# pos

id: cord-343632-cv3qgno3
pos: NNP
token:
  https://www.bgi.com/global/company/news/bgi-develops-realtime-dna-based-kit-for-detecting-the-2019-novel-coronavirus/

lemma:
  https://www.bgi.com/global/company/news/bgi-develops-realtime-dna-based-kit-for-detecting-the-2019-novel-coronavirus/


# ent

id: cord-271434-30nh2gc7
type: CHEMICAL
entity:
  N gene the whole sequence of E gene or the part sequence of O gene Table S1 Supporting Information online were constructed by Xiamen Zeesan Biotech China Briefly the recombinant plasmids encoding a RNA sequence


id: cord-258057-ti0rpt0q
type: CHEMICAL
entity:
  dNTPs 0 4 μM each of F3 and B3 2 5 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 1 μL template


id: cord-104321-fpoztmcl
type: DNA
entity:
  human testis determining factor 7 8 9 10 Methods for sex determination using PCR for amplification of SRY gene


id: cord-285088-krim73zt
type: CELL_LINE
entity:
  F3 TTT GGC CTT GTT GTT GTT GG B3 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


id: cord-285088-krim73zt
type: DNA
entity:
  backward loop primer LB 1 2 mM dNTPs 1 Bst DNA Polymerase Buffer Zhengzhou Shenxiang Industrial Co Ltd China 1 EvaGreen 1 Rox 1 pg pUC57 N DNA


id: cord-283415-1nu399lz
type: CHEMICAL
entity:
  MgCl 2 2 5 lL of 5 M Betaine 2 5 lL of 109 Thermo Buffer 1 5 lL of Bst DNA polymerase and 2 lL of DNA C83529


id: cord-004133-32w6g7qk
type: ORG
entity:
  Nondigital Polymerase Chain Reaction and Loop Mediated Isothermal Amplification Assays for Quantification of Human Cytomegalovirus Direct Serum Assay


id: cord-004133-32w6g7qk
type: WORK_OF_ART
entity:
  Endocervical Specimens by Polymerase Chain Reaction Genotyping Single Nucleotide Polymorphisms in Human Genomic


id: cord-004133-32w6g7qk
type: ORG
entity:
  PCR Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses


id: cord-264676-k531q3ir
type: CHEMICAL
entity:
  MgCl 2 pH 9 5 Subsequently the substrate nitroblue tetrazolium chloride 5 bromo 3 chloro 3indolyl phosphate NBT


id: cord-323845-s78t5qxj
type: CL
entity:
  stem lengths and cauliflower like structures of multiple loops formed by annealing between alternately inverted repeats of the target sequence


id: cord-333524-a6p6ma8r
type: ORG
entity:
  Coronavirus Using a Reverse Tran Scriptional Loop Mediated Isothermal Amplification RT LAMP Diagnostic Plat Form


id: cord-310657-04pp0o74
type: ORG
entity:
  DNA A Mismatch Tolerant Reverse Transcription Loop Mediated Isothermal Amplification Method and Its Application on Simultaneous Detection of All Four Serotype


id: cord-297974-sduz0j35
type: ORG
entity:
  Ebola Zaire RNA Development of Lyophilized Loop Mediated Isothermal Amplification Reagents for the Detection of Leptospira Detection


id: cord-332820-6qx6svs5
type: ORG
entity:
  Reverse Transcription Loop Mediated Isothermal Amplification SARS CoV 2 Detection Using an Isothermal Amplification Reaction


id: cord-296977-yzhsdz9c
type: DNA
entity:
  Ct 15 red 2 high Ct between 20 and 15 yellow 3 medium Ct between 20 and 25 green 4 low Ct 25 blue and 5 negative dark blue Color coding


id: cord-296977-yzhsdz9c
type: ORG
entity:
  Coronavirus Using a Reverse Transcriptional Loop Mediated Isothermal Amplification RT LAMP Diagnostic Platform Rapid


id: cord-296977-yzhsdz9c
type: ORG
entity:
  SARS CoV 2 Development of Reverse Transcription Loop Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus


id: cord-302663-gb2vgs97
type: DNA
entity:
  X174 Hinc II digest lanes 2 7 10 1 10 2 10 3 10 4 10 5 and 10 6 respectively B 0 5 M NaOH method lane 1 molecular size marker X174 Hinc II digest lanes 2 5 10 2 10 3 10 4 and 10 5 RT


id: cord-322238-8iwljdoi
type: CL
entity:
  stem lengths and cauliflower like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence


id: cord-322238-8iwljdoi
type: DISEASE
entity:
  Gastroenteritis Coronavirus TGEV strain H Porcine Reproductive and Respiratory Syndrome Virus PRRSV Pesudorabies PRV Porcine Parvovirus


id: cord-002081-vi6rth9o
type: DISEASE
entity:
  carcinogenic human papillomavirus and herpesvirus 12 and the detection of Middle East Respiratory Syndrome Coronavirus


id: cord-333220-tcvs4beg
type: DNA
entity:
  IB loopF 5 TACCTTGATAGTCCAGAAGAACC 3 and IB loopB 5 CTACGGACGGAAACTGCAC 3 0 4 mM dNTPs 1 M betaine 20


id: cord-321886-0b3ocoh9
type: CHEMICAL
entity:
  MgCl 2 1 l each of outer primers 10 M 1 l each of inner primers 40 M 1 l each of loop primers 20 M 2 5 l of dNTP mixture 2 5 mM each 2 5 l of betaine 5 M 1 l of extracted template


id: cord-271504-t3y1w9ef
type: ORG
entity:
  Development of Reverse Transcription Loop Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus


id: cord-280442-jtvez46y
type: PROTEIN
entity:
  glycoprotein S small membrane protein E membrane glycoprotein M and phosphorylated nucleocapsid protein N


id: cord-280442-jtvez46y
type: PROTEIN
entity:
  nucleocapsid N matrix protein M phosphoprotein P fusion protein F hemagglutinin neuraminidase protein HN


id: cord-280442-jtvez46y
type: DNA
entity:
  primer pairs 5 ATCACACTAGCCTTGC GCTAGA 3 5 GCAAAAGCATCAGCGTAATCC 3 and 5 AATCTGTATTACATGTCTAGG 3 5 AGA GAGAATATATCCTTTCGC 3


id: cord-284644-9k2oox64
type: CHEMICAL
entity:
  influenza D virus in cattle Rapid and specific detection of H3 swine influenza virus using reverse transcription


id: cord-304343-m7tbdfri
type: WORK_OF_ART
entity:
  Peptidomimetic Compounds for Use in the Treatment of Neuronal Diseases Highly Potent Peptides to Control Cancer


id: cord-304343-m7tbdfri
type: ORG
entity:
  Neurodegenerative Diseases Patent WO Methods for Reducing Neurodegeneration Method for Modulating Autophagy


# url

id: cord-343632-cv3qgno3
domain: www.bgi.com
url:
  http://www.bgi.com/global/company/news/bgi-develops-realtime-dna-based-kit-for-detecting-the-2019-novel-coronavirus/


# bib

id: cord-000049-rl7sdzd7
author: Lee, David
date: 2009-02-02
txt: ./txt/cord-000049-rl7sdzd7.txt
cache: ./cache/cord-000049-rl7sdzd7.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656497/
doi: 10.1186/1472-6750-9-7
summary:
  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. 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. Here we assess the LAMP protocol for the detection of GMOs using primers that target event-specific sequences for transgenic MS8 and RF3 oilseed rape (Brassica napus L.) and generic GMO sequences such as the cauliflower mosaic virus 35S promoter (P-35S) and the promoter and terminator for the nopaline synthase gene (P-nos and T-nos, respectively) from Agrobacterium spp. LAMP sensitivity was assessed by the detection of ten RoundUp Ready™ GMO targets in a background of 100 ng of genomic oilseed rape DNA (Figure 3 ).

title:
  Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

abstract:
  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 0.01% 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.


