Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2
Identifieur interne : 001203 ( Pmc/Corpus ); précédent : 001202; suivant : 001204Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2
Auteurs : Gun-Soo Park ; Keunbon Ku ; Seung-Hwa Baek ; Seong-Jun Kim ; Seung Il Kim ; Bum-Tae Kim ; Jin-Soo MaengSource :
- The Journal of Molecular Diagnostics : JMD [ 1525-1578 ] ; 2020.
Abstract
Epidemics of coronavirus disease 2019 (COVID-19) now have >100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. Herein, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19, were developed and evaluated. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay so that the tests potentially performed in higher throughput.
Url:
DOI: 10.1016/j.jmoldx.2020.03.006
PubMed: 32276051
PubMed Central: 7144851
Links to Exploration step
PMC:7144851Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2</title><author><name sortKey="Park, Gun Soo" sort="Park, Gun Soo" uniqKey="Park G" first="Gun-Soo" last="Park">Gun-Soo Park</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Research Group of Food Processing, Korea Food Research Institute, Wanju-gun, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Ku, Keunbon" sort="Ku, Keunbon" uniqKey="Ku K" first="Keunbon" last="Ku">Keunbon Ku</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Baek, Seung Hwa" sort="Baek, Seung Hwa" uniqKey="Baek S" first="Seung-Hwa" last="Baek">Seung-Hwa Baek</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Seong Jun" sort="Kim, Seong Jun" uniqKey="Kim S" first="Seong-Jun" last="Kim">Seong-Jun Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Seung Il" sort="Kim, Seung Il" uniqKey="Kim S" first="Seung Il" last="Kim">Seung Il Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Bum Tae" sort="Kim, Bum Tae" uniqKey="Kim B" first="Bum-Tae" last="Kim">Bum-Tae Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Maeng, Jin Soo" sort="Maeng, Jin Soo" uniqKey="Maeng J" first="Jin-Soo" last="Maeng">Jin-Soo Maeng</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Research Group of Food Processing, Korea Food Research Institute, Wanju-gun, Republic of Korea</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32276051</idno><idno type="pmc">7144851</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144851</idno><idno type="RBID">PMC:7144851</idno><idno type="doi">10.1016/j.jmoldx.2020.03.006</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">001203</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001203</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2</title><author><name sortKey="Park, Gun Soo" sort="Park, Gun Soo" uniqKey="Park G" first="Gun-Soo" last="Park">Gun-Soo Park</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Research Group of Food Processing, Korea Food Research Institute, Wanju-gun, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Ku, Keunbon" sort="Ku, Keunbon" uniqKey="Ku K" first="Keunbon" last="Ku">Keunbon Ku</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Baek, Seung Hwa" sort="Baek, Seung Hwa" uniqKey="Baek S" first="Seung-Hwa" last="Baek">Seung-Hwa Baek</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Seong Jun" sort="Kim, Seong Jun" uniqKey="Kim S" first="Seong-Jun" last="Kim">Seong-Jun Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Seung Il" sort="Kim, Seung Il" uniqKey="Kim S" first="Seung Il" last="Kim">Seung Il Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Bum Tae" sort="Kim, Bum Tae" uniqKey="Kim B" first="Bum-Tae" last="Kim">Bum-Tae Kim</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Maeng, Jin Soo" sort="Maeng, Jin Soo" uniqKey="Maeng J" first="Jin-Soo" last="Maeng">Jin-Soo Maeng</name><affiliation><nlm:aff id="aff1">Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Research Group of Food Processing, Korea Food Research Institute, Wanju-gun, Republic of Korea</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Molecular Diagnostics : JMD</title><idno type="ISSN">1525-1578</idno><idno type="eISSN">1943-7811</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Epidemics of coronavirus disease 2019 (COVID-19) now have >100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. Herein, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19, were developed and evaluated. