key: cord-1017653-o7bgf1ha authors: Luo, Zhen; Ye, Chunhong; Xiao, Heng; Yin, Jialing; Liang, Yicong; Ruan, Zhihui; Luo, Danju; Gao, Daolong; Tan, Qiuping; Li, Yongkui; Zhang, Qiwei; Liu, Weiyong; Wu, Jianguo title: Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis date: 2022-01-13 journal: Chem Eng Sci DOI: 10.1016/j.ces.2022.117430 sha: abd692832befc3a71b13934d5a9a4a03672f665d doc_id: 1017653 cord_uid: o7bgf1ha Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 μL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locating at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to a promising test in COVID-19 surveillance. The outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes a pandemic coronavirus disease 2019 (COVID-19) since late 2019. As of 19 December 2021, the number of COVID-19 patients has exceeded 273 million worldwide, and the cumulative number of deaths has been over 5.3 million (WHO, 2021) . SARS-CoV-2 is a beta coronavirus of group with a whole-genome resemblance of over 96%, including 5' untranslated region (UTR), S (Spike), E, M, N genes, replicase complex (orf1ab) 3'UTR, and other unspecified non-structural open reading frames (Zhou et al., 2020) . At present, the most important countermeasures for preventing human-to-human transmission of SARS-CoV-2 are early identification, proper isolation, and follow-up of patients (West et al., 2020) . To provide timely care and create a surveillance system to prevent the spread of COVID-19, the development of an on-site, rapid, and responsive diagnostic assay, especially in-vitro diagnosis (IVD), for SARS-CoV-2 is a top priority. Although the efforts on COVID-19 surveillance have been greatly made, emerging SARS-CoV-2 variants (eg. Alpha, Beta, and Delta variants), particularly the new variant Omicron, also known as B.1.1.529, have shown worrisome effects on this improvement (Karim and Karim, 2021; Yang et al., 2020) . Unfortunately, there are rarely current point-of-care (POC) testing modality allows specific identification on SARS-CoV-2 variants. SARS-CoV-2 infection is primarily diagnosed by reverse transcriptase quantitative realtime polymerase chain reaction (RT-qPCR) as the standard molecular diagnostic test in the clinical laboratory. To date, numerous kinds of methods by reverse transcription quantitative PCR (RT-qPCR) assays have been developed soon after the genome sequence of SARS-CoV-2 was published, and they are now the preferred assay for detecting viral RNA (Chu et al., 2020; Corman et al., 2020) . Undoubtedly, RT-qPCR assay in SARS-CoV-2 diagnosis with high sensitivity necessitates the extraction of viral RNA prior to the test, but the use of a precise thermal cycler instrument connected to a reliable source of electricity, and costs of expensive reagent along with extensive labor (Kudo et al., 2020) . Furthermore, due to overwhelmed healthcare systems and lack of rapid test of samples with a risk of viral RNA degradation during sample transport to the laboratory, there is an increase of false-negative rate in SARS-CoV-2 diagnosis (Yelin et al., 2020) . To control and track SARS-CoV-2 infections, it is urgent to establish a lean immediately applicable protocol that can be performed at the point-of-care, even in rural areas of developing countries where reliable energy, device, and technician may be inaccessible. Loop-mediated isothermal amplification (LAMP) is a fast and responsive DNA detection tool with fast procedures and simple equipment (Notomi et al., 2000) , which has received high concern to be widely used in the field of pathogenic microorganism such as Klebsiella pneumoniae (Nakano et al., 2015) , Staphylococcus aureus (Hanaki et al., 2011) , Ebola virus (Kurosaki et al., 2016a; Kurosaki et al., 2016b) , and Zika virus (Kurosaki et al., 2017) . The updating LAMP technology has been deployed for field surveillance in response to an early phase of in COVID-19 outbreaks, the detection of SARS-CoV-2 by RT-LAMP assays were initially explored (Baek et al., 2020; Yan et al., 2020) . However, as COVID-19 pandemic emphasizes rapidly adapted and deployed diagnostics in a variety of settings, the optimization of primers design and development of