id: cord-000625-cpjlzutk
title: Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method
author: Ablordey, Anthony
date: 2012-04-03
txt: ./txt/cord-000625-cpjlzutk.txt
cache: ./cache/cord-000625-cpjlzutk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317900/
doi: 10.1371/journal.pntd.0001590
summary:
  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 (60uC) provided by a disposable pocket warmer [34] . 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 IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans.

abstract:
  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 IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. PRINCIPAL FINDINGS: The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 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


id: cord-001762-dtvzwin8
author: Jeong, Joojin
date: 2015-09-30
txt: ./txt/cord-001762-dtvzwin8.txt
cache: ./cache/cord-001762-dtvzwin8.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564147/
doi: 10.5423/ppj.oa.03.2015.0044
summary:
  title: Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification 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. This study showed similar results in that the RT-LAMP assay included two loop primers and took only 15 min for detection of PVX. Simple and rapid detection of Potato leafroll virus (PLRV) by reverse transcription loop-mediated isothermal amplification (RT-LAMP) Reverse transcription loop-mediated isothermal amplification of DNA for detection of Potato virus Y Rapid detection and differentiation of Dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples

title:
  Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

abstract:
  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 30 min, compared to 120 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.


id: cord-002081-vi6rth9o
author: Zhang, Chao
date: 2016-06-01
txt: ./txt/cord-002081-vi6rth9o.txt
cache: ./cache/cord-002081-vi6rth9o.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887897/
doi: 10.1038/srep26533
summary:
  In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. The successful establishment of an inexpensive, rapid and real-time LAMP protocol for CYP2C19*2 and CYP2C19*3 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. In this study, the successful establishment of an inexpensive, rapid and real-time LAMP protocol for CYP2C19 SNP genotyping expanded the scope of application of this technique to human gene mutation detection. 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.

title:
  Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

abstract:
  Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) 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 CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 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 CYP2C19 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.


id: cord-002720-lrkscs71
author: Kurosaki, Yohei
date: 2017-10-18
txt: ./txt/cord-002720-lrkscs71.txt
cache: ./cache/cord-002720-lrkscs71.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647432/
doi: 10.1038/s41598-017-13836-9
summary:
  title: Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification The assay detected viral RNA at 14.5 TCID(50)/mL in virus-spiked serum or urine samples within 15 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 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. 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) 14, 18, 19 . 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 15 min) can be used as an indicator of the number of RNA copies in each reaction.

title:
  Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification

abstract:
  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 14.5 TCID(50)/mL in virus-spiked serum or urine samples within 15 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 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. 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.


id: cord-002982-zwvesrct
author: Thiessen, Lindsey D.
date: 2018-04-20
txt: ./txt/cord-002982-zwvesrct.txt
cache: ./cache/cord-002982-zwvesrct.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912203/
doi: 10.7717/peerj.4639
summary:
  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. 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., 2009b; Rogers, Atkins & West, 2009; Temple & Johnson, 2011; Thiessen et al., 2016) . 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).

title:
  Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator

abstract:
  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 4.4 enabled, Bluetooth-capable Nexus 7 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 >20 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.


id: cord-003342-wmmbkmrg
title: Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification
author: Wang, De-Guo
date: 2015-04-07
txt: ./txt/cord-003342-wmmbkmrg.txt
cache: ./cache/cord-003342-wmmbkmrg.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272222/
doi: 10.3390/molecules20046048
summary:
  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 10 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 11 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 2000 [1] , can specifically, sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing 6 independent sequences of a target gene under isothermal conditions.

abstract:
  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 10 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 11 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.


id: cord-004133-32w6g7qk
title: Advances in Directly Amplifying Nucleic Acids from Complex Samples
author: Walker, Faye M.
date: 2019-09-30
txt: ./txt/cord-004133-32w6g7qk.txt
cache: ./cache/cord-004133-32w6g7qk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955841/
doi: 10.3390/bios9040117
summary:
  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. 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 50% (v/v) serum, they achieved a very low LOD of 100 pg [119] . 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 50% (v/v) serum, they achieved a very low LOD of 100 pg [119] .

abstract:
  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 1989 to 2017, 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.


id: cord-004307-4sltubqk
author: Horiuchi, Sho
date: 2020-01-14
txt: ./txt/cord-004307-4sltubqk.txt
cache: ./cache/cord-004307-4sltubqk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010225/
doi: 10.3892/ijo.2020.4961
summary:
  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. 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. Following removal of normal lung tissues, DNA samples were extracted as aforementioned, and investigated using Therascreen EGFR PCR and a LAMP assay. 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 19 deletion by the primers of the similarly targeted mutation. A rapid, sensitive assay to detect EGFR mutation in small biopsy specimens from lung cancer Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay

title:
  A novel loop-mediated isothermal amplification method for efficient and robust detection of EGFR mutations

abstract:
  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 32/59 tumor samples (LAMP) and 33/59 tumor samples (Therascreen). Notably, the LAMP assay identified one tumor as wild-type, which had previously been identified as a deletion mutation in exon 19 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 19 deletion mutation using both methods. In addition, a novel deletion mutation in exon 19 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.


id: cord-005377-36io7zsm
title: Alternative Molecular Tests for Virological Diagnosis
author: Sidoti, Francesca
date: 2012-04-09
txt: ./txt/cord-005377-36io7zsm.txt
cache: ./cache/cord-005377-36io7zsm.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091206/
doi: 10.1007/s12033-012-9533-8
summary:
  The main isothermal methods reviewed here include loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and helicase-dependent amplification (HDA). Development and evaluation of a novel loop mediated isothermal amplification (LAMP) method for rapid detection of SARS Corona virus Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. 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 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 Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A

abstract:
  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.


id: cord-104321-fpoztmcl
author: Almasi, Mohammad Amin
date: 2017
txt: ./txt/cord-104321-fpoztmcl.txt
cache: ./cache/cord-104321-fpoztmcl.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359858/
doi: nan
summary:
  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. 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 4 ), calcium chloride (CaCl 2 ), 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 (37) (38) (39) (40) . Development of Loop mediated isothermal amplification (LAMP) of SRY gene in Almasi MA and Almasi G JRI human blood samples for sex determination. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Visual detection of potato leafroll virus by one-step reverse transcription loop-mediated isothermal amplification of DNA with hydroxynaphthol blue dye

title:
  Loop Mediated Isothermal Amplification (LAMP) for Embryo Sex Determination in Pregnant Women at Eight Weeks of Pregnancy

abstract:
  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 15 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 15 blood samples from pregnant women were seven male embryos (46.6%) and eight female embryos (53.4%). 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.


id: cord-252686-viz05uam
author: Dong, Qing
date: 2019-11-04
txt: ./txt/cord-252686-viz05uam.txt
cache: ./cache/cord-252686-viz05uam.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0003267019307755
doi: 10.1016/j.aca.2019.06.048
summary:
  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. 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). 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. 1D and Fig. S6 , in SI).

title:
  A signal-flexible gene diagnostic strategy coupling loop-mediated isothermal amplification with hybridization chain reaction

abstract:
  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 30 copies of NoV gene in 2% 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.


id: cord-256845-5pjam7em
author: Stranieri, Angelica
date: 2017-01-18
txt: ./txt/cord-256845-5pjam7em.txt
cache: ./cache/cord-256845-5pjam7em.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093416306656
doi: 10.1016/j.jviromet.2017.01.009
summary:
  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 2 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). Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis

title:
  Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

abstract:
  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 (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63 °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 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.