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay so that the tests potentially performed in higher throughput.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zhou, P" uniqKey="Zhou P">P. Zhou</name></author><author><name sortKey="Yang, X L" uniqKey="Yang X">X.L. Yang</name></author><author><name sortKey="Wang, X G" uniqKey="Wang X">X.G. Wang</name></author><author><name sortKey="Hu, B" uniqKey="Hu B">B. Hu</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L. Zhang</name></author><author><name sortKey="Zhang, W" uniqKey="Zhang W">W. Zhang</name></author><author><name sortKey="Si, H R" uniqKey="Si H">H.R. Si</name></author><author><name sortKey="Zhu, Y" uniqKey="Zhu Y">Y. Zhu</name></author><author><name sortKey="Li, B" uniqKey="Li B">B. Li</name></author><author><name sortKey="Huang, C L" uniqKey="Huang C">C.L. Huang</name></author><author><name sortKey="Chen, H D" uniqKey="Chen H">H.D. Chen</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J. Chen</name></author><author><name sortKey="Luo, Y" uniqKey="Luo Y">Y. Luo</name></author><author><name sortKey="Guo, H" uniqKey="Guo H">H. Guo</name></author><author><name sortKey="Jiang, R D" uniqKey="Jiang R">R.D. Jiang</name></author><author><name sortKey="Liu, M Q" uniqKey="Liu M">M.Q. Liu</name></author><author><name sortKey="Chen, Y" uniqKey="Chen Y">Y. Chen</name></author><author><name sortKey="Shen, X R" uniqKey="Shen X">X.R. Shen</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author><author><name sortKey="Zheng, X S" uniqKey="Zheng X">X.S. Zheng</name></author><author><name sortKey="Zhao, K" uniqKey="Zhao K">K. Zhao</name></author><author><name sortKey="Chen, Q J" uniqKey="Chen Q">Q.J. Chen</name></author><author><name sortKey="Deng, F" uniqKey="Deng F">F. Deng</name></author><author><name sortKey="Liu, L L" uniqKey="Liu L">L.L. Liu</name></author><author><name sortKey="Yan, B" uniqKey="Yan B">B. Yan</name></author><author><name sortKey="Zhan, F X" uniqKey="Zhan F">F.X. Zhan</name></author><author><name sortKey="Wang, Y Y" uniqKey="Wang Y">Y.Y. Wang</name></author><author><name sortKey="Xiao, G F" uniqKey="Xiao G">G.F. Xiao</name></author><author><name sortKey="Shi, Z L" uniqKey="Shi Z">Z.L. Shi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, F" uniqKey="Wu F">F. Wu</name></author><author><name sortKey="Zhao, S" uniqKey="Zhao S">S. Zhao</name></author><author><name sortKey="Yu, B" uniqKey="Yu B">B. Yu</name></author><author><name sortKey="Chen, Y M" uniqKey="Chen Y">Y.M. Chen</name></author><author><name sortKey="Wang, W" uniqKey="Wang W">W. Wang</name></author><author><name sortKey="Song, Z G" uniqKey="Song Z">Z.G. Song</name></author><author><name sortKey="Hu, Y" uniqKey="Hu Y">Y. Hu</name></author><author><name sortKey="Tao, Z W" uniqKey="Tao Z">Z.W. Tao</name></author><author><name sortKey="Tian, J H" uniqKey="Tian J">J.H. Tian</name></author><author><name sortKey="Pei, Y Y" uniqKey="Pei Y">Y.Y. Pei</name></author><author><name sortKey="Yuan, M L" uniqKey="Yuan M">M.L. Yuan</name></author><author><name sortKey="Zhang, Y L" uniqKey="Zhang Y">Y.L. Zhang</name></author><author><name sortKey="Dai, F H" uniqKey="Dai F">F.H. Dai</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Wang, Q M" uniqKey="Wang Q">Q.M. Wang</name></author><author><name sortKey="Zheng, J J" uniqKey="Zheng J">J.J. Zheng</name></author><author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name></author><author><name sortKey="Holmes, E C" uniqKey="Holmes E">E.C. Holmes</name></author><author><name sortKey="Zhang, Y Z" uniqKey="Zhang Y">Y.Z. Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, N" uniqKey="Zhu N">N. Zhu</name></author><author><name sortKey="Zhang, D" uniqKey="Zhang D">D. Zhang</name></author><author><name sortKey="Wang, W" uniqKey="Wang W">W. Wang</name></author><author><name sortKey="Li, X" uniqKey="Li X">X. Li</name></author><author><name sortKey="Yang, B" uniqKey="Yang B">B. Yang</name></author><author><name sortKey="Song, J" uniqKey="Song J">J. Song</name></author><author><name sortKey="Zhao, X" uniqKey="Zhao X">X. Zhao</name></author><author><name sortKey="Huang, B" uniqKey="Huang B">B. Huang</name></author><author><name sortKey="Shi, W" uniqKey="Shi W">W. Shi</name></author><author><name sortKey="Lu, R" uniqKey="Lu R">R. Lu</name></author><author><name sortKey="Niu, P" uniqKey="Niu P">P. Niu</name></author><author><name sortKey="Zhan, F" uniqKey="Zhan F">F. Zhan</name></author><author><name sortKey="Ma, X" uniqKey="Ma X">X. Ma</name></author><author><name sortKey="Wang, D" uniqKey="Wang D">D. Wang</name></author><author><name sortKey="Xu, W" uniqKey="Xu W">W. Xu</name></author><author><name sortKey="Wu, G" uniqKey="Wu G">G. Wu</name></author><author><name sortKey="Gao, G F" uniqKey="Gao G">G.F. Gao</name></author><author><name sortKey="Tan, W" uniqKey="Tan W">W. Tan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huang, C" uniqKey="Huang C">C. Huang</name></author><author><name sortKey="Wang, Y" uniqKey="Wang Y">Y. Wang</name></author><author><name sortKey="Li, X" uniqKey="Li X">X. Li</name></author><author><name sortKey="Ren, L" uniqKey="Ren L">L. Ren</name></author><author><name sortKey="Zhao, J" uniqKey="Zhao J">J. Zhao</name></author><author><name sortKey="Hu, Y" uniqKey="Hu Y">Y. Hu</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L. Zhang</name></author><author><name sortKey="Fan, G" uniqKey="Fan G">G. Fan</name></author><author><name sortKey="Xu, J" uniqKey="Xu J">J. Xu</name></author><author><name sortKey="Gu, X" uniqKey="Gu X">X. Gu</name></author><author><name sortKey="Cheng, Z" uniqKey="Cheng Z">Z. Cheng</name></author><author><name sortKey="Yu, T" uniqKey="Yu T">T. Yu</name></author><author><name sortKey="Xia, J" uniqKey="Xia J">J. Xia</name></author><author><name sortKey="Wei, Y" uniqKey="Wei Y">Y. Wei</name></author><author><name sortKey="Wu, W" uniqKey="Wu W">W. Wu</name></author><author><name sortKey="Xie, X" uniqKey="Xie X">X. Xie</name></author><author><name sortKey="Yin, W" uniqKey="Yin W">W. Yin</name></author><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name></author><author><name sortKey="Liu, M" uniqKey="Liu M">M. Liu</name></author><author><name sortKey="Xiao, Y" uniqKey="Xiao Y">Y. Xiao</name></author><author><name sortKey="Gao, H" uniqKey="Gao H">H. Gao</name></author><author><name sortKey="Guo, L" uniqKey="Guo L">L. Guo</name></author><author><name sortKey="Xie, J" uniqKey="Xie J">J. Xie</name></author><author><name sortKey="Wang, G" uniqKey="Wang G">G. Wang</name></author><author><name sortKey="Jiang, R" uniqKey="Jiang R">R. Jiang</name></author><author><name sortKey="Gao, Z" uniqKey="Gao Z">Z. Gao</name></author><author><name sortKey="Jin, Q" uniqKey="Jin Q">Q. Jin</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J. Wang</name></author><author><name sortKey="Cao, B" uniqKey="Cao B">B. Cao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, V M" uniqKey="Corman V">V.M. Corman</name></author><author><name sortKey="Landt, O" uniqKey="Landt O">O. Landt</name></author><author><name sortKey="Kaiser, M" uniqKey="Kaiser M">M. Kaiser</name></author><author><name sortKey="Molenkamp, R" uniqKey="Molenkamp R">R. Molenkamp</name></author><author><name sortKey="Meijer, A" uniqKey="Meijer A">A. Meijer</name></author><author><name sortKey="Chu, D K" uniqKey="Chu D">D.K. Chu</name></author><author><name sortKey="Bleicker, T" uniqKey="Bleicker T">T. Bleicker</name></author><author><name sortKey="Brunink, S" uniqKey="Brunink S">S. Brunink</name></author><author><name sortKey="Schneider, J" uniqKey="Schneider J">J. Schneider</name></author><author><name sortKey="Schmidt, M L" uniqKey="Schmidt M">M.L. Schmidt</name></author><author><name