LAMP detection for SARS-CoV-2 and main variants require further investigation. In this study, we optimized and designed the primers for loop-mediated isothermal amplification (LAMP) based on SARS-CoV-2 N gene, aiming to develop a robust and visual assay in SARS-CoV-2 and emerging variants diagnosis. All 41 suspected COVID-19 patients were subjected to the tests including clinical examination, Computed Tomography (CT) according to the "pneumonia diagnosis protocol for novel coronavirus infection (trial version 5)" (the published guidelines released by the National Health Commission of the People's Republic of China), and admitted in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China from Jan 27 to 31, 2020. All nasopharyngeal swab samples were collected to RNA extraction and reverse-transcription to cDNA for further examination. The cDNA of clinical samples was stored at -80°C until use. The sequence of SARS-CoV-2 N gene is derived from NCBI GenBank database (28274 to 29533 nt in complete genome from Wuhan-Hu-1 strain, accession number: NC_045512.2.). The target gene was synthesized (GenScript Co., Ltd., Nanjing, China) and then ligated to pcDNA3.1-HA vector (Addgene Inc, Watertown, MA, USA) to generate pcDNA3.1-N-HA plasmid. Subsequently, the pcDNA3.1-N-HA plasmid was transformed into Escherichia coli DH5α strain, cultured and propagated in LB medium supplement with ampicillin to achieve amplification. The pcDNA3.1-N-HA plasmid DNA was extracted, and DNA concentration was determined. The plasmid DNA carrying SARS-CoV-2 N gene was stored at 4°C until use. The mass concentration and total base pair (bp) number of the plasmid pcDNA3.1-N-HA carrying the SARS-CoV-2 N gene and were determined. The plasmid DNA copy number was calculated according to the following formula: DNA copy number = DNA mass concentration/(Number of bp × Relative molecular mass of one bp) × 6.02×10 23 . The solution of the calculated DNA copy number was subjected to 10-fold serial dilutions in ddH 2 O to the concentrations from 1 × 10 to 1 × 10 8 copies μL -1 . Subsequently, the prepared standards were stored at 4°C until use. According to the principle of the LAMP, one primer set contains three pairs of primers that can recognize six independent regions of the target sequence. Namely, inner primers pair: forward inner primer (FIP) and backward inner primer (BIP); outer primers pair: F3 primer and B3 primer; loop primer pair: loop primer forward (LF) and loop primer backward (BF). Six sets of primers were designed for LAMP targeting SARS-CoV-2 N gene using PrimerExplorer V5 software (Fujitsu Limited, Tokyo, Japan). Primers designed for detection of five kinds of coronavirus (CoV) N genes were used in reverse-transcription (RT)-PCR. The information of the primers is listed in Table 1 . template, and rest of ddH 2 O to make up to 25 μL. The LAMP reaction was performed in DNA amplification instrument. The DNA products were amplified with 1 μL of DNA template at different concentrations ranged from 10 1 to 10 7 copies μL -1 at temperature gradients (55°C, 58°C, 60°C, 62°C) for 30 min or at time gradients (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 min) , and then separated by agarose gel electrophoresis. Relative quantity of each DNA product was analyzed by ImageJ software. For each reaction, at least three independent replicates were performed. In a colorimetric LAMP reaction system, additional 1 μL of 625 μM Calcein (Lot#G424BA0029, purchased from Sangon Biotech, Shanghai, China) and 1 μL of 7.5 mM MnCl 2 (Lot#MKCJ8390, purchased from Sigma-Aldrich, St. Louis, MO, USA) was supplied, and then incubated in an optimal condition at 58°C for 40 min in either a PCR device (Lot#8110953843) (Eastwin Scientific Equipments Inc; Suzhou, China) or a constant temperature water bath (DK-8D) (Everone Precision Instrument Co., Ltd; Shanghai, China). The color change from orange to green was observed in a positive reaction. A serial of 10-fold dilutions of DNA standards ranged from 1 × 10 to 1 × 10 7 copies μL -1 was prepared. The sensitivity of LAMP assay was accessed by added 1 μL of DNA template in the 25 μL of volume reaction at 58°C for 0, 5, 10, 15, 20, 25, 30, 