id: cord-258057-ti0rpt0q
title: Establishment of a Porcine Parvovirus (PPV) LAMP Visual Rapid Detection Method
author: Zhao, Kai
date: 2020-07-01
txt: ./txt/cord-258057-ti0rpt0q.txt
cache: ./cache/cord-258057-ti0rpt0q.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32621958/
doi: 10.1016/j.jviromet.2020.113924
summary:
  A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. To verify the specificity of PPV detection by LAMP, the DNA (PPV, PCV2, PRV) and cDNA samples (PRRSV, CSFV) were amplified as sample templates by the LAMP reaction at 59.0℃ for 50 min and terminated at 80℃ for 3 min, respectively. 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 10 1 copies. Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification Rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (LAMP) method

abstract:
  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 frame1 (ORF1) gene. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °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 10 copies, which is 100-fold lower than conventional PCR. Our LAMP assay did not cross-react with other viruses. We used the established LAMP system to test 1100 field samples and detected 660 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.


id: cord-264676-k531q3ir
author: Liu, Yi
date: 2009-07-09
txt: ./txt/cord-264676-k531q3ir.txt
cache: ./cache/cord-264676-k531q3ir.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/19592299/
doi: 10.1016/j.jcv.2009.06.010
summary:
  title: In situ reverse-transcription loop-mediated isothermal amplification (in situ RT-LAMP) for detection of Japanese encephalitis viral RNA in host cells 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. When the in situ RT-LAMP was run with the DIG-labeled primer, no positive reaction was shown in either JE virus-infected (Fig. 5A ) or uninfected (Fig. 5B ) BHK-21 cells. Detection of Japanese encephalitis virus in mouse peripheral blood mononuclear cells using an in situ reverse transcriptase polymerase chain reaction Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification Rapid detection and differentiation of Dengue virus serotypes by 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

title:
  In situ reverse-transcription loop-mediated isothermal amplification (in situ RT-LAMP) for detection of Japanese encephalitis viral RNA in host cells

abstract:
  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-21 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.


id: cord-265172-rn9pkk52
author: Michiwaki, Yuhei
date: 2020-10-09
txt: ./txt/cord-265172-rn9pkk52.txt
cache: ./cache/cord-265172-rn9pkk52.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.wneu.2020.09.166
doi: 10.1016/j.wneu.2020.09.166
summary:
  title: Emergent carotid artery stenting following intravenous alteplase infusion after rapid negative diagnosis for COVID-19 by loop-mediated isothermal amplification assay: A case report 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. 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. Carotid artery stenting (CAS) is a standard treatment procedure for internal carotid artery stenosis (ICS) 8 ; however, the efficacy and safety of emergent CAS (eCAS) for hyperacute ischemic stroke (hAIS) due to ICS have not been sufficiently established. 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.

title:
  Emergent carotid artery stenting following intravenous alteplase infusion after rapid negative diagnosis for COVID-19 by loop-mediated isothermal amplification assay: A case report

abstract:
  Background During the coronavirus disease 2019 (COVID-19) pandemic, a rapid screening method for COVID-19 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 71-year-old man with hAIS caused by severe ICS was treated via intravenous alteplase infusion. The patient underwent screening for COVID-19 by the loop-mediated isothermal amplification (LAMP) assay shortly after arrival at our institution. The LAMP result was obtained within 90 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 (200 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 15 days following eCAS. Because of the rapid negative diagnosis for COVID-19 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-19 pandemic, the LAMP assay for COVID-19 detection might be a suitable diagnostic strategy preceding stroke treatment because of the rapid turnaround time.


id: cord-268627-nnx46nwf
author: Ren, Xiaofeng
date: 2011-02-01
txt: ./txt/cord-268627-nnx46nwf.txt
cache: ./cache/cord-268627-nnx46nwf.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21286798/
doi: 10.1007/s11262-011-0570-3
summary:
  In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene was achieved at 63°C for 50 min. 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. As shown in Fig. 2a , 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 63°C was stronger than that at other reaction temperatures, which was judged as the optimal temperature for RT-LAMP amplifying PEDV N gene. 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.

title:
  Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus

abstract:
  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 63°C for 50 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.


id: cord-268993-2sjh17mw
author: Rödel, Jürgen
date: 2020-08-31
txt: ./txt/cord-268993-2sjh17mw.txt
cache: ./cache/cord-268993-2sjh17mw.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S1386653220303589?v=s5
doi: 10.1016/j.jcv.2020.104616
summary:
  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-2 (SARS-CoV-2) 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-2 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-19) 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 75% compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results.

title:
  Use of the variplex(TM) SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis

abstract:
  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-2 (SARS-CoV-2) 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-2 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-19) 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 75% 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 0.52-0.56). 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: 76.3% for variplex™, 84.2% for Allplex™ and 68.4% for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100%. 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-19 infection.


id: cord-271434-30nh2gc7
title: A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing
author: Tian, Fei
date: 2020-07-27
txt: ./txt/cord-271434-30nh2gc7.txt
cache: ./cache/cord-271434-30nh2gc7.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1007/s11426-020-9800-6
doi: 10.1007/s11426-020-9800-6
summary:
  In this work, we develop an automated centrifugal microfluidic system (Figure 1 ) consisting of a microfluidic disc and a customized instrument for rapid sample-to-answer detection of SARS-CoV-2 armored RNA particles with high sensitivity and specificity. 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. To demonstrate the automated detection of viral nucleic acids by the centrifugal microfluidic system, we loaded different concentrations of SARS-CoV-2 armored RNA particles with N gene (0.5, 1, 10, 10 2 , 10 3 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.

abstract:
  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 2 (SARS-CoV-2) armored RNA particles. The estimated limit of detection for armored RNA particles is 2 copies per reaction, the throughput is 21 reactions per disc, and the assay sample-to-answer time is approximately 70 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 10.1007/s11426-020-9800-6 and is accessible for authorized users.


id: cord-271504-t3y1w9ef
author: Luo, Zichao
date: 2020-06-16
txt: ./txt/cord-271504-t3y1w9ef.txt
cache: ./cache/cord-271504-t3y1w9ef.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32607499/
doi: 10.34133/2020/6925296
summary:
  A confirmed case should have at least one of the following criteria: (i) a positive result for 2019-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 2019-nCoV; and (iii) serum samples positive for IgM or IgG to 2019-nCoV, or seroconversion in IgG, or a fourfold or more significant increase in IgG antibody titer to 2019-nCoV in the recovery phase than in the acute phase [25] . Using blood samples taken from alleged COVID-19 patients, the researchers detected antibodies targeting the spike protein that prevented the virus from killing cells in laboratory tests. showed a promising in vitro inhibitory effect of this serine protease inhibitor in SARS-CoV and 2019-nCoV on human lung cells, showing potential as a viable option for COVID-19 treatment [113] . 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-19, updated on 17 February 2020 [189] .

title:
  Combating the Coronavirus Pandemic: Early Detection, Medical Treatment, and a Concerted Effort by the Global Community

abstract:
  The World Health Organization (WHO) has declared the outbreak of 2019 novel coronavirus, known as 2019-nCoV, a pandemic, as the coronavirus has now infected over 2.6 million people globally and caused more than 185,000 fatalities as of April 23, 2020. Coronavirus disease 2019 (COVID-19) 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-19 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-19. 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-19 by targeting nucleic acids, antigens, or antibodies. We also summarize potential treatments and vaccines against COVID-19 and discuss ongoing clinical trials of interventions to reduce viral progression.


id: cord-271735-gprd79di
title: Detecting amplicons of loop‐mediated isothermal amplification
author: Shirato, Kazuya
date: 2019-08-13
txt: ./txt/cord-271735-gprd79di.txt
cache: ./cache/cord-271735-gprd79di.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1111/1348-0421.12734
doi: 10.1111/1348-0421.12734
summary:
  Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus 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 Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) Development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (H1N1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) 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

abstract:
  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.


id: cord-276718-3lujp0oy
author: Neeraja, M.
date: 2014-10-24
txt: ./txt/cord-276718-3lujp0oy.txt
cache: ./cache/cord-276718-3lujp0oy.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25455901/
doi: 10.1016/j.jviromet.2014.10.005
summary:
  title: Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India 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 NS1 gene using the Genie® II flourometer was carried out. 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 NS1 gene using a real-time flourometer (Genie ® II from Optigene, U.K.). 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). Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay

title:
  Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India

abstract:
  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 NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 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 = 1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% 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.


