagnostic and therapies against -ncov. it is of paramount importance for all countries to share essential information about -ncov to mitigate its spread. because of this strategic approach, research has been mobilized to rapidly develop diagnostic methods and worldwide implementation to minimize the impact of the pandemic. practical diagnostic tests have aided management and contact tracing of covid- cases in hotspot areas. in this regard, molecular virological techniques have assisted the scientific community in characterizing infectious agents for years. these include qrt-pcr, isothermal amplification, and crispr technology. on the other hand, serological assays for antibodies and antigens present essential tools to obtain valuable information about prior exposure to -ncov and the prevalence of infection. these include elisa and lfa technologies. serological screening also enables novel vaccines to be assessed and supports the design of functional vaccine approaches. other approaches, including chest computed tomography (ct) and transmission electron microscopy (tem), can boost existing detection approaches. notably, there has been a marked increase in the use of both ct and tem to detect -ncov and other coronaviruses. these complimentary tools reveal the progression of suspected infection, which cannot be accomplished by conventional diagnostic means. nonetheless, there is a pressing need for continuous development of rapid, accurate diagnostic devices and strategies to characterize unknown respiratory pathogens. despite these signs of progress, the present data suggest that current public health policies and improved diagnostic measures alone may not be sufficient to eradicate covid- in the short term. efficacious and novel treatments are desperately required. presently, large numbers of ongoing clinical trials of various drugs may succeed in minimizing morbidity and mortality. we have highlighted several of them in this review. some are highly promising, while others may require more time to demonstrate usefulness. while some drug candidates appear promising and have been used in treating covid- patients in desperation, it does not necessarily mean that they are proven safe and efficacious in the long run. as such, stringent criteria must be established by health regulatory agencies. however, in the long term, vaccines and prophylactics may be required to curb the spread of -ncov. the authors declare no conflicts of interest. covid- ) outbreak situation who, -ncov outbreak is an emergency of international concern who-director-general-s-openingremarks-at-the-media-briefing-on-covid more and more coronaviruses: human coronavirus hku discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus 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regulation of pro-and anti-inflammatory th and t regulatory cells in a murine model of polymicrobial sepsis re-du-ninginhalation solution exerts suppressive effect on the secretion of inflammatory mediatorsviainhibiting ikkα/β/iκbα/nf-κb, mapks/ap- , and tbk /irf signaling pathways in lipopolysaccharide stimulated raw . macrophages andrographolide sulfonate ameliorates lipopolysaccharide-induced acute lung injury in mice by down-regulating mapk and nf-κb pathways tanreqing injection attenuates lipopolysaccharide-induced airway inflammation through mapk/nf-κb signaling pathways in rats model action mechanism of shenfu injection by computational system biology analysis key: cord- -e is g authors: klein, steffen; müller, thorsten g.; khalid, dina; sonntag-buck, vera; heuser, anke-mareil; glass, bärbel; meurer, matthias; morales, ivonne; schillak, angelika; freistaedter, andrew; ambiel, ina; winter, sophie l.; zimmermann, liv; naumoska, tamara; bubeck, felix; kirrmaier, daniel; ullrich, stephanie; barreto miranda, isabel; anders, simon; grimm, dirk; schnitzler, paul; knop, michael; kräusslich, hans-georg; dao thi, viet loan; börner, kathleen; chlanda, petr title: sars-cov- rna extraction using magnetic beads for rapid large-scale testing by rt-qpcr and rt-lamp date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: e is g rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus (sars-cov- ). the standard diagnostic pipeline for testing sars-cov- presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral rna is extracted using commercial kits, followed by reverse transcription and quantitative pcr detection. as a result of the large demand for testing, commercial rna extraction kits may be limited and, alternatively, non-commercial protocols are needed. here, we provide a magnetic bead rna extraction protocol that is predominantly based on in-house made reagents and is performed in -well plates supporting large-scale testing. magnetic bead rna extraction was benchmarked against the commercial qiacube extraction platform. comparable viral rna detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (rt-lamp) using a primer set targeting the n gene, as well as rt-qpcr using a primer set targeting the e gene, showing that the rna extraction protocol presented here can be combined with a variety of detection methods at high throughput. importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot. severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of coronavirus disease (covid- ) , was first described in the city of wuhan in china in december and spread globally thereafter causing pandemic. to slow its spread, large-scale diagnostics and the enforcement of strict public health measures were implemented in many countries. the current standard test for sars-cov- detection and diagnosis is based on viral rna extraction from a pharyngeal swab followed by highly sensitive reverse transcription and quantitative pcr (rt-qpcr). several primer sets targeting one or more of the sars-cov- genes-nucleocapsid (n), envelope protein (e), s glycoprotein (s), or rna-dependent rna polymerase (rdrp)-have been used [ ] . a two-step testing procedure using primer sets targeting the e gene for initial screening followed by the rdrp gene to confirm positive samples is recommended by the german consiliary laboratory for coronaviruses [ ] . the unprecedented global demand for commercial rna extraction kits and ensuing shortage of these reagents [ ] led to the establishment of several diagnostic workflows performed on patient samples with or without an intermediate rna extraction step [ ] [ ] [ ] [ ] . viral rna isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization, and on the denaturation of omnipresent rnases [ ] . the latter may be accomplished by the use of chaotropic chemicals, such as guanidinium salts [ ] or non-specific proteases that are active on both native and denatured proteins, such as proteinase k. in either case, after virus particle lysis, rna must be purified, since guanidinium salts, proteinase k and organic solvents inhibit the subsequent rt-qpcr step. rna can be separated from proteins either by liquid phase separation using chloroform-aqueous emulsions after lysis with commercially available trizol (a mixture of guanidinium thiocyanate and acid phenol) or by means of solid-phase separation using silica [ ] . nucleic acid-binding to negatively charged silica (sio ) is facilitated by guanidinium salts and the basic ph of the lysis buffer [ ] . to achieve a high nucleic acid binding capacity, silica-based nucleic acid extraction methods use either porous silica matrices that are embedded in a column (spin column) [ ] , a tip (trutips) [ ] , or a suspension of microparticles. microparticles can be separated from the lysate either by centrifugation or by a magnetic field provided that the microparticles' dense iron-containing cores are coated with porous silica [ ] . the protocol established in this study aimed at extracting sars-cov- rna from respiratory patients' swabs (oropharyngeal and nasopharyngeal) and is based on the magnetic bead-based nucleic acid extraction protocol that was published by he et al. [ ] and on the protocol from chemicell gmbh, who provided the simag-n-dna magnetic beads. we opted for silica magnetic beads because of their relatively easy manufacturing and sustainable availability, and because specialized plasticware (spin columns, modified tips) is not required to perform their separation. magnetic bead rna extraction was performed in -well plates in combination with a magnet plate optimized for deep-well plates. the portable manual pipetting system liquidator , which is less expensive than automated pipetting robots, was used to minimize the pipetting and handling errors ( figure a ). here, we show that the magnetic bead-based protocol yields rna extracts comparable to the commercially available qiacube viral rna extraction kit, as determined by the commonly applied detection methods rt-qpcr and reverse transcription loop-mediated isothermal amplification (rt-lamp) [ ] . a selection of upper respiratory tract specimens (flocked swabs in amies medium, eswab copan, brescia, italy) sent to the diagnostics laboratory of the heidelberg university hospital between april and may for sars-cov- pcr were used for the study. surplus material from a total of swab samples were collected from sars-cov- positive and negative patients, which were used for sars-cov- testing by rt-qpcr. the swab samples were either used at the day of collection (two positive, negative) or were frozen, stored at − °c, and thawed just before the rna extraction ( positive, negative). positive and negative samples were further diluted and used in replica to generate positive and negative samples in order to facilitate replicate testing. the following steps were performed in a biosafety level (bsl- ) laboratory according to standard microbiological and diagnostic practices. to extract sars-cov- rna from pharyngeal swabs, a lysis buffer containing m guanidinium thiocyanate, mm dithiothreitol, µ g/ml glycogen, % triton x- , and buffered with mm sodium citrate to ph was used. internal control (ic) for rt-qpcr ( µl/sample) (tib-molbiol, berlin, germany) was added into the lysis buffer just before use. µ l lysis buffer and µ l sample were vigorously vortexed in a . ml eppendorf tube for sec and incubated for min at room temperature inside a bsl- laminar flow cabinet to ensure both rapid virus deactivation and rnase denaturation. lysates ( µl) were transferred into a deep-well plate (greiner ag, kremsmünster, austria) with a maximum working volume of µ l per well, which is compatible with the liquidator pipetting system. just before the rna extraction, simag-n-dna magnetic beads (chemicell, berlin, germany) were washed three times in rnase-free water. the aqueous magnetic bead stock solution of µ g/µ l a selection of upper respiratory tract specimens (flocked swabs in amies medium, eswab copan, brescia, italy) sent to the diagnostics laboratory of the heidelberg university hospital between april and may for sars-cov- pcr were used for the study. surplus material from a total of swab samples were collected from sars-cov- positive and negative patients, which were used for sars-cov- testing by rt-qpcr. the swab samples were either used at the day of collection (two positive, negative) or were frozen, stored at − • c, and thawed just before the rna extraction ( positive, negative). positive and negative samples were further diluted and used in replica to generate positive and negative samples in order to facilitate replicate testing. the following steps were performed in a biosafety level (bsl- ) laboratory according to standard microbiological and diagnostic practices. to extract sars-cov- rna from pharyngeal swabs, a lysis buffer containing m guanidinium thiocyanate, mm dithiothreitol, µg/ml glycogen, % triton x- , and buffered with mm sodium citrate to ph was used. internal control (ic) for rt-qpcr ( µl/sample) (tib-molbiol, berlin, germany) was added into the lysis buffer just before use. µl lysis buffer and µl sample were vigorously vortexed in a . ml eppendorf tube for sec and incubated for min at room temperature inside a bsl- laminar flow cabinet to ensure both rapid virus deactivation and rnase denaturation. lysates ( µl) were transferred into a deep-well plate (greiner ag, kremsmünster, austria) with a maximum working volume of µl per well, which is compatible with the liquidator pipetting system. just before the rna extraction, simag-n-dna magnetic beads (chemicell, berlin, germany) were washed three times in rnase-free water. the aqueous magnetic bead stock solution of µg/µl was added into absolute ethanol in order to obtain a working solution of µg/µl. using a multichannel pipette, µl of magnetic bead solution was transferred into the deep-well plate containing the pharyngeal sample lysates. to facilitate the adsorption of the nucleic acid onto the magnetic beads, the -deep-well plate was placed on an orbital shaker ms (ika, staufen, germany) for min at - rpm, before the mixture was resuspended ( ×) using a liquidator , model µl (mettler toledo, columbus, oh, usa) ( figure a ,b) and placed again on the orbital shaker for additional min. subsequently, the plate was placed on a magnet plate for -deep-well plates (magtivio, nuth, the netherlands) ( figure c ) for min to allow the magnetic beads to form rings on the bottom of the wells ( figure d ,e). the -well plate was visually inspected to ensure that all of the pellets were formed. the clear supernatant was discarded using the liquidator and magnetic beads were washed three times with µl % ethanol. for each washing step, the -well plate was removed from the magnet and pellets were resuspended ( ×) using a liquidator . the plate was placed back on the magnet for min until the beads formed visible rings. after three washing steps, the magnetic beads were briefly rinsed with µl rnase-free water in order to remove any residual ethanol while the plate was kept on the magnet. the -well plate was visually inspected to ensure that none of the pellets was removed. finally, nucleic acids adsorbed onto the surface of the magnetic beads were eluted: µl rnase-free water was added to each well, the -well plate was removed from the magnet, resuspended ( ×), and vortexed for - min. the -well plate was placed back on the magnet until rings of magnetic beads were formed and µl eluate was transferred to a new -well pcr plate. a detailed step-by-step procedure and a complete list of all materials and instruments used for this magnetic bead rna extraction protocol can be found in the supplementary materials. the qiaamp viral rna body fluid kit was carried out with manual lysis according to the manufacturer's protocol to compare the performance of the magnetic bead rna extraction (qiagen, hilden, germany). the sample input volume was µl, the volume of ic per sample was µl, and the elution volume was set to µl. for rt-qpcr detection of the sars-cov- envelope protein gene (e gene), we adopted a widely used protocol based on corman et al. [ ] . for the mastermix, . µl of primer/probe lightmix ® modular sars and wuhan cov e-gene (tib-molbiol, berlin, germany) and . µl of lightmix ® modular eav rna extraction control (tib-molbiol, berlin, germany) was mixed with . µl rnase-free water, µl lightcycler ® multiplex rna virus master (roche, basel, switzerland), and . µl reverse transcriptase enzyme (supplied with lightcycler ® multiplex rna virus master kit, berlin, germany) per sample. the following primers and probe were used (provided by tib-molbiol, germany): fwd -acaggtacgttaatagttaatagcgt- , rev -atattgcagcagtacgcacaca- , probe fam-acactagccatccttactgcgcttcg-bbq; these primers are specific for a bp amplicon from position - of the sars-cov- genome (genbank nc_ ). µl of mastermix was distributed into a -well pcr plate and µl of purified rna from patient samples was added using the liquidator , model µl. rt-qpcr was performed using a lightcycler ® instrument ii (roche, basel, switzerland) with min of reverse transcription at • c, initial denaturation at • c for min, and subsequent amplification cycles with • c for sec, • c for sec, • c for sec, and finally cooling to • c for sec. cycle threshold (ct) was determined, where the fluorescence signal of the amplification reaction was above the background fluorescence using the lightcycler software (roche). data analysis on raw cts was performed in excel and graphpad prism (graphpad software, san diego, ca, usa), and % "exact" clopper-pearson confidence intervals were calculated using medcalc (medcalc software, ostend, belgium). five µl diluted ms rna (roche, basel, switzerland) containing . × to . × molecules was spiked into µl lysis buffer before a µl patient sample was added. magnetic bead rna extraction was performed as described in section . . rt-qpcr for ms was performed with a one-step rt-qpcr reaction (tib-molbiol, berlin, germany) using the following primers and probe: fwd -gagtgtttacagttccgaa- , rev -cccctttctggaggtacatattcata- , and probe cy -aatagatcgggctgcctgtaaggagc-bbq. µl of rna was used per rt-qpcr, covering a range from to ms rna molecules per reaction. rt-qpcr cycling program for ms rna amplification was performed as described in section . . rt-lamp detection of the sars-cov- n gene was based on the protocol by dao thi et al. [ ] . the rt-lamp primer set used in this study was targeted against the sars-cov- n gene [ ] . the sequences and concentrations of all oligonucleotides in the x primer mix used for the rt-lamp assays can be found in our recent publication [ ] . for the colorimetric lamp assay, the master mix consisted of . µl warmstart ® colorimetric lamp x master mix m (new england biolabs, ipswich, ma, usa) and . µl × lamp primer mix targeting the n gene [ ] per reaction. immediately afterwards, . µl of the freshly prepared reaction mix was distributed into -well plates and µl of purified rna was added using a liquidator , model µl. the plate was sealed with a transparent adhesive foil and subsequently incubated at • c for min in a -well-pcr block with heated lid ( • c). absorbance was measured at nm and nm wavelengths in a spark ® cyto or infinite m plate reader (tecan, männedorf, switzerland). phenol red absorbance spectra change in response to the acidification of the reaction (the absorbance at nm wavelength is increased and the absorbance at nm wavelength is decreased). to measure ph changes during the reaction, the difference between optical densities was calculated (∆od = od nm − od nm ) and samples with ∆od < . were classified as negative. for the fluorescent lamp assay, the same primer set as described in . was used. the master mix consisted of . µl warmstart ® lamp kit x master mix e (new england biolabs, ipswich, ma, usa), . µl × lamp primer mix targeting the n gene [ ] and . µl of × fluorescent dye (syto- , supplied with the rt-lamp kit) per reaction. . µl of the mix was distributed in a -well plate and . µl purified rna was added before the plate was sealed and incubated at a constant temperature of • c using a lightcycler ® instrument ii (roche, basel, switzerland). real-time fluorescence was detected with intervals of min for a duration of min. time of amplification (toa) was determined based on a fluorescence threshold, where the fluorescence signal of the amplification reaction was above the background fluorescence, using the lightcycler software. samples with toa > min were classified as negative. the presented work was done with the intention to support sars-cov- diagnostics and improve emergency preparedness and response to covid- . pseudo-anonymized surplus material from samples that had been collected for sars-cov- testing were used to establish and validate the protocol presented here. this work complies with the german act concerning the ethical review of research involving humans, permitting use of patient samples collected to perform the testing in question, for development and improvement of diagnostic assays. the magnetic bead rna extraction protocol was established in a -well plate format as part of the detection workflow ( figure ). the complete workflow from rna extraction to rna detection can be conducted in less than - h. we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure a we first applied both protocols on one sars-cov- positive pharyngeal swab sample, which was diluted in rnase-free water prior to rna isolation in a -fold dilution series up to fold, in order to compare the magnetic bead rna extraction protocol with the commercial qiacube extraction method. the extracted rna was subjected to rt-qpcr using e gene primers. viral rna was detected in samples diluted up to fold after either qiacube or magnetic bead rna extraction ( figure a ,c). rt-qpcr of the extracted rna by either of the two methods showed approximately equidistant amplification curves with an interval of ct values for each -fold dilution step ( figure b ,d). a dilution series of rna from ms bacteriophage was added to the swab sample prior to magnetic bead rna extraction and subjected to rt-qpcr using ms primers to further evaluate linearity of the magnetic bead rna extraction method across a broad range of defined rna inputs ( figure e,f) . ct values were linear over five orders of magnitude with a goodness of fit of r = . . the lower limit of detection (~ct ) was less than rna copies/reaction ( figure e ). in order to evaluate the magnetic bead rna extraction protocol on larger sample sets, sars-cov- positive and negative samples were generated from sars-cov- positive patients and from persons negative for sars-cov- , respectively. the samples were subjected to three independent magnetic bead rna extractions as well as to qiacube extractions. ct values of positive samples obtained by rt-qpcr using primers for the e gene and ic values from either magnetic bead or qiacube extraction were compared. in the remaining six samples, ct values for the e gene were not detected after magnetic bead rna purification but detected ct values for ic confirmed successful rna extraction. noteworthy, all six samples were previously frozen and had a ct value higher than as determined by rt-qpcr after the qiacube rna extraction. the e gene ct values that were obtained from the magnetic bead and qiacube rna extraction were in good agreement as shown by the slope of the linear regression of . ( figure a ). the average ic ct of . (sd = . , n = ) after magnetic bead extraction was the same as compared to the average ic ct of . from the qiacube rna extractions (sd = . , n = ) ( figure b,c) , showing that the magnetic bead rna extraction protocol is robust. these results also illustrate that quickly washing the beads with water to remove residual ethanol (which could inhibit pcr) does not lead to a substantial loss of rna. the sd of ct values for ic should be calculated to determine false negatives due to rna loss and judge the quality of the rna extraction. the sd of the ct values for ic should be smaller than ct values and samples with ic ct values with × sd lower ic ct values than the average ic ct should be repeated. because assays performed in a -well plate format can be prone to cross-contaminations, we evaluated the sensitivity and specificity of the three independent magnetic bead rna extractions. the samples were distributed in small groups of positive and negative samples on the -well plate. after magnetic beads rna extraction and rt-qpcr or rt-lamp analysis the results were compared to the results of the rt-qpcr after qiacube extraction to determine true and false positives (tp and fp) and true and false negatives (tn and fn) . for the magnetic beads extracted samples, a ct cutoff of was used to determine if a sample is defined as positive. the sensitivity was calculated (tp/(tp+fn)) for subsets of samples in specific ct ranges ( - , - , - , - ) ( figure d ). for all subsets up to ct , no false negatives were detected, which leads to a sensitivity of % with a % confidence interval (ci) of - %. in the range between ct - , six false negatives were counted leading to a sensitivity of % (ci = - %). these results indicate that false negatives were samples with high ct values and the negative results might be due to fluctuation of the viral load around the limit of detection. in total, three false positives were detected, resulting in a specificity of % (ci = - %) ( figure e ). to determine sensitivity and specificity, qiacube rt-qpcr results were used as a reference. rt-qpcr after magnetic bead rna extraction provides close to % sensitivity and specificity at a cutoff ct of . data are shown as mean with indicated % clopper-pearson confidence intervals (supplementary table s ). we also recently explored the rt-lamp assay as a valid alternative rna detection method due to shortages in rt-qpcr reagents [ ] . we compared the standard pipeline qiacube rna extraction followed by rt-qpcr detection of the e gene with magnetic rna extraction followed by rt-lamp detection using primer sets targeting the n gene, with either colorimetric (figure a -c, supplementary table s ) or fluorescent ( figure d-f, supplementary table s ) read-out. in total, we tested rna extracted from positive and negative swab samples. to determine sensitivity and specificity, qiacube rt-qpcr results were used as a reference. rt-qpcr after magnetic bead rna extraction provides close to % sensitivity and specificity at a cutoff ct of . data are shown as mean with indicated % clopper-pearson confidence intervals (supplementary table s ). we also recently explored the rt-lamp assay as a valid alternative rna detection method due to shortages in rt-qpcr reagents [ ] . we compared the standard pipeline qiacube rna extraction followed by rt-qpcr detection of the e gene with magnetic rna extraction followed by rt-lamp detection using primer sets targeting the n gene, with either colorimetric (figure a -c, supplementary table s ) or fluorescent ( figure d-f, supplementary table s ) read-out. in total, we tested rna extracted from positive and negative swab samples. during dna synthesis, the formation of a phosphodiester bond results in the release of a molecule of pyrophosphate and a proton causing a gradual acidification of the reaction mix. the detection principle of colorimetric rt-lamp is based on monitoring ph changes during the dna amplification, which only occurs in positive samples. typically, phenol red, which changes color from red to yellow when the ph is lowered, is used as a ph indicator [ ] . the color change can be determined by measuring the difference between the wavelengths of the two absorbance maxima of phenol red. the majority of isolated rna samples with a ct ≈ value obtained from rt-qpcr using the e gene yielded a color change with ∆od values between . and . as shown in figure a . therefore, we used a ∆od value of . as a threshold for positive samples. the majority of samples with higher ct values, especially between ct and ct , scored negative (∆od < . ), while all negative rt-qpcr samples also scored negative in the rt-lamp assay. using a ct cutoff of , the overall sensitivity of the colorimetric assay with magnetic bead-isolated rna was % ( % ci = - %) ( figure b) , with a specificity of % ( % ci = - %) ( figure c ). table s ). during dna synthesis, the formation of a phosphodiester bond results in the release of a molecule of pyrophosphate and a proton causing a gradual acidification of the reaction mix. the detection principle of colorimetric rt-lamp is based on monitoring ph changes during the dna amplification, which only occurs in positive samples. typically, phenol red, which changes color from red to yellow when the ph is lowered, is used as a ph indicator [ ] . the color change can be determined by measuring the difference between the wavelengths of the two absorbance maxima of phenol red. the majority of isolated rna samples with a ct ≈ value obtained from rt-qpcr using the e gene yielded a color change with Δod values between . and . as shown in figure a . therefore, we used a Δod value of . as a threshold for positive samples. the majority of samples with higher ct values, especially between ct and ct , scored negative (Δod < . ), while all negative rt-qpcr samples also scored negative in the rt-lamp assay. using a ct cutoff of , the overall sensitivity of the colorimetric assay with magnetic bead-isolated rna was % ( % ci = - %) ( figure b) , with a specificity of % ( % ci = - %) ( figure c ). the fluorescent rt-lamp assay is based on a fluorescent dye that intercalates into amplifying dna strands, allowing for their detection in real time. magnetic bead isolated samples with ct ≈ table s ). the fluorescent rt-lamp assay is based on a fluorescent dye that intercalates into amplifying dna strands, allowing for their detection in real time. magnetic bead isolated samples with ct ≈ led to a fluorescent signal at early time points (approx. - min) as compared to negative samples (> min). therefore, we used the time point min as a threshold for positive samples. similar to the colorimetric read-out, the majority of samples with higher ct values, especially between ct and ct , scored negative (> min) ( figure d ). all negative rt-qpcr samples also scored negative (> min). using a ct cutoff of , the overall sensitivity of the fluorescent assay was % ( % ci = - %) ( figure e) , with a specificity of % ( % ci = - %) ( figure f ). these results are well in agreement with the recently reported sensitivity cutoff at ct for the rt-lamp assay [ ] and, thus, confirm that rna purified by the presented magnetic bead extraction is compatible with rt-lamp detection without lowering its sensitivity. within the last decades, the frequency of emerging virus outbreaks has increased globally [ , ] , possibly as a result of cumulative anthropogenic environmental changes that increase the risk of zoonotic transmission [ ] . due to globalization, many of the outbreaks have increased pandemic potential and pose a burden on society and health systems. the currently ongoing sars-cov- pandemic emphasizes the urgency of appropriate preparedness and response. before a therapeutic or vaccine exists, the early identification and isolation of positive patients remains the most effective way to inhibit further human-to-human spread and mitigate the disease outbreak. in the case of a respiratory viral disease, such as influenza or covid- , pharyngeal swabs are collected and tested for the presence of viral rna. rna isolation prior to detection is a pivotal step to ensure high specificity and sensitivity of detection methods. to this end, robust and high-throughput nucleic acid isolation methods with high manufacturing and distribution capacity must be available. however, the majority of commercially available rna purification kits are cost-ineffective and often rely on multiple components that are not easily replaceable, and their supply can often not be guaranteed. in addition, buffer compositions are not provided. thus, overall, commercial kits do not offer enough flexibility and availability when it comes to a large epidemic or pandemic. here, we provide a magnetic bead-based rna extraction protocol that is, to a large extent, producer independent, does not rely on unique components that are difficult to replace, and is scalable for mass testing. because of the large surface binding capacity and rapid separation in solution by magnetic field, silica coated magnetic beads are compatible with a variety of plasticware and they can be used in a high throughput multiwell format [ ] as well as in small scale testing [ ] . in addition, magnetic beads can be prepared from widely available chemicals in a laboratory with basic equipment [ ] . recently, a magnetic bead rna purification protocol has been established for automated, high throughput sars-cov- diagnostics by the covid- crick consortia [ ] . however, a sophisticated infrastructure is required to carry out the protocol, and the protocol is partially dependent on commercial buffers. our aim was to provide a robust protocol that can be rapidly implemented and carried out in most of the laboratories around the world equipped with a magnetic plate, a multichannel pipette, or with a manual pipetting device. we established a magnetic bead rna isolation protocol that takes approximately min using a single -well plate as part of a workflow for sars-cov- diagnostic. although it is feasible to perform four extractions per day for one person, the rate-limiting and the most labor-intensive step of the presented workflow is the sample transfer from swab tubes to -well plates. special care should be taken regarding sample handling prior to rna isolation, and the samples should be kept at • c and processed at the day of collection. a single freeze-thawing cycle of a sars-cov- positive swab sample that was × diluted results in decreased sensitivity of rt-qpcr by approximately ct values (ct frozen -ct fresh = . ; sd = . , n = ). in contrast to other magnetic bead based nucleic acid purification protocols [ , ] that rely on an air-drying step to remove residual ethanol before elution, we rinse magnetic beads with a small amount of rnase-free water. the introduction of this step made our protocol more robust, since air-drying in a -well plate is slow and uneven. furthermore, air-drying did not lead to complete removal of ethanol, which interfered with the subsequent rt-qpcr even at low concentrations. we report a similar rna extraction yield when compared to the commercial qiacube extraction kit and validate that the quality of the rna extract is suitable for rt-qpcr and rt-lamp detection. rt-lamp relies on a different dna polymerase than rt-qpcr and, thus, provides a supplier-independent alternative to rt-qpcr. additionally, rt-lamp is faster and cheaper when compared to rt-qpcr and it does not require a thermocycler [ ] . as we have recently reported [ ] , rt-lamp to detect sars-cov- rna has a decreased sensitivity compared to rt-qpcr: rt-lamp with the primer set against the n gene as used in this study failed to detect most of the samples with a ct value over measured by rt-qpcr using the e-sarbeco primers. efforts are underway to increase the sensitivity of the rt-lamp assay, but this is out of the scope of this work. here, we showed that the presented magnetic bead extraction is compatible with rt-lamp and does not lower its sensitivity when compared to a qiacube purification method performed in our previous study [ ] . either rt-qpcr or rt-lamp can be used as an independent detection method and both methods do not have to be run in parallel. due to higher sensitivity, we recommend using rt-qpcr as a detection method after magnetic bead rna extraction. if resources are limited or large-scale testing is needed, rt-lamp offers an alternative detection approach; however, its lower sensitivity must be considered for now. although it is possible to perform rt-qpcr [ , ] and rt-lamp on unpurified patient samples [ , ] , the combination with our magnetic bead rna extraction protocols increases the sensitivity. due to the low cost, high-throughput compatibility, and independence from sophisticated laboratory equipment such as automated rna extraction kits and rt-qpcr machines, the combination of magnetic bead rna extraction and rt-lamp offers a framework for diagnostic preparedness in the case of mass-scale testing. in conclusion, we report a detailed step-by-step rna extraction protocol from pharyngeal swabs, which is based on magnetic beads and does not depend on commercial extraction kits and reagents. our protocol was validated in a -well plate format by the detection of sars-cov- rna by rt-qpcr and rt-lamp and provides reliable rna extraction for a diagnostic pipeline that can be rapidly deployed during the sars-cov- pandemic and is also accessible to regions with insufficient laboratory capacities. this might be of importance in case of possible future shortages of commercial rna extraction kits and enables diagnostic laboratories to be well prepared in case of a new rise of sars-cov- cases. our protocol can be easily adapted to fully automated liquid handling robotic systems and is very likely suitable for isolation and downstream detection assays for any kind of rna virus isolated from pharyngeal swabs. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , list of materials and instruments for magnetic beads extraction, detailed step-by-step procedure for rna extraction using magnetic beads, supplementary table s : specificity and sensitivity of qpcr as a pool of independent magnetic bead rna extractions, supplementary analytical sensitivity and efficiency comparisons of sars-cov- rt-qpcr primer-probe sets detection of novel coronavirus ( -ncov) by real-time rt-pcr. eurosurveillance rna extraction kits for covid- tests are in short supply in us. the scientist. available online: /www.the-scientist.com/news-opinion/rna-extraction-kits-for-covid- -tests-are-in-short-supplyin-us- the crick covid- consortium scalable and robust sars-cov- testing in an academic center fast sars-cov- detection protocol based on rna precipitation and rt-qpcr in nasopharyngeal swab samples a simple magnetic nanoparticles-based viral rna extraction method for efficient detection of sars-cov- a colorimetric rt-lamp assay and lamp-sequencing for detecting sars-cov- rna in clinical samples a modular method for the extraction of dna and rna, and the separation of dna pools from diverse environmental sample types the single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on rapid and simple method for purification of nucleic acids multiphasic dna adsorption to silica surfaces under varying buffer, ph, and ionic strength conditions dna purification on homemade silica spin-columns high-throughput, automated extraction of dna and rna from clinical samples using trutip technology on common liquid handling robots preparation of iron oxide silica particles for zika viral rna extraction integrated dna and rna extraction using magnetic beads from viral pathogens causing acute respiratory infections loop-mediated isothermal amplification of dna rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp visual detection of isothermal nucleic acid amplification using ph-sensitive dyes global trends in emerging infectious diseases global rise in human infectious disease outbreaks the consequences of human actions on risks for infectious diseases: a review simple synthesis of functionalized paramagnetic beads for nucleic acid purification and manipulation rapid direct nucleic acid amplification test without rna extraction for sars-cov- using a portable pcr thermocycler massive and rapid covid- testing is feasible by extraction-free sars-cov- rt-qpcr sars-cov- detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank all diagnostics employees for their technical support and advice. we thank jan-philipp mallm, karsten rippe and vladimir benes for advice on magnetic bead nucleic acid purification and for lending us their instruments. we would also like to acknowledge susanne horner und fabian finger for it support. we would like to thank different institutes on heidelberg campus for providing devices and their employees for support, namely in alphabetical order: johannes backs, nina beil, marco binder, christine engeland, holger erfle, stefan fröhling, richard harbottle, joshua hartmann, christel herold-mende, angret joester, dominik niopek, simon john ogrodnik, katrin pfütze, steffi sandke, claudia scholl, rolf warta, ellen wiedtke. the authors declare no conflict of interest.viruses , , key: cord- -mjr u ak authors: hu, x.; deng, q.; li, j.; chen, j.; wang, z.; fang, z.; li, h.; zhao, y.; yu, p.; li, w.; wang, x.; li, s.; zhang, l.; hou, t. title: development and clinical application of a rapid and sensitive loop-mediated isothermalamplification test for sars-cov- infection date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mjr u ak background the outbreak of sars-cov- urgently requires sensitive and convenient covid- diagnostics to assure the containment and timely treatment of patients. we aimed to develop and validate a novel reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect sars-cov- in both qualified laboratories and point-of-care settings. methods patients with suspected covid- and close contacts between jan and april , , were recruited from two hospitals. respiratory samples were collected and tested with lamp and the results were compared with those obtained by rt-qpcr. the inconsistent samples between these two methods were subjected to next-generation sequencing for confirmation. in addition, we tested the rt-lamp on an asymptomatic covid- carrier and patients with other respiratory viral infections. results we finally collected a cohort of cases ( nasopharyngeal swabs) and the independent cohort of patients ( nasopharyngeal swabs and sputum samples). rt-lamp was validated to be accurate (overall sensitivity and specificity: . % and . %; positive and negative predictive values: . % and . %) and diagnostically useful (positive and negative likelihood ratios: . and . ). rt-lamp showed an increased sensitivity ( . % vs . %) and high consistency (kappa . ) compared with rt-qpcr for sars-cov- screening while requiring only constant temperature heating and visual inspection. the median time required for rt-lamp was less than h from sample to result. further analyses indicated that rt-lamp was feasible for asymptomatic patients and did not cross-react with other respiratory pathogen infections. conclusion the rt-lamp assay offers a rapid, sensitive and straightforward detection for sars-cov- infection, which could aid the expansion of covid- testing in the public domain and hospitals. the skyrocketing covid- outbreak has become a public health emergency of international concern. a total of , , confirmed cases and , deaths have been reported in countries as of may , , since early december of , according to the who covid- report. at present, there are still no effective drugs or vaccines reported for covid- , and prompt diagnosis, close contact tracking and quarantine management are the hallmarks for the containment of this new pandemic. accurate early diagnosis of sars-cov- infection is crucial to prevent virus transmission and provide appropriate treatment for patients. due to its nonspecific symptoms and radiological features overlapping with those of the common cold and influenza, the confirmation of sars-cov- infection entirely depends entirely on viral rna detection. [ ] [ ] rt-qpcr is the standard and widely used method for sars-cov- rna detection in clinical laboratories. despite its outstanding analytical performance, rt-qpcr detection for covid- still suffers from many limitations, such as long turnaround times (more than h), poor availability (it is currently restricted to public health laboratories), requirement of expensive instrumentation, and high proportion of false negatives or equivocal values (up to %) [ ] [ ] in upper respiratory samples due to insufficient viral materials. these limitations make the rt-qpcr test far from adequate to meet the current challenge of a tremendous undocumented infected population, asymptomatic transmission and convalescence with viral rna conversion, which highlights the pressing need for a more rapid, simple and sensitive approach to quickly identify infected patients in different settings. loop-mediated isothermal amplification (lamp) is regarded as a promising point-of-care test (poct) assay due to its advantages of high sensitivity and specificity, rapid reaction and low laboratory infrastructure requirements. reverse transcription-lamp (rt-lamp) is a kind of lamp method to detect target rna using the amv reverse transcriptase. this approach allows reverse transcription and dna amplification to be accomplished rapidly at a - ℃ constant temperature in less than an hour and in one step, and detailed amplification mechanisms have been reported previously. rt-lamp can be detected by visual turbidity or fluorescence in real time, which makes this method a practical near-patient assay. in recent years, rt-lamp has been widely used in specialized laboratory testing as well as field surveys to identify various pathogens, including mycobacterium tuberculosis, zika virus, mers-cov, and sars-cov. shirato et al. reported a useful rt-lamp assay for the diagnosis of mers that was developed in this way, with a detection limit of . copies per reaction and no cross-reaction with other respiratory viruses. hong et al. developed a real-time quantitative rt-lamp for the early and rapid diagnosis of sars-cov, which demonstrated -fold greater sensitivity than conventional rt-qpcr. to accelerate clinical diagnostic testing for covid- , we conducted a prospective cohort study to develop and validate a novel rt-lamp assay capable of detecting sars-cov- rna for potential use in centralized facilities and point-of-care settings. moreover, we compared rt-qpcr and rt-lamp on clinical samples and demonstrated that rt-lamp produced a higher sensitivity and cost effectiveness for sars-cov- detection. to the best of our knowledge, this study is the first to comprehensively assess a rapid rt-lamp test for both covid- patients and an asymptomatic carrier, with improved diagnostic value in addition to current diagnostics for sars-cov- infection. this study was designed as a prospective observational cohort study with three sequential phases. in the initial stage, we developed a visual and rapid rt-lamp assay for sars-cov- detection and assessed its anti-cross interface ability, stability and detection limit. subsequently, we evaluated the rt-lamp and standard rt-qpcr assays on nasopharyngeal swabs from a cohort of suspected covid- patients and on the serial upper respiratory samples from an asymptomatic carrier, and the insistent samples between rt-lamp and rt-qpcr were further subjected to next-generation sequencing (ngs) for sars-cov- confirmation. finally, we analyzed an additional patients with other viral infections, healthy individuals, and an independent cohort of cases suspected of having covid- to further validate the detective captivity of rt-lamp for sars-cov- . the overall study strategy is shown in figure . cohort i inpatients with clinical-radiological suspicion of covid- presenting to guangdong provincial people's hospital between january to april , were eligible for inclusion. close contacts with exposure to confirmed covid- cases were simultaneously enrolled in the present study. every participant underwent a standard sars-cov- set of investigations testing for covid- . the patients' demographic, clinical, laboratory and radiological findings were collected from their medical records. serial nasopharyngeal swabs were collected from patients during hospitalization and close contact screening. sample sizes for swabs were defined by their availability. at least one nasopharyngeal swab from suspected patients was simultaneously sent to the cdc for double checking as required, where rt-qpcr was standardly utilized for sars-cov- . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . cohort ii we enrolled an independent cohort of suspected patients from guangdong second provincial general hospital for validation. sars-cov- set testing and the diagnosis procedure for covid- were identical in the two hospitals. a nasopharyngeal swab and ml of morning sputum were collected from suspected patients to validate the diagnostic performance of rt-lamp for sars-cov- . in addition, nasopharyngeal swab samples obtained from healthy subjects and patients with other respiratory virus infections were used to test the specificity of rt-lamp for sars-cov- detection. swabs were preserved in μl of virus preservation solution (tianlong, china), which virtually inactivates the virus and preserves all rna in the specimen. the sputum samples were preprocessed by a standard nalc-naoh digestion. total rna was extracted from specimens within h using a magnetic bead-based viral rna isolation kit on the da system . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint instrument (daan gene, china) according to the manufacturer's instructions. the extracts were stored at - °c until use. the same extracted rna of each specimen was submitted in parallel to rt-qpcr and rt-lamp in a double-blind manner for testing sars-cov- in a biosafety level laboratory. the inconsistent samples between these two methods were further analyzed with ngs for verification. rt-qpcr was carried out using an officially approved clinical rt-qpcr kit for the abi covid- quantstudio dx tm real-time pcr system (applied biosystems, usa) following the manufacturer's protocol (daan gene). primer and probe sets targeting the orf ab and n genes of sars-cov- are provided in table s . a final -μl-volume reaction mixture for rt-qpcr included μl of reaction buffer, μl of enzyme solution, and μl of template rna. the cycling program started at °c for min for reverse transcription, followed by °c for minutes for pcr initial activation and cycles consisting of °c for s and ℃ for s. a cycle threshold value less than was defined as a positive test. patient were defined as having a laboratory-confirmed covid- when both targets (orf a/b and n gene) were positive, and repeated tests using another approved rt-qpcr kit were necessary for single-target-positive (orf a/b or n positive) samples. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint was selected as the target to design our rt-lamp primers because it is highly homologous among various covid- sequences and highly divergent from those of other coronaviruses examined. we designed sets of rt-lamp primers targeting the sars-cov- s gene sequence (no. mn . ) using the online primerexplorer v software (available at: https://primerexplorer.jp/e/). one set of rt-lamp primers with the best parameters was chosen, including two outer primers, f and b ; two inner primers, forward inner primer (fip) and backward inner primer (bip); and two loop forward (lf) and backward (lb) primers ( figure s ), and synthesized by invitrogen (shanghai, china). primer specificity was verified with a blast search of the genbank nucleotide database via comparison with other coronavirus and published sars-cov- sequences, and the percent mismatch result is offered in table s . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . potential cross-reactivity for rt-lamp. their rt-lamp products were assayed by % agarose gel electrophoresis. to determine the lower detection limit of the rt-lamp method for covid- , a -fold gradient dilution series of synthetic sars-cov- s gene cdna ( . × - . × - ng/reaction) was tested as a template for amplification with rt-lamp. the minimum concentration of the positive reaction was recorded. this dilution series was run in parallel with rt-qpcr using primers that targeted this same region of the covid- genome. the detection limit of rt-lamp was determined by comparing the lowest concentration of the positive reaction with that of rt-qpcr. the inconsistent samples between rt-lamp and rt-qpcr and samples from covid- patients that were rt-qpcr negative were further analyzed with multiplex pcr-based enrichment plus ngs to detect the sars-cov- genome. briefly, total rna was reverse transcribed to synthesize first-strand cdna with random hexamers and superscript iii reverse transcriptase kit (vazyme, china). two-step sars-cov- genome amplification was performed with two pooled mixtures of primer sets (designed by genskey medical technology co., ltd.). the pooled primer sets were designed to cover the entire sars-cov- genome. cdna was . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint mixed with the components of the first pcr following the manufacturer's instructions. the nd pcr was performed using the index primer and the constructed libraries were sequenced on the illumina novaseq pe platform. analysis was carried out based mainly on an in-house pipeline produced by genskey medical technology. raw sequencing data was quality trimmed and subsequently filtered if shorter than bases by fastp v . . . sequence reads were first filtered against the human reference genome and then aligned to a reference genome of sars-cov- (nc_ . ) using bowtie v . . . the mapped reads were assembled with spades v . . with kmers ranging from to to obtain the coronavirus genome sequences. the sensitivity, specificity, positive and negative predictive values, likelihood ratios and their respective % confidence intervals for rt-lamp and rt-qpcr testing of nasopharyngeal specimens were calculated, and agreement analysis was computed using kappa concordance coefficients (a value ≥ . was deemed good) and percentage agreement (≥ . was considered good). statistical analyses were performed in the r programming environment. written informed consents were obtained from all participants before the study, and the study was approved by the ethics committee of each participating institution. the analysis was conducted on samples collected during standard covid- tests, with no extra burden on patients. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint as described in the materials and methods, we developed a rapid and simple rt-lamp assay to detect sars-cov- rna, and positive reactions resulted in a color change from purple to blue due to decreased magnesium concentration in the presence of extensive bst dna polymerase activity, while negative reactions retained the purple color. figure s shows the overall procedure of the rt-lamp assay. rt-lamp primers for covid- were specific and had a . - . % nucleotide mismatch with sars, mers and other coronavirus sequences (table s ), and a cross-interface experiment further demonstrated that rt-lamp did not cross-react with other human-pathogenic coronaviruses and common virus pathogens, supporting the specificity of this assay for covid- ( figure s ). our dilution experiments of the synthetic sars-cov- s gene showed shown the analytic limit of detection (lod) of rt-lamp relative to that of rt-qpcr for the detection of sars-cov- ( figure s ). the resulting lod was approximately . × - ng per -μl reaction solution (i.e., . copies/reaction) for rt-lamp and . × - ng/reaction (i.e., . copies/reaction) for rt-qpcr. rt-lamp exhibited a -fold higher sensitivity than the rt-qpcr used in the current clinical test, similar to previous lamp studies. [ ] [ ] characteristics of the subjects is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint presenting primarily with fever, cough/expectoration, and muscle pain/fatigue ( table ) table . we first evaluated the clinical application of the rt-lamp assay on nasopharyngeal specimens from cohort i. of these nasopharyngeal swabs, swabs were confirmed as sars-cov- positive according to the combined criteria of rt-qpcr positive results ( samples) and ngs confirmation ( samples) (see table , table s and figure s ). thirty-one out of clinically positive samples were determined to be positive using the rt-lamp assay, (table ) . compared with rt-qpcr, rt-lamp had a significantly better sensitivity ( . % vs . %) and comparable specificity ( . % vs %) for the diagnosis of sars-cov- infection ( table . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint ). detection results obtained using rt-lamp were in good concordance with those obtained using rt-qpcr, with a cohen's kappa of . ( . - . ), % positive predictive agreement and . % negative predictive agreement. these observations are in line with data reported by the studies from baek et al. [ ] [ ] [ ] in addition to exploring the diagnostic potential of rt-lamp on active covid- patients, we also tested the rt-lamp assay on an asymptomatic covid- carrier. rt-lamp has a higher sensitivity in detecting sars-cov- , particularly for those samples with a low viral load, and also suggested that rt-lamp can be used for the diagnosis of asymptomatic covid- carriers. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint we next validated the rt-lamp assay on an independent cohort ii of covid- patients and covid- exclusion cases. both one nasopharyngeal swab and one sputum specimen were collected from every participant in cohort ii. a total of samples comprised of positive samples ( swabs and sputum) and negative samples ( table and table s ). table ), and the agreement between the two assays was excellent (kappa . ( . - . ), table ). these observations corroborate with the results from cohort i as well as the previous rt-lamp findings, [ ] [ ] [ ] suggesting that rt-lamp may improve the sensitivity of pathogenic diagnosis for covid- . to further assess whether the rt-lamp assay was covid- specific, swab specimens from patients with influenza (n = ) or respiratory viral infections (n = , representing mycoplasma pneumoniae, hpiv- / / , rsv-a/b, rsv, and hadv-b/e) and healthy individuals were subjected to the rt-lamp assay. no positive results were observed, which demonstrated that rt-lamp-based detection can distinguish sars-cov- with no crossreactivity for other respiratory viruses, similar to reports in recent studies. [ ] [ ] [ ] we summarized the rt-lamp assay reported here for sars-cov- detection in two cohorts. rt-lamp exhibited an overall sensitivity of . % (higher than the . % for rt-qpcr), an overall specificity of . %, high consistency (kappa . ) with the rt-qpcr, and a median turnaround time less than h from sample to result in the detection of clinical specimens . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint from two cohorts (figure ). additional advantages of rt-lamp include cost effectiveness, simple operation and visual determination capability, which facilitate sars-cov- screening in well-equipped labs as well as in the field (figure ). rapid and reliable diagnosis is of particular importance for the containment of covid- outbreak. we described a simple and sensitive rt-lamp approach to rapidly diagnose sars-cov- infection. the robustness of the present study was demonstrated, as this rt-lamp assay was useful to diagnose active covid- patients and asymptomatic carriers and generally not confounded by other respiratory pathogen infections using clinical samples from two hospitals. existing methods to detect sars-cov- are based mainly on rt-qpcr, ngs and igm and igg immunological tests. comparing the results between rt-lamp and rt-qpcr, rt-lamp provided a better sensitivity ( . % vs . %) than rt-qpcr for sars-cov- . this added sensitivity is important in consideration of a significant number of covid- patients that have presented with negative qpcr results or the "relapse after negative" phenomenon due to potentially large variability between clinical samples, low-virus-titer samples and even disrupted binding of rt-qpcr primers due to variation in the viral genome. we used the self-developed bst dna polymerase in this rt-lamp assays, which was demonstrated a higher polymerization activity than the commercial bst dna polymerase and ensured the high sensitivity of this rt-lamp method. based on these findings, we propose that the rt-lamp assay was able to detect viral rna not only in rt-qpcr positive samples but also in those inconclusive samples. we found that rt-lamp was less sensitive and informative than ngs in our study and other literature. [ ] [ ] [ ] [ ] ngs is a robust tool for obtaining extensive genetic information and completing whole-genome sequence with the lod as low as copies/ml for sars-cov- detection, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint ranking as one reference test for covid- , especially for those challenging samples containing low viral content. , [ ] [ ] however, compared to the complex and costly ngs platform, rt-lamp had the advantage of low-threshold of infrastructure, data processing requirement and cost effectiveness, which enabled this friendly assay to be immediately deployed in hospitals and communities. rt-lamp also showed no cross-reactivity with other viruses that manifest similar respiratory diseases, and thus, the specificity of rt-lamp was higher than that reported for the igm and igg detection. in addition, we described the accuracy of covid- rt-lamp by means of likelihood ratios. likelihood ratios are not affected by disease prevalence, and their values higher than ten and lower than one strongly support the diagnostic value of a test. based on this metric, the near-patient lamp assay used in this study is diagnostically useful for covid- . overall, the established rt-lamp in this study could be a powerful complementary method for monitoring massive numbers of exposed individuals as well as aiding with screening efforts in hospitals and public domains, especially in areas with limited laboratory capacities. additionally, nasopharyngeal swabs from covid- patients had a higher positive rate than sputum specimens in both the rt-qpcr and lamp assays. liu et al. reported that the detection rate of sars-cov- rna in nasopharyngeal swabs was lower than that in bronchoalveolar lavage fluid and sputum. this inconsistency is most likely due to poor sputum quality and fluctuation of virus rna during different stages of the disease course. despite this inconsistency, nasopharyngeal swabs are noninvasive and easy to acquire, and evidence has shown that sars-cov- replicates actively in upper respiratory tissue. therefore, we argue that nasopharyngeal swabs are suitable for the detection of sars-cov- at an early stage of infection. we note that four samples from non-covid- cases were rt-lamp positive but rt-qpcr negative (see table ), as reported previously. the four false positive results by rt-lamp . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . were caused by aerosol contaminants, as we retested these samples in another clean room and obtained the negative rt-lamp results as expected. contaminant issues are not rare for nucleic acid testing, even when the best available reference laboratory tests are used. precautions to prevent cross-contamination or aerosol contaminants during assay are highly recommended, including using a spray solution for the elimination of potential rna fragments and changing gloves frequently. the rna extraction-free rt-lamp assay could address this important question. since this study was completed, the sars-cov- rt-lamp test has been optimized further, with the use of lyophilized reagents and direct detection of sars-cov- without any need to conduct rna extraction. this one-step single-tube rt-lamp hastens reaction time and minimizes false positive reactions and would be an ideal poct for covid- if validated in future studies. one limitation of our study was the relatively small sample size of positive covid- cases, which has resulted in widened confidence intervals for our estimates of diagnostic accuracy. we tested the samples using rt-lamp in a blind manner, and the designation of the real status of sars-cov- infection in clinical samples was based on a set of combined criteria of rt-qpcr and subsequent ngs confirmation to obviate potential false negative or positive results. we further validated the diagnostic potential of rt-lamp in another independent cohort with nasopharyngeal swabs and sputum samples. therefore, despite our small sample size, our study provided sufficient robustness for the rt-lamp assays. in conclusion, we developed a simple and rapid rt-lamp assay for sars-cov- detection and demonstrated its high diagnostic sensitivity and specificity among clinical samples. our findings suggest that rt-lamp can be an appropriate auxiliary assay for the diagnosis and epidemiologic surveillance of covid- in different hospital and community settings. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . notes: sen, sensitivity; spe, specificity; ppv, positive predictive value; npv, negative predictive value; plr, positive likelihood ratio; nlr, negative likelihood ratio. inconsistent samples between rt-lamp and rt-qpcr assays were further determined by next-generation sequencing . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint • positive • negative ngs positive . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint world health organization diagnosis and treatment of novel coronavirus pneumonia (trial version sixth) clinical management of severe acute respiratory infection (sari) when covid- disease is suspected genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention antibody responses to sars-cov- in patients of novel coronavirus disease sars-cov- viral load in upper respiratory specimens of infected patients positive rt-pcr test results in patients recovered from covid- diagnostic accuracy of loop-mediated isothermal amplification as a near-patient test for meningococcal disease in children: an observational cohort study loop-mediated isothermal amplification of dna loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples rapid and specific detection of asianand african-lineage zika viruses detection of middle east respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (rt-lamp) development and evaluation of a novel loopmediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus poor concordance between sequential transbronchial lung cryobiopsy and surgical lung biopsy in the diagnosis of diffuse interstitial lung diseases rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay rt-lamp for rapid diagnosis of coronavirus sars-cov- development of a reverse transcription-loopmediated isothermal amplification as a rapid early-detection method for novel sars-cov- the spectrum of severe acute respiratory syndromeassociated coronavirus infection enhancement of polymerase activity of the large fragment in dna polymerase i from geobacillus stearothermophilus by site-directed mutagenesis at the active site nanopore target sequencing for accurate and comprehensive detection of sars-cov- and other respiratory viruses. medrxiv preprint genomic epidemiology reveals multiple introductions of zika virus into the united states multiplex pcr-based next-generation sequencing and global diversity of seoul virus in humans and rats multiple approaches for massively parallel sequencing of hcov- (sars-cov- ) genomes directly from clinical samples. biorxiv preprint molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes diagnostic tests : likelihood ratios positive rate of rt-pcr detection of sars-cov- infection in cases from one hospital in viral load of sars-cov- in clinical samples this work was supported by the "peak project" scientific research special funding all the authors have declared no competing interests. notes: ppv, positive predictive value; npv, negative predictive value; plr, positive likelihood ratio; nlr, negative likelihood ratio.in cohort i, out of nasopharyngeal swabs from covid- patients were confirmed as sars-cov- positive according to the criteria of rt-qpcr ( samples) and ngs confirmation ( samples, table s ). in cohort ii, out of samples (paired nasopharyngeal swabs and sputum samples) from covid- patients were determined as sars-cov- positive accordingly ( were rt-qpcr-positive and were ngs-positive, table s ). key: cord- -tcvs beg authors: lee, szu-yuan; huang, jhen-gang; chuang, tsung-liang; sheu, jin-chuan; chuang, yi-kuang; holl, mark; meldrum, deirdre r.; lee, chun-nan; lin, chii-wann title: compact optical diagnostic device for isothermal nucleic acids amplification date: - - journal: sens actuators b chem doi: . /j.snb. . . sha: doc_id: cord_uid: tcvs beg we recently reported the successful use of the loop-mediated isothermal amplification (lamp) reaction for hepatitis b virus (hbv) dna amplification and its optimal primer design method. in this study, we report the development of an integrated isothermal device for both amplification and detection of targeted hbv dna. it has two major components, a disposable polymethyl methacrylate (pmma) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of dna amplification by-product, magnesium pyrophosphate. we have established a correlation curve (r( ) = . ) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. for the applications of rapid pathogens detection, we also have established a standard curve (r( ) = . ) by using lamp reaction with a standard template in our device. moreover, we also have successfully used the device on seven clinical serum specimens where hbv dna levels have been confirmed by real-time pcr. the result indicates that different amounts of hbv dna can be successfully detected by using this device within h. around the world, the hepatitis b virus is one of the most common viral pathogens, having infected more than million people [ ] . at present, serologic diagnosis of hbv infections is based on the viral antigens and antibodies. however, the reveal-hbv (risk evaluation of viral load elevation and associated liver disease/cancer-hepatitis b virus) study indicates that the serum level of hbv dna ( , copies/ml) is a strong risk predica-tor of hepatocellular carcinoma or cirrhosis [ , ] . it would thus be clinically valuable to have a molecular diagnostic method for screening and monitoring the progress of hepatitis. however, the long reaction time of traditional pcr amplification and the high cost of thermalcycler are still major issues for such a screening application. therefore, it is important to develop a rapid and accurate diagnostic device for field applications as soon as possible [ ] . even though many nucleic acid amplification methods are currently available, a low cost, yet rapid method would be extremely useful, especially in developing countries. there are several pcr-based amplification methods [ ] , such as nucleic acid sequence-based amplification [ ] and self-sustained sequence replication, for nucleic acid amplification. however, these methods require a precise instrument to provide the efficient thermal cycles and to shorten the total amplification time for a test run. in general, it takes up to . h for a pcr test with the present state of art equipment. alternatively, isothermal amplification methods, e.g., strand displacement amplification [ ] , branched dna amplification, invader, rolling circle amplification [ ] and loop-mediated amplification method (lamp) [ ] , have been proposed for amplifying the targeted nucleic acid sequence under a single working temperature condition with special designs for the buffer system and primers to prevent non-specific amplification. it would thus allow for the development of a low-cost device for rapid pathogen detection. loop-mediated isothermal amplification (lamp), originally developed by notomi et al. [ ] , utilizes a designed set of six primers, termed inner and outer primers, to recognize specific gene sequences, and a polymerase with strand displacement activity to generate large amounts of amplified product (> copies) within h [ ] . moreover, it has been shown that the well-known pcr inhibitors in the blood (e.g., heme) have little impact on the lamp reactions [ ] . it is believed that the bacillus stearothermophilus (bst) dna polymerase used in the lamp reactions is more resistant to these inhibitors. therefore, the lamp reaction has been successfully applied for fast genetic screening tests in many acute infectious diseases, including mycobacterium tuberculosis [ ] , severe acute respiratory syndrome virus [ ] , human influenza viruses [ , ] , avian influenza viruses [ ] and herpes viruses [ , ] , with special primer design and buffer adjustment. among these, the lamp method has been shown to have great promise for the amplification of hbv dna. in addition, the dna yield of the lamp reaction ( g/ l) is much higher than that of the traditional pcr ( . g/ l) [ ] . recently, we have successfully demonstrated that the level of turbidity, which is due to the by-product (magnesium pyrophosphate) of dna polymerization, has a high correlation to the amounts of amplified dna. then, we decided to work with a total of l of lamp reaction volume because of the practical limitations of turbidity detection. in addition to the use of the lamp protocol, we have adapted a simulated chemical reaction to mimic the by-product production without using expensive polymerase and primers [ ] . it can serve as an internal quality check for the system validation. therefore, it is our intention to design and implement a compact integrated device with a disposable chip and simple quantitative optical read-out for low-cost applications. in this study, the goal is to develop an integrated isothermal device for real-time detection of hbv viral dna via the lamp amplification method. it has two major components, a disposable pmma micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to precipitation of dna amplification by-product, magnesium pyrophosphate. we decided to work with a total of l of lamp reaction volume after adding l of dna sample because of the practical limitations of turbidity detection. the performance of this integrated isothermal device has been tested by using within and between runs. for the applications of rapid pathogens detection, we have successfully used the device on seven clinical serum specimens and confirmed hbv dna levels with real-time pcr. the results of using this device indicate that different amounts of hbv dna can be successfully detected within h with a threshold level of , copies/ml, which is the recommendatory quantity of reveal-hbv study. this integrated isothermal device can be advantageous in a wide spectrum of field applications, including pathogen detection and gene testing. the design methodology of primers used has been discussed in our previous study [ ] . in brief, the target dna sequences and the partial hbv polymerase gene sequences are collected from a public data base and then aligned to find highly conserved fragments. then, the primer design can be executed by using the available software, gene runner (hastings software, inc., hudson, ny, usa), to check design parameters. the melting temperature (t m ) of each primer is calculated by the nearest-neighbor t m theory. finally, the designed primers are synthesized by the contract services (quality systems, inc., taipei city, taiwan, roc) followed by having its concentration optimized with a reaction buffer in the amplified test. the partial hbv polymerase gene was directly cloned into pgem-t easy vector (promega, madison, wi, usa) as standard template dna. the lamp assay was performed in a total of l of the mixtures, which contain pmol each of ib-fip ( -tggaattagaggacaaacgggtgctgctatgcctcatctt- ) and ib-bip ( -gctcaaggaacctctatgtttcgatgatgggatgggaataca- ), pmol each of ib-f ( -ggcgttttatcatcttcct- ) and ib-b ( -aggttacttgcgaaagcc- ), pmol each of ib-loopf ( -taccttgatagtccagaagaacc- ) and ib-loopb ( -ctacggacggaaactgcac- ), . mm dntps, m betaine, mm tris-hcl (ph . ), mm kcl, mm (nh ) so , mm mgso , . % triton x- , units of the bst dna polymerase large fragment (new england biolabs, ipswich, ma, usa), and l of dna standard template-a partial hbv polymerase gene cloned into a pgem-t easy vector or purified dna. we utilized a set of six primers to recognize specific hbv gene sequences. this mixture was incubated in a mastercycler ® gradient pcr machine (eppendorf, hamburg, germany) or our miniaturized device at • c for h. the white precipitate of reaction by-product can be observed by naked eye. aliquots of . l of lamp products were electrophoresed in % agarose gels ( × tbe) and then stained with sybr green i dye for verification by fluorescent imager (geldoc-it imaging system, uvp, upland, ca, usa). in addition, the sequences of lamp product were confirmed by the abi -avant dna sequencer (applied biosystems, foster city, ca, usa). our integrated isothermal device has two major components, a disposable polymethyl methacrylate (pmma) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of dna amplification by-product, magnesium pyrophosphate. the disposable micro-reactor is constructed from two pmma parts to create a reaction chamber of -mm optical path length by using uv-light curing adhesives (ultrawide gn , everwide chemical company, yunlin county, taiwan, roc). for the lamp reaction, the reaction chamber is filled with l reagent manually via micropipette and then is sealed with gluey aluminum foil. the base apparatus consists of an optical detection unit, a thin-film heater, a temperature controller, and a power supply. the optical detection unit (fs-v g, keyence corporation, osaka, japan) employs a light emitting diode (led) light source at nm and a phototransistor detector with an extension of collimated optical fibers for the collection of forward scattering light. the temperature controller has a w power supply and two in. × in. thin-film kapton tm heaters (minco, minneapolis, mn, usa), which is controlled by a proportional-integral-derivative (pid) controller (anly electronics, taipei county, taiwan, roc) with one thermal coupler feedback. after the insertion of assembled micro-reactor chips into the base apparatus, we can initiate the hbv lamp reaction at • c through out the whole experimental time course. at the same time, the scattering light intensity is measured by the optical detection unit. in general, turbidity refers to the scattering of light by particles and has to be measured and calculated indirectly from eq. ( ) [ ] assuming no absorption in the path length: where i is the intensity of incident light and i is the intensity of transmitted light. during the dna polymerization process, white precipitation of magnesium pyrophosphate will be produced in the presence of magnesium ions and pyrophosphate ions as shown in the following equations: instead of using dntp and dna polymerases to initiate the precipitation, we have adapted a simulated reaction, as shown in eq. ( ), to mimic the production of magnesium pyrophosphate which is used for evaluating the performance of our device: this reaction is performed in a total of ml solution containing the following reagents, × thermolpol buffer ( mm tris-hcl (ph . ), mm kcl, mm (nh ) so , mm mgso , . % triton x- ), mm mgso , gradient concentrations of k p o or k po from . to . mm and double-distilled water. the mixture is incubated in the disposable micro-reactor at • c for h to measure the end-point turbidity by our system. in addition, this experimental result was confirmed by a uv-visible spectrometer (varain, palo alto, ca, usa) at nm. it also can be used to establish the correlation curve between the concentration of pyrophosphate ions and the level of turbidity. a series of seven serum specimens were obtained from patients at national taiwan university hospital (ntuh). the serum hbv viral dna was extracted by using the qiaamp viral dna mini kit (qiagen, valencia, ca, usa). briefly, l of viral particles lysis buffer (avl buffer) was mixed with l of serum. the avl buffer will lyse viral particles under highly denaturing conditions to inactivate dnases and to provide optimum binding buffering conditions. the mixture was incubated at room temperature for min and then mixed with l of ethanol. then, l of the mixture was transferred to the qiaamp spin column and was centrifuged at × g for min. five-hundred microliters of washing buffer (aw buffer) was added to the spin column and centrifuged at × g for min. then, l of washing buffer (aw buffer) was added to the spin column and centrifuged at , × g for min. the dna was eluted from the column by adding l of rnase-and dnase-free water. after centrifuging at × g for min, the supernatant contained the dna and was ready for use. in