35, 40 , 45 min, respectively. The positive reactions were observed by agarose gel electrophoresis or color change. The specificity of the LAMP assay was first examined by Xho I restriction endonuclease (Takara Biomedical Tech Co., Ltd., Beijing, China) at target DNA product. Then, several kinds of common pathogenic microorganism DNA were prepared for LAMP assay. The genome DNAs of ADV-3 from our previous study (Yan et al., 2021) , Streptococcus pneumonia and Staphylococcus aureus (obtained from CCTCC, Wuhan, China) was extracted, while genome RNA of H3N2 (HongKong/498/97 strain) from our previous study (Ge et al., 2017) The concordance rate between both assays was calculated by the formula: Concordance rate = Number of consistent results by both assays/Total number × 100%. In a comparison of two assays, the Chi-square (χ2) test was used for the difference between two groups of enumeration data by SPSS software version 25.0 (IBM Corp.; Armonk, NY, USA). A P value < 0.05 was defined as statistically significant. LAMP is one of most concerned point-of-care testing (POCT) assays in rapid SARS-CoV-2 diagnosis (Chaouch, 2021) . In the principle of the LAMP, one set of primers contains three pairs of primers, namely inner primers pair, outer primers pair and loop primer pair, which recognize six independent regions of the target sequence ( Figure 1A ). SARS-CoV-2 N gene is considered to be the main positive component in of SARS-CoV-2 nucleic acid detection . Six sets of primers were designed for LAMP targeting SARS-CoV-2 N gene using Primer Explorer V5 software in different regions. Two sets of primers (PS2 and 3; PS4 and 5) shared the same inner and outer primers but different loop primer, while PS1 and PS6 resided at 441-635 nt and 12-213 nt of the N gene, respectively (Table 1 and Figure 1B ). To investigate the sensitivity of LAMP assay with designed primers, a serial dilution of SARS-CoV-2 N gene DNA standards ranged from 10 0 to 10 8 copies μL -1 were used in 25 μL of volume reaction for 30 min at different temperatures. Except PS1, PS2 to PS5 primers were engaged to generate an obvious typical S-shaped DNA amplification curve at 55, 58, and 60 °C, whereas the detectable template concentrations were limited to 10 5 copies μL -1 (Figure 2A suggesting low sensitivity of LAMP reaction with PS2 to PS5. PS6 primers were engaged to an obvious S-shaped DNA amplification curve at 58°C with detectable template concentration reached to 10 1 copies μL -1 ( Figure 2F ), indicating the optimal primers in LAMP reaction. To further identify the sensitivity of LAMP assay with SP6 primers, the reaction was performed at indicated time at 58°C. The obvious S-shaped DNA amplification curves were observed with template concentrations from 10 7 to 10 1 copies μL -1 within 20 min, and entered a plateau phase within 30 to 45 min ( Figure 3A) , suggesting the assay is sensitive and rapid. Considering the specific sequence of amplificated DNA unit in LAMP, the DNA products with SP6 primers were digested by a distinct Xho I restriction endonuclease ( Figure 3B ). The amplified DNA by SP6 primers was significantly reduced by Xho I digestion ( Figure 3C ). Then, several kinds of commonly respiratory pathogenic microorganism DNA were prepared for LAMP assay. The LAMP reaction system displayed a strong positive detection on SARS-CoV-2 N gene as expected, but shown no cross action on neither ADV-3, Streptococcus pneumonia and Staphylococcus aureus genomic DNA, nor H3N2 cDNA ( Figure 3D ). Similarly, in the presence of various N genes of coronaviruses amplificated by specific PCR primers, an obvious positive LAMP assay was showed in the detection of cDNA of SARS-CoV-2, but not in common human coronavirus HCoV-OC43 and HCoV-NL63, or pangolin coronavirus GX_P2V, or swine acute diarrhea syndrome coronavirus SADS-CoV ( Figure 3E ), implying the specificity of DNA amplification of the LAMP assay. Emerging SARS-CoV-2 variants have shown worrisome effects on viral identification and require rapidly adapted and deployed diagnostics in a variety of settings (Kumar et al., 2021) . To develop a visual detection of SARS-CoV-2 LAMP for portable application, a metal ion