id: cord-276957-pk33dl8q
author: Hu, Xuejiao
date: 2020-08-26
txt: ./txt/cord-276957-pk33dl8q.txt
cache: ./cache/cord-276957-pk33dl8q.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32848011/
doi: 10.1128/msphere.00808-20
summary:
  To accelerate clinical diagnostic testing for COVID-19, we conducted a prospective cohort study to develop and validate a novel RT-LAMP assay capable of detecting SARS-CoV-2 RNA for potential use in centralized facilities and point-of-care settings. The detection results obtained using the RT-LAMP assay showed good concordance with those obtained using the RT-qPCR In Cohort I, 35 of 37 nasopharyngeal swabs from 24 COVID-19 patients were confirmed to be SARS-CoV-2 positive according to the criteria of RT-qPCR (28 samples) and NGS confirmation (7 samples) (see Table S3 in the supplemental material). Subsequently, we evaluated the RT-LAMP and standard RT-qPCR assays on 329 nasopharyngeal swabs from a cohort of 129 suspected COVID-19 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-2 confirmation.

title:
  Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

abstract:
  The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 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-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. 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-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 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-2 infection and may aid the expansion of COVID-19 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-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.


id: cord-279229-2226jnfl
author: Savan, R
date: 2005-11-22
txt: ./txt/cord-279229-2226jnfl.txt
cache: ./cache/cord-279229-2226jnfl.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16302951/
doi: 10.1111/j.1365-2761.2005.00670.x
summary:
  In this paper, we review a novel method of DNA amplification known as loop-mediated isothermal amplification (LAMP) of a target nucleic acid. 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 2 Loop-mediated isothermal amplification reaction amplifying the haemolysin gene from Edwardsiella tarda isolates. Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP) 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) Rapid diagnosis of herpes simplex virus infection 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) Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

title:
  Loop‐mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens

abstract:
  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.


id: cord-280442-jtvez46y
author: Wu, Xuan
date: 2019-11-01
txt: ./txt/cord-280442-jtvez46y.txt
cache: ./cache/cord-280442-jtvez46y.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31265112/
doi: 10.3382/ps/pez372
summary:
  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 13 IBV strains, 7 NDV strains, and the PCR and LAMP target sequences of 6 NDV and 1 turkey coronavirus strains (TCoV) synthesized by Sangon Biotech (Shanghai, China) Co, as well as 6 other avian virus strains, were used for the determination of the specificities of RT-PCR and RT-LAMP assays. 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 < 0.05). The mean IBV and NDV detection rates of different samples, detected by mRT-LAMP-LFD, were both 95%, and were significantly higher than those detected by conventional RT-PCR and qRT-PCR (P < 0.05, Figure 6B) .

title:
  Simultaneous and visual detection of infectious bronchitis virus and Newcastle disease virus by multiple LAMP and lateral flow dipstick

abstract:
  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 5′-untranslated region (5′-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 100.8 IBV RNA copies/reaction and 100.7 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 98.65% and 97.25%, respectively. In the IBV and NDV co-infected sample study, the mean detection rates of IBV and NDV were both 95%. This study showed that mRT-LAMP-LFD was a promising qualitative detection tool suitable for field single or multiple IBV and NDV detection.


id: cord-282126-gmjnbnx5
author: Yang, Limin
date: 2012-08-07
txt: ./txt/cord-282126-gmjnbnx5.txt
cache: ./cache/cord-282126-gmjnbnx5.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1007/s11262-012-0798-6
doi: 10.1007/s11262-012-0798-6
summary:
  We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). 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. To detect the limit of the RT-LAMP and RT-PCR assay, DHAV-1 total RNAs were extracted from the serially 10-fold diluted allantoic fluid, ranging from 10 4 to 10 -3 50 % egg lethal dose (ELD 50 ) per 100 ll. 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 10-fold serial dilutions (from 10 4 to 10 -3 ELD50 per 100 ll) of allantoic fluid. A one-step RT-LAMP assay with high specificity and sensitivity was developed for rapid diagnosis of DHAV-1, which has no cross-reaction with DEV, MPV, AIV, R.

title:
  Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of Duck hepatitis A virus type 1

abstract:
  We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). The amplification could be finished in 1 h under isothermal conditions at 63 °C by employing a set of four primers targeting the 2C gene of DHAV-1. The RT-LAMP assay showed higher sensitivity than the RT-PCR with a detection limit of 0.1 ELD(50) 0.1 ml(−1) of DHAV-1. 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-1.


id: cord-283415-1nu399lz
author: Jiang, Kuiyu
date: 2012-08-14
txt: ./txt/cord-283415-1nu399lz.txt
cache: ./cache/cord-283415-1nu399lz.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22890294/
doi: 10.1007/s00284-012-0204-6
summary:
  title: Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC) The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. However, the invention of Loop-mediated isothermal amplification (LAMP) provides new ideas and technologies for establishing a rapid detection method of F5 fimbriae gene. The result of detecting by established LAMP were positive only for F5 fimbriae gene, and no positive DNA products of the LAMP assay were observed when these control strains (F4ab, F6, F41, and F18ab) were used as templates (Fig. 3) . Development of loop-mediated isothermal amplification (LAMP) for detection of Escherichia coli producing Shiga toxin II variant Rapid and sensitive detection of heat-labile and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification

title:
  Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC)

abstract:
  The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10(2) cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC.


id: cord-284644-9k2oox64
author: Sharma, Vikrant
date: 2017-12-15
txt: ./txt/cord-284644-9k2oox64.txt
cache: ./cache/cord-284644-9k2oox64.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29253497/
doi: 10.1016/j.jviromet.2017.12.005
summary:
  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. 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. The objectives of the current study were to (1) optimize RT-LAMP assay for detection of influenza A viruses and their subtypes (H1N1, H3N2 and pdm09/H1N1); (2) determine sensitivity and specificity of RT-LAMP assay; (3) clinical evaluation of RT-LAMP assay and conventional one-step RT-PCR in comparison to WHO recommended rRT-PCR taken as standard. Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus

title:
  Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses

abstract:
  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 (H1N1, H3N2) and novel pandemic H1N1 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 H1N1, H3N2 and pdm09/H1N1 subtypes. One hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with RT-LAMP assay, detecting 30 (17.9%) samples positive for Influenza A virus. Out of 30 samples, 21, 7 and 2 were found positive for pdm09/H1N1, H3N2 and seasonal H1 respectively. Conventional one-step RT-PCR detected a total of 27 (16.2%) samples for influenza A and further subtyping showed 20 and 7 samples positive for pdm09/H1N1 and H3N2 virus respectively whereas none was found positive for seasonal H1N1. RT-LAMP assay demonstrated higher sensitivity (93.8%) than conventional RT-PCR (84.4%) 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.


id: cord-285088-krim73zt
author: Wang, Deguo
date: 2020-04-21
txt: ./txt/cord-285088-krim73zt.txt
cache: ./cache/cord-285088-krim73zt.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.04.21.052530
doi: 10.1101/2020.04.21.052530
summary:
  title: One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay 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-19. The analytic sensitivities of the newly developed real-time RT-LAMP assay and visual RT-LAMP assay were determined with pUC57-N DNA ranging from 2-0.02 fg, and the reaction mixtures were heated at the optimal temperature for 60 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 detection limits of the one-pot real-time RT-LAMP assay and the one-pot visual RT-LAMP assay were determined using pUC57-N DNA ranging from 2-0.02 fg at 59 °C for 60 min in a StepOneTM System (Applied Biosystems, Foster City, CA, USA) or in a water bath.

title:
  One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay

abstract:
  Background Rapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by COVID-19. 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-19. Methods Six specific LAMP primers targeting the N gene of COVID-19 were designed, the RT-LAMP reaction system was optimized with plasmid pUC57 containing N gene sequence, the detection limit was determined with a serial dilution of the plasmid pUC57 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-19 were established. Results Our results showed that the one-pot RT-LAMP assays can detect COVID-19 with a limit of ≥ 6 copies per μl−1 of pUC57 containing N gene sequence. Conclusion This study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of COVID-19.