addition, the hbv dna viral load was determined by real-time pcr which used the reagent of quantiplex tm hbv dna assay (chiron corporation, emeryville, ca, usa) and the detection system of abi prism sequence detection system (applied biosystems, foster city, ca, usa). the hbv viral load of these seven samples ranged from an undetectable level to more than × copies/ml for the testing of our device. following the principle of lamp reaction, the amplified products elongated to a length of several kbp and when generated they showed complex cauliflower-like structures [ ] . we demonstrated that the positive sample reveals many bands of different sizes after agarose gel electrophoresis (fig. a) . with an increase of lamp the structure of the disposable micro-reactor. the disposable micro-reactor has a reaction chamber and -mm optical pathway. the reaction chamber is mm-long, mm-wide and mm in height. (b) the structure of the base apparatus. the base apparatus is made with pmma to create a detection pathway and sliding track. it has two slices of flexible heaters on the top and on the bottom sides. the light source fibers and the optical sensor are lined on the detection pathway and put close (about mm) to each other. the reaction box is mm-long, mm-wide and mm high. the detection pathway is mm-long, mm-wide, mm-height and the sliding track is mm-long, mm-wide, mm-height. (c) picture of the disposable micro-reactor. this micro-reactor is just a home-made product. the disposable micro-reactor is constructed from two components of pmma and glass slide cover. it has a reaction chamber and a -mm optical pathway without heaters or sensors. (d) picture of the base apparatus. for the lamp reaction, the reaction chamber is filled with -l reagent and then sealed by gluey aluminum foil. the disposable micro-reactor component can be inserted into the base apparatus and the hbv lamp reaction can be started under isothermal condition. this system can provide appropriate reaction conditions for hbv lamp dna amplification. products, a large amount of by-product, magnesium pyrophosphate (mg p o ), is produced and precipitated in the reaction mixture. the white precipitate of lamp reaction by-product can be observed by naked eye (fig. b) . other than the optimization of primers for the lamp reaction, the reaction volume is a critical issue in the design of such a miniaturized device for amplification and detection. we decided to work with a total of l of lamp reaction volume because of the practical limitations of turbidity detection. the prototype of our integrated isothermal device has two components: a disposable micro-reactor ( fig. a) and a temperature-regulated optical detection unit (base apparatus) (fig. b) . for the lamp reaction, the disposable pmma micro-reactor is filled with l reagent (fig. c ) and then sealed with gluey aluminum foil. it can be inserted into the base apparatus and the hbv lamp reaction can be started under isothermal conditions. simultaneously, we can detect the turbidity of the lamp by-product in the base apparatus (fig. d ). to evaluate the performance of the base apparatus for dna amplification and detection, we have adapted a simulated chemical reaction to produce magnesium pyrophosphate. the simulated reaction utilizes potassium pyrophosphate and magnesium sulfate to synthesize magnesium pyrophosphate, as established in our previous study [ ] . white precipitates can be observed when the concentration of pyrophosphate ions exceeds . mm. fig. a shows the results of a h end point turbidity measurement by using a spectrometer (r = . ) or our own system (r = . ) at • c. fig. b shows the reproducibility results of within run for turbidity detection in our device. it shows that the coefficient of variation (cv%) within run is very low (< %). however, our system shows larger fluctuations between runs than does the spectrometer. this might be due to minor uncertainties in our fabricated devices, which will be able to control within acceptable limits with mass production of disposable chip. with a total of l of reagents and sample dna in sealed reaction chamber, we can start the reaction under isothermal condition ( • c) and monitor the intensity of scattering light by the optical unit. the real-time data on turbidity, decreases during the first min, and then increases until reaching a plateau at min. fig. a shows the superimposed plots of turbidity data from different initial concentrations of dna template. all of these samples show obvious changes in min. from these results, it would be reasonable to take turbidity value of min as a critical point for determining the end point of nucleic acid amplification. a linear relationship with a good correlation coefficient (r = . ) between measured values of turbidity and the initial concentration of dna template is shown in fig. b . the hbv lamp reaction is also performed in the thermalcycler at • c and then the amplified products are analyzed by electrophoretic analysis to confirm the consistency of the experi- mental results between our new system and the traditional system (fig. ) . in our previous study, we demonstrated that our hbv lamp reaction has great specificity and sensitivity ( copies/ l) [ ] . to confirm the results of the lamp reaction for hbv dna template amplification and detection in the integrated isothermal device, seven clinical serum specimens were obtained from patients with chronic hepatitis b at national taiwan university hospital. all of the serum specimens have been quantified by realtime pcr analysis as well. table shows the quantitative results of turbidity measurements when using the integrated isothermal device in the lamp reagents containing the different amounts of dna template from the clinical specimens. following the results of the reveal-hbv study [ ] , the serum level of hbv dna ( , copies/ml) is a strong risk predicator of hepatocellular carcinoma or cirrhosis. our quantitative results are good indicators for distinguishing the reported hbv dna threshold level in serum in h. in our previous study [ ] , we have demonstrated that the hbv lamp reaction can amplify specific dna sequences in less than h with high specificity and sensitivity ( copies/ l). unlike traditional pcr results, lamp products consist of several inverted-repeat structures. after agarose gel electrophoresis, the positive lamp reaction reveals many bands of different sizes. the sequences of lamp products were confirmed by abi -avant dna sequencer. moreover, in a comparison of the lamp reaction with pcr, the most significant advantage of the lamp reaction is its ability to amplify specific dna sequences under isothermal conditions ( • c) without thermo-cycling. it thus has great potential for the development of an integrated isothermal device with disposable micro-reactor for low-cost applications. following the principle of lamp reaction, both its yield of dna and magnesium pyrophosphate precipitate are greater than pcr reaction [ ] . in our system, an oven-like space has been designed and implemented to provide appropriate temperature conditions. an optical detection unit with a green led light source is used to detect the lamp reaction by turbidity derived from the mag-nesium pyrophosphate formation. in general, turbidity refers to the scattering of light by particles. usually, turbidity is not measured directly, but it is estimated from the optical density (od). it is assumed that the intensity of the input light is constant and that the samples have no absorbing constituents (dna absorption peak: nm) and that there are no changes in other loss mechanisms for the transmission of light, such as surface coatings, alignment, etc. then the changes in detected light intensity can be ascribed to changes in total scattering. thus, we interpret changes in detected light intensity as having resulted from changes in turbidity. after the integrated isothermal device is constructed, the performance of the base apparatus should be confirmed. first, we check the efficiency of the heating and optical detection unit. we have adapted a simulated chemical reaction to mimic the by-product (magnesium pyrophosphate) production without using expensive polymerase and primers. then, the results of the turbidity measurement from using a spectrometer are compared with the base apparatus. the reaction curves also have a similar trend between turbidity and initial amounts of potassium pyrophosphate. it shows that our system can provide appropriate reaction conditions for dna amplification and detection. second, we demonstrate that the results of within runs exhibit greater reproducibility of turbidity detection through the use of this base apparatus. this shows that our system has good stability. however, the results of between runs from our system show greater fluctuations than the results from the spectrometer, because of the disposable micro-reactor being a home-made product. in the future, we can use plastic molding for mass production to improve the quality and consistency of the micro-reactor and develop multi-channel micro-reactor type to achieve large-scale quantification test. the stability of light source and the precision of light-alignment are also important for the improvement of overall system performance. after the performance of micro-reactor system is confirmed, hbv lamp reactions can be transplanted into this system. the reaction curves decrease rapidly and achieve the lowest level of turbidity in min. then, the reaction curves increase quickly and achieve the highest level of turbidity in min. we think that this phenomenon is related to the principle of brownian motion [ ] . when the reaction mixture is initially raised from room temperature to • c, some microscopic clusters or particles may dissolve, causing an increase in the intensity of transmitted light. in min, the turbidity of hbv lamp reaction obviously changes. the standard curve shows that the numeric of the turbidity are highly correlated to the initial amounts of hbv template dna (r = . ). after min, gravity causes the numeric of turbidity to continually decrease because of the magnesium pyrophosphate precipitation. therefore, the numeric of turbidity in min could be a critical point for judging the performance of the hbv lamp reaction. in addition, the hbv lamp reaction is also performed in the thermalcycler. the amplified products can be analyzed by electrophoresis to confirm the consistency of the lamp reaction between our device and thermalcycler. in fig. , we show that the electrophoretic result of the hbv lamp reaction is performed in the thermalcycler (lane ) or in our device (lane ). it seems that there are many small fragments (< bp) of hbv lamp reaction as shown in lane . we presume that this phenomenon is related to the slow heat transfer in our device, which causes bst dna polymerase cannot elongate the lamp product under stable condition, initially. the reveal-hbv study indicates that serum level of hbv dna ( , copies/ml) is a strong risk predicator of hepatocellular carcinoma or cirrhosis [ ] . seven serum specimens with chronic hepatitis b were collected from national taiwan university hos-pital. these clinical specimens had triplicate tests by hbv lamp reaction with this integrated isothermal device. the hbv viral load of these seven samples ranged from an undetectable level to more than × copies/ml for the testing of our device. the frequency of positive results in triplicate test represents by fractional number. in addition, our hbv lamp reaction has good sensitivity ( copies/ l). even though the quantitative results from our device are higher than the data from real-time pcr, these results (table ) are valid to effectively distinguish the hbv dna threshold level in the serum samples according to current protocol. thus, using the integrated isothermal device provides great assistance for early diagnosing and monitoring the progress of chronic hepatitis b. however, there are several inhibitors from the blood samples that might potentially interfere with the amplification processes require further elucidations. in this study, the serum hbv viral dna was extracted by using the qiaamp viral dna mini kit. the purified dna is free of protein, nucleases, and other contaminants and inhibitors. in , a study demonstrated that the lamp assay does not require purified dna for efficient dna amplification [ ] . these pcr inhibitors have little impact on the lamp reactions even though there are inhibitors (e.g., heme) in blood could severely affect the amplification of dna in pcr assays. as dna polymerase have different susceptibilities to pcr inhibitors, they suspect that the bacillus stearothermophilus (bst) dna polymerase used in the lamp reactions is more resistant. in the future, the influence of various inhibitors, e.g., elevated levels of triglycerides, bilirubin, hemoglobin, and non-specific human dna, will be addressed when we directly detect dna from serum or heat-treated blood by lamp. it would also be necessary for us to show that systemic lupus erythematosus (sle) and rheumatoid arthritis have no impact on the method during large-scale clinical experiment. we will also try to improve the sensitivity and further reduce the reaction volume to l by using surface mode of optical detection [ ] [ ] [ ] . in conclusion, we have successfully demonstrated the feasibility of the lamp reaction for hbv dna amplification and detection within h in this novel integrated isothermal device. using the lamp reaction in the disposable micro-reactor component described here can amplify hbv dna with high specificity and efficiency under isothermal conditions. the base apparatus component can also provide appropriate reaction conditions as well as a steady optical detection system for detecting turbidity derived from magnesium pyrophosphate formation without fluorescence labeling. thus, using this integrated isothermal device greatly assists early diagnosing and monitoring the progress of chronic hepatitis b. it can improve the prognosis of patients and enormously save medical expenses. in the future, we hope to provide a multichannel, portable, label-free, real-time monitoring medical device for rapid identification and quantification of pathogenic organisms and point-of-care applications. epidemiology and natural history of hepatitis b, semin. liver dis predicting cirrhosis risk based on the level of circulating hepatitis b viral load correlation of hbv dna levels in serum and liver of chronic hepatitis b patients with cirrhosis micro total analysis system (micro-tas) in biotechnology enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia nucleic acid sequence-based amplification strand displacement amplification-an isothermal, in vitro dna amplification technique mutation detection and single-molecule counting using isothermal rolling-circle amplification loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation detection of human influenza a viruses by loop-mediated isothermal amplification development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method sensitive and rapid detection of herpes simplex virus and varicella-zoster virus dna by loop-mediated isothermal amplification detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation efficient, specific, compact hepatitis b diagnostic device: optical detection of the hepatitis b virus by isothermal amplification optimal hepatitis b virus primer sequence design for isothermal amplification turbidity measurements of bacterial cultures in some available commercial instruments brownian motion, fluctuation and life admittance loci design method for multilayer surface plasmon resonance devices design and fabrication of an alternating dielectric multi-layer device for surface plasmon resonance sensor a miniaturized germanium-doped silicon dioxide-based surface plasmon resonance waveguide sensor for immunoassay detection she made a dive to m below sea level on the the study was supported by grants from national science and technology program (nsc - -b- - ) for biotechnology and pharmaceuticals of national science council. the authors thank the national science council for its assistance in facilitating international cooperation with the university of washington, seattle, usa. key: cord- -yle z z authors: murnain, kaila l; ooi, ju‐lee; sharma, neil s title: evaluation of the slit lamp shield to reduce droplet exposure date: - - journal: clin exp optom doi: . /cxo. sha: doc_id: cord_uid: yle z z nan editor: the current covid- global pandemic has brought infection control measures to the forefront of international attention. patients with sars-cov- infection may be asymptomatic but infectious. the face-to-face proximity of clinicians and patients during slitlamp examination potentially places eyecare providers at a high risk of aerosolised particles from respiratory droplets. similarly, patients may be at risk from an unknowingly infected clinician, which could have disastrous consequences, especially in a busy clinic with a large proportion of elderly patients. recognising this potential risk, we recently designedand had urgently manufactured the slit lamp shield, made in australia from clear acrylic (plexiglass). our slit lamp shield is distinct from other slitlamp breath guards by having angled side-wing panels that provide a large physical barrier while still allowing access to the slitlamp controls. the use of slitlamp barriers has become increasingly common during the current covid- global pandemic. the american academy of ophthalmology has recommended the use of commercially manufactured barriers that can be regularly disinfected. we decided to evaluate the ability of the slit lamp shield to reduce potential droplet exposure. in our simulation (video s ), a clinician attired in personal protective equipment including surgical mask and face shield was positioned in the examination position. a staff member in the patient position executed a single release of a commercially available fluorescent dye spray (vericlean; diversey inc, fort mill, sc, usa). this was performed both without and with the slit lamp shield. without the shield, dye was found on the clinician's face shield, mask, gown, gloves, desk and the machine itself. when the experiment was repeated with the shield in position, most of the dye was located on the outside of the shield, with smaller amounts on the clinician gloves, desk and machine ( figure ) . importantly, there was no dye on the clinician's face shield or mask. we repeated the experiment on several occasions and obtained similar results. we acknowledge the limitations of our methodology including that it is not validated for the projectile direction, speed and turbulence of a true cough and is performed in an artificial experimental setting. nevertheless, this demonstration illustrates the potential benefit of using a barrier shield during slitlamp examination. it is important to remember to continue to use other personal protective equipment as guided by local protocols, and that frequent disinfection of the shield, equipment and surfaces is still required. preparedness among ophthalmologists: during and beyond the covid- pandemic alert: important coronavirus updates for ophthalmologists additional supporting information may be found in the online version of this article at the publisher's website:video s . a simulated evaluation of the effectiveness of the slit lamp shield. key: cord- - b ocoh authors: zhang, chao-fan; cui, shang-jin; zhu, chao title: loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: b ocoh a loop-mediated isothermal amplification (lamp) assay was developed specifically for detection and differentiation of pseudorabies virus (prv). one group of primers was designed to detect wild-type strains (i.e., strains with the ge gene) and the other group of primers was designed to detect both prv ge-vaccine and wild-type strains (i.e., strains with the gg gene and with or without the ge gene). after amplification by bst enzyme at a constant temperature of °c, a laddering of bright products was visible following electrophoresis on a % agarose gel. lamp was – -fold more sensitive than the standard pcr. the assay was specific in that it did not amplify other porcine viruses including porcine parvovirus, porcine circovirus type , porcine circovirus type , porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine transmissible gastroenteritis coronavirus, and porcine epidemic diarrhea virus. because of its sensitivity, specificity, and simplicity, the lamp assay could be a useful method for early and rapid differentiation of swine vaccinated with prv ge-deleted vaccine from swine infected with wild virus. pseudorabies virus (prv) is a member of the herpesviridae family and causes aujeszky's disease, which is characterized by neurological signs and death in young piglets, respiratory disorders in older pigs, and abortion in pregnant swine. in addition to swine, prv infects many other animals including sheep, cattle, dogs, and rodents. wild-type strains cause a peripheral neuropathy characterized by violent pruritus in dead-end hosts like sheep, cattle, and dogs but not in pigs. prv has caused substantial damage to the swine stockbreeding worldwide (thomsen et al., ; nauwynck, ; rooij et al., ). an improved system for detecting prv is needed (pejsak and truszczyni, ) . traditionally, prv detection has been based on direct virus isolation or detection of antigens by immunohistological methods. however, both methods are time-consuming. pcr facilitates the rapid detection of prv but requires specialized equipment and an elaborate method for detecting the amplified product (osorio, ; balasch et al., ) . although prv vaccine containing attenuated virus prevents the expression of clinical signs, such vaccination does not eliminate wild-type prv in pigs infected previously nor does it prevent sub-sequent infection by wild-type strains. these latent prv infections can be activated and cause the spread of the wild-type virus. therefore, it is very important to develop a method to identify pigs infected with wild-type virus or immunized with the prv gevaccine (prv lacking the ge gene). compared to the standard pcr, loop-mediated isothermal amplification (lamp) is a simple and rapid nucleic acid amplification method. lamp has very high specificity because it uses four to six primers that recognize six to eight regions of the target dna. lamp is used increasingly for clinical diagnosis of infectious diseases including those caused by bacteria, viruses, and parasites. for example, lamp is being used for detecting newcastle disease virus (hang et al., ) , salmonella enterica (kayoko et al., ) , plasmodium spp. (han et al., ) , porcine circovirus , and porcine parvovirus (chen and cui, ) . although a lamp assay for prv has been described previously (en et al., ) , that assay does not allow genetic diva. the use of lamp is described for determining whether swine are infected with wild-type prv or have been vaccinated with prv ge-and remain uninfected by wild-type strains. the prv ge-vaccine (strains prv-bartha-k and prv-plus), st cells, and the following viruses were obtained from the - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . table primers for prv-ge and prv-gg. type sequence reverse gtgagcccgtgcttcatg harbin veterinary research institute of chinese academy of agricultural sciences: prv wild-type strains (strains prv-jx and prv-hlj), porcine parvovirus (strain ppv-bq), porcine circovirus type (strain pcv -hlj), porcine circovirus type (strain pcv -sh), porcine reproductive and respiratory syndrome virus (strain prrsv-hb), classical swine fever virus (strain csfv-sm), swine transmissible gastroenteritis coronavirus (strain tgev-hlj), and porcine epidemic diarrhea virus (pedv-hlj). samples from the cerebrum were collected from piglets suspected of being infected with prv in hei long jiang and ji lin provinces. these piglets exhibited the clinical manifestations of disease such as high fever, diarrhea, depression, disgorging, loose stools, ataxia, epilepsy, circling, hindquarter paralysis, and opisthotonos. the disease caused rapid death of the piglets. because the piglets showed some clinical signs of prv and csfv, swine fever infection was also suspected, but csfv was not detected by rt-pcr (unpublished data). the infected piglets that were sampled were - days old. although all the sows on these farms had been treated with the prv ge-vaccine about months before the samples were collected, none of the piglets had been treated with prv ge-vaccine. piglets are generally not immunized with the prv ge-vaccine until they are - weeks old. dna and rna were extracted from referenced virus and clinical samples with a tianamp virus genomic dna/rna kit (beijing tiangen biotech co., beijing, china) in accordance with the manufacturer's instructions. the extracted dna was eluted in a total volume of l of rnase-free ddh o and stored at − • c until use. cdna synthesis reaction was performed by the transcript firststrand cdna synthesis supermix (beijing transgen biotech co.) in accordance with the manufacturer's instructions. because ge has been deleted from all vaccines used in china but have gg, these two genes were targeted for lamp detection. the lamp primers were designed using the primer explorer v software based on both the glycoprotein e gene (ge) and the glycoprotein g gene (gg) (genbank accession number nc ) ( table ). pigs that have been vaccinated but that have not been infected with wild-type prv will have virus with the gg gene but not the ge gene whereas pigs that have been infected with wild-type prv will be infected with virus with both genes whether or not they have been vaccinated. the complete coding sequences of the ge and gg genes were inserted into the vector pmd -t (takara biotechnology co., dalian, china). the resulting pmd -t-prv-ge and pmd -t-prv-gg were amplified in e. coli dh ɑ, and the recombinant plasmids were purified using the axypreptm plasmid miniprep kit (axy-gen biotechnology co., hangzhou, china). the products were kept at − • c until used. except for the primers in the thermal water bath, the reaction mixtures for the prv-ge-lamp and the prv-gg-lamp were identical. the -l reaction mixture contained . l of × ther-mopol buffer, u of bst dna polymerase (new england biolabs (beijing) co., ltd.), l of mm mgcl , l each of outer primers ( m), l each of inner primers ( m), l each of loop primers ( m), . l of dntp mixture ( . mm each), . l of betaine ( m), l of extracted template dna or cdna in a . -ml eppendorf tube, and sufficient distilled water to increase the volume to l. in initial optimization tests, the amplification reaction was performed at , , or • c for min and then terminated by heating at • c for min. lamp products were subjected to electrophoresis on a % agarose gel. in subsequent tests, the amplification reaction was performed at • c (see section ). the reaction mixtures for prv-ge-pcr and prv-gg-pcr were identical except for the primers. the prv-pcr reaction mixture contained . l of × gc buffer, l of dntp ( . mm each), . l of each of forward and reverse primer ( m) (table ) , l of extracted template dna, . l of la taq polymerase (takara biotechnology co., dalian, china), and sufficient distilled water to increase the volume to l. the amplification conditions were • c for min; followed by cycles of • c for s, • c for s, and • c s; and a final extension of • c for min. pcr products were subjected to electrophoresis on a % agarose gel. the sensitivity of prv-ge-lamp and prv-gg-lamp was compared with prv-specific real-time pcr by using serially diluted plasmids with × , × , × , × , × , × , × copies/l. the specificity of prv-ge-lamp and prv-gg-lamp was examined using dna of pcv , pcv , and ppv, and cdna of prrsv, csfv, pedv, and tgev. dna of the prv-jx strain was used as the positive control, and dnas of st cells and three samples from each of five uninfected animals were used as the negative control. the prv ge-vaccines (strains prv-bartha-k and prv-plus) and prv wild-type strains (strains prv-jx and prv-hlj) were subjected to the prv-ge-lamp and prv-gg-lamp assay. the results were visualized by electrophoresis on a % agarose gel. the applicability of the prv-ge-lamp and prv-gg-lamp assay for clinical diagnosis of prv was determined in with brain samples collected from piglets in the hei long jiang and ji lin provinces. the sows, the mothers of the infected piglets, had been immunized routinely with prv ge-vaccine. as noted earlier, the piglets had not been vaccinated and exhibited signs of prv infection. lamp results were compared with those of typical prv-ge-pcr and typical prv-gg-pcr, which were run in parallel. prv-ge-lamp and prv-gg-lamp products from the lamp reactions carried out on the clinical material were purified using the axypreptm dna gel extraction kit (axygen biotechnology co., hangzhou, china) and sent to takara company for sequencing. dnastar software and blast searching of genblank were used to determine the homology with known prv-ge and prv-gg gene sequences. the prv-ge-lamp and prv-gg-lamp reactions with l of template dna extracted from prv-jx strain and with or without loop primers were conducted at , , and • c for min in a water bath. the results were optional at • c because at this temperature the prv-lamp reactions produced bright, ladder-like bands (fig. ) . the detection limits were copies per sample for both the prv-ge-lamp and the prv-gg-lamp, copies for the prv-ge-pcr, and , copies for the prv-gg-pcr ( fig. a-d) . table detection of prv in clinical samples by lamp and pcr. primers were specific for the ge gene or the gg gene of prv. pigs vaccinated with the prv ge-deleted vaccine will lack the ge gene. pigs infected with wild-type prv will contain both kinds of genes. positive negative the prv-ge-lamp and the prv-gg-lamp amplified prv-jx but did not amplify seven other porcine viruses, dna from st cells, or samples from uninfected animals (fig. ) . the prv-gg-lamp detected the prv ge-vaccine strains (prv-bartha-k and prv-plus) and the prv wild-type strains (prv-jx and prv-hlj), but the prv-ge-lamp only detected the wild-type strains (fig. ) . the results for prv-lamp and prv-pcr were similar (table ) , although two samples that were negative for prv based on prv- - contained × , × , × , × , × , × , × , and × copies/ml of recombinant plasmid, respectively. lane was the negative control. ge-pcr were positive for prv based on prv-ge-lamp. based on prv-gg-lamp, of the piglets were infected with wild-type prv and may or may not have been vaccinated. based on both the prv-gg-lamp and the prv-ge-lamp, three of the piglets had antibody from the mothers but had not been infected with wild-type pcr. sequencing of ge and gg products from the lamp reactions was carried out on virus that was isolated from the clinical material and then propagated in tissue culture. the sequences showed completely homology with the sequences of a known prv strain (accession number nc ). the prv-lamp assays described above should be useful for the management of infection and study of prv. in viral assays, specificity and sensitivity are essential in cases where low concentrations of virus are expected. prv-lamp is specific analytically (it did not amplify seven other porcine viruses) and is much more sensitive analytically and easier to perform than classical prv-pcr. although typical pcr is inferior to the prv-specific real-time pcr, the sensitivity of the prv-lamp and the prv-specific real-time pcr was compared in another study; the sensitivity of the two assays was similar, i.e., both assays detected copies per sample (unpublished data). although our unpublished results indicate that the prv-lamp is as sensitive as the prv-specific real-time pcr, the prv-lamp is easier and requires less time to perform than the prvspecific real-time pcr. these characteristics should make lamp very useful for field tests and in other situations where a rapid, simple test is required. eradicating latent infections of prv wild-type strains in swine is very difficult, and producers therefore vaccinate routinely their swine two to three times each year with the prv ge-deleted vaccine to reduce economic losses. many different pcr assays can differentiate between the wild-type prv and gene-deleted virus vaccines (liu et al., ) , but lamp is easier to perform and provides more rapid results. en et al. ( ) described a lamp system for prv detection but that lamp system could not differentiate between swine infected with wild-type prv and swine vaccinated with prv ge-deleted. all the vaccines used in china now are ge-deleted and gg-retained. in our experience, the gg primers used in this study are more specific and more sensitive than primers aimed at gb and gd (data not shown), and so the gg gene was used in the lamp assay. the development of a sensitive antigen-detection method that can distinguish between wild-type strains and attenuated vaccine strains will help producers identify and eliminate swine infected by wild-type prv strains. the nature of the problem is exemplified by the data obtained from clinical samples in this study. although these samples were obtained from piglets produced by vaccinated sows, % of the samples were positive for wild-type prv. the identify of the virus in the clinical samples was confirmed by isolation and sequencing and also by injecting the isolated virus into rabbits, which developed subsequently neurological signs of prv including the chewing of the leg at the injection site, ataxia, hindquarter paralysis, and opisthotonos (data not shown). the prv-ge-lamp in particular should help producers eliminate wild-type prv from their herds. aujeszky's disease (pseudorabies) virus detection in cerebrospinal fluid in experimentally infected pigs detection of porcine parvovirus by loop-mediated isothermal amplification rapid detection of porcine circovirus type by loop-mediatedisothermal amplification loop-mediated isothermal amplification establishment for detection of pseudorabies virus loop-mediated isothermal amplification for rapid detection of newcastle disease virus detection of four plasmodium species by genus-and species-specific loop-mediated isothermal amplification for clinical diagnosis detection of salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of salmonella isolates microarray-based detection and differentiation of virulent and attenuated pseudorabies virus functional aspects of aujeszky's disease (pseudorabies) viral proteins with relation to invasion diagnosis of pseudorabies (aujeszky's disease) virus infections truszczynsk: aujeszky's disease (pseudorabies) a dna vaccine coding for gb and gd of pseudorabies virus (suid herpes type ) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines pseudorabies virus as a live virus vector for expression of foreign genes the study was supported in part by funding from the national high-tech r&d program ( program- aa ) and the chinese national key laboratory of veterinary biotechnology fund (nklvbp ). key: cord- -jtvez y authors: wu, xuan; song, zengxu; zhai, xiwen; zuo, lei; mei, xueran; xiang, rong; kang, zhuangzhuang; zhou, long; wang, hongning title: simultaneous and visual detection of infectious bronchitis virus and newcastle disease virus by multiple lamp and lateral flow dipstick date: - - journal: poultry science doi: . /ps/pez sha: doc_id: cord_uid: jtvez y abstract infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are both important viruses seriously affecting poultry industry worldwide. in this study, reverse-transcription lamp (rt-lamp) was combined with lateral flow dipstick (lfd) forming a novel detection tool which could simultaneously detect ibv and ndv visually. primers targeted the ′-untranslated region ( ′-utr) of ibv genome and the conserved region of ndv large polymerase gene (lp). the specificity and sensitivity of this multiple reverse transcription-lamp-lfd (mrt-lamp-lfd) assay were compared with those of conventional rt-pcr, nested rt-pcr (nrt-pcr), quantification rt-pcr (qrt-pcr), and rt-lamp monitored by electrophoresis. no non-specific amplifications were observed when the assays were tested with unrelated viruses. according to the sensitivity study, when detecting ibv or ndv alone, the lowest detection limits of mrt-lamp-lfd were . ibv rna copies/reaction and . ndv rna copies/reaction. furthermore, when detecting ibv and ndv simultaneously, the lowest detection limit was the same as that of the single detection assays. in the clinical sample study, mrt-lamp-lfd performed the best among these assays. when tested with ibv or ndv single infected samples, the mean detection rates were . % and . %, respectively. in the ibv and ndv co-infected sample study, the mean detection rates of ibv and ndv were both %. this study showed that mrt-lamp-lfd was a promising qualitative detection tool suitable for field single or multiple ibv and ndv detection. infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are of the most important viruses seriously affecting the poultry industry and causing huge economic losses worldwide (bande et al., ; brown and bevins, ) . ibv and ndv belong to the gammacoronavirus of the coronaviridae family and the avulavirus of the paramyxoviridae family, respectively (http://www.ictv.global). the genome of ibv is about . kb in length. it encodes non-structural proteins, and structural proteins: spike glycoprotein (s), small membrane protein (e), membrane glycoprotein (m), and phosphorylated nucleocapsid protein (n). at the and ends of the genome, there is an untranslated region (utr) each (armesto et al., ). ndv possesses a kb long genome comprising genes c poultry science association inc. received april , . accepted june , . corresponding author: whongning@ .com which individually encode the nucleocapsid (n), matrix protein (m), phosphoprotein (p), fusion protein (f), hemagglutinin-neuraminidase protein (hn), and large polymerase protein (lp) (de leeuw and peeters, ) . ibv and ndv both have high mutation rates, making their prevention and control difficult. quick and accurate detection of ibv and ndv is important for preventing the viruses from spreading. a wide variety of diagnostic assays for ibv and ndv have been developed, including virus isolation, and serological and molecular assays (bande et al., ; brown and bevins, ) . costs, requirements of stringent techniques, and time required limit the use of virus isolation as a routine virus detection assay (bande et al., ) . serological assays, such as hemagglutination inhibition and elisa, are faster and simpler than virus isolation, but tend to lack specificity and sensitivity, especially in the case of ibv, and poor crossreactions between serotypes makes serological tests less applicable (cavanagh, ; miller et al., ) . in view of their high sensitivity, specificity, and reduced flow time, molecular assays are the most commonly used methods for ibv and ndv monitoring. according to previous studies, both ibv and ndv quantification rt-pcr (qrt-pcr) detection methods were highly specific, and the lowest detection limits were - genome copies indicating that these qrt-pcr methods were highly sensitive (callison et al., ; farkas et al., ; wise et al., ) . another highly specific and sensitive molecular method is nested rt-pcr (nrt-pcr) which involves rounds of pcr amplifications. as previously reported, the lowest detection limits of ibv and ndv nrt-pcr assays were . and . eid /ml, respectively (nguyen et al., ) . while pcr assays are widely applied in pathogen detection, the conduct of