sortKey="Mulders, D G" uniqKey="Mulders D">D.G. Mulders</name></author><author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B.L. Haagmans</name></author><author><name sortKey="Van Der Veer, B" uniqKey="Van Der Veer B">B. van der Veer</name></author><author><name sortKey="Van Den Brink, S" uniqKey="Van Den Brink S">S. van den Brink</name></author><author><name sortKey="Wijsman, L" uniqKey="Wijsman L">L. Wijsman</name></author><author><name sortKey="Goderski, G" uniqKey="Goderski G">G. Goderski</name></author><author><name sortKey="Romette, J L" uniqKey="Romette J">J.L. Romette</name></author><author><name sortKey="Ellis, J" uniqKey="Ellis J">J. Ellis</name></author><author><name sortKey="Zambon, M" uniqKey="Zambon M">M. Zambon</name></author><author><name sortKey="Peiris, M" uniqKey="Peiris M">M. Peiris</name></author><author><name sortKey="Goossens, H" uniqKey="Goossens H">H. Goossens</name></author><author><name sortKey="Reusken, C" uniqKey="Reusken C">C. Reusken</name></author><author><name sortKey="Koopmans, M P" uniqKey="Koopmans M">M.P. Koopmans</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chu, D K W" uniqKey="Chu D">D.K.W. Chu</name></author><author><name sortKey="Pan, Y" uniqKey="Pan Y">Y. Pan</name></author><author><name sortKey="Cheng, S M S" uniqKey="Cheng S">S.M.S. Cheng</name></author><author><name sortKey="Hui, K P Y" uniqKey="Hui K">K.P.Y. Hui</name></author><author><name sortKey="Krishnan, P" uniqKey="Krishnan P">P. Krishnan</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Ng, D Y M" uniqKey="Ng D">D.Y.M. Ng</name></author><author><name sortKey="Wan, C K C" uniqKey="Wan C">C.K.C. Wan</name></author><author><name sortKey="Yang, P" uniqKey="Yang P">P. Yang</name></author><author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name></author><author><name sortKey="Peiris, M" uniqKey="Peiris M">M. Peiris</name></author><author><name sortKey="Poon, L L M" uniqKey="Poon L">L.L.M. Poon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nayak, S" uniqKey="Nayak S">S. Nayak</name></author><author><name sortKey="Blumenfeld, N R" uniqKey="Blumenfeld N">N.R. Blumenfeld</name></author><author><name sortKey="Laksanasopin, T" uniqKey="Laksanasopin T">T. Laksanasopin</name></author><author><name sortKey="Sia, S K" uniqKey="Sia S">S.K. Sia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Niemz, A" uniqKey="Niemz A">A. Niemz</name></author><author><name sortKey="Ferguson, T M" uniqKey="Ferguson T">T.M. Ferguson</name></author><author><name sortKey="Boyle, D S" uniqKey="Boyle D">D.S. Boyle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Notomi, T" uniqKey="Notomi T">T. Notomi</name></author><author><name sortKey="Okayama, H" uniqKey="Okayama H">H. Okayama</name></author><author><name sortKey="Masubuchi, H" uniqKey="Masubuchi H">H. Masubuchi</name></author><author><name sortKey="Yonekawa, T" uniqKey="Yonekawa T">T. Yonekawa</name></author><author><name sortKey="Watanabe, K" uniqKey="Watanabe K">K. Watanabe</name></author><author><name sortKey="Amino, N" uniqKey="Amino N">N. Amino</name></author><author><name sortKey="Hase, T" uniqKey="Hase T">T. Hase</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shirato, K" uniqKey="Shirato K">K. Shirato</name></author><author><name sortKey="Yano, T" uniqKey="Yano T">T. Yano</name></author><author><name sortKey="Senba, S" uniqKey="Senba S">S. Senba</name></author><author><name sortKey="Akachi, S" uniqKey="Akachi S">S. Akachi</name></author><author><name sortKey="Kobayashi, T" uniqKey="Kobayashi T">T. Kobayashi</name></author><author><name sortKey="Nishinaka, T" uniqKey="Nishinaka T">T. Nishinaka</name></author><author><name sortKey="Notomi, T" uniqKey="Notomi T">T. Notomi</name></author><author><name sortKey="Matsuyama, S" uniqKey="Matsuyama S">S. Matsuyama</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Oscorbin, I P" uniqKey="Oscorbin I">I.P. Oscorbin</name></author><author><name sortKey="Belousova, E A" uniqKey="Belousova