id: cord-287104-4k8pqbc0
author: Lee, J. Y.
date: 2019-04-04
txt: ./txt/cord-287104-4k8pqbc0.txt
cache: ./cache/cord-287104-4k8pqbc0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1007/s12088-019-00803-3
doi: 10.1007/s12088-019-00803-3
summary:
  title: Development of Rapid and Specific Detection for the Human Aichivirus A Using the Loop-Mediated Isothermal Amplification from Water Samples In this study, developed a LAMP method to achieve a rapid, specific and highly sensitive detection of AiV-A. A newly developed method was more rapid (approximately 2–8 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. A method for detecting AiV-A specific genes by using reverse transcription nested polymerase chain reaction (RT-PCR) assay, have been reported [15] [16] [17] . In this study, developed a rapid, specific and highly sensitive detection of AiV-A by using a LAMP assay. In this study, developed a LAMP assay that could rapid, specific, and sensitive detection of AiV-A from water samples.

title:
  Development of Rapid and Specific Detection for the Human Aichivirus A Using the Loop-Mediated Isothermal Amplification from Water Samples

abstract:
  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 1989. 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 2–8 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 (10.1007/s12088-019-00803-3) contains supplementary material, which is available to authorized users.


id: cord-288887-lshsgex3
author: Yoda, Tomoko
date: 2007-01-23
txt: ./txt/cord-288887-lshsgex3.txt
cache: ./cache/cord-288887-lshsgex3.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17245722/
doi: 10.1002/jmv.20802
summary:
  The loop-mediated isothermal amplification (LAMP) assay originally described [Notomi et al., 2000] is based on the principle of autocycling strand displacement DNA synthesis for the detection of a specific DNA sequence with specific characteristics: (1) all reactions can be conducted under isothermal conditions ranging from 60 to 658C; (2) the specificity of the reaction is extremely high because it uses six primers [Nagamine et al., 2002] recognizing eight distinct regions on the target nucleotides; and (3) a simple detection method such as visual judgment using Fluorescent Detection Reagent (Eiken Chemical Co., Ltd.) is possible. 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.

title:
  Evaluation and application of reverse transcription loop‐mediated isothermal amplification for detection of noroviruses

abstract:
  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, 88 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. 79:326–334, 2007. © 2007 Wiley‐Liss, Inc.


id: cord-295491-zlah6u5s
author: Günther, Sonja
date: 2018-03-11
txt: ./txt/cord-295491-zlah6u5s.txt
cache: ./cache/cord-295491-zlah6u5s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29540320/
doi: 10.1016/j.jviromet.2018.03.003
summary:
  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. 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. The FIP group (n = 34) 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 26/34 samples putative disease-causing mutations could be detected.

title:
  Detection of feline Coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification

abstract:
  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 71 cats, including 34 samples from cats with a definitive diagnosis of FIP, and 37 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 (61 °C–62 °C). Using the Isothermal Mastermix of OptiGene Ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. Using the PCRun™ Molecular Detection Mix of Biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. 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.


id: cord-296977-yzhsdz9c
author: Soares, R. R. G.
date: 2020-11-06
txt: ./txt/cord-296977-yzhsdz9c.txt
cache: ./cache/cord-296977-yzhsdz9c.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.11.04.20225888v1?rss=1
doi: 10.1101/2020.11.04.20225888
summary:
  ; https://doi.org/10.1101/2020.11.04.20225888 doi: medRxiv preprint imaging camera and SYBR Green I derived fluorescence transduction by naked eye or smartphone camera 27 ; (3) 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 30 ; (4) a hermetic container providing power-free chemical-based heating for LAMP amplification followed by detection using a smartphone flashlight and camera for fluorometric detection 32 ; (5) 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 32 ; (6) Centrifugal platform with automated bead-beating lysis followed by direct RT-LAMP by continuous measurement of fluorescence with UVC illumination and a standard camera 22 ; and (7) 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 24 .

title:
  Point-of-care detection of SARS-CoV-2 in nasopharyngeal swab samples using an integrated smartphone-based centrifugal microfluidic platform

abstract:
  With its origin estimated around December 2019 in Wuhan, China, the ongoing 2020 SARS-CoV-2 pandemic is a major global health challenge, resulting in more than 45 million infections and 1.2 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-19 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 1 hour from sample collection and a detection limit between 100 and 1000 RNA copies in 10 L reaction volume. Furthermore, direct detection of non-extracted SARS-CoV-2 RNA in nasopharyngeal swab samples from patients with Ct values below 26 (n=25 plus 6 PCR negative samples) was achieved with ~94% sensitivity and 100% 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-19 diagnostics closer to resource-limited settings.


id: cord-297974-sduz0j35
author: Bokelmann, L.
date: 2020-08-06
txt: ./txt/cord-297974-sduz0j35.txt
cache: ./cache/cord-297974-sduz0j35.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.08.04.20168617v1?rss=1
doi: 10.1101/2020.08.04.20168617
summary:
  Here we describe a method to detect SARS-CoV-2 RNA of a single infected individual within a bulk sample comprised of up to 26 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 1A) . To investigate whether it is possible to detect single infected individuals in pools of gargle lavage samples, we created eleven pools of 25 patient samples each, all of which had been tested negative in RT-qPCR assay and in the Cap-iLAMP assays for the Orf1a and the N gene ( Figure 2D ). All tested gargle lavages from single healthy individuals (n=6) and 11 pools of 25 healthy individuals (n=275) correctly tested negative for both the Orf1a gene and N gene in the Cap-iLAMP assay ( Figure 2C and E), indicating that false positive results which were sometimes observed when samples are added directly into the iLAMP reaction (Supp.

title:
  Rapid, reliable, and cheap point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP (Cap-iLAMP)

abstract:
  Efforts to contain the spread of SARS-CoV-2 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-2 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 25 uninfected samples, thus reducing the technical cost per test to ~1 Euro per individual.


id: cord-302663-gb2vgs97
title: Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP)
author: Mekata, Tohru
date: 2006-04-04
txt: ./txt/cord-302663-gb2vgs97.txt
cache: ./cache/cord-302663-gb2vgs97.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093406000656
doi: 10.1016/j.jviromet.2006.02.012
summary:
  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 development of a loop-mediated isothermal amplification (LAMP) assay for detection of white spot disease virus (WSDV) DNA was described by Kono et al. Ten-fold serial dilutions (10 −1 to 10 −8 diluted) of RNA extracted from YHV-infected shrimp was used as a template for RT-LAMP according to determined conditions. In order to determine the sensitivity of detection limit, RT-LAMP and nested RT-PCR were carried out using various concentrations (10 −1 to 10 −8 dilution) of RNA extracted from YHV-infected shrimp as template.

abstract:
  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 60 min at 65 °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.


id: cord-302829-1o1jo8uk
title: Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification
author: Chen, Hao-tai
date: 2008-03-19
txt: ./txt/cord-302829-1o1jo8uk.txt
cache: ./cache/cord-302829-1o1jo8uk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18355932/
doi: 10.1016/j.jviromet.2008.01.023
summary:
  A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). DNA was extracted from blood, lymph nodes, lung, liver, kidney, heart and spleen samples taken from PCV2-infected and healthy pigs, using a DNeasy Tissue Kit (Qiagen) according to the manufacturer''s instructions. In order to evaluate the optimal tissues for viral detection and to compare the sensitivity of PCV2 detection by LAMP and PCR, DNAs from tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from PCV2-infected pigs were extracted and subjected to LAMP and PCR. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction

abstract:
  A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 °C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 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.


id: cord-304343-m7tbdfri
author: Khandia, Rekha
date: 2019-07-03
txt: ./txt/cord-304343-m7tbdfri.txt
cache: ./cache/cord-304343-m7tbdfri.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/cells8070674
doi: 10.3390/cells8070674
summary:
  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 [194] . 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 [253] . (5) A novel anti-cancer molecule, HA15, which targets HSPA5/BIP was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. (5) A novel anti-cancer molecule, HA15, which targets HSPA5/BIP was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. In addition, the novel anti-cancer molecule HA15, which targets HSPA5/BIP, was shown to induce ER stress and increase the unfolded protein response, resulting in cancer cell death via autophagy and apoptosis [304] .