pcr requires sophisticated laboratory equipment and observation of pcr product requires electrophoresis, making pcr assays unsuitable for point-of-care and visible detections, especially in some low-resource regions. loop-mediated isothermal amplification (lamp) amplifies dna under isothermal conditions by the bst dna polymerase large fragment (notomi et al., ) . numerous studies have demonstrated that the amplification efficiency of lamp is quite high (khan et al., ; zhang et al., ) . moreover, the specificity of lamp is also satisfactory as there are specially designed primers recognizing distinct regions on the target dna (asiello and baeumner, ; zhang et al., ) . furthermore, unlike conventional pcr assays, only simple devices are needed during lamp, such as a water bath or a heat block. lamp is thought to revolutionize molecular biology not only because of its excellent performance on dna amplification but also due to its diverse, simple, and intuitional reaction monitoring methods. several naked eye monitoring approaches have been applied, such as adding color indicators into reactions and combining with immunochromatographic techniques (parida et al., ; zhang et al., ) . lateral flow dipstick (lfd), an immunochromatographic technique, utilizes antibody capture followed by secondary antibody labeling (chen et al., ; zhang et al., ) . lamp combined with lfd (lamp-lfd) could be used for highly sensitive, simple, visual, and multiple pathogen detections (chen et al., ) . lamp products can be labeled by employing biotin/fitc modified fip/bip primers, and subsequently, these biotin-fitc double labeled lamp products can be captured by biotin-antibodies and immobilized at specific locations on lfd strips (test line). subsequently, fitc at the other end of the products can specifically combine with gold particles labeled with fitc-antibodies, thus making the results readable using the naked eye (nimitphak et al., ) . however, no previous studies have reported multiple detection of avian pathogens using lamp-lfd. both ibv and ndv are pathogens that cause avian respiratory diseases, and single or multiple infection by them may cause similar clinical signs. studies have shown that multiple conventional rt-pcr could be used for detecting and differentiating respiratory dis-ease pathogens in poultry diseases (pang et al., ; rashid et al., ) . however, the sensitivity of multiple conventional rt-pcr is not satisfactory. multiple nested rt-pcr is much more sensitive than multiple conventional rt-pcr but time-consuming (nguyen et al., ) . furthermore, these assays are not suitable for on-site pathogen detection, because products of rt-pcr need to be monitored by electrophoresis and qrt-pcr need to be conducted with highly accurate instruments. here, we developed a visual multiple rt-lamp-lfd (mrt-lamp-lfd) assay which could simultaneously detect ibv and ndv and be easily carried out and monitored by the naked eye. to evaluate this novel detection method, pcr assays (including conventional rt-pcr, qrt-pcr and nrt-pcr) and reverse-transcription lamp (rt-lamp) monitored by electrophoresis were also conducted and the specificity and sensitivity of the assays were compared with those of the mrt-lamp-lfd assay. a total of ibv strains, ndv strains, and the pcr and lamp target sequences of ndv and turkey coronavirus strains (tcov) synthesized by sangon biotech (shanghai, china) co, as well as other avian virus strains, were used for the determination of the specificities of rt-pcr and rt-lamp assays. the genbank numbers of ibv and ndv strains were labeled in figure s (supplementary information); the tcov strain and the other avian virus strains used in the specificity study were listed in table s (supplementary information). tissue samples used in this study were stored at − • c. rna in the samples was extracted with trizol (invitrogen, carlsbad, ca). subsequently, cdna was synthesized using primescript rt reagent kit (takara, beijing, china) following the manufacturer's instructions. briefly, the reverse transcription reaction mixture consisted of μl × primescript buffer, . μl primescript rt enzyme mix i, . μl oligo dt primer, . μl random mers, the rna of the virus, and rnase free dh o, thus creating a final volume of μl. complete genome sequences of ibv strains and ndv strains available in genbank were aligned using mega software. subsequently, to determine conserved regions in the ibv and ndv genomes, aligned results were used for similarity plotting analysis with the simplot program . . . primers for rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays were designed tgcatgtgccacatgagact nd-b ctttcctctgtattctctctcc on base of ibv and ndv genome conservative regions: ibv primers targeted the -utr region, and ndv primers located in the conserved region of lp gene. primers for rt-pcr, nrt-pcr, and qrt-pcr were designed by primer premier software (premier inc., palm desert, ca); primers for rt-lamp assays were designed by primerexplorer v software (fujitsu, tokyo, japan). in mrt-lamp-lfd reactions, modified fip and bip primers were used: bio-ib-fip was modified by biotin on the -end, dig-nd-fip was modified by digoxigenin on the -end, and fitc-ib-bip and fitc-nd-bip were modified by fitc on the -end. the sequences of the primers are listed in table . the most appropriate annealing temperatures for each pair of primers were determined using gradient pcr and listed in table . conventional rt-pcr reaction mixture consisted of . μl × m pfu pcr mas-termix (mei , beijing, china), pmol of each primer, μl template and double distilled water (ddh o) creating a final volume of μl. the pcr parameters included an initial denaturation step for min at • c followed by cycles of denaturation at • c for s, annealing for s, extension at • c for to s depending on the sizes of the products and a final extension step at • c for min. nrt-pcr involved rounds of amplifications. the reaction mixture and parameters of each round of amplification were the same as that of conventional rt-pcr. the products of conventional rt-pcr and nrt-pcr were monitored by electrophoresis in % agarose gels. qrt-pcr assays were conducted with μl × sso-fast evagreen supermix (bio-rad, hercules, ca), pmol of each primer, μl template and ddh o making the final volume to μl. the parameters were: initial denaturation for s at • c followed by cycles of denaturation at • c for s, annealing/extension for s, and a final melting curve at to • c with increment . • c/ s. data was analyzed by bio-rad cfx maestro . software (bio-rad, hercules, ca). plate read was added during the extension and melting curve steps. to establish ibv and ndv qrt-pcr standard curves, fragments amplified using qib-f/r and qnd-f/r were individually cloned into peasy-t vector (transgen, beijing, china). plasmids were extracted using tianprep mini plasmid kit (tiangen, beijing, china). subsequently, ibv plasmids were -fold serial diluted from . × to . × copies/μl and ndv plasmids were -fold serial diluted from . × to . × copies/μl. diluted plasmids were used as standard samples during the establishment of figure . principle of mrt-lamp-lfd. ibv and ndv lamp amplify products were labeled with fitc/biotin and fitc/digoxigenin, respectively. gold particles modified with fitc-antibodies can combine with the labeled ibv and ndv products. subsequently, ibv products will be captured by biotin-antibodies immobilized on the test line , ndv products will be captured by digoxigenin-antibodies immobilized on the test line , and the free gold particles will be immobilized on the control line. thus, the products are visualized. ibv and ndv qrt-pcr standard curves. slopes and intercepts of standard curves, amplification efficiency (e) and corresponding correlation coefficients (r ) were generated using bio-rad cfx maestro . software (bio-rad, hercules, ca). the rt-lamp reactions were conducted under gradient temperatures ( to • c) to determine optimal reaction temperatures. the concentration of mgso ( to mm) and dosage of bst . warmstart dna polymerase (new england biolabs, ipswich, ma) ( to u) were also optimized. the optimal reaction mixture contains . μl × isothermal amplification buffer to evaluate the specificities of the assays, the phylogenetic analyses on pcr and lamp target sequences of ibv and ndv strains were conducted. according to the phylogenetic trees, ibv strains were grouped into clades, and ndv strains were grouped into clades. a total of ibv strains were used to determine the specificities of the assays distribute in all clades, likewise, ndv strains were used to determine the specificities distribute in all clades ( figure s , supplementary information) . in addition, cdna of avian reovirus (arv), infectious bursal disease virus (ibdv), and avian influenza virus (aiv), dna of gallid alphaherpesvirus (gahv- ) and fowl adenovirus (fadv), as well as synthesized sequence of tcov strain were used as templates for further evaluation of the specificities of the assays (table s , supplementary information) . to determine the lowest detection limits of the assays in terms of rna copy numbers, -fold serial diluted in vitro-transcribed rna of target regions was used as templates. briefly, fragments containing ibv -utr and ndv lp target regions were separately amplified using primer pairs -atcacactagccttgc gctaga- / -gcaaaagcatcagcgtaatcc- and -aatctgtattacatgtctagg- / -aga gagaatatatcctttcgc- . subsequently, the fragments were separately ligated downstream t promoter. in vitro-transcriptions were conducted using hiscribe t high yield rna synthesis kit (neb, beijing, china). the concentrations of the rna transcripts were measured using nano drop (thermo, shanghai, china) , and the copy numbers of ibv and ndv rna molecules were calculated following the formula reported previously (fronhoffs et al., ) . the copy numbers of ibv and ndv rna molecules were . and . copies/μl, respectively. ibv rna was -fold serial diluted into . to . copies/μl; ndv rna was -fold serial diluted into . to . copies/μl. whereafter, μl rna from each concentration was used in reverse transcription reaction ( μl reaction volume). after reverse transcription, μl cdna was used in the pcr and lamp assays. thus, the concentrations of serial diluted ibv and ndv rna, which was finally used as templates, were separately . to − . copies/reaction and . to − . copies/reaction. furthermore, serial diluted ibv and ndv rna was mixed to test the lowest detection limit of mrt-lamp-lfd when simultaneously detecting ibv and ndv. negative control reactions in specificity and sensitivity studies were conducted with total rna extracted from allantoic fluid of health specific pathogen-free (spf) chick embryo as templates. ibv-positive samples, including tissue samples ( tracheas and lungs), swabs ( oral swabs and cloacal swabs), ndv-positive samples, including tissues ( tracheas and lungs), and swabs ( oral swabs and cloacal swabs) were used to examine the performance of the assays in detecting ibv and ndv in clinical samples. in addition, to further investigate the specificities of the assays when detecting clinical samples, negative tissues (including tracheas, lungs, and kidneys), fadv positive livers, and aiv h n positive lungs were also tested by the assays. all these samples were collected from chicken farms distributed in provinces, china, during our routine monitor on avian diseases. to investigate whether the mrt-lamp-lfd assay could detect ibv and ndv in co-infected samples, both accurately and simultaneously, ten -wk-old spf chickens were inoculated with . eid ibv m and . eid ndv f e by the nasal route to mimic ibv-ndv co-infected chickens. oral and cloacal swabs were collected on and days-post-infection (dpi) from each bird. on dpi, all the chickens were sacrificed, and lungs and tracheas were collected. the animal experiment in this study was approved by the animal ethics committee of the college of life sciences, sichuan university (license: syxk-chuan- - ). all experimental procedures and animal welfare standards strictly followed the guidelines of animal management at sichuan university. statistical significance differences in the mean detection rates of the assays, when detecting different kinds of samples, were evaluated by one-way anova using graphpad prism version (graphpad software inc., san diego, ca). differences were considered to be significant at * p < . . under optimal annealing temperatures, rt-pcr and nrt-pcr amplified specific products of the expected lengths, and there were no non-specific bands observed in the negative controls ( figure a ). both in ibv and ndv qrt-pcr assays, fluorescent signals were detected with the target ibv or ndv templates, while no fluorescent signals were detected in negative control reactions ( figure b ). according to the standard curves, c t values (y), and log of copy numbers (x) were linearly correlated (ibv: y = − . x + . , e = . %, r = . ; ndv: y = − . x + . , e = . %, r = . ). the melting curve showed a single peak indicating no primer dimer formed ( figure c ). after ibv and ndv rt-lamp amplification, symbolic ladder-like bands were observed in a % agarose gel ( figure a ). as for mrt-lamp-lfd assays, ibvpositive reactions generated test line and the control line; ndv-positive reactions generated test line and the control line; and test lines , , and the control line appeared when both ibv and ndv were present; only the control line was generated when neither virus was present ( figure b ). as figure a shows, there were no positive reactions observed when ibv assays were tested with other pathogen templates except for tcov. the results are not unexpected, because tcov and ibv are very closely related in terms of both antigenic and genomic characterizations (guy, ) . target sequence ( -utr) nucleotide identities between tcov and ibv strains are . to . % (data not shown). moreover, according to the new taxonomy of viruses published by international committee on taxonomy of viruses, ibv, and tcov are classified as specie (http://www.ictv.global). ndv detection assays were specific to the ndv templates ( figure b ). conventional rt-pcr, nrt-pcr, and rt-lamp assays yielded specific products only when tested with target ndv templates. in mrt-lamp-lfd assays, test lines were observed when target templates were contained in the reaction mixture. as for qrt-pcr assays, fluorescent signals were detected only when tested with the ndv templates. the lowest detection limits of ibv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, when detecting ibv alone, were . , . , . , . , and . copies/reaction, respectively ( figure a ). the lowest detection limits of ndv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, detecting ndv alone, were individually . , . , . , . , and . copies/reaction ( figure b ). when simultaneously detecting ibv and ndv, mrt-lamp-lfd produced clear visible test lines at concentration of . ibv + . ndv copies/reaction, and the lowest detection limit was the same as that of mrt-lamp-lfd when detecting ibv or ndv alone ( figure c ). as figure a shows, mrt-lamp-lfd exhibited the highest mean detection rates in the detection of different types of clinical samples when conducting ibv or ndv single detection, . % for ibv and . % for ndv. statistical significance difference studies showed that the mean detection rates of mrt-lamp-lfd were significantly higher than that of conventional rt-pcr assays when detecting ibv or ndv alone (p < . ). no positive results were observed when the assays were tested with negative tissues, fadv positive livers, and aiv positive lungs. to further evaluate mrt-lamp-lfd, chickens were experimentally co-infected with ibv and ndv. results showed that mrt-lamp-lfd could not only detect pathogens simultaneously, but also showed higher mean detection rates than the other assays presented here. the mean ibv and ndv detection rates of different samples, detected by mrt-lamp-lfd, were both %, and were significantly higher than those detected by conventional rt-pcr and qrt-pcr (p < . , figure b) . . specificity study of pcr and lamp assays of (a) ibv and (b) ndv. in specificity study, cdna of ibv, ndv, arv, ibdv, and aiv and dna of gahv- and fadv, as well as the synthesized pcr and lamp target sequences including ndv and tcov were used as templates. in the ibv detection assays, positive results were detected with ibv and tcov templates. in ibv qrt-pcr, other viruses include ndv, arv, gahv- , ibdv, fadv, and aiv. as for ndv detection assays, positive results were observed only when ndv templates existed. in ndv qrt-pcr, other viruses refer to ibv, arv, gahv- , ibdv, fadv, tcov, and aiv. nc means negative control reactions which were conducted using total rna extracted from allantoic fluid of healthy spf chick embryo as template. markers in the electrophoresis were the same as that in figure a . timely and accurate diagnostic methods are very important for the control of infectious diseases, especially for ibv and ndv which are of the most important contagious viruses seriously affecting the poultry industry. furthermore, ibv and ndv produce clinical picture somewhat resembling each other, it is very much crucial not only to detect but also differentiate simultaneously. existing ibv and ndv diagnostic methods, including virus isolation and pcr assays, are specific and sensitive. however, they are not suitable for timely onsite pathogen detection. although portable pcr machines are gradually applied in the field, in most areas, especially in undeveloped and developing countries, these sophisticated equipments are too expensive to be popularized, and the sensitivity of pcr is not satisfactory. multiple rt-lamp-lfd developed in this study could detect and differentiate ibv and ndv, both simultaneously and accurately. when ibv and ndv cdna co-exist in the same reaction system, an ibv and ndv double-positive result was observed. to evaluate the sensitivity of mrt-lamp-lfd, conventional rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays detecting ibv or ndv alone were also conducted to compare with mrt-lamp-lfd. when detecting ibv or ndv alone, mrt-lamp-lfd performed as sensitive as nrt-pcr and rt-lamp did, in a directly visual way. it is always thought that qrt-pcr methods provided high sensitivity during pathogen diagnoses. according to the result of detection limit study, nrt-pcr, and lamp assays, established in this study, possessed times higher sensitivity than qrt-pcr. in the first round of amplification of nrt-pcr, the original template was amplified with the outer primers. thus, the number of the fragments containing the inner primer target sequence is greatly improved compared with the original template. as a result, the number of templates in the second round of amplification of nrt-pcr is much higher than that in qrt-pcr. this is the reason why nrt-pcr could detect lower concentration of original templates than qrt-pcr do. similarly to our results, previous study conducted by weng and chen indicated that npcr showed higher sensitivity than real-time pcr when detecting phytophthora infestans (khan et al., ) . lamp is one of the most widely used isothermal nucleic acid amplification techniques (inats) . several studies on the diagnostic methods of other pathogens had showed that these inats possessed equal or even higher sensitivities compared with qpcr assays (gao et al., ; khan et al., ; yang et al., ) . our results indicated that lamp assays possessed higher sensitivities than qpcr when detecting ibv and ndv ( figure ). several multiple rt-pcr assays detecting avian respiratory pathogens have been developed in previous studies, while the sensitivity of these multiple assays was lower than single pathogen detection assays (nguyen et al., ; pang et al., ) . this may be due to the competition among different sets of primers. however, when detecting with ibv and ndv co-existing samples, the lowest detection limit of mrt-lamp-lfd was the same as that of mrt-lamp-lfd when detecting a single pathogen (i.e., . copies/reaction for ibv and . for copies/reaction for ndv), indicating that the sensitivity of mrt-lamp-lfd was not affected when the components of the reaction system became more complex. the purpose of this study was to develop a novel detection tool which could simultaneously accurately detect ibv and ndv on site. previous studies on pcr methods detecting ibv and ndv showed that these pcr methods are specific and sensitive, but the need for expensive thermal cycling equipments makes them not suitable for on-site ibv and ndv detection. portable pcr machines are gradually applied in the field. while, in most areas, especially in undeveloped and developing countries, these sophisticated to further evaluate mrt-lamp-lfd, chickens were experimentally co-infected with ibv and ndv. the mean detection rates of the assays, when detecting different kinds of samples, are labeled above the histograms. the fraction numbers under the x-axis represent (positive sample numbers detected by the assays)/(total sample numbers). * indicate p < . . equipments are too expensive to be popularized. thus, simpler and visible ibv and ndv detecting methods are urgently needed. high specific and sensitive single ibv and ndv rt-lamp assays have been developed in previous studies (chen et al., ; pham et al., ) . in these assays, products were visualized by electrophoresis or by adding color indicators. when detecting multiple pathogens, the products must be easily differentiated. when visualized by electrophoresis, lamp products could be distinguished by observing bands with different molecular weights, however, this method is not suitable for on-site pathogen detection. by adding color indicators, the change in color could be easily observed, but this change is non-specific. obviously, these two lamp monitoring methods are not applicable for on-site multiple pathogens detection. multiple lamp-lfd has been applied in the detection of some pathogens, and it showed great advances compared with common pcr and lamp assays, such as the requirement for little equipment, short reaction time, and the ability to detect multiple genes or pathogens (chen et al., ; lalle et al., ) . to our knowledge, rt-lamp-lfd has not been applied to ibv or ndv detection and no studies on an mrt-lamp-lfd technique that detects multiple avian respiratory viruses have been reported. in this study, the products generated in ibv and ndv rt-lamp were differentiated by biotin/fitc and digoxigenin/fitc labeling, respectively. the products could bind with biotin-or digoxigenin-antibodies fixed on different test lines on the lfd strip, and then products were visualized by combining with gold particles modified with fitc-antibodies (figure ). when tested with clinical samples, the mean detection rate of mrt-lamp-lfd was higher than that of the other assays. these results indicate that mrt-lamp-lfd is not only able to detect ibv and ndv simultaneously, but is also suitable for field testing in both technical (more sensitive than pcr and lamp assays) and practical aspects (more simple to put out and no specialized equipment needed). this study combined rt-lamp and lfd conducting a novel ibv and ndv mrt-lamp-lfd detection assay which is specific and sensitive in detecting ibv and ndv simultaneously. furthermore, mrt-lamp-lfd does not require specialized instrumentations, making it suitable for on-site detection. in conclusion, mrt-lamp-lfd is a promising qualitative detection tool, and is even applicable in some low-resource locations. supplementary data are available at poultry science online. table s . the tcov strain and other avian virus strains used in the specificity study figure s . phylogenetic analyses based on pcr and lamp target sequences of (a) ibv and (b) ndv. strains used in specificity study are in bigger font. wild strains detected during our avian virus monitoring, of which genome sequences have not been submitted to genbank, were labeled by solid circles; artificially synthesized sequences were labeled by solid triangles. according to the trees, ibv strains were grouped into clades, and the ibv strains used in this study distribute in all clades; ndv strains were grouped into clades, and the ndv strains used in this study distribute in all clades. the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity 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of taq-man minor groove binder probes a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of salmonella in shellfish turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review evaluation of different pcr-based assays and lamp method for rapid detection of phytophthora infestans by targeting the ypt gene loopmediated isothermal amplification-lateral-flow dipstick (lamp-lfd) to detect toxoplasma gondii oocyst in ready-to-eat salad newcastle disease: evolution of genotypes and the related diagnostic challenges multiplex nested rt-pcr for detecting avian influenza virus, infectious bronchitis virus and newcastle disease virus shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick loop-mediated isothermal amplification of dna development and application of a multiplex polymerase chain reaction for avian respiratory agents loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases loopmediated isothermal amplification for rapid detection of newcastle disease virus multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of salmonella in produce brief review of monitoring methods for loop-mediated isothermal amplification (lamp) this research is funded by the national key r&d program of china ( yfd ), the china agriculture research system (cars- ), the national system for layer production technology (cars- -k ), and the project for science and technology support program of sichuan province ( nz ). we declare that we have no conflict of interest. key: cord- - lujp oy authors: neeraja, m.; lakshmi, v.; lavanya, vanjari; priyanka, e.n.; parida, m.m.; dash, p.k.; sharma, shashi; rao, p.v. lakshmana; reddy, gopal title: rapid detection and differentiation of dengue virus serotypes by ns specific reverse transcription loop-mediated isothermal amplification (rt-lamp) assay in patients presenting to a tertiary care hospital in hyderabad, india date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lujp oy early and rapid detection of dengue virus (denv) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. in the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (rt-lamp) for rapid detection and serotyping of the denv targeting ns gene using the genie® ii flourometer was carried out. the performance of the rt-lamp was compared to rt-pcr, cdc - real time pcr and the ns antigen elisa, igm and igg anti denv antibodies. acute denv infection was confirmed in / patients suspected clinically of denv infection. rt- lamp and cdc - real time pcr assay was positive in / patients, while / patients were positive for anti- dengue igm and igg antibodies. the rt-lamp assay and the cdc real-time rt-pcr assay showed high concordance (k = . ). the detection rate of acute denv infection improved to % ( / ) when the results of rt-lamp were combined with ns ag, igm and igg elisa. the rt-lamp had a detection limit of copies for den- and den- , copies for den- and den- compared to copies for den- and den- , copies for den- and den- by the conventional rt-pcr. the assay showed % specificity. the rt-lamp assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of denv infection in endemic countries such as india. dengue is a mosquito borne flaviviral infection, affecting the tropical and subtropical regions of the world and is one of the major emerging global public health problems. there are four antigenically distinct dengue virus serotypes den- , den- , den- and den- and each serotype contains phylogenetically distinct genotypes (teoh et al., ) . the dengue virus (denv) infection induces a lifelong protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes. therefore, multiple and sequential infections with the four denv serotypes would be expected for people living in a region where the infection is hyper endemic due to the lack of cross-protective neutralizing antibodies. seroepidmiological studies have shown that the secondary infection is a major risk factor for dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) through antibody-dependent enhancement (halstead et al., ; monath and heinz, ) . diagnosis of denv infection on the basis of clinical signs and symptoms is not reliable as more than half of the infected individuals either are asymptomatic or have a mild undifferentiated fever (burke et al., ; endy et al., ) . early diagnosis of dengue infection can reduce the number of cases of dhf and dss. therefore, there is a great demand for the rapid detection of the infection and differentiation of denv serotypes for timely clinical management and disease control, respectively. the most common methods for laboratory diagnosis of denv include serological methods detecting antibodies (igm and igg) against denv and additionally various methods are used in detecting denv rna or antigens: non-structural protein (ns ) and envelope protein (e). the serological methods are vulnerable to cross reactions caused by antibodies against related flaviviruses and are therefore not denv-specific tests like denv ns antigen and rna detection methods. the detection of dengue specific secretory ns (non-structural protein ), a highly conserved glycoprotein represents a new approach to the diagnosis of acute denv infection, in recent times. enzyme-linked immunosorbent assays (elisa) directed against ns antigen (ns ag) have demonstrated its presence at high concentrations in the sera of dv infected patients during the early clinical phase of the disease (dussart et al., ) . assays based on the detection of nonstructural protein (ns ) tend to be specific for denv infection. ns antigen levels correlate well with viremia and it circulates at high levels during the first few days of illness especially in patients with dhf. ns antigen remains circulating in patients' blood for longer periods than does viral rna and is reported to be detectable even up to the th day of illness. the first isothermal amplification methods introduced in s included the transcription mediated amplification (tma), nucleic acid sequence based amplification (nasba) and the strand displacement amplification (sda). the loop-mediated isothermal amplification (lamp) is a novel nucleic acid amplification method and has the potential to replace pcr because of its simplicity, rapidity, specificity, sensitivity and cost-effectiveness without the need of specialized equipment (notomi et al., ; parida et al., ; tomita et al., ) . the rt-lamp assay is being increasingly used by various investigators for rapid detection and typing of emerging viruses (chan and fox, ; mori et al., ; parida et al., ) . these earlier reports, however, evaluated their rt-lamp assays for the detection of denv infection with a small clinical sample size (< ) and using the c-prm gene (lu et al., ) or serotype-specific regions of the untranslated region (utr) (parida et al., ; li et al., ; sahni et al., ) . the c-prm gene, however, was relatively less conserved among all the four denv serotypes (inter-serotype) in comparison to the utr (teoh et al., ) . however, we have targeted a highly conserved region of ns , revealing > % sequence identity among various genotypes within each serotype. as such genotyping of dengue serotypes can be done employing many gene including ns and ns (klungthong et al., ) . in the present study, the rt-lamp assay was developed for the detection and serotyping of denv infection targeting the serotype specific regions of the ns gene using a real-time flourometer (genie ® ii from optigene, u.k.). the detection sensitivity and the specificity of the reported denv ns serotype specific rt-lamp in freshly obtained blood samples from patients suspected clinically of denv infection, is compared with available test system for suitable algorithm. to the best of our knowledge this is the first report of the detection and differentiation of dengue using ns rt-lamp with real time fluorometer (genie ® ii from optigene, u.k.) from south india. the study was approved by the institutional ethics committee of nizam's institute of medical sciences (ec/nims/ / ). written informed consent was obtained from each patient. reference strains of the four dengue virus serotypes den- , rr (kf ), den- , gwl (ay ), den- , nd (fj ), den- , nd (hm ) were used in this study (dash et al., (dash et al., , neeraja et al., ) . patients suspected clinically of dengue/dhf/dss, who either reported directly or were referred to a tertiary care institute for treatment from the regions in and around hyderabad, from july to december , were included in the study. the dengue/dhf/dss case proformas prepared as per the who protocol (world health organization, ) for denv infection was filled by the treating clinicians. acute phase and early convalescent serum and plasma samples based on reporting time were collected from patients with a history of sudden onset of fever, and the presence of two or more of the symptoms viz. headache, eye pain, nausea, vomiting, rash, myalgia, abdominal pain suggestive of denv infection. samples collected within days of fever were categorized as acute phase samples and those collected after days of fever were considered as convalescent phase sample. in order to check the cross-reactivity, within serotypes and with other closely related members of flavivirus family i.e., je, wnv archived samples from drde gwalior and hcv positive samples from our tertiary care hospital were included in the study. confirmed chikungunya (chikv) rna positive samples were also included as symptoms of denv and chikv mimic each other. in addition, a panel of samples collected from healthy individuals was included as negative controls. before performing the rt-lamp assay, all the samples were also screened for denvspecific rna by rt-pcr (lanciotti et al., ; neeraja et al., ) and ns antigen by panbio dengue early elisa assay (inverness medical innovations, australia), dengue igg and igm capture elisa (pan bio, queensland, australia). denv serotype specific oligonucleotide primers were designed from the ns region of denv genome. the nucleotide sequence of the ns gene of denv, representative of respective genotype and serotype strain was retrieved from gen bank (den- , accession no. eu ; den- , accession no. af ; den- , accession no. eu ; and den- , accession no kc ) and was aligned with the available ns gene sequences from global denv strains including the circulating strains in india, to identify the conserved regions using dnasis software (hitachi, japan). the primers were selected based on criteria described by notomi et al. percent gene homology among each serotype were found to be ≥ %. the potential target region corresponding to the genome positions was selected from the aligned sequences, and the rt-lamp primers were designed from conserved region of each serotype using the primer explorer version software (eiken chemical co., tokyo, japan). a set of six primers comprising two outer (f and b ), two inner (fip and bip), and two loop primers (flp and blp) that recognize eight distinct regions on the target sequence was designed. the primers were selected based on criteria described previously by notomi et al. all the primers were assessed for specificity before use in lamp assays with a blast search with sequences in the gen bank (table ) . the viral rna was extracted from l of the serum/plasma samples by using the qiaamp viral rna mini kit (qiagen, germany). the rna was eluted from the qia spin columns in a final volume of l of the elution buffer and stored at − • c until testing. the rt-lamp was carried out in a final reaction volume of l. the reaction mixture contained l of isothermal master mix iso- (optigene, u.k.) containing, geobacillus species dna polymerase, thermostable inorganic pyrophosphatase, optimized buffer including mgcl , dntps and ds-dna dye (optigene, u.k.), l primer mix consisting of primers each for denv- , denv- , denv- , and denv- (f and b primers at . m, fip and bip primers at . m, lf and lb primers at . m), . units amv reverse transcriptase (promega, madison, wi.), . l nuclease free water and l extracted nucleic acid. the rt-lamp assay was run at temperatures between and • c and time between min and min in the real-time fluorometer (genie® ii from optigene, u.k.) to determine the optimal temperature with the shortest amplification time and the highest fluorescence reading. all the rt-lamp assays were subsequently run at • c for min followed by a heating and cooling step to • c to • c ( . • c/s) to allow re-annealing of amplified dna and display of the annealing curve. the genie ii displays amplification signals in real time and at the end of the run displays the time to positivity that is expressed in terms of plots of fluorescence signals (real time curves) and t m for each specimen. the analysis of each sample was done in a set of four tubes, with serotype specific primer mixture. the t m for denv- was . • c, denv- was . • c, denv- was . • c and denv- was . • c. positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. amplification of the dna leads to an increase in fluorescence emitted from a dna intercalating dye. this increase was monitored in real time using the genie® ii fluorometer. following incubation at • c for min, a l of aliquot of the rt-lamp assay products was electrophoresed on % nusieve : agarose gel (biowhittaker molecular applications, rockland, maine) in trisborate buffer, followed by staining with ethidium bromide and visualization on a uv transilluminator at nm. in order to facilitate the field application of the rt-lamp assay, monitoring of amplification was done visually with an unaided eye. following amplification in genie ® ii flourometer l of sybr green i intercalating dye was added to the reaction tube. the rt-lamp amplification was visually monitored for colour change. positive reaction turned the reaction mix green and fluoresces under the white light and uv irradiation, respectively. the reaction mix remained orange and non-fluorescent in the absence of amplification. this change of color is permanent and thus can be kept for record purposes. in order to compare the sensitivity and specificity of the rt-lamp assay, one-step rt-pcr was done by employing the two outer primer pairs ( pmol of f and b ) targeting the ns gene of each serotype. amplification of the rna was carried out in l reaction volume with the pcr mix containing primescript tm step enzyme mix and its buffer along with respective sense (f ) and anti sense (b ) primer in a thermal cycler (applied biosystems, usa). the thermal profile of the rt-pcr reaction was-reverse transcription at • c for min, initial denaturation at • c for min, followed by cycles of denaturation at • c for min, annealing at • c for min, extension at • c for min and final extension at • c for min (neeraja et al., ) . the real-time rt-pcr assay from cdc was used as a standard test for the denv serotype specific identification in abi quantitative pcr system (abi, usa). the assay is based on taqman chemistry including a panel of oligonucleotide primers and dual labeled hydrolysis probe sets [d , d , d , d ] employing invitrogen super script tmiii platinum® one step quantitative kit. the amplification was carried out in a l reaction volume. instruction and standard thermal profile for sample screening was as follows, reverse transcription • c for min, initial