indicator, Calcein, of which the color was modulated by manganese chloride (MnCl 2 ) from orange to green was introduced in the reaction system ( Figure 4A ). The reaction gave clear color change from orange to green for 40 min in the samples with template concentrations from 10 5 to 10 1 copies μL -1 ( Figure 4B ). In addition, the colorimetric LAMP assay was effectively operated in either PCR device ( Figure 4C and D) or water bath ( Figure 4E and F), indicating a convenient and sensitive assay regardless of isothermal heating instruments. To evaluate the clinical diagnostic effect of the LAMP assay, 41 clinical samples of nasopharyngeal swabs from suspected COVID-19 patients were tested in comparison to a commercial qPCR assay. Of these samples, 23 and 18 were detected as SARS-CoV-2 positive and negative by LAMP assay, respectively ( Figure 5A and Table 2 ). Additionally, the colorimetric LAMP displayed a specific positive reaction to DNA samples of SARS-CoV-2 N gene, but not to commonly respiratory pathogens ( Figure 5B ) or other kinds of coronaviruses ( Figure 5C ). In comparison, 21 and 20 were detected as SARS-CoV-2 positive and negative by qPCR assay, respectively ( Figure 5D and Table 2 ). The LAMP assay behaved a comparative positive rate in SARS-CoV-2 diagnosis to qPCR assay ( Figure 5E and Table 3 ). Notably, two samples were detected as positive by LAMP assay but negative by qPCR. The concordance rate between both assays was 95.1% (Table 3) , demonstrating LAMP assay present a reliable detection and matchable viability to qPCR for SARS-CoV-2 diagnosis. In recent years, the LAMP technology has been recognized by many aspects in infectious disease analysis and diagnosis, owing to no need of high temperature denaturation of the template and repeated heating and cooling steps. The LAMP operation process is simple, and the amplification is rapid and efficient, which is suitable for testing in the cases of imperfect experimental equipment, and wide use in the grassroots, laboratories with poor experimental conditions and on-site use (Wong et al., 2018) . Now, the accessible nature of the nucleic acid test by LAMP extends its potential to make huge impact in the fight against COVID-19. Compared to RT-qPCR, LAMP have been developed to detect SARS-CoV-2 RNA targeting nucleocapsid (N), open reading frame 1ab (ORF1ab), and spike (S) gene, demonstrating an excellent agreement with other SARS-CoV-2 nucleic acid amplification methods (Bulterys et al., 2020) . Nevertheless, emerging SARS-CoV-2 variants commonly cause genetic mutations, which comes to the potential impacts on performance genetic variants evading detection by specific viral diagnostic tests (Thompson et al., 2021) . Unfortunately, the lack of current pointof-care (POC) testing modality hardly allows specific identification on SARS-CoV-2 variants. In this study, the optimization of the primers and LAMP assay was developed for the detection of SARS-CoV-2 and emerging variants. The optimal conditions at isothermal 58℃ within 40 min of LAMP reaction system were established among six sets of primers targeting SARS-CoV-2 N gene. Although similar studies reveal a compatible conditions of LAMP assay for SARS-CoV-2 nucleic acid detection, the developed LAMP assay here reached a limit of detectable template of 10 to100 copies in each reaction within 40 min, suggesting a superior sensitivity and timesaving to previously reported 100 to 1000 copies per reaction within 40 to 60 min (Baek et al., 2020; Lu et al., 2020; Pang et al., 2020; Quino et al., 2021) . It could be explained that the optimal primers targeting at 12-213 nt of SARS-CoV-2 N gene contributed the improvement of the LAMP assay. More importantly, there specific sequences of primers were highly conservative according to the alignments of 12-213 nt region of N gene pf main emerging SARS-CoV-2 variants, behaving a perfect compatibility on specific identification on SARS-CoV-2 variants. As the COVID-19 epidemic, the great efforts have been made to adopt the methods of test for SARS-CoV-2 variants. FnCas9-based CRISPR and novel SHERLOCK diagnosis of SARS-CoV-2 variants arise rapid test in COVID-19 surveillance (de Puig et al., 2021; Kumar