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

abstract:
  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.


id: cord-305399-98sqovwb
author: Li, Hao
date: 2019-04-22
txt: ./txt/cord-305399-98sqovwb.txt
cache: ./cache/cord-305399-98sqovwb.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2019.04.019
doi: 10.1016/j.jviromet.2019.04.019
summary:
  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 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. The final volume of 25 μl reaction mixtures for RT-LAMP was prepared, which contained 1 μl of Bst DNA polymerase (NEB, USA) (8000 U/ml), 2.5 μl of 10 × Isothermal Amplification Buffer, 5 μl of Betaine (5 M), 1 μl of MgSO 4 (100 mM), 5 μl of dNTP (2.5 mM), 2 μl of each inner primers FIP and BIP (10 μmol), 0.25 μl of each outer primers F3 and B3 (10 μmol), 0.25 μl of each loop primers LF and LB (10 μmol), 1.25 μl of AMV reverse transcriptase (TaKaRa, China) (40 U/μl), 2 μl of RNA template, and the sterile distilled water was set as a negative control template.

title:
  Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

abstract:
  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 NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 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.


id: cord-307068-360qs3ov
author: Hagiwara, Masanori
date: 2007-03-26
txt: ./txt/cord-307068-360qs3ov.txt
cache: ./cache/cord-307068-360qs3ov.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17385684/
doi: 10.1002/jmv.20858
summary:
  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. In order to evaluate the reliability of HPV type‐specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. 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. Type-specific real-time PCR was used to measure the quantity of the DNAs of HPV-6, -11, -16, and -18 in each sample. The sensitivity of HPV-6, -11, -16, and -18 type-specific LAMP determined by turbidity assay were 1,000 copies/tube (Fig. 3) . The sensitivity of amplification of LAMP for detection of viral DNA was nearly the same as that of real-time PCR.

title:
  Loop‐mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

abstract:
  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 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV‐6 DNA and HPV‐11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV‐16 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‐11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 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. 79:605–615, 2007. © 2007 Wiley‐Liss, Inc.


id: cord-310657-04pp0o74
author: Lu, Renfei
date: 2020-04-18
txt: ./txt/cord-310657-04pp0o74.txt
cache: ./cache/cord-310657-04pp0o74.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32325642/
doi: 10.3390/ijms21082826
summary:
  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-2 detection based on its N gene. 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-2 RNA, and evaluated the novel assays using clinical samples. The novel RT-LAMP assay has a high sensitivity, with a LOD of 118.6 copies per reaction, and shows no cross-reactivity with 17 common human respiratory viruses, including four other human coronaviruses (OC43, 229E, HKU-1 and NL63). In view of a mean viral load of 1.4 × 10 6 copies/mL in nasal swabs of COVID-19 patients, the novel assay is sufficiently sensitive for the detection of SARS-CoV-2 at an early stage of infection using nasal swab specimens. The RT-LAMP assay has a high sensitivity, with a LOD of 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction, and good specificity regarding common respiratory viruses.

title:
  A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

abstract:
  COVID-19 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-19 virus (SARS-CoV-2). 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-2 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 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.


id: cord-312222-aw5849rc
author: Österdahl, Marc F.
date: 2020-10-20
txt: ./txt/cord-312222-aw5849rc.txt
cache: ./cache/cord-312222-aw5849rc.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1186/s12879-020-05484-8
doi: 10.1186/s12879-020-05484-8
summary:
  METHODS: This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 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. Since then, a number [13] 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-2 (Severe acute respiratory syndrome coronavirus 2) within RNA extracted from nose -throat swabs and endotracheal secretions/bronchoalveolar lavage fluid [5, 14, 15] .

title:
  Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

abstract:
  BACKGROUND: A cost effective and efficient diagnostic tool for COVID-19 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 30 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 21 residents of a high dependency care home, with two index COVID-19 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 8/10 RT-PCR positive cases and identified a further 3 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 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. CONCLUSIONS: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 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.


id: cord-316816-yjbcvf3o
author: Cardoso, Tereza C.
date: 2010-08-21
txt: ./txt/cord-316816-yjbcvf3o.txt
cache: ./cache/cord-316816-yjbcvf3o.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0890850810000563
doi: 10.1016/j.mcp.2010.08.003
summary:
  title: Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. 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 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. Embryo tissues (ileum and ileumecaecal junction portions) were prepared by infecting 25-day-old embryonated turkey eggs with 0.3 ml 10 2.4 EID 50 TCoV via the amniotic sac, as described previously [9, 10] . Reverse transcription loop-mediated isothermal amplification for rapid detection of infectious bronchitis virus in infected chicken tissues

title:
  Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

abstract:
  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 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 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; 100% 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.


id: cord-318120-vfznyyz6
author: Dauner, Allison L.
date: 2015-05-15
txt: ./txt/cord-318120-vfznyyz6.txt
cache: ./cache/cord-318120-vfznyyz6.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0732889315001571
doi: 10.1016/j.diagmicrobio.2015.05.004
summary:
  title: Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loopmediated isothermal amplification assay Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus

title:
  Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus

abstract:
  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 4 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 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7–94.8%) and specificity of 93.0% (95% CI, 83.0–98.1%) 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.


id: cord-321886-0b3ocoh9
author: Zhang, Chao-fan
date: 2010-08-04
txt: ./txt/cord-321886-0b3ocoh9.txt
cache: ./cache/cord-321886-0b3ocoh9.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2010.07.034
doi: 10.1016/j.jviromet.2010.07.034
summary:
  title: Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). 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. Based on PRV-gG-LAMP, 26 of the 70 piglets were infected with wild-type PRV and may or may not have been vaccinated. Many different PCR assays can differentiate between the wild-type PRV and gene-deleted virus vaccines (Liu et al., 2007) , but LAMP is easier to perform and provides more rapid results. (2008) 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.

title:
  Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines

abstract:
  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 65 °C, a laddering of bright products was visible following electrophoresis on a 2% agarose gel. LAMP was 100–1000-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 1, porcine circovirus type 2, 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.


id: cord-322238-8iwljdoi
author: Chen, Qin
date: 2010-08-29
txt: ./txt/cord-322238-8iwljdoi.txt
cache: ./cache/cord-322238-8iwljdoi.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20799985/
doi: 10.1186/1743-422x-7-206
summary:
  Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR. 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 [5] . As a kind of nucleic acid amplification method, LAMP could not only qualitatively detect the TGEV, but also quantitatively analyze the virus. 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 10 pg RNA, which was 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.

title:
  Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification

abstract:
  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 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × 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 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.


id: cord-323845-s78t5qxj
author: Soliman, H.
date: 2006-05-31
txt: ./txt/cord-323845-s78t5qxj.txt
cache: ./cache/cord-323845-s78t5qxj.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0378113505004463
doi: 10.1016/j.vetmic.2005.11.063
summary:
  title: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS) A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS) 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 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. 5) . Detection of viral hemorrhagic septicaemia virus (VHS) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

title:
  Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS)

abstract:
  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, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63 °C for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 63 °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 10(6). 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.


id: cord-324094-23kzr8rq
title: Rapid and real-time detection technologies for emerging viruses of biomedical importance
author: Parida, M. M.
date: 2008-11-01
txt: ./txt/cord-324094-23kzr8rq.txt
cache: ./cache/cord-324094-23kzr8rq.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/19208986/
doi: 10.1007/s12038-008-0079-7
summary:
  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. 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. 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. 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

abstract:
  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.