denaturation and enzyme inactivation • c for min, cycles of extension at • c for sec and • c for min of denaturation and annealing extension respectively (chien et al., ) . briefly, the reagents include × buffer (invitrogen one-step rt-pcr kit, usa) . l, enzyme mix . l, d /d both forward and reverse primers . l ( nm), d /d both forward and reverse primers . l ( nm) and d -d probe . l ( nm) each and depc treated water added up to a total volume of l. finally, l of viral rna elute extracted from different samples was added for real-time rt-pcr assay. . . performance parameters of denv rt-lamp . . . sensitivity of serotype-specific dengue virus-specific rt-lamp assay the sensitivity of the ns serotype specific rt-lamp assay was determined through serial dilutions of in vitro transcribed denv with known copy number. the specificity of the primers for detecting denv serotypes was validated by testing samples that were positive for other flavivirus including je, wnv, hcv and chikv. in addition, the authenticities of the amplified products were also established by nucleotide sequencing of amplified products with outer (f ) and inner (b ) primers (parida et al., ) . nucleotide sequencing of the ns gene of randomly selected dengue viruses from the clinical samples, that included one den- (vl ) and two den- (vl , vl ), was carried out by employing the big dye terminator cycle sequencing ready reaction kit with an abi sequencer (applied biosystems, usa) for identifying the genotype of the denv serotype by following the standard protocol (dash et al., ) the sequences were initially subjected to blast to find the closest sequence identity. further, phylogenetic analyses based on the ns gene junction of den- and den- were carried out by including a large number of geographically diverse denv gene sequences, by using the neighbour-joining (nj) method of the mega software version . . the sequences of vl , vl , and vl were submitted to genbank under the accession numbers kc , kj , kf , respectively. inter-assay variability for reproducibility was assessed by testing sample of each serotype in separate lamp runs and recording time and t m for each serotype. the intra-assay variability for repeatability was assessed by simultaneously testing samples of each serotype that included strong positive and weak positive of each serotype. the degree of agreement between rt-lamp and the cdc real time pcr test results was measured by kappa value (k). fisher's exact test (two tailed) was done to calculate p value, p value < . was used to suggest significant results. the diagnostic performance of rt-lamp assay and the cdc real time pcr assay as compared with ns ag and ns rt-pcr assay was calculated using med calc easy to use statistical software (http://www.medcalc.org/ calc/diagnostic test.php). / patients suspected clinically of denv infection, were confirmed as acute denv infection by detection of the ns ag, anti-igm, conventional rt-pcr, and the real-time rt-pcr either alone or in combinations. / patients were positive by ns ag alone. out of the remaining patients were positive only for anti-dengue igm and igg antibodies. ns serotype specific rt-pcr assay was positive in / patients, ns serotype specific rt-pcr assay detected patients that were negative for ns ag by elisa test. rt-lamp assay detected additional patients that were negative for ns ag by elisa and ns rt-pcr assay. / patients were identified as past denv infection as dengue igg antibodies alone were tested positive among them (table ) . all the four denv serotype-specific primers were highly specific for the detection and differentiation of the appropriate serotypes with no cross reaction. none of the serotype-specific primer sets amplified or cross reacted with any of the je, wnv, hcv or chikv viral rna template and samples from healthy individuals, there by indicating their specificity. as depicted in fig. , the size of the resultant product by rt-pcr using outer primers f and b was in good agreement with the predicted size for each serotype, i.e., bp for denv- , bp for denv- , bp for denv- , and bp for den- , (fig. ) . % sequence homology was also observed between the primers and the corresponding nucleotide sequences. the sensitivity of the in house developed denv ns serotype specific rt-lamp assay was same as that of the cdc - real time rt-pcr assay. these two methods showed high concordance with kappa value of . . the diagnostic accuracy improved to % ( / , % confidence interval = . - . ) when the results of the denv rt-lamp or the cdc real time assay were combined with the results of the ns antigen and anti -dengue igg and igm elisa (table ) the diagnostic performance of rt-lamp compared to ns ag by elisa and the ns rt-pcr is summarized in table . the sensitivity of the ns serotype specific rt-lamp assay as a function of the timing of the test (days after onset of fever) was studied and it was found that the sensitivity was optimal, at . % ( % ci, . % to . %), between days and for ns serotype specific rt-lamp assay (table ) . sensitivity of the ns serotype-specific dengue virus rt-lamp assay compared to ns ag, igg + igm antibody, ns rt-pcr and the cdc real time rt-pcr assay. the cdc-real time pcr assay was considered as gold standard control. a ns rt-pcr detected samples that were negative for ns ag by elisa. b rt-lamp and cdc real time rt-pcr assay detected samples which were negative for ns ag by elisa and ns rt pcr assay. the diagnostic performance of the rt-lamp assay against ns ag and the rt-pcr assay in dengue patients in tertiary care hospital in hyderabad. table the sensitivity of the ns serotype specific rt-lamp assay related to number of days after onset of fever (n = ). the rt-lamp assay was positive more in the patients with primary infections ( / ) compared to patients with secondary infections ( / ). p value for the detection of primary infections by rt-lamp was statistically significant when compared with secondary infections (p < . , by fishers exact test). rt-lamp was positive among patients with df, patients with dhf and patients with dss. the rt-lamp assay detected copies of den- and den- and copies of den- and den- rna, respectively, as shown in fig. a and b and the sensitivity of rt-pcr was copies of den- and den- and copies of den- and den- as shown in fig. c and d. the optimized amplification time of samples from patients ( of each serotype) by denv rt-lamp was min. as shown in table . the mean time to positivity for all positives was min fig. . agarose gel electrophoresis of denv serotype-specific rt-pcr assay products on a % agarose gel employing f and b primers of respective serotypes. d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product bp. comparative sensitivity of rt-lamp versus rt-pcr for detection of the ns gene of denv. sensitivity of the rt-lamp assay as monitored by real-time measurement of fluorescence. shown from left to right are the curves of decreasing concentrations of virus from × to × copy numbers of the template in a serial -fold dilution. the detection limit for the assay was copy numbers for denv and denv (a) and copy numbers for denv and denv (b). (c and d) sensitivity of rt-pcr for the detection of the denv ns gene as observed by agarose gel analysis with a detection limit of copy numbers for denv and denv and copy number for denv and denv . lane m, -bp dna ladder (sigma); lanes to , different concentrations of virus ranging from × to × − copy numbers in a serial -fold dilution pattern. the real-time amplification of each dengue virus serotype in genie ® ii fluorometer is shown in fig. a and b, that shows different amplification times and annealing temperatures (c and d). on %agarose gel electrophoresis the amplification product was detected as a ladder-like pattern due to the formation of a mixture of stem-loop dnas with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand (fig. ) . the visual detection of the rt-lamp results is shown in fig. . the most predominant serotype documented in our study was den- ( patients), followed by den- ( patients), den- ( patients) and den- ( patients). den- and den- serotypes were confirmed by sequencing, with accession numbers kj and kf for den- and kc for den- . phylogenetic analysis of den- showed that the strain belonged to genotype iv and den- belonged to genotype iii (fig. ) . precision of the rt-lamp for identification and serotyping of denv was determined by testing samples of each serotype that included strong positive and weak positive of each serotype. the mean amplification times of the replicates for strong and weak positives for den- was . min (standard deviation [sd], . ) and . min (sd, . ), den- was . min (sd, . ) and . min (sd, . ), den- was . min (sd, . ) and . min (sd, . ) and den- was . min (sd, . ) and . min (sd, . ), respectively. to further assess the reproducibility of rt-lamp, we tested sample of each serotype in separate lamp runs and recorded the time to positivity and t m for each serotype. the difference in the amplification times for each serotype across separate runs were within . min for each serotype and sds ranging from . to . indicating that the rt-lamp for denv identification and serotyping is highly reproducible (table ) . the nonstructural protein (ns ), of dengue viral genome has been shown to be a useful tool for the early diagnosis of acute dengue infections (cdc-laboratory guidance dengue) and was found to be highly conserved for all dengue serotypes. the denv ns antigen elisa, a widely used test in recent times, is highly sensitive and specific (young et al., ; alcon et al., ) but it can be compromised by pre-existing ns -igg immunocomplexes in the acute stage of secondary denv infection, a common feature in dengue endemic regions (lapphra et al., ; hang et al., ) . although, who (world health organization, ) recommends the detection of denv rna, as the most effective diagnostic method in the acute phase of the illness, its use is limited due to lack of infrastructure and technical expertise. more recently, molecular techniques to detect virus genomic rna sequence by the reverse transcription-polymerase chain reaction (rt-pcr) and the real-time quantitative rt-pcr (qrt-pcr) are gradually being accepted as new standards over virus isolation for the detection of denv in the acute sera (lanciotti et al., ; shu et al., ) . these pcr-based methods require either high-precision instruments for the amplification or elaborate methods for detection of the amplified products. in addition, these methods are often cumbersome to adapt to routine clinical use, especially in the peripheral health care settings and the private clinics (parida et al., ) . the rt-lamp assay has emerged as a powerful gene amplification tool for rapid identification of microbial infections and is being increasingly used by various investigators for rapid detection and typing of emerging viruses, such as the west nile, severe acute respiratory syndrome, dengue, and japanese encephalitis viruses (hong et al., ; parida et al., parida et al., , parida et al., , parida et al., , . a four tube denv ns serotype specific rt-lamp assay was developed in this study for the rapid detection and differentiation of dengue serotypes in this study with high sensitivity and specificity. using the primer concentrations (f and b primers at . m, fip and bip primers at . m, lf and lb primers at . m), together with the use of commercially available isothermal master mix from optigene, uk, containing an engineered large fragment dna polymerase (gspssd), amplification time of min and temperature of c was optimized for rapid detection and serotyping of the denv. geobacillus dna pol enzyme demonstrated superior lamp amplification speed compared to bst dna pol i (parida et al., ; sahni et al., ; boon-teong et al., ) . this isothermal amplification mix allows fluorescence detection of the product on the genie ® ii platform but may also be used on generic qpcr instrumentation (www.optigene.co.uk, ). very few rt-lamp assays have been described for the detection and serotyping of denv. recently teoh et al. ( ) developed single tube rt-lamp assay by targeting utr of denv using nine sets of primers for serotyping of denv. however no information on the serotypes detected from the clinical samples was reported in fig. . phylogenetic tree of studied sequences with geographical strains of serotype and generated by neighbor-joining method. the tree is based on ns regions (nt - bp for denv- and nt - bp for denv- ) of the selected strains. this study. our experience revealed that because of emergence of many genotypes across multiple denv serotypes, the designing of pan-dengue primers is an impossible task and is most likely to miss some genotypes. the reproducibility of rt-lamp assay in this study was low and sensitivity was slightly lower than q rt-pcr. the specificity of the rt-lamp assay developed by teoh et al. was tested by single site restriction enzyme digestion that is not cost effective and also laborious. these reported methods for detection and differentiation of lamp results were done by real time monitoring of turbidity (at nm) with a loopamp real-time turbidimeter, "ladder-like feature" on agarose gel electrophoresis and color change from orange to green caused by fluorescent detection reagent. however real time monitoring of amplification for the detection and differentiation of denv serotypes in a fluorometer has rarely been reported. a few studies have reported the lamp assay for rapid detection of viruses by real-time fluorometer (genie ii from optigene, u.k.) (mahony et al., a,b) . in this study we established real-time fluorescence monitoring of isothermal method using simpler and less costly genie ® ii instrument. the most common method for real-time fluorescence monitoring of lamp reactions uses intercalating dyes such as sybr green (maeda et al., ; ohtsuka et al., ) . fluorescence detection using intercalating dyes has the advantage of allowing further analysis in terms of the temperature at which amplification products melt or anneal. lamp products contain structures of differing lengths containing catenated repeats of the target sequence which melt/anneal at a specific temperature determined by the length and g/c content of the target. after amplification, the reactions can be subjected to a gradual melting or annealing step with fluorescence monitoring to discriminate the specific amplification products from the non-specific artefacts in genie® ii instrument. this eliminates the need for gel electrophoresis or turbidity detection and allows for a closed-tube system thus reducing the cost of the assay. in our study the lack of overlap of t m allowed for the easy identification of all serotypes of dengue. the color change from orange to green in the positive control and the samples was evident in first - min in this study. the denv ns serotype specific rt-lamp assay developed in this study was significantly faster with a mean amplification time of min compared with earlier studies (parida et al., ; sahni et al., ; teoh et al., ) . the speed of the assay was probably due to the use of improved polymerase. this is the first report for evaluation of rt-lamp employing ns region of viral genome with larger clinical samples size of in a genie ® ii flourometer. the performance of the rt-lamp assay was validated by testing the samples simultaneously by the cdc real time pcr that is most sensitive and specific method for detection and differentiation of the denv (cdc dengue). both the rt-lamp and the cdc real time assay for detection and differentiation of the denv showed comparable sensitivity (k = . ). the rt-lamp scored over rt-pcr in terms of sensitivity and specificity and reproducibility compared with study done by teoh et al. in developing countries such as india where dengue is endemic and resources are limited, serological assays are most common methods used to confirm denv infection. in our study using actual clinical samples, the ns antigen by elisa was positive in / of patient's samples which were collected in acute phase of illness when antibodies were absent. the rt-lamp or cdc real time assay when used in combination with ns antigen and anti -dengue igg and igm elisa increased the diagnostic coverage of febrile patients to % ( / ). this is in concordance to the study done by teoh et al. ( ) . the most predominant serotype documented in this study was den- and den- which belonged to the genotype iii and genotype iv respectively. co-circulation of more than one serotype of denv is also known to cause hyperendemicity (dash et al., ) . the ns serotype specific rt-lamp assay developed in this study may be utilized as a rapid, simple, easy, cost effective, isothermal, highly sensitive and specific field applicable technique for detection and differentiation of the dengue virus serotypes which overcomes the deficiencies present in existing techniques. its applicability in tertiary care institutes is emphasized by its ability in viral quantification and evaluation of viraemia in patients. it can suitably be introduced in zonal/peripheral hospitals as an adjunct to existing immunological tests acting as parallel controls. the rt-lamp assay in optigene genie ii instrument may be employed as the new gold standard for timely and accurate diagnosis and serotyping of dengue in the 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pathogenesis of dengue hemorrhagic fever. iv. relation of disease severity to antibody response and virus recovered diagnostic accuracy of ns elisa and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses development and evaluation of a novel loop mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome corona virus molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes mega : integrated software for molecular evolutionary genetics analysis and sequence alignment rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction evaluation of an ns antigen detection for diagnosis of acute dengue infection in patients with acute febrile illness simultaneous detection and differentiation of dengue virus serotypes - , japanese encephalitis virus, and west nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay rapid identification of chikungunya and dengue virus by a real-time reverse transcription-loopmediated isothermal amplification method detection of periodontal pathogen porphyromonas gingivalis by loop-mediated isothermal amplification method development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in minutes multiplex loop-mediated isothermal amplification (m-lamp) assay for the detection of influenza a/h , a/h and influenza b can provide a specimen-to-result diagnosis in min with single genome copy sensitivity flaviviruses detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation investigation of acute dengue infection in southern india employing dengue specific serological and molecular assays loop-mediated isothermal amplification of dna detection of salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of salmonella isolates real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay development and evaluation of reverse transcription loop mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus rapid and real-time detection of chikungunya virus by rtlamp reverse transcription loop-mediated isothermal amplification (rt-lamp) for diagnosis of dengue development of group-and serotype-specific one-step sybr green i-based real-time reverse transcription-pcr assay for dengue virus detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products guidelines for diagnosis, treatment, prevention and control an antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein ns in the sera of infected patients authors acknowledge financial support by the drde, gwalior, india, through sanction no. tc/ /proj /task- / to carry out this work. the authors would like to thank ms. deepal pandya of ampligene india biotech pvt. ltd. for her valuable help in the rt-lamp technique. the authors declare that there is no conflict of interests regarding the publication of this article. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- - k oox authors: sharma, vikrant; chaudhry, dhruva; kaushik, samander title: evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of influenza a viruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k oox background: influenza a viruses (iavs) have always remain a serious concern for the global economy and public health. a rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management. objectives: to develop rt-lamp assays for detection of influenza a viruses, their further subtyping into seasonal (h n , h n ) and novel pandemic h n viruses and to evaluate clinical applicability of optimized rt-lamp assays on patients’ samples. study design: in this study, we optimized rt-lamp assay to detect iavs by using primers against matrix gene and subtyping of iavs was done by using primers against hemagglutinin gene. optimized rt-lamp assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step rt-pcr and real-time rt-pcr. results: rt-lamp assays successfully detected and differentiated iavs into h n , h n and pdm /h n subtypes. one hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with rt-lamp assay, detecting ( . %) samples positive for influenza a virus. out of samples, , and were found positive for pdm /h n , h n and seasonal h respectively. conventional one-step rt-pcr detected a total of ( . %) samples for influenza a and further subtyping showed and samples positive for pdm /h n and h n virus respectively whereas none was found positive for seasonal h n . rt-lamp assay demonstrated higher sensitivity ( . %) than conventional rt-pcr ( . %) for influenza a viruses detection in clinical samples. conclusions: rt-lamp assay is rapid, sensitive, specific and cost effective method for detection of influenza a viruses than conventional one-step rt-pcr and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries. influenza a viruses (iavs) pose a serious threat to the world and are responsible for severe morbidity and mortality among human population globally. seasonal outbreaks, epidemics and pandemics have been witnessed for iavs and have resulted in immense loss to public health and economy (wright and neumann, ) . the devastating potential of influenza is shown by the facts that annually there are estimated - million cases of severe illness and , - , deaths around the globe (who, ) . influenza viruses belong to the family orthomyxoviridae, characterized by segmented, negative-sense, single stranded rna genome (shaw and palese, ) . the family contains seven genera viz., influenzavirus a, influenzavirus b, influenzavirus c, influenzavirus d, isavirus, quaranjavirus and thogotovirus (ictv, ) . out of the four genera of influenza viruses, influenza a and b are the main cause of seasonal epidemics while influenza c causes mild reparatory illness and influenza d affects cattle and not known to cause any infection in humans (hause et al., ; cdc, ; ferguson et al., ) . influenza or "flu" is a respiratory illness characterized by high grade fever, myalgia, headache, malaise, sore throat, rhinitis and cough (cox and subbarao, ). generally influenza is a self limiting disease but may become life-threatening in high risk group individuals including young children, pregnant women, aged people and people with compromised immunity (cox and subbarao, ; cox and subbarao, ) . influenza vaccines are updated annually according to the circulating strains and this information is provided by the surveillance system of who's global influenza surveillance and response system (gisrs) for both the northern and southern hemispheres (who, ; webster and govorkova, ) . antivirals treatment proved effective against influenza if given within - h of the onset of disease (moscona, ) . hence, early and accurate diagnosis of influenza plays a crucial role for timely intervention of therapy, clinical management and thus controlling the spread of disease. clinical symptoms of influenza and other respiratory infections can be very similar, making it hard to differentiate. only laboratory diagnosis can provide an accurate influenza diagnosis. these include conventional virus isolation, antigen/antibody detection and molecular methods like reverse transcription polymerase chain reaction (rt-pcr) (poddar, ) and real time rt-pcr (rrt-pcr) (spackman and suarez, ; carr et al., ; wang and taubenberger, ) . virus isolation and serological assays are time consuming and take days to weeks to get results making them unsuitable during epidemics and also these methods require highly sophisticated biosafety laboratories. molecular methods such as rt-pcr and rrt-pcr are although rapid, sensitive and specific but need costly equipments and trained laboratory persons making these assays hard to use in resource-limited laboratories of developing countries. loop-mediated isothermal amplification (lamp) is a specific, efficient and rapid technique that is similar to pcr amplification but the target dna amplification is under isothermal conditions. this method makes use of a dna polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target dna (notomi et al., ) . the method is applicable to amplification of rna templates when combined with a reverse transcription reaction (rt-lamp). rt-lamp assay has been successfully applied for the detection of various rna viruses including chikungunya (parida et al., ; lu et al., ) , dengue (lu et al., ; parida et al., ) , japanese encephalitis (parida et al., ; toriniwa and komiya, ) , severe acute respiratory syndrome (thai et al., ) and west nile virus . influenza viruses and their novel subtypes like h , h , h and h , have been effectively detected by rt-lamp assay (poon et al., ; ito et al., ; imai et al., ; chen et al., ; zhang et al., ; luo et al., ) . the specificity of rt-lamp is extremely high due to use of four primers that recognize six distinct regions on the target dna. it is faster than pcr because of single temperature is needed for amplification and also cost effective as costly equipments are not required. the amplification results can be viewed directly by using fluorescent dye like sybr green, reducing the time of the assay. the objectives of the current study were to ( ) optimize rt-lamp assay for detection of influenza a viruses and their subtypes (h n , h n and pdm /h n ); ( ) determine sensitivity and specificity of rt-lamp assay; ( ) clinical evaluation of rt-lamp assay and conventional one-step rt-pcr in comparison to who recommended rrt-pcr taken as standard. viral rna was extracted from an aliquot of each reference strain of influenza viruses, using the genejet viral dna/rna purification kit (thermo fisher scientific, usa) by following the manufacturer's instructions. rna was eluted in a final volume of μl in elution buffer (provided with kit) and divided into aliquots of μl each and stored at − °c until used. rna extraction was performed in class ii biosafety cabinets. for detection of influenza a viruses, published primers were taken from matrix (m) gene (poon et al., ) . for subtyping of iavs, primers were selected from hemagglutinin (ha) gene. published primers were used to detect pandemic influenza h n virus (pdm /h n ) (nakauchi et al., ) . for seasonal h n and h n virus, ha gene sequence of the respective reference strain was retrieved from genbank database (genebank accession numbers cy and kc ) and primers were designed by using primer explorer version (https:// primerexplorer.jp/e/). four primers consisting of two internal primers (forward internal primer or fip and backward internal primer or bip) and two external primers (f and b ) were designed for the study. all primers were chemically synthesized by sigma-aldrich (sigma-aldrich, bangalore, india) and were of hplc grade purity (table ) . twenty five microliters of rt-lamp reaction cocktail was prepared in . ml tubes using . μm of each outer primers (f and b ); . μm of each inner primers (fip and bip); . mm of each deoxynucleotides (dntps); mm tris-hcl (ph . ); mm (nh ) so ; mm kcl; mm mgso ; . m betaine; . % tween ; u of bst dna polymerase (new england biolabs); . u of amv reverse transcriptase (invitrogen, usa) and μl of sybr green i (thermo fisher scientific, usa). two microliters of each reference strain's rna was added to their respective tubes (containing strain specific primers) making final volume μl. no template or negative controls were also included in the assay. the reaction tubes were incubated at °c for h either in a water bath or a heating block. the tubes were heated at °c for min to stop the reaction. amplified products were visualized both by electrophoresis using . % agarose gel and by visual detection of green fluorescence due to added sybr green i stain (thermo fisher scientific, usa) under ultra violet (uv) light. conventional one-step rt-pcr was performed on rna samples extracted from the reference strains of iavs. for detection of iavs, primer pair was taken from the matrix (m) gene while subtyping primer pairs were taken from hemagglutinin (ha) gene. for diagnosis of pandemic influenza virus, primer pair was selected from the conserved region of ha gene (genebank accession number ku ) using primer blast software (https://www.ncbi.nlm.nih.gov/tools/primerblast/). details of primer sets were given in table . one-step rt-pcr reaction was carried out in final reaction volume of μl by using verso -step rt-pcr kit (thermo fisher scientific, usa) by following manufacturer's instructions. briefly, reaction mixture was comprised of . μl of x rt-pcr buffers; μl of rt enhancer; . μm of each forward and reverse primer; . μl of enzyme mix and μl of extracted rna. the protocol for optimization of one-step rt-pcr was as follows: °c for min, °c for min, followed by cycles of °c for s, °c- °c for s, °c for s and final extension at °c for min. pcr products were separated by electrophoresis using % agarose gel and visualized by uv transilluminator. influenza virus a/california/ / (h n ) strain with known virus titre as plaque forming units per microliters (pfu/μl) was used to compare sensitivities of rt-lamp and conventional rt-pcr with who recommended taqman real-time rt-pcr (rrt-pcr). ten-fold serial dilutions were made from virus strain in separate tubes ranging from to − pfu per reaction using molecular grade water. rna was extracted from each dilution and used separately for rt-lamp, conventional rt-pcr and rrt-pcr. matrix gene specific primers and μl of rna from each dilution were used in all assays. likewise, sensitivities of rt-lamp, conventional rt-pcr and rrt-pcr were also assessed for subtyping of influenza viruses using ha gene specific primers on h n and h n reference strains. for specificity evaluation, each individual rt-lamp assay was applied on influenza a viruses (pdm /h n , a/ h n and a/h n ), influenza b virus along with other additional respiratory viruses including respiratory syncytial virus (rsv) and human metapneumovirus (hmpv), and cross-reactivity of the assay was checked. a total of clinical samples were collected from patients with influenza like illness (high grade fever (> . °c), cough, sore throat and myalgia) from post graduate institute of medical sciences (pgims), rohtak, haryana, india from november to december . one throat and one nasal swab was collected in ml of viral transport medium (hanks' balanced salt solution supplemented with % bsa, u/ml penicillin and μg/ml streptomycin) from each patient and transported to the laboratory under cold conditions at °c. the samples were processed and rna was extracted in class ii biosafety cabinets. clinical samples were evaluated by using optimized rt-lamp and conventional one-step rt-pcr and detection results were compared with who recommended taqman real-time rt-pcr (rrt-pcr) considering the later as standard. standardized rt-lamp and conventional one-step rt-pcr were applied on clinical samples and amplified products were analyzed by gel electrophoresis. taqman rrt-pcr assay was applied on extracted rnas from clinical samples for detection of influenza a viruses and further subtyping into h n , h n and pdm / h n subtypes. taqman rrt-pcr assay was performed in a μl reaction volume using who recommended protocol and primers/probes sequences (who, ). all three reference strains of influenza a virus were detected by matrix gene specific rt-lamp reaction (fig. a) . on further subtyping, each respective subtype was also detected by ha gene specific rt-lamp assay (fig. a) . optimum amplification temperature and incubation time were found to be °c and h respectively. amplification results were visualized by both gel electrophoresis and green fluorescence detection under uv light ( fig. b and b) . conventional one-step rt-pcr was able to detect all three influenza a virus strains using matrix gene primers. optimum annealing temperature and time was found to be °c for s. amplicons of bp were visualized using % agarose gel electrophoresis (fig. c) . for subtyping conventional rt-pcr, annealing at °c for s was found to be optimum for both pdm /h n and h n subtypes whereas for seasonal h n subtype, annealing temperature of °c for s was optimized. amplicons of bp, bp and bp were detected for pdm /h n , h n and h n respectively, after % agarose gel electrophoresis (fig. c) . sensitivity of rt-lamp assays were assessed for detection of influenza a viruses using m gene primers and for detection of three subtypes i.e. h n , h n and pdm /h n , using ha gene primers (table ) . the results were compared with that of conventional one-step rt-pcr and rrt-pcr. the detection limit of m gene specific rt-lamp was found to be ten times higher ( . pfu/reaction) than conventional one-step rt-pcr ( . pfu/reaction) (fig. a) and ten times lower than rrt-pcr ( . pfu/reaction) ( table ). the detection limits of ha gene specific rt-lamp assays for pdm /h n , h n and h n were . , . and . pfu per reaction respectively. the detection limits of conventional one-step rt-pcr for pdm /h n , h n and h n were , . and pfu per reaction respectively, making it - times less sensitive than rt-lamp. rt-lamp demonstrated similar sensitivities as that of rrt-pcr for the detection of pdm /h n and h n while times less sensitivity for detection of h n subtype (table ) . specificity of each rt-lamp primer set was evaluated against different influenza viruses and other respiratory viruses. all the rt-lamp primer sets were found to be highly specific for their respective influenza viruses as there was no amplification with other influenza viruses or different respiratory viruses. as shown in fig. b , pdm /h n rt-lamp (ha gene) was found to be highly specific for detecting pandemic iavs as only pdm /h n reaction showed positive amplification while there was no amplification with other viruses. all clinical samples were analyzed for presence of iavs and their subtypes (seasonal h n , h n and pdm /h n ) using who recommended taqman real-time rt-pcr, rt-lamp assay and conventional rt-pcr assay. real-time rt-pcr results showed that out of swab samples, ( . %) were positive for influenza a viruses. further subtyping of (n = ) positive samples by real-time rt-pcr showed that ( . %) samples were positive for pdm /h n , ( . %) were positive for h n and ( . %) were positive for h n virus. matrix gene specific rt-lamp assay detected ( . %) samples positive for influenza a viruses which were further sub-typed using ha gene specific rt-lamp. results showed that out of ( %) samples were positive for pdm /h n , ( . %) samples were positive for h n , and ( . %) samples were positive for h n virus. these results showed the comparable sensitivity and specificity of real-time rt-pcr with that of rt-lamp assay (table ). conventional rt-pcr detected ( . %) samples positive for influenza a viruses out of which ( . %) were found positive for pdm /h n and ( . %) were positive for h n virus where as no sample was detected positive for h n virus. all samples which were found to be negative by realtime rt-pcr also come negative by both rt-lamp and conventional rt-pcr assay. influenza surveillance has importance not only in diagnosis of circulating strains of the virus but also to upgrade the available vaccine on annual basis to provide protection against influenza. control of novel influenza a viruses mainly depends of early and accurate identification of virus strain followed by early intervention of antiviral therapy. so, a rapid, sensitive, specific and cost effective method of detection and subtyping of influenza a viruses is always seems fruitful in this scenario. virus isolation using egg embryo culture or cell culture is considered as 'gold standard' for influenza virus detection but the whole process is labour intensive and lengthy taking - days for obtaining results (ellis and zambon, ) . serological methods are also not very reliable for influenza surveillance as these are time consuming and require analysis of serum samples from both acute phase and recovering phase. rapid detection kit based assays are although fast but are not very consistent, have low sensitivity and may produce false results (wang and taubenberger, ; ellis and zambon, ; kim and poudel, ) . molecular detection methods have emerged as very table limit of detection of rt-lamp, conventional one-step rt-pcr and real-time rt-pcr assay using virus titre as plaque forming units (pfu). useful tools in comparison to time consuming conventional virus isolation and serological methods. molecular methods based on conventional rt-pcr and real-time rt-pcr have been proved useful in diagnosis and surveillance of influenza viruses, but most of these assays require highly sophisticated instruments and are not feasible to conduct in resource-limited settings of developing countries. therefore, present study was focused on rt-lamp assay for detection of influenza a viruses and further subtyping into seasonal influenza (h and h ) and novel pandemic influenza a virus (pdm /h n ). rt-lamp assay has been successfully applied to detect influenza a viruses like swine flu (h n ), pdmh n , avian influenza h , h , h and h subtypes (poon et al., ; ito et al., ; imai et al., ; chen et al., ; luo et al., ; kubo et al., ; parida et al., ; bao et al., ) . in this study, we used matrix gene based primers for detection of influenza a viruses (poon et al., ) and hemagglutinin gene based primers for their further subtyping. matrix gene based rt-lamp assay was able to amplify all three reference strains of influenza a virus and hemagglutinin gene based rt-lamp assay successfully detected the respective subtypes. no cross-reactivity was observed for other related viruses and influenza subtypes making the reaction highly specific. sensitivity of a detection assay is an important deciding factor when it comes to applicability of the assay. in this study we have compared rt-lamp with conventional rt-pcr and taqman real-time rt-pcr. in our study, detection limit of taqman rrt-pcr was found to be ten times more than rt-lamp for influenza a and its subtype h n whereas for detection of pdm /h n and h n both assays showed similar values. sensitivity comparison of rt-lamp with conventional rt-pcr showed that rt-lamp was - times more sensitive than conventional rt-pcr. previous studies have also established that rt-lamp is more sensitive than conventional rt-pcr and has comparable sensitivity with real-time rt-pcr for detection of influenza viruses (poon et al., ; kubo et al., ; parida et al., ; bao et al., ; sharma and kaushik, ) . in the present study, a total of clinical samples were analyzed