et al., 2021) . Although multiply tests to variants of concern (VOC), including Delta and Omicron, are now being developed and utilized such as next-generation sequencing (NGS), it is neither practical nor sustainable for in most POC tests. Thus, the rapid identification of viral nucleic acids using technologies leveraging PCR or isothermal amplification targeting beyond S gene is encouraged (Karim and Karim, 2021; Thomas et al., 2021) . However, as no specialists are needed to operate it and results are easy to interpret, LAMP assay is still primary fast test SARS-CoV-2 infection diagnosis. Next, we accessed the rapid and visual detection with the introduction of manganese chloride-calcein, a typically metal ion indicator in the LAMP reaction system (Tomita et al., 2008) . In this colorimetric LAMP reaction, the precisely visual color change from orange to green were observed for 40 min at limitation as many as 10 copies template. Of note, the visual LAMP assay could be well performed regardless of isothermal heating instruments, which is reliably carried out the on-site use with imperfect experimental equipment and poor experimental conditions. In a comparison to a commercial qPCR assay using 41 clinical samples, the LAMP assay was tested a comparative positive rate in SARS-CoV-2 diagnosis to qPCR assay with no crossreactivity with nucleic acid from other four human respiratory microorganisms, including ADV-3, Streptococcus pneumonia, Staphylococcus aureus, and H3N2, revealing that it possesses a high sensitivity and specificity in SARS-CoV-2 detection in nasopharyngeal swabs samples. Generally, the nasopharyngeal swabs samples were primary selection for the early diagnosis of COVID-19, especially for mild and asymptomatic patients, but its false negative rate was usually reported to be lower than that in other types of clinical specimens such as sputum . The mean viral load of in nasal swabs of COVID-19 patients is 1.4 × 10 6 copies/mL , our developed LAMP assay with a detectable viral cDNA template of 10000 copies (equal to 4 × 10 5 copies/mL) is competent for the early SARS-CoV-2 diagnosis combined with easy sampling of nasal swab specimens. However, there are still some procedures in this LAMP assay to be improved, eg. the nucleic acid extraction-free protocol before LAMP (Lalli et al., 2021) , one-step reverse transcription (RT) with LAMP Wong Tzeling et al., 2021) , or the LAMP-based quantitative detection of SARS-CoV-2 (Gonzalez-Gonzalez et al., 2021), which polishes the developed LAMP reaction system and facilitates the generalization of the simple, fast, and efficient in-vitro diagnosis. In sum, we optimized primers targeting SARS-CoV-2 N gene and established a robust and visual LAMP reaction system at isothermal 58℃ within 40 min. The superiority of the LAMP assay exerted excellent compatibility on specific detection on SARS-CoV-2 variants and a limit of detectable template of 10 copies regardless of isothermal heating instruments. We believe the developed LAMP assay present matchable effectiveness to qPCR for SARS-CoV-2 infection diagnosis, which is expected to be performed at the point-of-care (POC) in control of COVID-19 pandemic. We frankly thank patients, researchers, and clinical staff who provided significant contributions to this study. We also appreciate Dr Jincun Zhao of Guangzhou Medical University for kindly All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. All authors declare that there are no competing interests. Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the Ethics Committee and Institutional Review Board of the Tongji Hospital of Huazhong University of Science and Technology (file no. TJ-IRB20200201). Written informed consent was obtained from each enrolled patient. Primer Name Sequence (5' to 3') positive (in red) rates were displayed by LAMP and qPCR assays, respectively.  