id: cord-328042-e1is656g
title: SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP
author: Klein, Steffen
date: 2020-08-07
txt: ./txt/cord-328042-e1is656g.txt
cache: ./cache/cord-328042-e1is656g.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/v12080863
doi: 10.3390/v12080863
summary:
  The standard diagnostic pipeline for testing SARS-CoV-2 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. 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. 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) [16] .

abstract:
  Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 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 96-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.


id: cord-331641-u27ohm5p
author: Liu, Xiaonan
date: 2018-09-15
txt: ./txt/cord-331641-u27ohm5p.txt
cache: ./cache/cord-331641-u27ohm5p.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.bios.2018.05.021
doi: 10.1016/j.bios.2018.05.021
summary:
  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. 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 C677T and ALDH2 Glu504Lys, respectively, were used to determine the detection limit. 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. 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.

title:
  A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types

abstract:
  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 30 min from sample collection to provision of results. The accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) C677T and aldehyde dehydrogenase-2 (ALDH2) Glu504Lys, 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.


id: cord-332820-6qx6svs5
title: Standard operating procedures for SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay
author: Buck, M. D.
date: 2020-07-01
txt: ./txt/cord-332820-6qx6svs5.txt
cache: ./cache/cord-332820-6qx6svs5.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.06.29.20142430v1?rss=1
doi: 10.1101/2020.06.29.20142430
summary:
  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-2 by RT-qPCR. Herein, we describe the use of loop mediated isothermal amplification PCR coupled with reverse transcription (RT-LAMP) as a robust method for SARS-CoV-2 detection in clinical specimens 6 . Our results demonstrate that within the CCC pipeline, RT-LAMP can readily replace RT-qPCR as a means for detecting SARS-CoV-2 transcripts within RNA extracted from nosethroat swabs and endotracheal secretions/bronchoalveolar lavage fluid. The RT-LAMP assay reproducibility and precision were determined by extracting RNA 5 times from a confirmed COVID-19 positive patient sample through the CCC pipeline and assessing by N gene and 18S RT-LAMP in 5 independent experiments, performed by two different operators ( Figure 4D ).

abstract:
  The ongoing pandemic of SARS-CoV-2 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 40,000 RT-qPCR results since its commencement at the beginning of April 2020. 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-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.


id: cord-332961-ebr623rm
author: Zhang, Qingli
date: 2017-11-30
txt: ./txt/cord-332961-ebr623rm.txt
cache: ./cache/cord-332961-ebr623rm.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0022201115300112
doi: 10.1016/j.jip.2015.09.001
summary:
  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. 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. For CMNV quantitative detection of samples, a standard curve was generated for CMNV qRT-LAMP by plotting a graph between different concentrations of pMD19-T-CMNV plasmids, ranging from 10 8 to 10 0 copy numbers, to cycle threshold (Ct) values, obtained through real-time monitoring of the amplification. The lowest detection limit of the newly developed CMNV RT-LAMP method was 6.2 pg of total RNA when the reactions were tested using 1 lL of 10-fold serially diluted RNA from shrimp artificially infected with CMNV (Fig. 3A) . 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

title:
  Reverse transcription loop-mediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp

abstract:
  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 6mM MgCl2 and 1.6mM dNTPs, an incubation temperature of 65°C and a reaction time of 50min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3pg total RNA of CMNV-infected shrimp and 27 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 96.4% and 94.4%, 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 2 =0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5×102 to 6.7×106 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.


id: cord-333220-tcvs4beg
title: Compact optical diagnostic device for isothermal nucleic acids amplification
author: Lee, Szu-Yuan
date: 2008-08-12
txt: ./txt/cord-333220-tcvs4beg.txt
cache: ./cache/cord-333220-tcvs4beg.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0925400508001998
doi: 10.1016/j.snb.2008.03.008
summary:
  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. 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. 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. 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.

abstract:
  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(2) = 0.99) 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(2) = 0.96) 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 1 h.


id: cord-333331-ddcz7zck
title: Detection of hepatitis C virus by an improved loop-mediated isothermal amplification assay
author: Yang, Jin
date: 2011-05-12
txt: ./txt/cord-333331-ddcz7zck.txt
cache: ./cache/cord-333331-ddcz7zck.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1007/s00705-011-1001-4
doi: 10.1007/s00705-011-1001-4
summary:
  An improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting the 5′ untranslated region (UTR) was developed to detect hepatitis C virus (HCV) infection. A variety of molecular diagnostic assays, such as reverse transcriptase PCR [12] , nucleic-acid-sequence-based amplification [13] , transcription-mediated amplification [29] , branched-chain DNA assay [26] , and in-house realtime PCR [8] , have been developed for the detection of HCV RNA. 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. 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. Reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis E virus

abstract:
  An improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting the 5′ 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 84 IU/ml, and no cross-detection was observed. The assay was evaluated further with 126 clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of HCV RNA.


id: cord-333524-a6p6ma8r
author: Khan, Pavana
date: 2020-09-23
txt: ./txt/cord-333524-a6p6ma8r.txt
cache: ./cache/cord-333524-a6p6ma8r.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32966744/
doi: 10.1021/acssynbio.0c00359
summary:
  19 The current most common diagnostic method used to identify SARS-CoV-2 infection is a molecular technique for detecting viral RNA through nucleic acid amplification, RT-PCR. Nucleic acid amplification tests (NAATs) are the most common diagnostic tests used to detect pathogens, and many of the current SARS-CoV-2 detection techniques are primarily based on NAATs. 21 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. 34 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-2 samples. 55 The technique first uses RT-LAMP for reverse transcription and isothermal amplification of viral RNA, and then employs the Cas12a enzyme to identify sequences of SARS-CoV-2, allowing cleavage of a reporter molecule ( Figure 5 ).

title:
  Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings

abstract:
  [Image: see text] The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 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-2 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.


id: cord-334518-mjr6u7ak
author: Hu, X.
date: 2020-05-23
txt: ./txt/cord-334518-mjr6u7ak.txt
cache: ./cache/cord-334518-mjr6u7ak.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.05.20.20108530
doi: 10.1101/2020.05.20.20108530
summary:
  To accelerate clinical diagnostic testing for COVID-19, we conducted a prospective cohort study to develop and validate a novel RT-LAMP assay capable of detecting SARS-CoV-2 RNA for potential use in centralized facilities and point-of-care settings. Subsequently, we evaluated the RT-LAMP and standard RT-qPCR assays on 329 nasopharyngeal swabs from a cohort of 129 suspected COVID-19 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-2 confirmation. . https://doi.org/10.1101/2020.05.20.20108530 doi: medRxiv preprint As described in the Materials and Methods, we developed a rapid and simple RT-LAMP assay to detect SARS-CoV-2 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.

title:
  Development and clinical application of a rapid and sensitive loop-mediated isothermalamplification test for SARS-CoV-2 infection

abstract:
  Background The outbreak of SARS-CoV-2 urgently requires sensitive and convenient COVID-19 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-2 in both qualified laboratories and point-of-care settings. Methods Patients with suspected COVID-19 and close contacts between Jan 26 and April 8, 2020, 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-19 carrier and patients with other respiratory viral infections. Results We finally collected a cohort of 129 cases (329 nasopharyngeal swabs) and the independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). RT-LAMP was validated to be accurate (overall sensitivity and specificity: 88.89% and 99.00%; positive and negative predictive values: 94.74% and 97.78%) and diagnostically useful (positive and negative likelihood ratios: 88.89 and 0.11). RT-LAMP showed an increased sensitivity (88.89% vs 81.48%) and high consistency (kappa 0.92) compared with RT-qPCR for SARS-CoV-2 screening while requiring only constant temperature heating and visual inspection. The median time required for RT-LAMP was less than 1 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-2 infection, which could aid the expansion of COVID-19 testing in the public domain and hospitals.