for the presence of iavs and their further subtyping was done. we have showed that rt-lamp assay can be applied successfully for the diagnostic and surveillance studies of influenza viruses. detection time is a crucial factor of any diagnostic assay. rt-lamp assays were completed in h and results were shown after min of agarose gel electrophoresis. so rt-lamp assay gave efficient results within one and a half hours of sample processing (rna extraction) making this assay fast when compared with conventional rt-pcr and real time rt-pcr which gave results within - h (gu et al., ) . previous studies have reported that by using two accessory loop primers the amplification time can be reduced from min to - min (luo et al., ; kubo et al., ; gu et al., ; imai et al., ; peng et al., ) . for the sake of simplicity and making our reaction more cost effective we stuck to the basic protocol of rt-lamp assay and use only four primers for amplification which didn't compromise with sensitivity of the assay. rt-lamp assay can be modified to detect samples in real-time by using turbidity measurement by turbidimeter as turbidity of reaction increases due to release of pyrophosphates during amplification. moreover, addition of a florescent dye like calcein or sybr green in the reaction can be used for direct visualization of the amplification products under uv light thus eliminating the need of gel electrophoresis making the reaction more rapid and less prone to crosscontaminations (luo et al., ; kubo et al., ; bao et al., ; gu et al., ; peng et al., ) .we used sybr green i dye (sigma-aldrich, usa) for direct visualization under uv light and both visual detection and agarose gel electrophoresis produced similar results. this showed that rt-lamp assay can be applied as a point-of-care test during influenza outbreaks due to its simplicity, high sensitivity and less turn-around time. in conclusion, this study showed that rt-lamp assay can be used for detection and surveillance studies during the time of influenza outbreaks as a good alternative for conventional rt-pcr and real-time rt-pcr. rt-lamp assay is highly cost effective and easy to perform as sophisticated instruments like a thermocycler and fluorescent data analyzing computers (as in real-time rt-pcr) are not needed and the whole reaction can be done in a controlled temperature water bath or dry heating block. the results of the study clearly showed that rt-lamp assay can be applied successfully to the clinical samples and the results were compatible with that of the who's recommended real-time rt-pcr. the rt-lamp assay is a simple, rapid, specific, sensitive and cost effective method for detection and subtyping of influenza a viruses and may prove useful in the resource-poor laboratories of the developing countries during influenza outbreaks. vs has designed the study, performed the experiments, analyses and writing, dc participated in designing the study, sk designed the study, performed analyses and writing. this work was funded by a major research project grant ( - / ) of university grants commission (ugc), new delhi, india. this study was approved by the human ethical committee (letter no. phy/ / ), maharshi dayanand university, rohtak, haryana, india and detailed informed consent was taken from each patient before taking swab samples. development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype h types of influenza viruses development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections development of reverse transcription loop-mediated isothermal amplification for rapid detection of h avian influenza virus global epidemiology of influenza: past and present molecular diagnosis of influenza pathogenesis of influenza d virus in cattle rapid and specific detection of h swine influenza virus using reverse transcription loop-mediated isothermal amplification method characterization of a novel influenza virus in cattle and swine: proposal 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sensitive detection of h n avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification none declared. key: cord- -m tbdfri authors: khandia, rekha; dadar, maryam; munjal, ashok; dhama, kuldeep; karthik, kumaragurubaran; tiwari, ruchi; yatoo, mohd. iqbal; iqbal, hafiz m.n.; singh, karam pal; joshi, sunil k.; chaicumpa, wanpen title: a comprehensive review of autophagy and its various roles in infectious, non-infectious, and lifestyle diseases: current knowledge and prospects for disease prevention, novel drug design, and therapy date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: m tbdfri autophagy (self-eating) is a conserved cellular degradation process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. autophagy dysfunction can have various pathological consequences, including tumor progression, pathogen hyper-virulence, and neurodegeneration. this review describes the mechanisms of autophagy and its associations with other cell death mechanisms, including apoptosis, necrosis, necroptosis, and autosis. autophagy has both positive and negative roles in infection, cancer, neural development, metabolism, cardiovascular health, immunity, and iron homeostasis. genetic defects in autophagy can have pathological consequences, such as static childhood encephalopathy with neurodegeneration in adulthood, crohn’s disease, hereditary spastic paraparesis, danon disease, x-linked myopathy with excessive autophagy, and sporadic inclusion body myositis. further studies on the process of autophagy in different microbial infections could help to design and develop novel therapeutic strategies against important pathogenic microbes. this review on the progress and prospects of autophagy research describes various activators and suppressors, which could be used to design novel intervention strategies against numerous diseases and develop therapeutic drugs to protect human and animal health. phosphatidylinositol- -kinase (pi k) inhibitors and beclin inhibits the starvation-induced mitochondrial autophagy, but not the neurotoxin ( -methyl- -phenylpyridinium)-mediated autophagy [ ] [ ] [ ] . although autophagy was discovered over years ago [ ] , its molecular mechanisms were only understood in the late s following a genetic screening in yeast, which revealed mutations in autophagy-related genes. at least yeast autophagy genes (atgs) have been identified, many of which have mammalian cell homologs [ ] . many molecular mechanisms have been explored to reveal the basic processes underlying autophagy. multiple signaling pathways focus on two protein complexes to initiate autophagy, the ulk (unc -like autophagy activating kinase ) protein kinase complex and the pi kc -c (class iii phosphatidylinositol -kinase complex i) lipid kinase complex [ ] . novel autophagy regulators with rna-related activities have also been shown to be involved in this process [ ] . furthermore, upstream signaling pathways common to both autophagy and apoptosis are known to be induced by er stress via signaling molecules such as perk/atf , ire α, atf , and ca + [ ] . the details of these mechanisms will shed light on the different forms of autophagy and the numerous intermediates involved. three types of autophagy [macroautophagy, microautophagy, and chaperone-mediated autophagy (cma)] are depicted in figure . autophagy refers to the process of delivering cytoplasmic or extracellular components to the lysosomes of an animal cell or the vacuoles of plant or yeast cells [ ] . the production and maturation of autophagosomes are directly regulated by location, timing, and intensity [ ] . the phosphoinositide-binding protein, hs bp , is a negative regulator of autophagosome biogenesis that regulates the lipid composition and phosphatidic acid (pa) levels of autophagosome precursor membranes [ ] . increased levels of systemic autophagy have been reported in caenorhabditis macroautophagy is initiated when a portion of cytoplasm containing a cellular organelle is sequestered to form the autophagosome [ ] . the autophagosome fuses with the lysosome or late endosomal multivesicular bodies (mvbs) to degrade the materials within it. atg (microtubule-associated protein a/ b-light chain , lc , is an atg homolog in humans) was the first autophagosomal protein to be characterized [ ] . macroautophagy can be classified as cargo-specific or non-selective [ , , ] . mitophagy has been observed in yeasts when a shift occurs between non-fermentable and fermentable carbon sources, such as glucose, following which the surplus mitochondrial population undergoes mitophagy [ , ] . the first protein identified to cause mitophagy in yeast was uth p, a member of the sun family, which is present in the outer mitochondrial membrane and allows excessive mitochondria to be removed during starvation [ ] . the mitochondrial outer membrane protein, atg , is a receptor for selective autophagy [ ] that is not conserved in mammalian species; instead, fundc and bnip , bnip l/nix, and sqstm /p act as mitophagy receptors, and are dependent upon hypoxia, erythrocyte maturation, and damage-induced mitophagy, respectively [ , , ] . pexophagy is also induced in saccharomyces cerevisiae and pichia pastoris via the atg and ppatg receptors, respectively, when the fungal medium is switched from an oleic acid or methanol to a glucose or nitrogen starvation medium [ , ] . starvation has also been shown to induce non-selective macroautophagy [ ] , whereas mitochondrial phospholipids have been demonstrated to be required for autophagy [ ] . the machinery required for selective autophagy has been studied extensively using yeast cells, revealing that the cytoplasm-to-vacuole targeting (cvt) pathway is used to specifically transport vacuolar hydrolases into the vacuole of budding yeast cells [ ] . a high degree of curvature in the initiating membranes (phagophores or isolation membranes) is a prominent feature of cvt vesicles during mammalian autophagy [ ] . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [ ] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein (hsc ). hence, microautophagy involves both endocytic and autophagic components [ , ] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [ , ] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp- a [ , ] . lamp- a must form a multiprotein complex to translocate the substrate [ ] ; lamp- a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp- a for degradation, following which lamp- a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp- a tightly regulate the rate of cma at the lysosomal membrane [ , ] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p adaptor-mediated autophagic viral protein clearance induces cell survival [ ] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [ ] [ ] [ ] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [ ] . the process of cma is depicted in figure . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [ ] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein (hsc ). hence, microautophagy involves both endocytic and autophagic components [ , ] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [ , ] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp- a [ , ] . lamp- a must form a multiprotein complex to translocate the substrate [ ] ; lamp- a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp- a for degradation, following which lamp- a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp- a tightly regulate the rate of cma at the lysosomal membrane [ , ] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p adaptormediated autophagic viral protein clearance induces cell survival [ ] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [ ] [ ] [ ] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [ ] . the process of cma is depicted in figure . autophagy is an evolutionarily conserved process induced via multiple signaling pathways by numerous stimuli including nutrient starvation [ , ] , hypoxia [ , ] , oxidative stress [ , ] , pathogen infection [ , ] , and er stress [ ] . in the presence of nutritional substances and cytokines, mechanistic/mammalian target of rapamycin (mtor) can prevent apoptosis and stimulate cell growth [ ] , whereas stress and nutrient starvation inhibit mtor to initiate autophagy via at least four molecular complexes, including the unc- -like kinase (ulk) complex, consisting of ulk- , atg , atg , and fak-family interacting protein (fip ); the pi k complex, consisting of atg , vacuolar protein sorting (vps) , vps , beclin , and beclin -regulated autophagy protein (ambra ) [ ] [ ] [ ] ; transmembrane protein complexes, including atg and wipi; and two ubiquitin-like protein conjugation systems (atg and lc ) [ , ] . autophagy is initiated by the assembly of the ulk complex, which phosphorylates ambra and leads to activation of the pi k complex [ , ] . class iii pi k is known to participate in various membrane trafficking events, whilst pi k and beclin mediate membrane nucleation. the atg -atg -atg complex is recruited to the pre-autophagosomal structure (pas) where it associates with the outer membrane of the phagophore, essentially preventing the premature fusion of vesicles and lysosomes [ ] . the second ubiquitin-like system stimulates the binding of phosphatidylethanolamine (pe) and atg /microtubule-associated protein light chain (lc ). lc has a high affinity for the lysosome when bound to the phagosome (laposome); thus, any engulfed pathogens will be killed and degraded at a higher rate [ ] . atg , atg , and atg process lc into lc -ii, a molecular marker for autophagosomes [ ] that is present on both its inner and outer surfaces and is essential for the expansion and completion of the autophagic membrane. following autophagosomal closure, the atg -atg -atg complex dissociates from the autophagosome. atg is required for the formation of intraluminal vesicles and is localized within the autolysosome for acidification [ ] ; atg is also translocated to the site of autophagosome formation where it provides a membrane to elongate the limiting membrane, known as the phagophore [ ] . the autophagosome then fuses to the lysosome to form the autolysosome, which is regulated by lysosomal membrane proteins and cytoskeletal proteins [ ] . the lamp- / protein controls autophagosomal maturation. genetic mutations in lamp- are known to cause danon disease, a glycogen storage disorder linked to hypertrophic cardiomyopathy, skeletal muscle weakness, and intellectual disability [ ] . within the autolysosome, hydrolytic enzymes digest the internalized cargo and the internal autophagosome membrane, then the digested products such as amino acids are released into the cytosol to be recycled. autophagosomes are also directly related to cell trafficking pathways. recently, holland et al. identified that the phosphoinositide-binding protein hs bp negatively regulates autophagosome production [ ] . hs bp is thought to reduce phospholipase d (pld ) activity and its localization to atg l and transferrin receptor (tfrc)-positive vesicles. it is also known to regulate the levels of pa and the lipid content of autophagosome precursor membranes [ ] . two large families of e ubiquitin ligases, trim and cullin, have been recognized as important autophagy regulators which promote or inhibit the process, respectively [ ] . the gtpase ras-related protein in brain (rab ) also plays a key role in autophagy regulation, particularly in modulating its flux [ ] . knockdown of the small gtpase rab has been shown to inhibit pterostilbene-induced autophagy in vascular endothelial cells (vecs), whilst its upregulation stimulates autophagy in vecs [ ] . under basal autophagy conditions in humans, proteomic analysis of the autophagy interaction network (ain) revealed a network of interactions between candidate proteins [ ] . in order to identify the proteins modulating starvation-induced autophagy, genome-wide screening of sirna in a gfp-lc -expressing human cell line was carried out [ ] , shortlisting nine proteins. one of these, short coiled-coil protein (scoc), forms an essential starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified genes that promote autophagy when inactivated [ ] . long ncrnas (lncrnas), which are longer than nucleotides and do not encode proteins, often possess regulatory functions; for example, mir - has been found to regulate atg expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc b and lamp- c, have been found to bind rna [ ] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genome-wide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure . starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified genes that promote autophagy when inactivated [ ] . long ncrnas (lncrnas), which are longer than nucleotides and do not encode proteins, often possess regulatory functions; for example, mir - has been found to regulate atg expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc b and lamp- c, have been found to bind rna [ ] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genomewide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure . liu and levine [ ] described a novel form of non-apoptotic autophagic gene-dependent cell death, termed autosis, which is mediated by the na + /k + -atpase pump. autosis involves enhanced cell-substrate adhesion, focal ballooning of the perinuclear space, and dilation and fragmentation of the endoplasmic reticulum. the tat-beclin peptide complex may initiate autosis, with the fusion of the evolutionarily conserved, -amino acid-long beclin domain with amino acids from the hiv tat protein transduction domain aiding the cellular entry of the fusion peptide [ ] . the tat-beclin fusion peptide has been shown to inhibit the replication of hiv, chikv, sindbis (sinv), and wnv, as well as intracellular bacteria such as listeria monocytogenes [ ] . tat-beclin treatment also reduced mortality in neonatal mice infected with chikv and wnv, demonstrated using a tunel assay, and cleared mutant huntingtin protein aggregates [ ] . autosis can be partially rescued by knocking down atg or atg or using -methyladenine. under serum/amino acid starvation, approximately % of dying cells exhibit a morphology similar to that of cells treated with tat-beclin and autosis is selectively blocked when the na + /k + -atpase pump is inhibited [ ] . during cerebral hypoxia or ischemia, the neonatal brain releases cardiac glycosides (ouabain or endobain), which inhibit the na + /k + -atpase pump and reduce autosis [ ] . autosis has also been observed in patients with severe liver anorexia nervosa who display focal ballooning of the perinuclear space, convoluted nucleus, dilated and fragmented er, empty vacuoles, and several autolysosomes in their hepatocytes [ ] . ischemic injury can also lead to autosis in other organs, including the kidney and heart, which is attenuated in beclin +/− mice [ , ] . autophagy can promote or inhibit cell death depending on the cellular context; many other death mechanisms are intricately involved in the processes, with several mechanistic links elucidated between autophagy and other death mechanisms. autophagy and apoptosis regulation overlap when the bh domain of the beclin autophagy protein interacts with anti-apoptotic proteins of the bcl- family, including bcl- , bcl-xl, and mcl- [ ] [ ] [ ] [ ] . the bh domain has a critical role in the interaction between these proteins and has been shown to interact with the receptor domain of the bcl- family in nutrient-rich cells [ ] . beclin -mediated autophagy is inhibited by er-localized bcl- [ ] ; the transgenic expression of bcl- was shown to inhibit autophagy in mouse heart muscles. beclin mutants, which are unable to bind to bcl- , induce higher levels of autophagy than their wild-type counterparts [ , ] ; hence the physical beclin -bcl-xl/bcl- interaction regulates beclin -mediated autophagy [ ] . abt , a compound which mimics the bh domain and thus inhibits this interaction, increases the aggregation of lc , an autophagy marker which is present on autophagosomes [ ] , in both nutrient-rich and nutrient-deprived media. furthermore, the knockdown of beclin and other essential atg proteins using sirna heteroduplexes was shown to reduce abt -stimulated lc aggregation [ ] . atg is a dual-functioning protein that participates in both autophagy and apoptosis [ ] ; non-conjugated atg can bind and inhibit mcl- and bcl- via a bh -like domain to positively regulate mitochondrial apoptosis. atg knockout inhibits the release of cytochrome c from the mitochondria and apoptosis, whilst abnormal atg expression represses the anti-apoptotic activity of mcl- [ ] . autophagy promotes apoptosis by degrading a negative regulator of the fas ligand [ ] ; however, it can also protect cells against apoptosis induced by tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail) by altering the concentrations of bcl family members [ ] . similarly, components were found to be degraded by autophagy during developmental apoptosis [ ] , whilst it was recently shown that inhibiting autophagy increased apoptosis and accelerated mortality in murine sepsis models with inadequate autophagy pathways in cd + t cells, indicating that autophagy has a functional role against apoptosis and immunosuppression in t cells in sepsis [ ] . furthermore, trail combined with a novel chalcone derivative, chal- , was found to remarkably increase lung cancer cell cytotoxicity via autophagy-mediated apoptosis [ ] . necroptosis is often associated with inflammation [ ] . the relationship between autophagy and necroptosis is complex, elusive, and slightly controversial since reports have indicated that necroptosis may promote [ ] , inhibit [ , ] , or do not affect autophagy [ ] . in several cell lines, including l cells, lymphocytes, and cancer cells, autophagy is activated in the presence of tnfα and under starvation to suppress necroptosis [ ] . the apoptosis-inhibiting peptide, carbobenzoxy-val-ala-asp (zvad), prevents autophagy and induces necroptosis in response to tnfα by regulating lysosomal cathepsins, highlighting the pro-survival function of autophagy against necroptosis [ , ] . similarly, inhibiting the mtor signaling pathway can prevent apoptosis and even enhance necroptosis, whereas starvation, which induces autophagy, protects cells from zvad-mediated necroptotic death [ ] . sirtuins (sirt) are nad + -dependent protein deacetylases which are actively involved in both autophagy and necroptosis, as well as transcription, stress resistance, and aging. sirt- deacetylates various components of the autophagy pathway, including atg , atg , and atg [ ] , thus promoting autophagy. in cancer cells, dissociation of the foxo transcription factor from sirt- during oxidative stress or starvation results in the acetylation and binding of foxo to atg , which subsequently induces autophagy [ ] . the binding of sirt- to receptor-interacting protein (rip) mediates rip deacetylation in response to tnfα; rip and rip then form a complex, which triggers necroptosis [ ] . the switch between necroptosis and apoptosis is achieved by recruiting necrosome components to autophagy machinery. atg knockdown reduced the association between ripk and mlkl, suggesting that atg is important in trail-induced necrosome activation. furthermore, atg knockout in the atg -/-df- cell line inhibited autophagy but promoted apoptosis [ ] . autophagy machinery also affects the mechanism of cell death by promoting efficient necrosomal activation and mlkl phosphorylation, thus inducing necroptosis [ ] . several anti-cancer agents, including sorafenib, cause deficient autophagosome formation and facilitate the interaction between p and ripk, resulting in cell death by necroptosis [ ] ; however, there is still much to be elucidated about the interplay between these two processes. necrosis refers to the increase in cell volume caused by organelle swelling, which results in plasma membrane rupture and the loss of intracellular contents. when atp is depleted, the cell is unable to undergo apoptosis and undergoes necrosis instead [ ] . poly adp ribose polymerase (parp ) is an enzyme with roles in dna repair, transcriptional regulation, and chromatin modification [ ] . parp over-activation decreases the atp reservoir and induces necrotic cell death by bypassing energy-dependent apoptotic cell death [ ] . atp depletion also activates amp-activated kinases (ampk) [ ] , which induce autophagy by activating the ulk complex or inhibiting mtor signaling [ ] . thus, dna damage-induced parp activation leads to a decline in atp levels, ampk activation, mtor inhibition, and autophagy induction [ ] . parp plays a dual role in autophagy and necrosis since autophagy is a pro-survival mechanism, whilst necrosis is a pro-death mechanism. the fate of the cell depends on the balance between autophagy and necrosis, where autophagy represents the final attempt of the cell to survive before necrosis. autophagy plays a beneficial role against infectious diseases by simultaneously degrading pathogens and activating the host immune system [ ] . this enables infections to be countered directly, by killing infectious agents, and indirectly, by inducing host immunity against pathogens. autophagy provides an excellent intracellular defense system against bacterial pathogens, including salmonella enterica serovar typhimurium [ , ] , listeria monocytogenes [ , ] , and shigella flexneri [ ] . anti-bacterial autophagy is termed xenophagy [ , , ] . numerous cellular, membrane-associated, or cytoplasmic moieties modulate xenophagy; and those cells unable to carry out xenophagy, exhibit higher rates of infection. bcl-xl regulates xenophagy, and bcl-xl knockout cells are more susceptible to streptococcus pyogenes infection [ ] . the infection of non-phagocytic cells by shigella flexneri is dependent upon type-iii secretion system (t ss) effector proteins [ ] which reorganize the host cell cytoskeleton, ruffle the cell membrane, and cause bacterial uptake. following internalization, bacterial peptidoglycans are detected by nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) which trigger a pro-inflammatory immune response [ ] (figure ). the bacteria-sensing nod proteins interact with atg l to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [ ] . intracellular bacterial sensing either by nlrs or sequestosome- -like receptors (slrs) recruits autophagy proteins, including unc- -like kinase (ulk) / and lipid kinase complexed with beclin and atg l , to initiate phagophore membrane nucleation and engulf invading bacteria [ ] . mutant c. elegans with defective autophagy genes exhibit increased susceptibility to bacterial infection [ ] . in addition, it has been reported that hlh- /tfeb-mediated autophagy and autophagy pathways can regulate the tolerance of c. elegans to bacillus thuringiensis infection by protecting against its pore-forming toxins [ ] , suggesting a novel association between intrinsic epithelial defenses and hlh- -mediated autophagy against in vivo bacterial attacks. liang et al. [ ] reported that beclin overexpression inhibits sindbis virus replication, indicating that autophagy protects against infectious pathogens. autophagy may activate innate immunity against mycobacteria via pattern recognition receptors (prrs) or non-receptor-mediated processes [ ] . infection with group a streptococcus species (gas; streptococcus pyogenes) induces anti-apoptotic bcl-xl expression which inhibits autophagy directly by suppressing autophagosome-lysosome fusion, and indirectly by interacting with beclin -uvrag to suppress gas internalization [ ] . in addition, mycobacterium tuberculosis is known to induce mir expression in human macrophages and monocytes and adversely affect their antimicrobial activities and innate host immune responses against the bacterial infection by targeting dram (dna damage-regulated autophagy modulator ), which is a critical element of the autophagy response [ ] . the ubiquitin ligase, smurf , has also been shown to control m. tuberculosis replication in human macrophages by associating with bacteria in the lungs of patients with pulmonary tuberculosis. the murine macrophage cell line, raw . , has been used to study bacillus amyloliquefaciens sc -induced autophagy and its anti-bacterial response against escherichia coli; b. amyloliquefaciens stimulated autophagy by increasing the expression of beclin and the atg -atg -atg complex, but not activating the akt/mtor signaling pathway [ ] . several autophagy-inducing drugs have been used to treat microbial infections; for example, ar- [ -amino-n-[ -[ -( phenanthrenyl)- -(trifluoromethyl)- h-pyrazol- -yl] phenyl]-acetamide] inhibits phosphoinositide-dependent kinase- and eliminates salmonella typhimurium in murine macrophages and francisella tularensis in human leukemic thp- macrophages [ , ] . furthermore, α, -dihydroxycholecalciferol, a form of vitamin d, can enhance autophagy and inhibit human immunodeficiency virus (hiv) replication in macrophages [ ] . the bacteria-sensing nod proteins interact with atg l to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [ ] . intracellular bacterial sensing either by nlrs or sequestosome- -like receptors (slrs) recruits autophagy proteins, including unc- -like kinase (ulk) / and lipid kinase complexed with beclin and atg l , to initiate phagophore membrane nucleation and engulf invading bacteria [ ] . many bacteria have evolved mechanisms to overcome autophagy and allow them to replicate within infected cells or even within autophagosomes. these bacteria may express receptors to prevent or enhance phagosome formation, capture nutrient containing phagosomes, subvert autophagy machinery, prevent fusion, or resist autophagy. certain bacteria hijack autophagosomes and use the by-products of autophagic degradation for microbial replication [ ] . anaplasma (formerly ehrlichia) phagocytophilum, the causative agent of human anaplasmosis, uses the effector anaplasma translocated substrate (ats- ) to enhance autophagosome formation and acquire nutrients from inside the autophagosome [ ] . after entering the cell, a. phagocytophilum replicates inside a double-lipid bilayer membrane associated with lc (atg ), beclin , and atg but lacking lysosomal markers. inhibiting autophagy with -methyladenine did not prevent bacterial internalization but arrested its growth [ ] , indicating that the autophagic machinery had been subverted to facilitate bacterial proliferation. another bacterium, yersinia pseudotuberculosis, replicates intracellularly inside specific compartments called yersinia-containing vacuoles (ycvs), which contain autophagy markers; however, ycvs are not acidified and sustain bacterial replication [ ] . during y. pestis infection, lc -i is conjugated with pe to recruit lc -ii, a marker of autophagy progression, to the phagosomal membrane [ ] . a similar mechanism is used by coxiella burnetii, the causative organism of q fever. coxiella-replicative vacuoles contain lc , beclin , and rab ; overexpression of these proteins increases the number of coxiella-replicative vacuoles [ ] . brucella abortus replicates within brucella-containing vacuoles (bcvs) which traffic from the endocytic compartment to the endoplasmic reticulum, where the bacteria proliferate. bacterial proliferation requires the autophagy-initiation proteins ulk , beclin , and atg l; however, atg , atg l , atg b, atg , and lc b are not required [ ] . pathogens such as brucella spp. and porphyromonas gingivalis have evolved to survive inside autophagosomes by preventing its fusion with the lysosome, thus escaping host innate immunity mechanisms [ , ] . salmonella typhimurium studies have revealed that autophagy targets invading intracellular bacterial pathogens for degradation [ ] ; s. typhimurium regulates the sirt /lkb /ampk complex of the mtor pathway by targeting sirt , lkb , and ampk to lysosomes for rapid degradation, restricting autophagy and disrupting ampk-mediated mtor regulation [ ] . autophagy is differentially regulated in tuberculoid and lepromatous leprosy [ ] ; in tuberculoid skin lesion cells, autophagy controls mycobacterium leprae, whereas in lepromatous cells, the blocking of bcl- -mediated autophagy promotes bacterial persistence. ifn-γ may counteract the m. leprae-mediated inhibition of autophagy in lepromatous macrophages as autophagy levels were restored in lepromatous patients who developed the reversal reaction, an inflammatory state associated with augmented ifn-γ and rapamycin treatment, indicating that autophagy is an important innate mechanism associated with m. leprae control in skin macrophages [ ] . autophagy has a beneficial role in cellular defense against invasion by viruses; therefore, it has been used for antiviral immunity [ , , ] . autophagy helps to clear viral pathogens during infection via various molecular mechanisms, regulates immune responses, and prevents harmful overactivation and inflammation [ ] . for example, autophagy increases the presentation of endogenous viral antigens in the peptide grooves of major histocompatibility complex (mhc) class i molecules on the cell surface during herpes simplex virus type (hsv- ) infection. studies of viral peptides have suggested a complex interaction between vacuoles and mhc class i presentation pathways in autophagosomes [ ] . in contrast, mhc class ii molecules continuously accept input from autophagosomes, which facilitates antigen presentation by mhc class ii molecules [ , ] . autophagy is a major component of drosophila immunity against vesicular stomatitis virus (vsv) [ ] as it can deliver viral antigens to tlrs for presentation. during anti-viral signaling, pattern recognition receptors (prrs) at the plasma membrane (i.e., toll- ) that are engaged by vsv stimulate an autophagy-dependent innate immune response mediated by pi k-akt-signaling [ , ] . furthermore, toll/tlr signaling has been shown to regulate the rift valley fever virus (rvfv) replication in both flies and mammals [ ] , whilst sirt , an nad(+)-dependent deacetylase, modulates the activation of dendritic cells and autophagy during induced immune responses against respiratory syncytial virus (rsv) [ ] , thereby directing an effective anti-viral immune response. furthermore, autophagy is stimulated by the salicylamide derivatives against cytopathic bovine viral diarrhea virus (cp-bvdv), a flaviviridae pestivirus [ ] . foot-and-mouth disease virus (fmdv) infection suppresses autophagy and nf-κb anti-viral activities by degrading atg -atg using the viral protein, c pro , suggesting that atg -atg positively modulates anti-viral nf-κb and irf pathways during fmdv infection to limit fmdv proliferation [ ] . however, autophagy is often hijacked by viral pathogens and can be modulated to their own benefit. subverting the autophagic pathway can have adverse consequences by giving pathogens access to nutrients for growth and reproduction [ ] . autophagy plays an important role in viral replication and pathogenesis [ ] , with coronaviruses [ ] , coxsackievirus b [ ] , poliovirus [ ] , hepatitis c virus (hcv) [ , [ ] [ ] [ ] , and denv [ ] all known to stimulate and require autophagy for accelerated replication. for instance, autophagy has been demonstrated to be actively involved in the replication of influenza a virus (iav), which induces autophagosome formation during the early phase of infection and later inhibits autophagosomal maturation by preventing autophagosomal-lysosomal fusion and promoting autophagosomes to accumulate in virus-infected cells [ ] . autophagy-deficient cells are more susceptible to apoptosis upon influenza infection [ , ] , while using pharmacological reagents or rna interference to alter cellular autophagy can impair viral protein accumulation [ ] . human single-chain antibody variable fragments (scfvs) which bind to the influenza a virus ion channel protein (m ) and inhibit viral replication [ ] were found to restore autophagosome maturation suppressed by the infecting virus (personal communication). it has also been reported that hcv can trigger autophagy via immunity-related gtpase m, which promotes hcv replication [ ] . paramyxoviruses such as newcastle disease virus (ndv) have been shown to trigger autophagy in u glioma cells to enhance viral replication [ ] . in addition, modulating ndv-induced autophagy using rapamycin, chloroquine, or small interfering rnas which target genes critical for autophagosome formation (atg and beclin ) affects virus production, suggesting that ndv may utilize autophagy to promote its replication [ ] . human immunodeficiency virus (hiv) uses multiple methods to regulate autophagy and enhance its replication [ , ] . hiv induces the early stages of autophagy but inhibits the later stages which would suppress the production of new virions. the hiv- accessory protein, nef, inhibits autophagosomal maturation by interacting with beclin [ ] , whilst the hiv protein vpr can trigger autophagy in transfected thp- macrophages, indicating that autophagy may be involved in maintaining hiv reservoirs in macrophages [ ] . hsv- [ ] , kaposi's sarcoma-associated herpesvirus (kshv) [ ] , and mouse herpesvirus (mhv- ) encode proteins that bind beclin to prevent autophagy initiation [ ] . during poliovirus (pv) infection, vesicle acidification, which can mature autophagosomes, has been shown to induce the maturation of virions into infectious particles [ ] . one of the most important characteristics of high-risk human papillomavirus (hrhpv) etiopathogenesis is that inhibiting host autophagy could cause cervical cancer via hrhpv [ ] . in epithelial cells, flavivirus ns a-induced autophagy protects infected cells and induces viral replication [ ] . autophagy also plays a critical role in the replication of coronaviruses and the generation of their replicative structures [ ] . coronavirus nonstructural proteins (nsp ) induce the formation of omegasomes and autophagosomes from the er via an omegasome intermediate [ ] . in addition, autophagy has been shown to induce the replication of infectious spleen and kidney necrosis virus (isknv) in the chinese perch brain (cpb) cell line, suggesting complex interactions between isknv and host cells during viral pathogenesis and for anti-viral treatment strategies [ ] . treating fmdv-infected cells with rapamycin, an autophagy inducer, was shown to increase viral replication, whilst inhibiting the autophagosomal pathway using -methyladenine or small-interfering rnas decreased viral replication [ ] . furthermore, disrupting autophagy using the knockdown approach in hepatitis c virus (hcv)-infected hepatocytes stimulated the interferon signaling pathway and induced apoptosis, indicating that hcv-induced autophagy can impair the innate immune response [ ] . suppressing hcv-induced autophagy could be a promising approach for inhibiting exosome-mediated viral transmission [ ] , besides autophagy has been shown to reduce hcv clearance following ifn-α/ribavirin (rbv)-based anti-viral therapy [ ] . a denv study revealed that autophagy inhibitors are better candidate targets than conventional anti-viral therapies using interferons (ifns) [ ] ; upregulating cellular autophagy was reported to inhibit rlr-mediated type-i ifn-independent signaling and cause the antibody-dependent enhancement (ade) of denv [ ] . suppressing autophagic vacuoles has been demonstrated to stimulate the maturation of infectious bursal disease virus [ ] . adenoviral infection may be favored by autophagy via an increase in atp, essential to increase anabolism of the infected cells and amino acid pools for the synthesis of viral proteins. in the later stages of adenoviral