The colorimetric LAMP tool is rapid (40 min) and sensitive (10 copies)  The LAMP assay can be stably performed in portable heating devices  This LAMP assay acts matchable effectiveness to qPCR detection  This POC test is optimal in the diagnosis of main SARS-CoV-2 variants Dr Huahan Fan of Beijing University of Chemical Technology for providing GX_P2V and SDAS-CoV cDNA, and Dr Junhua Li of BGI-Shenzhen This work was supported by the National Natural Science Foundation of China Conceptualization, Data curation, Funding acquisition, Validation, Writingoriginal draft, Writing -review & editing Chunhong Ye: Data curation, Formal analysis Data curation, Formal analysis Jialing Yin: Formal analysis, Investigation, Methodology Formal analysis, Investigation, Methodology. Zhihui Ruan: Formal analysis, Investigation, Methodology Formal analysis, Methodology, Validation Daolong Gao: Investigation, Resources, Validation. Qiuping Tan: Investigation, Resources, Validation Formal analysis, Investigation, Resources World Health Organization (WHO), 2021. COVID-19 Weekly Epidemiological Update Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2 Comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of SARS-CoV-2 Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2 Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia -nCoV) by real-time RT-PCR Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants HERP Binds TBK1 To Activate Innate Immunity and Repress Virus Replication in Response to Endoplasmic Reticulum Stress Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number Loop-mediated isothermal amplification assays for identification of antiseptic-and methicillin-resistant Staphylococcus aureus Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic Detection of SARS-CoV-2 RNA by multiplex RT-qPCR FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV Deployment of a Reverse Transcription Loop-Mediated Isothermal Amplification Test for Ebola Virus Surveillance in Remote Areas in Guinea Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification A novel One-pot rapid diagnostic technology for COVID-19 A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2 Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop Loop-mediated isothermal amplification of DNA Using Loop-Mediated Amplification and CRISPR Technology Evaluation of reverse transcription-loop-mediated isothermal amplification for rapid detection of SARS-CoV-2 SARS-CoV-2 and Variant Diagnostic Testing Approaches in the United States Rapid Emergence and Epidemiologic Characteristics of the SARS-CoV-2 B.1.526 Variant Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Detection of SARS-CoV-2 in Different Types of Clinical Specimens Applying principles of behaviour change to reduce SARS-CoV-2 transmission One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Adenovirus Vaccine Within an Adenovirus Vector Analysis of genomic distributions of SARS-CoV-2 reveals a dominant strain type with strong allelic associations Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools The N gene of SARS-CoV-2 was the main positive component in repositive samples from a cohort of COVID-19 patients in Wuhan, China E) Each viral cDNA samples (10000 copies) were subjected to 25 μL of volume LAMP reaction and observed by agarose gel electrophoresis. Various coronavirus N genes were amplified by PCR with specific primers. M, DNA marker (F) The alignment of amplification of target region by SP6 primers of LAMP assay in SARS-CoV-2 N gene from various variants. The sequences of Alpha variant: B.1.1.7; Beta variant: B.1.351; Gamma variant: P1; Delta variant: B.1.617.2 were released from public NCBI database Conceptualization, Data curation, Funding acquisition, Validation, Writingoriginal draft, Writing -review & editing Chunhong Ye: Data curation, Formal analysis Data curation, Formal analysis Jialing Yin: Formal analysis, Investigation, Methodology Formal analysis, Investigation, Methodology. Zhihui Ruan: Formal analysis, Investigation, Methodology Formal analysis, Methodology, Validation Daolong Gao: Investigation, Resources, Validation. Qiuping Tan: Investigation, Resources, Validation Formal analysis, Investigation, Resources Qiwei Zhang: Investigation, Resources, Validation Conceptualization, Resources, Validation Conceptualization, Funding acquisition, Supervision, Writing -review & editing All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission All authors declare that there are no competing interests.