id: cord-338899-qt17jhg0
title: Clinical Features and Molecular Diagnosis of Chikungunya Fever from South India
author: Lakshmi, Vemu
date: 2008-05-01
txt: ./txt/cord-338899-qt17jhg0.txt
cache: ./cache/cord-338899-qt17jhg0.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18419449/
doi: 10.1086/529444
summary:
  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. We report clinical observations and laboratory investigations involving virus isolation methods and molecular assays performed for 296 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. All 132 patients who had clinically suspected CHIK virus but whose RT-PCR and RT-LAMP results were negative presented 17 days after the onset of fever; this may be the reason for the negative test 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.

abstract:
  An epidemic of Chikungunya fever of unprecedented magnitude occurred in many parts of India in early 2006 after an interval of 33 years, and there has been a resurgence in some parts of South India since June 2007. 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 20 additional Chikungunya virus—positive cases, compared with reverse-transcriptase polymerase chain reaction. Chikungunya virus was isolated from 20 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.


id: cord-338942-q4neat3x
title: LAMP-on-a-chip: Revising microfluidic platforms for loop-mediated DNA amplification
author: Zhang, Haoqing
date: 2019-01-31
txt: ./txt/cord-338942-q4neat3x.txt
cache: ./cache/cord-338942-q4neat3x.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32287531/
doi: 10.1016/j.trac.2019.01.015
summary:
  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. 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. A microfluidic disk-based LAMP chip, integrating sample preparation and detection, was developed [44] (Fig. 2B ). Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens An integrated rotary microfluidic system with DNA extraction, loop-mediated isothermal amplification, and lateral flow strip based detection for point-ofcare pathogen diagnostics An integrated microfluidic loop-mediated-isothermal-amplification system for rapid sample pre-treatment and detection of viruses Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples

abstract:
  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.


id: cord-342145-cq6xe5r7
title: A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples
author: Dao Thi, Viet Loan
date: 2020-08-12
txt: ./txt/cord-342145-cq6xe5r7.txt
cache: ./cache/cord-342145-cq6xe5r7.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32719001/
doi: 10.1126/scitranslmed.abc7075
summary:
  The SARS-CoV-2 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. During the early phase of the COVID-19 pandemic (early March 2020) in Germany, we tested the sensitivity and specificity of a colorimetric RT-LAMP assay for detecting SARS-CoV-2 RNA in clinical RNA samples isolated from pharyngeal swab specimens collected from individuals being tested for COVID-19 (and provided by the Heidelberg University Hospital''s diagnostic laboratory after removal of an aliquot for SARS-CoV-2 RNA testing by RT-qPCR) (fig. For samples with a CT ≤ 30 as measured by RT-qPCR with E-Sarbeco primers, we found overall satisfactory sensitivity and specificity values for SARS-CoV-2 RNA detection by the RT-LAMP assay using RNA samples isolated from pharyngeal swab specimens ( Fig. 3 and Table 1 ).

abstract:
  The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 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-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab–to–RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) 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.


id: cord-343136-kftffes0
title: Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2
author: Mohon, Abu Naser
date: 2020-09-15
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url: https://www.ncbi.nlm.nih.gov/pubmed/32941977/
doi: 10.1016/j.jviromet.2020.113972
summary:
  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-2 has been developed. Limit of detection of the LAMP assay was evaluated by using a nasopharyngeal (NP) swab sample infected with SARS-CoV-2 for which the viral load was quantified using digital droplet PCR (see Supplementary Methods). Twenty four replicates from a serial dilution containing 25-50 copies of SARS-CoV-2 which equates to 1X LOD (patient sample NP swab in VTM viral load confirmed by digital droplet PCR) per reaction were tested using dual-target RT-LAMP (Table 3) . The dual-target RT-LAMP test for SARS-CoV-2 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.

abstract:
  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-2 has been developed. The LAMP assay achieves a comparable limit of detection (25 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 98.48% (95% CI 91.84% to 99.96%) and NPA 100.00% (95% CI 93.84% to 100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.


id: cord-343632-cv3qgno3
title: Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP
author: Zhang, Yinhua
date: 2020-02-29
txt: ./txt/cord-343632-cv3qgno3.txt
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url: https://doi.org/10.1101/2020.02.26.20028373
doi: 10.1101/2020.02.26.20028373
summary:
  Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. Here we describe a molecular diagnostic approach for SARS-CoV-2 RNA detection using loop-mediated isothermal amplification (LAMP) and simple visual detection of amplification for potential use in rapid, field applications. This study describes testing and validation of 5 sets LAMP primers targeting two fragments of the SARS-CoV-2 genome using short (~300bp) RNA fragments made with in vitro transcription and RNA samples from patients. https://doi.org/10.1101/2020.02.26.20028373 doi: medRxiv preprint With current diagnostic methods, e.g. RT-qPCR, purified RNA is used in the input. Although a small number of samples were tested here, the colorimetric LAMP assay enables reliable SARS-CoV-2 detection without sophisticated instrumentation, matching the RT-qPCR performance in field and point-of-care settings. /2020 In conclusion, colorimetric LAMP provides a simple, rapid method for SARS-CoV-2 RNA detection.

abstract:
  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-2 (COVID-19) 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-19 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.


id: cord-344636-go5cw92q
title: RT‐LAMP for rapid diagnosis of coronavirus SARS‐CoV‐2
author: Huang, Wei E.
date: 2020-04-25
txt: ./txt/cord-344636-go5cw92q.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/32333644/
doi: 10.1111/1751-7915.13586
summary:
  In this work, we developed a COVID-19 diagnosis kit for the rapid detection of SARS-CoV-2, using one-step reverse transcription and loop-mediated isothermal amplification (RT-LAMP). Positive amplification products were obtained even for 2 copies of the synthetic viral DNA fragment template in 30 min when using the S17 primers (lane 4 in Fig. 1D ), demonstrating that the LAMP reaction was rapid and sensitive. To assess the potential of RT-LAMP in detecting RNA virus of SARS-CoV-2, we then tested the performance of these primers with synthesized RNA fragments of the N gene, S gene and Orf1ab gene obtained from in vitro transcription (Appendix S1). 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-2 virus directly from a throat swab sample.

abstract:
  The pandemic coronavirus SARS‐CoV‐2 in the world has caused a large infected population suffering from COVID‐19. 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‐2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS‐CoV‐2 in the regions of orf1ab, 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 80 copies of viral RNA per ml in a sample. We validated the RT‐LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 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.


id: cord-346846-yle3z5z2
title: Evaluation of the Slit Lamp Shield to reduce droplet exposure
author: Murnain, Kaila L
date: 2020-05-25
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url: https://www.ncbi.nlm.nih.gov/pubmed/32452068/
doi: 10.1111/cxo.13096
abstract: nan
summary:
  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. The use of slitlamp barriers has become increasingly common during the current COVID-19 global pandemic. 2 We decided to evaluate the ability of the Slit Lamp Shield to reduce potential droplet exposure. In our simulation (Video S1), a clinician attired in personal protective equipment including surgical mask and face shield was positioned in the examination position. 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 1) . Importantly, there was no dye on the clinician''s face shield or mask.


id: cord-348243-e5tdb08v
title: Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay
author: Schermer, Bernhard
date: 2020-11-02
txt: ./txt/cord-348243-e5tdb08v.txt
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url: https://doi.org/10.1371/journal.pone.0238612
doi: 10.1371/journal.pone.0238612
summary:
  METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). To allow for the comparison of different nucleic acid detection methods for SARS-CoV-2 we collected redundant material from nasopharyngeal swabs obtained for qPCR testing in clinical routine due to suspected COVID-19. We first tested two recently described assays for SARS-CoV-2 detection on isolated RNA from patient samples. In summary, our multiplex RT-LAMP protocol is a simple and sensitive way to detect SARS-CoV-2 RNA from clinical samples. 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.

abstract:
  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 2 (SARS-CoV-2) 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-2 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.