infection, atg -atg complex is significantly upregulated as an evidence of enhanced autophagy [ ] ; therefore, autophagy may improve the virulence of some viruses. autophagy genes are involved in the regulation and execution of autophagy [ ] . beclin was the first mammalian gene identified to stimulate autophagy [ ] . some viruses, such as α-, βand γ-herpesviruses, encode the neurovirulence protein, icp . , which associates with atg /beclin and inhibits autophagy by preventing the formation of the pi kinase complex [ ] . the autophagy genes fip , beclin , atg , atg l , atg , atg , and atg have been found to promote the reactivation of latent murine gamma-herpesvirus by inhibiting virus-induced systemic inflammation molecules, such as ifn-γ [ ] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv- escapes innate immunity, resulting in fatal disease [ ] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h n influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [ ] . activating the pi k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type [ ] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [ ] . hsv- mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [ ] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [ ] . the proviral and anti-viral actions of autophagy are illustrated in figure . [ ] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv- escapes innate immunity, resulting in fatal disease [ ] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h n influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [ ] . activating the pi k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type [ ] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [ ] . hsv- mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [ ] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [ ] . the proviral and anti-viral actions of autophagy are illustrated in figure . initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg /beclin gene was found to be lost monoallelically in - % of human prostate, breast, and ovarian cancers [ ] , whereas excessive autophagy stimulation by beclin overexpression has been reported to inhibit tumor progression [ , ] . autophagy causes necrosis and chronic inflammation by inhibiting the release of proinflammatory hmgb , which is involved in tumorigenesis [ ] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [ ] . p (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg -deficient mice exhibit enhanced accumulation of p and ubiquitinated protein aggregates in hepatocytes and neuron [ ] . autophagy has also been implicated in benign hepatomas [ ] , and the inactivation of beclin and atg was shown to increase the incidence of initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg /beclin gene was found to be lost monoallelically in - % of human prostate, breast, and ovarian cancers [ ] , whereas excessive autophagy stimulation by beclin overexpression has been reported to inhibit tumor progression [ , ] . autophagy causes necrosis and chronic inflammation by inhibiting the release of pro-inflammatory hmgb , which is involved in tumorigenesis [ ] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [ ] . p (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg -deficient mice exhibit enhanced accumulation of p and ubiquitinated protein aggregates in hepatocytes and neuron [ ] . autophagy has also been implicated in benign hepatomas [ ] , and the inactivation of beclin and atg was shown to increase the incidence of cancer in mice [ ] . atg -and atg -deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [ ] . heterozygous beclin (beclin +/-mutant) was shown to have a high incidence of spontaneous tumors [ , ] , whilst beclin inhibited tumor growth in cell lines such as the breast cancer cell line, mcf- , in which beclin expression was lower than in normal epithelial breast cells [ ] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [ ] (figure ). cancer in mice [ ] . atg -and atg -deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [ ] . heterozygous beclin (beclin +/mutant) was shown to have a high incidence of spontaneous tumors [ , ] , whilst beclin inhibited tumor growth in cell lines such as the breast cancer cell line, mcf- , in which beclin expression was lower than in normal epithelial breast cells [ ] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [ ] (figure ). ( ) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and the ribosomal s kinases (s ks) and , which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. ( ) a novel anti-cancer molecule, ha , which targets hspa /bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [ ] [ ] [ ] [ ] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and ribosomal s kinases (s ks) and ) promote cell cycle progression [ ] . mtor, which is inhibited by rapamycin [ ] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [ , ] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi k/akt/mtor axis [ ] . in addition, the novel anti-cancer ) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and the ribosomal s kinases (s ks) and , which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. ( ) a novel anti-cancer molecule, ha , which targets hspa /bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [ ] [ ] [ ] [ ] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and ribosomal s kinases (s ks) and ) promote cell cycle progression [ ] . mtor, which is inhibited by rapamycin [ ] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [ , ] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi k/akt/mtor axis [ ] . in addition, the novel anti-cancer molecule ha , which targets hspa /bip, was shown to induce er stress and increase the unfolded protein response, resulting in cancer cell death via autophagy and apoptosis [ ] . trichosanthin (tcs), a kda protein from the bioactive component of the root tuber of trichosanthes kirilowii (chinese cucumber plant; gua lou in mandarin), also exhibited anti-cancer properties against different human ovarian cancer cells via a pathway common to both autophagy and apoptosis [ ] . various factors and mechanisms are involved in autophagy-mediated tumor progression. tumor-induced nutrient shortage, cell debris (degraded proteins), inflammation, and oxidative cascades are all molecular mechanisms affecting tumor progression. during nutrient starvation, autophagy induction promotes the survival of normal cells and may also promote tumor cell survival; however, hypoxia, metabolic stress, energy shortage, oxidative stress-damaged mitochondria, and organelles can be caused by cancer-causing genes or cancer treatments [ ] . the undifferentiated colon cancer cell line, ht- , and other transformed cells have shown an increased tendency to degrade autophagic proteins [ , , ] . the elevated expression of the autophagy signature protein, bnip , a pro-apoptotic bcl- member, has been demonstrated in colorectal and gastric epithelial carcinomas, suggesting that bnip expression may be important for the development of these cancers [ ] . the activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) was shown to protect colon cancer cells against apoptosis induced by the interaction between butyrate and docosahexaenoic acid (dha) in a cell type-dependent manner [ ] . additionally, an atg /lc family member implicated in autophagy and tumor suppression was associated with alterations to cell death and cytokine secretion in mice lacking gamma-aminobutyric acid receptor-associated protein (gabarap) [ ] . it has been well documented that inhibiting autophagy in cancer cells increases their death, with this strategy proving most useful in tumors that behave like ras-activated tumors [ ] . inhibiting autophagy is expected to cause ubiquitinated proteins to accumulate and p levels to increase; in hepatic tumors, autophagy suppresses spontaneous tumorigenesis via cell-intrinsic pathways whilst p accumulation promotes tumor formation [ ] . the elimination of damaged organelles via autophagy may allow cancer cells to survive despite the stress caused by chemotherapeutic agents [ ] . it has also been reported that upregulating autophagy after chemotherapy causes cancer cells to enter a dormant state, which may then propagate at a later stage [ , ] . the state of cell cycle arrest, termed senescence, has been postulated to underlie autophagy-induced tumor cell dormancy [ ] . ras-induced senescence is mediated by autophagy, with autophagy inhibition delaying senescence [ ] . moreover, it has been reported that psmd /gankyrin stimulates autophagy in hepatocellular carcinoma (hcc) in response to starvation or stress. a physical association occurs between psmd and atg , and is translocated to the nucleus to bind to atg promote and upregulates atg expression [ ] . there is growing evidence that autophagy performs both physiological and pathological functions in the nervous system, and that autophagic stimulation plays critical roles in neuronal survival and activity. autophagy is the only method by which neurons degrade and excrete expired organelles, and is responsible for clearing abnormal intracytoplasmic contents from normal cells which would otherwise cause protein accumulation and damage neuronal activity, inducing severe functional impairment. autophagy also clears protein aggregates from old neurons; thus, inhibiting autophagy can lead to neuronal degeneration and intraneuronal protein accumulation. mutations in atg confined to neural tissues can cause impaired growth, progressive motor and behavioral deficits, prominent neurodegeneration, and axonal swelling in regions of the brain with increased levels of ubiquitinylated proteins, indicating that autophagy has a neuroprotective role [ ] . in mammals, the absence of atg and atg can cause severe neurodegeneration, further supporting the neuroprotective role of autophagy [ , ] . furthermore, neuronal death can be attributed to the loss of basal autophagy or an imbalance in autophagic flux. in some neurodegenerative diseases, such as alzheimer's, parkinson's, and huntington's, as well as in the brain or spinal cord trauma, the damaged neurons exhibit abnormally high numbers of autophagosomes. therefore, understanding the interaction between pathophysiological mechanisms and autophagy could be a promising approach for therapies against neurological disorders [ ] . prenatal alcohol exposure has been shown to increase the number of autophagic vacuoles in the cortical micro-vessels of human fetal and mouse neonatal brains, impairing autophagy [ ] . furthermore, autophagy can modulate notch degradation, stem cell development, and neurogenesis [ ] . several reviews have evaluated the relationship between autophagy and neurodegenerative diseases [ , ] . autophagy is vital for neuronal homeostasis [ ] , and its deregulation is highly associated with numerous neurodegenerative effects, such as the accumulation of damaged and toxic molecules with pathological consequences in neurodegenerative disorders such as alzheimer's, parkinson's, and huntington's diseases [ , ] . lysosomal system inactivation is responsible for the accumulation of autophagosomes observed in alzheimer's disease [ ] , whilst the disease is thought to be due to either excessive or impaired autophagosomal degradation, or the activation of autophagy genes in response to temporary injury/stress in neuronal tissues. alzheimer's [ ] , parkinson's [ , ] , and huntington's diseases [ ] are key examples where autophagosomal accumulation and anomalies in the endosomal-lysosomal pathway have been observed in post-mortem human brain tissues via electron microscopy. autophagy deficiency resulted in neuronal loss in the cerebral and cerebellar cortices in a mouse model [ ] . dysfunction or abnormalities in autophagy, including mutations in autophagy-regulating genes, are accompanied by neurodegenerative diseases across the age spectrum with exceptional frequency. atg -deficient mice exhibited ubiquitin accumulation in their cns, causing nervous symptoms, neurodegeneration, and ultimately death [ , ] , whilst atg -deficient mice developed cytoplasmic inclusions and exhibited motor dysfunctions [ ] , and ambra -deficient mouse embryos displayed neuronal tube defects [ ] . autophagy is involved in the cytoplasmic clearance of α-synuclein (α-syn), which is observed in parkinson's disease [ ] . in a mouse model, beclin overexpression was found to reduce the clearance of α-syn, leading to pathological neuron abnormalities [ ] . pharmacological and genetic pathways are involved in the degradation of α-syn by polo-like kinase via autophagy, suggesting that these two proteins are concomitantly co-degraded [ ] . the pink and parkin genes regulate mitophagy [ ] , indicating that mutations in these genes can cause defects in mitophagy which have been correlated with parkinson's disease [ , ] . beclin expression is lower in the brains of patients with alzheimer's disease and not only affects autophagy but also increases the deposition of β-amyloid proteins causing neurodegeneration [ ] . huntington's disease is caused by the extension of the polyglutamine (polyq) proteins aggregate intracellularly, which causes neuronal death. atg-knockout c. elegans exhibit increased polyq toxicity [ ] , whilst in drosophila the autophagy-enhancing small molecule -( -phenylphenyl)- , -dihydroimidazo[ , -b] [ , ] thiazole, also known as autophagy enhancer- (auten- ), has been shown to prevent the symptoms of neurodegenerative diseases [ ] . the dual role observed for autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. the differences between physiological and pathological autophagy may help design therapeutic strategies specifically targeting pathological autophagy without hindering its physiological roles [ ] . for example, the apoptosis-stimulating protein p - (aspp / bp l) was reported to have different effects on autophagy in neurons stimulated with different levels of gp , a soluble envelope glycoprotein of hiv- that interacts with chemokine receptors such as cxcr and ccr . thus, regulating autophagy in the cns could be a potential therapeutic approach against hiv-associated neurocognitive disorders [ ] . . . autophagy in the immune system and autoimmune diseases . . . autophagy in the immune system the roles of cellular autophagy in immunological processes and autoimmune diseases have been reviewed extensively [ , , ] . autophagy plays important roles in both innate and adaptive immunity [ ] , modulates cellular and humoral immune responses [ ] [ ] [ ] , and has roles in the non-metabolic and metabolic functions of immune cells [ ] . furthermore, autophagy is involved in innate immune cell differentiation, degranulation, phagocytosis and extracellular trap formation involving neutrophils, eosinophils, mast cells, and natural killer cells, and plays an essential role in the renewal, differentiation, and homeostasis of immune cells [ ] . autophagy also regulates the functional responses of immune cells, such as phagocytosis, antigen presentation, cytokine production, control of inflammasome activation, tolerance, and their consequences on overall host defense via monocytes, macrophages, dendritic cells, and antigen presentation [ ] . additionally, autophagy plays important roles in b cell development, activation, and differentiation, which enables b cells to adapt to various events, and determines their fate, survival, and function [ ] . since b cells produce antibodies, autophagy can determine humoral immune responses. in one study, the b cells of atg -deficient mice had defective antibody responses, indicating that autophagy has a role in antibody production [ ] . several studies have documented interplay between autophagy and the nf-κb signaling pathway. members of the nf-κb family of transcription factors regulate the transcription of genes involved in cell proliferation, survival, differentiation, and development, whilst activation of the inhibitor of nf-κb (iκbα) kinase complex is essential for autophagy induction. t-cell receptor-mediated nf-κb activation in b-cell lymphoma/leukemia is linked with the autophagy adaptor p /sqstm [ ] , which modulates the nlrp -inflammasome activation and il- β production in macrophages [ ] . il- α secretion is enhanced in atg -deficient macrophages, whilst inhibiting autophagy results in il- β overexpression [ ] . the anti-inflammatory cytokine, il- , inhibits autophagy by activating mtor complex [ ] and inhibits starvation-and ifn-γ-induced autophagy via bcl- and beclin in various autoimmune and inflammatory disorders [ ] . autophagy has predisposing, pathogenic, and therapeutic roles in autoimmunity. defects in autophagy pathways and/or autophagy-related genes have been implicated in numerous autoimmune and autoinflammatory diseases, including multiple sclerosis, systemic lupus erythematosus (sle), rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, diabetes mellitus, crohn's disease, and vitiligo [ , [ ] [ ] [ ] . abnormalities in the maintenance of homeostasis via autophagy result in the accumulation of dysfunctional or defective cellular organelles, abnormal proteins, infectious agents, and metabolite accumulation. this predisposes cells to the generation of autoantibodies and proinflammatory mediators and exposes vital and susceptible cellular structures to deleterious agents that can cause disease [ ] [ ] [ ] [ ] . a recent study revealed a correlation between the expression pattern of autophagy-related genes and the type of lupus nephritis (ln); thus, autophagy could indicate of the type of ln when formulating a treatment regimen [ ] . several atgs are known to be involved in autoimmune disorders, including atg , pr domain zinc finger protein (prdm ; also known as blimp- ), and dna-damage regulated autophagy modulator (dram ) in sle patients and atg l and immunity-related gtpase m (irgm) in crohn's disease and ulcerative colitis. autophagy defects have been observed in t cells, b cells, and macrophages [ ] . mhc class ii antigen presentation by macrophages occurs via cma; lysosomal proteins have a central role in antigen processing, which is essential for a correct immune system function. studies in mrl/lpr mice which develop a full panel of lupus autoantibodies revealed that increased lysosomal ph might be an important lysosomal malfunction involved in autoimmunity, and that perturbed lysosomal turnover may lead to hyperactive antigen presentation by antigen presenting cells (apc) in autoimmune disorders [ ] . in innate immunity, reduced atg and mtor expression result in defective autophagy, affecting the clearance of dead cells, increasing levels of nucleic acid remnants and self-antigens, increasing type ifn by dcs, and inducing b cell hyper-differentiation and autoantibody production [ , , ] . it has also been reported that autophagy-related gene knockdown can have therapeutic effects on autoimmune diseases [ ] . modulating autophagy can manage immunity-related and inflammatory diseases [ , , , ] by regulating cytokine and antibody production against immunogenic insults to prevent autoimmune diseases. therefore, regulating autophagy has clinical potential in cancer immunotherapy [ ] , whilst autophagy and adenoviral combinations are proving beneficial in adenoviral-based oncolytic virotherapy [ ] . under normal conditions, the myocardium exhibits low levels of autophagy, whilst stressful conditions can increase the level of autophagy to increase cell survival [ , ] . patients with congestive heart failure, coronary artery disease, hypertension, and aortic valvular disease display increased autophagosomal accumulation in their myocardial biopsies [ ] . autophagy levels vary in normal and affected or stressed hearts, with constitutive autophagy maintaining normal structure and function, and upregulated autophagy occurring during cardiac disease or stress [ ] . in atg -deficient mice, contractile dysfunction and hypertrophy have been observed during cardiomyopathy [ ] , whilst cell culture studies have revealed that autophagic gene deficiency can cause the accumulation of unwanted proteins and contribute to myocardial disease [ ] . similarly, lamp- -deficient mice displayed increased autophagic vacuole accumulation and could not degrade proteins, thereby promoting cardiomyopathy [ , ] . it has been postulated that during early life, between birth and suckling, autophagy provides the energy required for cardiac cells [ ] , whilst mitophagy protects cardiac muscles under ischemic stress [ ] . increased autophagy can cause heart failure [ ] , with autophagy-induced degeneration resulting in the death of cardiomyocytes. this knowledge has helped our understanding of the pathogenic role of autophagy in cardiac failure models and helped devise therapeutic targets [ , ] . autophagy can cause myocardial cell damage via parp , which promotes autophagy in cardiomyocytes by modulating foxo a transcription [ ] . increased autophagy causes pathological remodeling of the heart, whilst decreased autophagy reduces remodeling [ ] . thus, it has both protective and destructive roles in the cardiovascular system. iron homeostasis involves a form of macroautophagy known as ferritinophagy, wherein ferritin, an iron storage protein, is degraded in the lysosome [ ] . iron levels are tightly regulated in cells; nutrient deficiency induces autophagy, during which cellular proteins and organelles are engulfed by the autophagosome, which then fuses with the lysosome. the degradation of these contents provides essential resources that either promote cell survival or lead to cell death. iron (fe), copper (cu), zinc (zn), and aluminum (al) react with molecular oxygen to produce reactive oxygen species (ros) and reactive nitrogen species (rns). besides acting as a cofactor for metalloprotein enzymes involved in redox reactions, iron also plays a major role in mitochondrial atp metabolism and other cellular processes. the fenton and haber-weiss redox reaction is highly involved in ros production and alzheimer's progression [ ] . to maintain iron homeostasis, storage and recycling are critical. when engulfed by macrophages, the iron within erythrocytes is either stored as a ferritin complex or exported from the cell by the ferroportin iron-exporter [ ] . hsp and ferritin bind iron in the cytosol and autophagocytosis of these proteins can sequester redox-active iron in the lysosomes [ ] . cells rich in these proteins exhibit increased resistance to oxidative stress; therefore, autophagy plays a major role in maintaining cellular redox status [ , ] . the autophagy inhibitor ncoa , which is a substrate of pi k, has been shown to physically bind to the ferritin protein complex and direct it to autolysosomes for degradation [ ] . ncoa knockdown prevents the localization of ferritin in the lysosomes and increases the levels of iron-responsive element-binding protein (irp ), which is a free intracellular fe antagonist that prevents cell death via exogenous ros [ ] . ncoa also acts as an autophagy receptor for ferritin and delivers it to the lysosome to maintain iron homeostasis. experimentally simulating low-iron conditions by chelating iron revealed that ferritin is degraded to release the stored iron [ ] . autophagy is involved in obesity [ ] and diabetes mellitus [ ] . improper lipid and glycogen processing can affect the liver activity and thus, insulin synthesis, resulting in diabetes. studies have shown that hepatocytes from mouse models of obesity display reduced autophagy [ ] , with decreased atg expression causing er stress and affecting insulin signaling [ ] . mutant atg mice also exhibit reduced β cell mass, reduced insulin circulation, and glucose intolerance, indicating that autophagy defects can reduce insulin levels and cause hyperglycemia [ , ] . a genetic mosaic screen for mutations that increase lysosomal and/or autophagic activity in d. melanogaster larva revealed that autophagy-lysosome pathways underlie novel cytoprotective features in drosophila [ ] . obesity impairs autophagy in the liver via s-nitrosylation, a process induced by nitric oxide (no). s-nitrosylation of the lysosomal enzymes cathepsin b (ctsb) and hexosaminidase subunit β (hexb) impairs normal lysosomal functioning and is carried out by denitrosylation enzymes, particularly s-nitrosoglutathione reductase (gsnor) and thioredoxin [ ] . obesity inhibits the denitrosylation ability of the liver, impairing hepatic autophagy and insulin resistance [ ] . in obese animals, hepatic insulin signaling is impaired by no-induced hepatic autophagy repression, which ultimately causes the progression of type diabetes [ ] . the potential roles of autophagy in ameliorating diseases while maintaining homeostasis have been enumerated in table . details of applied/granted patents for treating autophagy-related ailments and dysfunctions are presented in table . human nuclear ribonucleoprotein k (hnrnp-k) and ubiquilin (ubqln ) help in viral replication. ndp human autophagy receptor interacts with chikv nsp and acts as proviral factor wong and chu, [ ] increased autophagy-associated protein lc and bnip -linked to colorectal and gastric cancers; elevated expression of nip (a pro-apoptotic member of the bcl- family of cell death factor) in gastric carcinomas lee et al., a [ ] autophagy inhibition leads to cell death in tumors acting like an ras-activated tumor guo et al., [ ] in the absence of autophagy -accumulation of ubiquitinylated protein aggregates and higher p level-responsible for liver tumor takamura et al., [ ] activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) protect colon cancer cells against apoptosis tylichová et al., [ ] in ras-activated tumors, inhibition of autophagy leads to increased cancer cell death guo maintaining homeostasis is the most important role of autophagy, which is the final survival mechanism used to escape cell death. mutations or genetic dysfunctions in autophagy-associated genes can have a variety of pathological consequences, as described below: . . . static encephalopathy of childhood with neurodegeneration in adulthood (senda) senda is a recently discovered type of neurodegeneration associated with iron accumulation in the brain [ ] . it begins with early-onset spastic paraplegia and mental retardation during childhood, followed by symptoms of parkinsonism and dystonia during adulthood along with eye movement abnormalities, dysautonomia, and sleep disorders. whole-exome next generation sequencing has revealed that senda is associated with a mutation in the wipi gene (also known as wdr ), located at xp . [ , , ] . the disease is characterized by iron accumulation in the globus pallidus [ ] . wipi , which is the mammalian homolog of yeast atg , is involved in autophagosome formation [ ] by binding phosphatidylinositol -phosphate, which is recruited to the autophagosome formation site [ ] . a severe decline in wipi expression has been reported in lymphoblastoid cell lines derived from patients with senda [ ] . as the wipi gene is found on the x chromosome, one of which undergoes inactivation in females, the loss of wipi function is expressed in a mosaic pattern in females. crohn's disease is a major inflammatory bowel disease whose symptoms include abdominal pain, diarrhea, vomiting, and weight loss. atg l mutations have been found to be linked with crohn's disease [ ] . atg l forms a complex with atg -atg which initiates autophagosome formation by inducing the conjugation of lc with pe [ ] . a study on the t > a atg l mutation revealed that the mutation is differentially involved in crohn's disease and canonical autophagy [ ] . in mice, the mutations that eliminated or reduced atg l expression indicated a relationship between atg l mutations and crohn's disease [ ] . other autophagy-associated proteins, including irgm- , , and and nod - , , and have also been linked to crohn's disease in humans [ ] . autophagy is thought to be involved in the pathogenesis of crohn's disease, but it can also control disease severity by reducing the levels of inflammatory mediators [ ] . hsp is a neurodegenerative disorder that causes axonal degeneration in the corticospinal or pyramidal motor and sensory tracts that control distal organs and is caused by a recessive mutation in the tecpr gene. tecpr interacts with six human atg orthologs to positively regulate autophagy, with tecpr knockdown in hela cells reducing autophagic activity and indicating its role in autophagy [ ] . the zinc-finger protein spastizin interacts with the beclin -uvrag-rubicon complex and is involved in autophagosome maturation. in fibroblasts derived from patients with hsp, knockout of the spastizin gene reduced autophagy and caused autophagosome accumulation by impairing lysosomal fusion [ ] . the zfyve /spastizin and spg /spatacsin mutations were recently associated with hsp [ ] ; however, there are currently no treatments available to reverse nerve degeneration in hsp, with efforts instead directed towards reducing symptoms by physiotherapy and improving spasticity using medication. danon disease is a rare cardiomyopathic disease caused by lamp- deficiency and characterized by the accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscles, cardiomyopathy, and intellectual dysfunction [ ] . the lamp- gene is alternatively spliced into three isoforms (lamp- a, - b, and - c) which have different functions in autophagy. lamp- a acts as a receptor for cma, in which proteins carrying the kferq motif are selectively trafficked into the lysosome and degraded. lamp- b has been suggested to play a prominent role in macroautophagy and is responsible for the danon phenotype. lamp- b-deficient mice exhibit higher mortality and autophagic vacuole accumulation in the liver, kidney, pancreas, and cardiac and skeletal muscles [ ] . whole genome sequencing identified a nonsense mutation (codon c>t in exon ) in lamp in a family with danon disease [ ] . lamp- knockout resulted in failed autophagic progression, as evidenced by inappropriate cathepsin d processing in the autophagic vacuoles and abnormally high numbers of mannose- -phosphate receptors that limited the degradation of long-lived proteins during starvation [ ] . . . . x-linked myopathy with excessive autophagy (xmea) xmea is a rare x-linked recessive skeletal myopathy caused by a single-nucleotide substitution (c. - t > g) in vma , whose protein product assembles proton pumps in the lysosome which generate and maintain the acidity required for the activity of various lysosomal hydrolases [ ] . impaired hydrolase activity can block the final step of autophagy or induce autophagy by inhibiting mtor. if autophagy is impaired, a feed-forward pathogenic loop is activated, which results in the accumulation of autophagolysosomes containing incompletely-digested products [ ] . xmea is characterized by progressive muscle weakness, particularly in the proximal muscles of the legs, and muscle degeneration (atrophy) in adulthood. recently, two vma non-coding microdeletions [ ] , one intronic (c. - _ - del) and the other in the utr (c.* _* del), were reported to result in more severe clinical manifestations with extra-ocular and upper extremity involvement and earlier disease onset. besides long-lived proteins and membranes, xmea also affects membrane repair, interrupts sarcolemmal membrane homeostasis, and increases serum creatine phosphokinase (cpk) levels [ ] . sibm is an age-related progressive muscle disorder which presents in the elderly and is characterized by the presence of autophagic vacuoles and ubiquitinylated misfolded multiprotein aggregates. two major lysosomal proteases, cathepsins d and b, exhibit decreased activation in sibm muscle fibers [ ] whilst lc -ii is increased, indicating that autophagosome formation is increased due to reduced cathepsin d and b activity during autophagosome maturation [ ] . t cells may have a role in sibm [ ] , whilst allelic variants of the hla-dr locus, other genetic factors, and the hla genotype are also thought to play major roles in the progression and severity of the disease [ ] . dysphagia (difficulty in swallowing) may occur due to weakness in the neck muscles of patients with sibm, whilst myalgia (muscle pain) and difficulty in manipulating objects may occur due to weakness in the fingers. an overview of diseases caused by genetic defects in autophagy genes is presented in table . to alleviate autophagy-associated diseases, a variety of drugs, biomolecules, chemicals, and epigenetic strategies have been developed to either promote or inhibit autophagy. autophagy can be suppressed during any stage of autophagic flux. the pro-apoptotic role of autophagy may lead to hyperstimulation-induced excessive activation, which results in detrimental self-cannibalism that can go beyond the limit of cellular recovery. numerous chemical inhibitors have been identified and tested in various cell and animal models. most autophagy inhibitors are poorly selective, limiting their wider applications. -methyladenine ( -ma), a class-iii pi k inhibitor, is commonly used to inhibit autophagy [ ] . class iii pi k and beclin are essential during the first step of autophagy induction [ ] ; thus, inhibiting pi k reduces autophagosome formation. bafilomycin a is a vacuolar h + -atpase inhibitor, which functions at concentrations as low as nm to inhibit both the early and late stages of autophagy by activating the mtor pathway [ ] . in early , bafilomycin a was reported to inhibit autophagic vacuoles in rat hepatoma [ ] by dissociating the beclin -vps complex and preventing autolysosome formation, thus attenuating functional autophagy. bafilomycin a has been tested in a mouse model and found to be safe [ ] . furthermore, disruption of the vacuolar-type h + -atpase complex in mouse liver cells has been shown to induce the rapamycin complex (mtorc )-independent aggravation of autophagic vacuoles and lysosomes [ ] . concanamycin a belongs to the same class of vacuolar h + -atpase inhibitors as bafilomycin a and in the presence of autophagosomes causes autophagic bodies to accumulate in the central vacuole and cytoplasm of tobacco by- cells [ ] . therefore, vacuolar-type h + -atpases have been proposed as potential therapeutic agents. cycloheximide is a protein biosynthesis inhibitor that is frequently used in biomedical research. in mouse pancreatic acinar cells, cadmium chloride-or hyperosmotic sucrose-stimulated autophagy was found to be inhibited by cycloheximide [ ] , which likely inhibits autophagy at the segregation step. cycloheximide is used to prevent the autophagy-lysosome pathway [ ] and its effects can be rapidly reversed by its removal [ ] . chloroquine, hydroxychloroquine, nh cl, and neutral red are chemicals that can rapidly neutralize the acidic environment of the lysosome; therefore, they are used to block autophagosome maturation. chloroquine and hydroxychloroquine are repurposed drugs that have been used to treat malaria, sle, and rheumatoid arthritis [ ] ; however, higher hydroxychloroquine concentrations are required to induce active autophagy as a cancer treatment, which are usually not achievable in cancer patients. chloroquine-mediated lysosomal dysfunction is thought to have increased anti-cancer functions when combined with nutrient deprivation [ ] . lys , a dimeric form of chloroquine in which each molecule is separated by the spacer molecule n-bis( -aminoethyl)-methylamine, has been reported to inhibit autophagy at a level -fold higher than chloroquine lys , a water-soluble salt of lys , effectively accumulates within the lysosome to concurrently deacidify and inhibit autophagy [ ] . lysosomal proteases are active at an acidic ph. the protease inhibitor leupeptin inhibits cysteine, serine, and threonine peptidases to block protein degradation and thus inhibits the final step of autophagy, as evidenced by the accumulation of vacuolar autolysosomes [ ] . cathepsins are aspartic, cysteine, and serine proteases that are transported to lysosomes via mannose- -phosphate receptors [ ] and can be inhibited by the lysosomal protease inhibitors e d and pepstatin a. cathepsins b, h, and l are inhibited by e d, whilst cathepsins d and e are inhibited by pepstatin a. cells treated with e d and pepstatin a exhibit increased lc -ii levels [ ] . bafilomycin and chloroquine, which inhibit autophagy by targeting lysosomes, have been reported to increase levels of the autophagosome marker lc -ii, block key aspects of autophagy, decrease mitochondrial quality, and increase mitochondrial dna damage in primary neurons [ ] . the atg genes are crucial in autophagy; therefore, the inactivation or knockdown of these genes may be a useful way to manipulate the process [ ] . mir- a has been reported to downregulate beclin and atg expression, whilst mir- can inhibit basal and rapamycin-induced autophagy. autophagy is induced by the activation of the ulk complex, which consists of ulk / , atg , fip , and atg . in melanoma cells, mir- - clusters can inhibit ulk and atg expression to suppress autophagic cell death [ ] . mir- - p targets ulk to regulate autophagy [ ] , whereas mirna- a/b, mirna- b, mir- a, mir- a, and mir- - p inhibit beclin expression to suppress vesicle nucleation [ ] [ ] [ ] . recently, the leucine-rich repeat kinase gene (lrrk ), which is associated with crohn's disease, parkinson's disease, and leprosy, was shown to inhibit the non-canonical autophagy cascade, indicating that the negative regulation of this cascade could directly induce disease [ ] . in addition, the atg l t > a polymorphism has been demonstrated to disrupt unconventional tmem . -mediated autophagy in mice [ ] . rapamycin, also known as sirolimus, is a natural mtor inhibitor [ ] that stimulates autophagy both in vitro and in vivo; however, its long-term use is often complicated as it suppresses ribosome biogenesis and protein translation [ ] . the rapamycin ester cci- /temsilorimus, which is also an mtor inhibitor, prevents the development of intracellular tau protein inclusions (abundant protein that stabilizes microtubules in cns neurons) which are known to accumulate in neurons in alzheimer's disease and other tauopathies and can also reduce the density of neurofibrillary tangles by stimulating mtor-dependent autophagy [ ] . rad and ap are rapamycin derivatives with comparatively high safety and low toxicity [ , ] . during the early stages of carotid atherosclerosis, which is an inflammatory step in the primary pathogenesis of cerebrovascular diseases, mirna- plays an important role in the activation of autophagy by rapamycin [ ] . rapamycin has also been shown to increase autophagy, decrease cyclin d expression, and attenuate aggressive iga nephropathy progression in a rat model [ ] . the immunosuppressive activities of rapamycin limit its frequent use; therefore, safer molecules are required. three smers were identified from , compounds that can induce autophagy [ ] ; smers , , and can reduce the pathogenesis associated with polyglutamine aggregation and a t α-synuclein, which are linked to huntington's disease and familial parkinson's disease, respectively [ ] , and induce autophagy in an mtor-independent manner. in addition, the small molecule auten- activates autophagy in cell cultures and animal models and inhibits the progression of neurodegenerative symptoms [ ] . trehalose is a natural disaccharide known to stimulate autophagy via ampk [ ] . in cultured cells from an animal model of huntington's disease, trehalose has been shown to stimulate autophagy and reduce huntingtin protein aggregation during starvation by inhibiting a family of glucose transporters known as the solute carrier or the glucose transporter family [ ] . as well as being an autophagy activator and a non-reducing disaccharide, trehalose can also reduce the levels of aggregate-prone proteins and ameliorate cytotoxicity in neurodegenerative disease models [ ] . trehalose treatment has been shown to increase the conversion of lc -i to lc -ii via an mtor-independent pathway, whilst sucrose and raffinose are also known to induce autophagy [ ] . it has been shown that trehalose can be used to safely induce autophagy in neurodegenerative disorders such as parkinson's disease [ ] , alzheimer's disease [ ] , and prion disease [ ] . the bioavailability of trehalose in the body for autophagy induction is low due to the hydrolytic enzyme, trehalase, which is expressed in the intestine and kidney; therefore, the enzyme-stable trehalose analogs, lentztrehaloses a, b, and c, were synthesized and found to be as effective as trehalose [ ] . lithium chloride (licl) can induce autophagy by inhibiting inositol monophosphatase (impase), which reduces inositol and inositol- , , -triphosphate (ip ) levels [ ] . since lithium has already been approved by the fda for patients with bipolar disorder, it can easily be adapted for other diseases [ ] . long-term oral lithium administration enhanced autophagy in a tauopathy mouse model by inhibiting glycogen synthase kinase- . l- , , a bisphosphonate inhibitor of impase, exhibits a similar function to lithium by clearing mutant synucleins and egfp-hdq [ ] . valproic acid is an angio-suppressive compound which suppresses the akt/mtor signaling pathway by acting as a histone deacetylase inhibitor and promotes autophagy, as evidenced by the increased concentrations of lc -ii and beclin after its administration in prostate cancer cell lines [ ] . carbamazepine and valproic acid can both reduce intracellular inositol- , , trisphosphate levels [ ] . therefore, other impase inhibitors could also be incorporated into therapeutic strategies. anacardic acid, curcumin, garcinol, and spermidine increase autophagy by reducing the level of acetylation in cultured human cells, as evidenced by depleted sequestosome- levels and mtorc inhibition [ ] . spermidine also inhibits ep and the major autophagy proteins, atg , atg , atg , and lc to repress autophagy [ ] . supplementing the diet of mice with coffee beans rich in polyphenols concomitantly increased autophagy and decreased acetylation levels [ ] , whilst natural polyamines can inhibit acetyltransferases, and their dietary intake can improve the life span of short-living mouse strains and the health of long-living ones [ ] . fluoxetine, which selectively improves the secretion of the pro-inflammatory cytokine tnf-α, and gefitinib, which inhibits the epidermal growth factor receptor (egfr), can also enhance autophagy to impart neuroprotection and restrict m. tuberculosis growth [ ] . the antiepileptic compound, carbamazepine, has been found to protect cells against m. tuberculosis infection, likely by inducing autophagy [ ] . elevated intracytosolic ca + levels can inhibit autophagy; penitrem a, which inhibits ca + -dependent k + channels, is known to induce autophagy and has been used to treat neurodegenerative disorders [ , ] . furthermore, bacterial pore-forming toxins can induce the expression of the transcription factor hlh- (tfeb) in c. elegans, which stimulates autophagy genes and thus inhibits bacterial infection [ ] . metformin, an ampk activator, has been shown to induce autophagy and reduce the risk of hepatocellular carcinoma (hcc) in diabetic patients [ ] . a patent studied by gorski and qadir [ ] revealed the potential of a thioxanthone-based autophagy inhibitor for suppressing atg gene expression by using sirna-directed therapy against cancer cells receiving anti-cancer therapies, and for treating cancer in combination with other therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [ ] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)-a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [ ] . imidazo [ - f] [ , ] phenanthroline derivatives ( - ) have been evaluated for their anti-cancer effects, with their use of upregulating lc -ii and beclin expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [ ] . in nanosilica stimulated raw . cells, melatonin was found to increase lc expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [ ] . sasanquasaponin iii obtained from schima crenata korth was found to upregulate lc -ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [ ] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [ ] . compounds that inhibit and activate autophagy are depicted in figure . therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [ ] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [ ] . imidazo [ - f] [ , ] phenanthroline derivatives ( - ) have been evaluated for their anti-cancer effects, with their use of upregulating lc -ii and beclin expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [ ] . in nanosilica stimulated raw . cells, melatonin was found to increase lc expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [ ] . sasanquasaponin ΙΙΙ obtained from schima crenata korth was found to upregulate lc -ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [ ] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [ ] . compounds that inhibit and activate autophagy are depicted in figure . compounds that inhibit and activate autophagy. autophagy inhibitors: -methyladenine inhibits pi k. bafilomycin a causes dissociation of the beclin -vps complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad and ap are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc -i to lc -ii conversion in an mtor-independent pathway. valproic acid increases lc -ii and beclin concentrations. compounds that inhibit and activate autophagy. autophagy inhibitors: -methyladenine inhibits pi k. bafilomycin a causes dissociation of the beclin -vps complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad and ap are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc -i to lc -ii conversion in an mtor-independent pathway. valproic acid increases lc -ii and beclin concentrations. autophagy is a fast-moving area of science which has had an excellent positive impact on animal and human health-related issues and threatening diseases and has considerable future potential. autophagy is a highly complex process, and understanding the complexity of its mechanisms and internal regulations will be highly beneficial for developing novel methods such as simple ameba-based experimental models. emerging studies have established many interesting connections in the field of autophagy research. since autophagy is one of the most conserved evolutionary processes, present in lower organisms and all highly evolved mammals, much work has been carried out to understand its physiological and pathological features. pathogenesis-associated autophagy is involved in degenerative, inflammatory, infectious, and neoplastic diseases, whereas physiological autophagy is associated with maintaining liver iron homeostasis, cns development, endothelial cell alignment, atheroprotection, and preventing infection. numerous mechanisms of action involving various signaling pathways have been linked to autophagy, with novel mechanisms currently being investigated. mtor acts as a nutrient sensor in autophagy; however, the process may also be regulated via mtor-independent pathways. intracellular ca + regulates autophagy, with intracellular ca + mobilization via the ip receptor stimulating calmodulin and erk pathways to inhibit autophagy. notably, ca + also inhibits autophagy by increasing mitochondrial atp levels, which inhibits the camkkβ/ampk pathway, yet promotes autophagy by promoting the ampk-dependent inhibition of mtor. autophagy is linked with other cellular mechanisms such as apoptosis, necrosis, autosis, and necroptosis, and can take the form of microautophagy, macroautophagy, and cma. autophagy can be selective, neutralizing specific substances, or non-selective, degrading different materials irrespective of their nature. it can play physiological and pathological roles by maintaining homeostasis and causing disease, thereby affecting both health and disease. autophagy encounters infectious and non-infectious diseases, either mediating protection or aggravating the disease. autophagy has dual roles in an anti-bacterial, anti-viral, and tumor suppressing capacity and by favoring bacterial infections, viral infections, and tumor progression. among its dual physio-pathological roles, autophagy can promote brain development, immunity, and cardiovascular and endocrine development, or cause neurodegeneration, autoimmune diseases, cardiovascular diseases, obesity, and diabetes. autophagy has a genetic basis and many diseases are caused by genetic defects in autophagy genes, including senda, crohn's disease, hsp, danon disease, xmea, and sibm. efforts are being made to explore the positive aspects of autophagy to overcome various health and disease problems. novel therapeutics and interventions are being investigated to counter autophagy-associated diseases, including apoptosis inhibitors and activators. excessive autophagy stimulation may cause self-destruction which could be controlled with drugs such as vacuolar-type h (+)-atpase inhibitors, cycloheximide, lysosome alkalizers (chloroquine, hydroxychloroquine, nh cl, and neutral red), and acidic protease inhibitors (e d and pepstatin a), whilst knocking down beclin and atg using mir- a could be used to inhibit the excessive cannibalism caused by autophagy. upregulating autophagy could therapeutically benefit a range of diseases caused by reduced neuronal apoptosis, including the neurodegeneration-associated impairment of learning/memory capabilities, motor dysfunction, seizures, adult stroke, neonatal asphyxia, cardioskeletal myopathy, and cancers. autophagy could also prevent various bacterial and viral diseases, inflammatory and autoimmune conditions, and also increase lifespan. rapamycin, small-molecular enhancers of rapamycin (auten- ), trehalose, impase inhibitors (carbamazepine and valproic acid), epigenetic modulators (anacardic acid, curcumin, garcinol, and spermidine), and chemicals (fluoxetine, penitrem a, and metformin) are all autophagy stimulators which help to ameliorate disorders associated with reduced autophagy. the apparent dual role of autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. understanding the differences between physiological and pathological autophagy may help us design therapeutic strategies to target pathological autophagy without hindering its physiological effects. studies on mouse models combined with human genetic studies provide an important insight into the role of autophagy in neurological diseases like parkinson's and inflammatory disorders like crohn's disease. some critical issues have yet to be addressed regarding the role of autophagy in therapeutics and diagnostics. there are few efficient markers for studying autophagy modulation and those that exist have limitations. these markers are important for determining the effect of autophagy on disease initiation and progression. furthermore, autophagy modulating drugs are imprecise and nonspecific; hence, more specific drugs are required. similarly, 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neurodegenerative diseases metformin promotes apoptosis in hepatocellular carcinoma through the cebpd-induced autophagy pathway phenanthroimidazole derivatives act as potentinducer of autophagy by activating dna damage pathway melatonin enhances autophagy and decreases apoptosis induced by nanosilica in raw . cells sasanquasaponin iii from schima crenata korth induces autophagy through akt/mtor/p s k pathway and promotes apoptosis in human melanoma a cells epigallocatechin- -gallate stimulate autophagy and reduces apoptosis levels in retinal müller cells under high-glucose conditions this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: all the authors acknowledge and thank their respective institutes and universities. all authors declare that there exist no commercial or financial relationships that could in any way lead to a potential conflict of interest. key: cord- -vfznyyz authors: dauner, allison l.; mitra, indrani; gilliland, theron; seales, sajeewane; pal, subhamoy; yang, shih-chun; guevara, carolina; chen, jiann-hwa; liu, yung-chuan; kochel, tadeusz j.; wu, shuenn-jue l. title: development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . . sha: doc_id: cord_uid: vfznyyz during dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. to facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay that detects all serotypes of dengue virus (denv). we used a quencher to reduce nonspecific amplification. the assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at °c. results can be visualized using uv fluorescence, handheld readers, or lateral flow immunochromatographic tests. we report a sensitivity of . % ( % confidence interval [ci], . – . %) and specificity of . % ( % ci, . – . %) using a panel of clinical specimens characterized by denv quantitative reverse transcription–polymerase chain reaction. this pan-serotype denv rt-lamp can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need. dengue is one of the fastest growing global health problems today, caused by a single-stranded, positive-sense enveloped rna virus that belongs to the flaviviridae family (trent et al., ; rico-hesse, ) . the human immune system recognizes antigenically distinct serotypes of dengue virus (denv) (i.e., denv - ) (george and lum, ; gubler, ) . symptoms can range from mild fever, headaches, myalgia, and rashes (henchal et al., ; trent et al., ) to severe disease characterized by plasma leakage and hemorrhage. severe dengue can be fatal, especially without intensive medical care. the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes and is endemic in most tropical and subtropical areas where these vectors are found. an estimated million denv infections are believed to occur each year, resulting in million symptomatic cases (bhatt et al., ) . neither vaccines nor antiviral treatments are available to reduce denv-associated morbidity or mortality. the only available treatment option is supportive bed rest, fluids, and symptomatic relief with analgesics. diagnosing dengue is important for guiding appropriate supportive care and for alerting the physician to disease-specific warning signs that may require hospitalization; however, it is not possible to make an accurate differential diagnosis of dengue based on clinical features alone, as many symptoms of dengue resemble those of other diseases, such as malaria, chikungunya, measles, influenza, and rickettsial infections (teles et al., ; shu and huang, ) . traditional laboratory techniques for dengue diagnosis include viral isolation followed by indirect immunofluorescence assay or serological assays such as plaque reduction neutralization test, hemagglutination inhibition, and igm antibody capture enzyme-linked immunosorbent assay (teles et al., ; shu and huang, ; kao et al., ) . some of these techniques can take days or weeks to complete. furthermore, serological methods require convalescent samples demonstrating an increase in antibody titer from pre-exposure samples in order to make a definitive diagnosis, making it impossible to confirm a diagnosis in the acute stage, limiting the value of serological assays for informing patient management. reverse transcription-polymerase chain reaction (rt-pcr) and quantitative rt-pcr (qrt-pcr) can be used to identify denv rna, which enables diagnosis during the acute phase of dengue infection. this can be an advantage because it facilitates earlier detection of dengue (within - days post onset of symptoms), before an immune response has been mounted and serology-based diagnostics can detect igm or igg levels (typically detectable - days post onset of symptoms). however, pcr equipment is expensive, requires trained personnel, and is best suited to diagnostic reference laboratories. other nucleic acid detection strategies based on isothermal amplification such as nucleic acid sequence-based amplification (nasba) and reverse transcription loop-mediated isothermal amplification (rt-lamp) have been developed to address these diagnostic microbiology and infectious disease j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / d i a g m i c r o b i o challenges and move molecular assays closer to point-of-care use (jittmittraphap et al., ; neeraja et al., ) . here, we have explored a denv diagnostic solution using the rt-lamp technology. the technology was originally developed by eiken chemical company (notomi et al., ) and is based on the principle of strand displacement. briefly, bst polymerase has both polymerase and strand displacement activity and can be used to amplify nucleic acids using a series of primers that initiate a specific stem loop structure following binding to a target sequence (notomi et al., ; nagamine et al., ) . this structure allows the enzyme to polymerize nucleic acids continuously at permissive temperatures beyond - °c. a variety of methods can be used to visualize the amplified dna product or the magnesium pyrophosphate by-product. the dna amplicon can be labeled with an intercalating agent such as sybr green and visualized using the naked eye or under uv light. alternatively, the magnesium pyrophosphate causes turbidity, which can be visualized by the naked eye or using a turbidimeter (mori et al., ) . another benefit of this assay is that it does not require expensive instrumentation and takes less than an hour to perform, making lamp and rt-lamp suitable for diagnostic applications in low-resource settings. the technology has been adapted to detect a variety of microorganisms ranging from bacteria such as mycobacterium (iwamoto et al., ) ; parasites such as plasmodium (sattabongkot et al., ) ; and viruses such as human immunodeficiency virus (curtis et al., (curtis et al., , , severe acute respiratory syndrome-coronavirus (hong et al., ) , and hepatitis b virus (nagamine et al., , . rt-lamp has also been evaluated for diagnosing other members of the flaviviridae family, including yellow fever virus (kwallah et al., ) , japanese encephalitis virus (parida et al., ; toriniwa and komiya, ) , west nile virus , and tick-borne encephalitis virus (hayasaka et al., ) . in this study, we developed an rt-lamp assay to detect all serotypes of denv in a single reaction. other groups have explored this concept, with several assays detecting the serotypes of denv in separate reactions (neeraja et al., ; parida et al., ) . some groups have developed pan-serotype denv rt-lamp assays (teoh et al., ) , and others have expanded this concept to a pan-flaviviral assay that detects denv - , japanese encephalitis virus, and west nile virus in a single reaction (li et al., ) . we report a pan-serotype denv rt-lamp assay that has been optimized and evaluated with an extensive panel of clinical samples obtained from febrile patients in south america. we have additionally explored a method of reducing assay variability in a denv quantitative rt-lamp (qrt-lamp) that uses a quencher to reduce false positives. finally, we have also examined methods for streamlining sample preparation and evaluated visualization of the qrt-lamp product using lateral flow immunochromatographic tests (icts) and a handheld fluorescence reader. together, this represents a complete assay, from sample to result, which can be used to identify denv with high accuracy in low-resource settings. the procedures applied in this study were done in accordance with the ethical standards of the naval medical research center (nmrc) institutional review board and with the helsinki declaration of , as revised in . study protocols were approved by the nmrc institutional review board (nmrcd. . and nmrc. . ) in compliance with all applicable federal regulations governing the protection of human subjects. clinical serum samples of denv viremic patients or those determined to have other febrile illness were collected during ongoing febrile surveillance studies (years - ) at the naval medical research unit- peru at regional sites in piura, tumbes, madre de dios, and iquitos. the samples were deidentified and shipped to the nmrc. additional serum samples from denv-negative individuals were obtained from a dengue vaccine trial at the nmrc and a blood bank at the walter reed army medical center, both in the united states. all of these specimens were obtained with institutional review board approval. tissue culture-derived laboratory virus stocks propagated in vero african green monkey kidney cells at the nmrc were used as positive controls. the following denv strains were used to cover all serotypes: denv , wp ; denv , ; denv , ch ; and denv , . rna extraction for these denv stocks as well as clinical serum samples was performed using qiaamp viral rna mini kit (qiagen, valencia, ca, usa) following the manufacturer's protocol. rna was eluted in μl of nuclease-free water. samples were characterized as positive or negative for denv using a qrt-pcr assay, modified from one previously described (mcavin et al., ) . briefly, the sequences of the qrt-pcr primers used were as follows: denv forward: ′-ggttagaggagacccctc- ′, denv reverse: ′-cagagatcctgctgtctc- ′, probe: ′fam-cagcatattga cgctggga-tamra ′. we utilized the taqman® ez rt-pcr core reagents kit (life technologies, carlsbad, ca, usa) with a final volume of μl, with the following reaction concentrations: . μmol/l forward primer, . μmol/l reverse primer, . μmol/l probe, . mmol/l dntp, mmol/l mn(oac) , x abi buffer, . u rtth polymerase, and . u amperase. the reaction was held at °c for minutes and °c for minutes, followed by cycles of denaturation at °c for seconds and polymerization at °c for seconds. to quantify rna copies, the denv ′utr was cloned into the pcr_script amp vector (stratagene, la jolla, ca, usa), and the transcript was transcribed in vitro using the riboprobe t transcription kit (promega, madison, wi, usa). rna was quantified using a spectrophotometer, and the rna copy number was calculated. this rna standard with known copy numbers was serially diluted and run alongside all qrt-pcr reactions to enable quantification of rna copies in the reaction. rt-lamp primers were designed against the conserved regions of denv using eiken's primerexplorer version (http://primerexplorer. jp/e/) utilizing denv genome (genbank accession number fm . ) as the initial template. these primers were then aligned with denv , , and , and degenerate primers were manually created to improve sequence consensus. these included outer primers (f and b ), inner primers (forward inner primer [fip] and backward inner primer [bip]), and loop primer (loopb). these primers were then aligned with up to published strains each of denv , denv , and denv . six additional primers were added to broaden detection of these denv strains: fipdegen / , fipdegen , bipdegen / , bipdegen , b degen / , and b degen . the rt-lamp reactions were carried out in a -μl volume with the following concentrations: pmol/l fip, fipdegen , bip, and bipdegen ; pmol/l fipdegen / , bipdegen / , and loopb; . pmol/l f and b (integrated dna technologies, coralville, ia, usa); x thermopol buffer (new england biolabs, ipswich, ma, usa); . mol/l betaine (sigma-aldrich, st. louis, mo, usa); . mmol/l each dntp (life technologies, grand island, ny, usa); mmol/l total mgso (sigma-aldrich); u bst dna polymerase (new england biolabs); . u amv rt (life technologies, grand island, ny); and μl of template. reactions were held at °c for minutes unless otherwise indicated. a positive control template corresponding to copies/reaction denv rna was used in rt-lamp reactions, unless otherwise stated. completed reactions were run on a % agarose gel containing x sybrsafe (life technologies). the rt-lamp product banding pattern was also compared against those from true positives. to visualize rt-lamp product in the tubes, x sybrsafe was added. the agarose gels and tubes were visualized using an e-gel imager system (life technologies). fluorescence in reaction tubes was also measured using the picoflour tm model number - (turner biosystems, sunnyvale, ca, usa). the method of visualizing rt-lamp product on lateral flow icts was adapted from previous reports (puthawibool et al., ; ding et al., ) . a home-made ict strip, which had streptavidin on the detection zone and the mouse anti-fitc antibody conjugated with colloidal gold in the sample pad of the strip, was used for detection of the lamp product. for visualizing the rt-lamp product on this system, the reaction was performed as described above except with the addition of biotin- -dutp (life technologies) and fitc- -dutp both to a final concentration of μmol/l. following a lamp reaction, the mixture was heated to °c for minutes and then cooled on ice for minutes. approximately . μl of the biotinylated rt-lamp product was applied to the binding pad of the lateral flow ict. running buffer ( μl of . % tween ) was added to facilitate capillary flow, and the lateral flow icts were visually inspected after minutes. the presence of a visible purple line was considered reactive. sequence-specific detection was performed as above, but incorporating a fam-labeled-loopb primer instead of the unlabeled primer. to reduce visualization of nonspecific amplification, pmol/l of black hole quencher (bhq) -labeled antisense loopb primer was added to the rt-lamp reaction tube following isothermal amplification. both the fam-and bhq-labeled primers were obtained from integrated dna technologies. the tubes were quenched for approximately hours at room temperature, followed by visualization using a uv source as described above. we designed four sets of lamp primers to detect all serotypes and multiple strains of denv in a single reaction. among these, set of primers was down-selected based on faster time to positivity in a pilot experiment (data not shown). table summarizes the primers used. the primers target a ′ untranslated region of denv that is highly conserved between all serotypes and multiple strains. unlike traditional lamp assays, which require - primers, this pan-dengue rt-lamp uses primers, including fip, bip, and b degenerate primers. using a positive control template, the rt-lamp reaction was tested within a range of temperatures ( - °c, fig. a ) using a simple water bath as the heat source. nucleic acid amplification took place at all the temperatures tested, and subsequent experiments were performed at °c. next, various assay characteristics were systematically optimized, including primer, enzyme, and betaine concentrations, in order to achieve the fastest time to result (data not shown). under optimized conditions, copies/reaction of denv rna was detectable in approximately minutes when using gel electrophoresis or uv as a readout (fig. b) . the assay was designed to be reactive to all serotypes of denv, and this was confirmed using tissue culture-derived positive strains (fig. c) . using a serial dilution of denv , we determined a limit of detection of copies/reaction where samples were detected reliably a majority (n %) of the time, comparable to qrt-pcr ( fig. a) . fig. b illustrates how time to positivity is affected by the number of copies of denv template present in the reaction. based on these results, we selected a reaction time of minutes to enable reliable detection of n copies/reaction for all denv serotypes while minimizing nonspecific amplification. we also saw positive rt-lamp reactions using denv-positive samples that had been heat treated for minutes at °c. boiling samples can eliminate the need for formal rna extraction steps as has been previously reported with dna-based pathogen targets (mikita et al., ; hopkins et al., ) . a panel of clinical specimens pcr confirmed as denv positive was used to determine the clinical sensitivity of our optimized rt-lamp reaction. the panel included denv , , and naturally circulating in northern peru at the time of sample collection. denv-negative samples, including human serum samples from individuals without fever symptoms as well as samples from individuals presenting fever symptoms of unknown origin that tested negative for denv by qrt-pcr, were used to determine clinical specificity (fig. ) . our optimized rt-lamp assay demonstrated . % sensitivity ( % confidence interval [ci], . - . % using binomial confidence interval (clopper and pearson, ) ) and . % specificity ( % ci, . - . %). the false-negative samples consisted of denv and denv specimens. overall, the assay demonstrated a . % agreement with denv qrt-pcr, underscoring the potential clinical utility of this assay. as has been reported by others, we occasionally observed falsepositive results when using our denv rt-lamp assay (curtis et al., ). precautionary measures were implemented to reduce risk of contamination from template, and these substantially reduced initial false-positive results. however, another kind of nonspecific amplification was also infrequently observed, which was distinguishable from contamination-derived false-positive results based on a different banding pattern on agarose gels (fig. ) . it has been hypothesized that this is due to non-template-driven self-priming. other groups were able to limit this phenomenon by attaching a fluorescent label to their loop primer, followed by the addition of a probe with a fluorescence quencher (curtis et al., ) . as the increased number of primers in our pan-serotype assay may facilitate nonspecific amplification, we examined whether sequence-specific detection was a feasible solution. at the end of the rt-lamp reaction, any unbound fam-loop probe was quenched by bhq-labeled probe (which consists of the reverse complement sequence of the fam-loop probe with a bhq modification.) we optimized this formulation and found no difference in the permissible temperature range, time to positivity, or number of serotypes detected when including a fluorescently labeled loop primer ( fig. a-c) . in fig. . characteristics of a pan-dengue rt-lamp assay. amplicons generated via rt-lamp were visualized via the incorporation of sybrsafe using agarose gel electrophoresis or uv illumination. a) copies/reaction denv at - °c, b) copies/reaction denv held at °c for - min, c) copies/reaction denv , , , or held at °c for min. ntc = no template control. fig. . comparison of limit of detection between pan-serotype rt-lamp and rt-pcr. ten-fold limiting dilutions of denv beginning at copies/reaction were used to compare the limit of detection of our optimized pan-dengue rt-lamp to an in-house rt-pcr assay. addition, this formulation was able to reduce the nonspecific amplification (fig. d ). in our hands, rt-lamp readout was not prone to operator error, and the positives and negatives were always clearly distinguishable. however, various methods to visualize rt-lamp results including real-time pcr thermocyclers, agarose gel electrophoresis, turbidity measurements, and uv illumination are not practical in resourcelimited settings. we thus explored the feasibility of determining rt-lamp positivity via an inexpensive lateral flow ict. we found % correlation between our agarose gel and ict results when using denv -, denv -, or denv -positive specimens. one specimen that was false negative when visualized via agarose gel electrophoresis was also negative via ict (fig. ) , suggesting that ict has sensitivity comparable to other methods. our data demonstrate that rt-lamp results can be reliably determined without the aid of expensive or bulky equipment. we further evaluated the ability of our rt-lamp assay to be visualized using a battery-operated handheld fluorescent reader and observed the kinetics of quenching over time. true-positive samples remain above a fluorescent threshold of units following hour of quenching. diagnosing dengue accurately during acute infection is critical for informing patient management decisions. lateral flow icts targeting the denv nonstructural protein (ns ) antigen have had some success in enabling early diagnosis at the point of need; however, nucleic acid tests (nats) may detect denv rna earlier than ns antigen, and nats can be more sensitive and specific. the primary disadvantage with pcr-based nats involves the need for thermocyclers, which are often bulky and too expensive to be used in resource-limited settings. commercially available pcr kits cost $ - /test and are generally unsuitable for high-throughput usage in developing economies (dineva et al., ; fiscus et al., ) . in addition, thermocyclers can cost $ , -$ , , which may be prohibitive in many dengueendemic countries (dineva et al., ) . training personnel to operate the instrument and interpret results also requires resources typically only found in a reference laboratory. pcr is also subject to inhibition by substances such as heme compounds, heparin, and edta often present in a clinical samples and blood collection tubes, leading to falsenegative results (klein et al., ; fredricks and relman, ) ; removing pcr inhibitors can lead to increased overhead costs and turnaround time to results. conversely, pcr is also often subject to reporting false positives from the presence of contaminating nucleic acids (fredricks and relman, ) , necessitating the use of template-free "clean rooms" for preparing a master mix, which is impractical in the field. to develop a diagnostic solution that overcomes these limitations, we developed a pan-denv rt-lamp assay. we demonstrate that the reaction can proceed continuously within a wide temperature range - °c, making it suitable for a water bath or electricity-free heater (labarre et al., ) . in addition, we have explored various methods of visualizing the assay results. electrophoresis gel confirmation of reaction products may be performed in a reference laboratory, but in the field, the reaction can be visualized in real time by measuring an increase in turbidity from magnesium pyrophosphate by-products (mori et al., ) . we propose a method involving the measurement of color change by the incorporation of sybr green adapted from a previous report, which was easier to read with the naked eye (parida et al., ) . we also explored visualization using lateral flow icts and demonstrated % concordance between rt-lamp visualized on a gel versus those visualized using icts. this format is likely to be easier to read in resource-limited settings where a uv light source is not available. finally, we explored the feasibility of using a handheld uv fluorescence reader, which when coupled with a quencher, can provide higher specificity detection of denv. the handheld uv reader that we used was approximately $ , and such readers could improve accuracy of readout as well as provide some level of quantitation to rt-lamp results. lamp has several other advantages over traditional pcr, including less sensitivity to inhibitors such as ethanol, isopropanol, edta, and sodium acetate (mori et al., ; kaneko et al., ) . the use of loop-specific primers can reduce assay times to provide faster sample to result than pcr, and other groups have reported higher sensitivity (nagamine et al., ) . lamp also does not require the template to be denatured , and contrasted with nasba (a similar isothermal nat), lamp does not require a separate nucleic acid extraction step (francois et al., ) . this robustness further reduces the cost as well as time to result. like qpcr, qlamp is amenable to multiplexing (tanner et al., ) , and we adapted it to detect all serotypes of denv in reaction. our data demonstrate that the use of degenerate primers is a feasible method by which to design assays for targets of interest even when sequence conservation is limited. the assay was able to detect a majority of denv pcr-positive clinical samples obtained from south america (sensitivity of . %), representing multiple circulating strains of denv - ; however, the clinical performance is limited with only dengue-positive clinical specimens from region and lack of inclusion of denv -positive specimens. the assay was also specific to denv and did not react to other pathogens that cause similar fever-like symptoms. this demonstrates the assay's effectiveness for enabling differential diagnosis in a clinical situation with multiple strains and serotypes. since this is a molecular assay, it will likely be able to detect denv in mosquito samples and have further applications for vector surveillance. due to the robustness of lamp, it may be used without explicit sample preparation in applications where having false negatives for low copy number samples are acceptable and with integrated sample preparation where it is not. acute denv viremic patients have - plaque-forming units/ml (vaughn et al., ) , and such high titers are detectable by lamp without explicit sample preparation. the assay reagents can also be lyophilized for field expediency and provided in a single tube to which one may add hydration buffer and test sample. the assay can be run at a range of temperatures and can be performed using a water bath or other available heat source. we also envision the assay readout adapted to specific applications and present uv visualization, handheld fluorescence readers, and lateral flow icts as viable alternatives. in conclusion, this work adds to the growing body of isothermal amplification assays available to detect denv. more than previous reports, we have explored all aspects of denv detection from sample to result, with an emphasis on visualizing the rt-lamp product, as readout is critical for ensuring accuracy. the assay can be adapted to use in dengue-endemic countries where it is most needed to reduce morbidity and mortality associated with this widespread disease. the views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the department of the navy, the department of defense, or the us government. dr allison dauner, ms indrani mitra, mr theron gilliland jr, ms sajeewane seales, and dr subhamoy pal are (or were) employed by the henry m. jackson foundation for the advancement of military medicine and were funded to do this work by the us government. dr shuenn-jue wu and dr tadeusz kochel are employees of the us government or members of the us military. this work was prepared as part of their official duties. title u.s.c. § provides that "copyright protection under this title is not available for any work of the united states government." title u.s.c. § defines a us government work as a work prepared by military service members or employees of the us government as part of those persons' official duties. this work was funded by the military infectious diseases research program, l _ _nm. the work was supported by work unit number .rad .l.a . fig. . sequence-specific detection. amplicons generated via sequence-specific rt-lamp were visualized via agarose gel electrophoresis or uv illumination minutes after the addition of a reverse complement loop quencher. a) copies/reaction denv at - °c, b) copies/reaction denv held at °c for - minutes, c) copies/reaction denv , , , or held at °c for minutes, d) replicates each of ntc (lanes - ) or denv (lanes - ) after minutes at °c. reactions were quenched for hours prior to uv illumination. e. quenching reaction measured using picflour shows detection of rt-lamp product following quenching. ntc = no template control. the global distribution and burden of dengue the use of 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general de epidemiologia and diresa piura, tumbes, madre de dios, and iquitos.