E">E.A. Belousova</name></author><author><name sortKey="Zakabunin, A I" uniqKey="Zakabunin A">A.I. Zakabunin</name></author><author><name sortKey="Boyarskikh, U A" uniqKey="Boyarskikh U">U.A. Boyarskikh</name></author><author><name sortKey="Filipenko, M L" uniqKey="Filipenko M">M.L. Filipenko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goto, M" uniqKey="Goto M">M. Goto</name></author><author><name sortKey="Honda, E" uniqKey="Honda E">E. Honda</name></author><author><name sortKey="Ogura, A" uniqKey="Ogura A">A. Ogura</name></author><author><name sortKey="Nomoto, A" uniqKey="Nomoto A">A. Nomoto</name></author><author><name sortKey="Hanaki, K" uniqKey="Hanaki K">K. Hanaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Miyamoto, S" uniqKey="Miyamoto S">S. Miyamoto</name></author><author><name sortKey="Sano, S" uniqKey="Sano S">S. Sano</name></author><author><name sortKey="Takahashi, K" uniqKey="Takahashi K">K. Takahashi</name></author><author><name sortKey="Jikihara, T" uniqKey="Jikihara T">T. Jikihara</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tanner, N A" uniqKey="Tanner N">N.A. Tanner</name></author><author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y. Zhang</name></author><author><name sortKey="Evans, T C" uniqKey="Evans T">T.C. Evans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nie, K" uniqKey="Nie K">K. Nie</name></author><author><name sortKey="Qi, S X" uniqKey="Qi S">S.X. Qi</name></author><author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y. Zhang</name></author><author><name sortKey="Luo, L" uniqKey="Luo L">L. Luo</name></author><author><name sortKey="Xie, Y" uniqKey="Xie Y">Y. Xie</name></author><author><name sortKey="Yang, M J" uniqKey="Yang M">M.J. Yang</name></author><author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y. Zhang</name></author><author><name sortKey="Li, J" uniqKey="Li J">J. Li</name></author><author><name sortKey="Shen, H" uniqKey="Shen H">H. Shen</name></author><author><name sortKey="Li, Q" uniqKey="Li Q">Q. Li</name></author><author><name sortKey="Ma, X J" uniqKey="Ma X">X.J. Ma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nyan, D C" uniqKey="Nyan D">D.C. Nyan</name></author><author><name sortKey="Ulitzky, L E" uniqKey="Ulitzky L">L.E. Ulitzky</name></author><author><name sortKey="Cehan, N" uniqKey="Cehan N">N. Cehan</name></author><author><name sortKey="Williamson, P" uniqKey="Williamson P">P. Williamson</name></author><author><name sortKey="Winkelman, V" uniqKey="Winkelman V">V. Winkelman</name></author><author><name sortKey="Rios, M" uniqKey="Rios M">M. Rios</name></author><author><name sortKey="Taylor, D R" uniqKey="Taylor D">D.R. Taylor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, D" uniqKey="Lee D">D. Lee</name></author><author><name sortKey="Shin, Y" uniqKey="Shin Y">Y. Shin</name></author><author><name sortKey="Chung, S" uniqKey="Chung S">S. Chung</name></author><author><name sortKey="Hwang, K S" uniqKey="Hwang K">K.S. Hwang</name></author><author><name sortKey="Yoon, D S" uniqKey="Yoon D">D.S. Yoon</name></author><author><name sortKey="Lee, J H" uniqKey="Lee J">J.H. Lee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jung, Y J" uniqKey="Jung Y">Y.J. Jung</name></author><author><name sortKey="Park, G S" uniqKey="Park G">G.-S. Park</name></author><author><name sortKey="Moon, J H" uniqKey="Moon J">J.H. Moon</name></author><author><name sortKey="Ku, K" uniqKey="Ku K">K. Ku</name></author><author><name sortKey="Beak, S H" uniqKey="Beak S">S.-H. Beak</name></author><author><name sortKey="Kim, S" uniqKey="Kim S">S. Kim</name></author><author><name sortKey="Park, E C" uniqKey="Park E">E.C. Park</name></author><author><name sortKey="Park, D" uniqKey="Park D">D. Park</name></author><author><name sortKey="Lee, J H" uniqKey="Lee J">J.-H. Lee</name></author><author><name sortKey="Byeon, C W" uniqKey="Byeon C">C.W. Byeon</name></author><author><name sortKey="Lee, J J" uniqKey="Lee J">J.J. Lee</name></author><author><name sortKey="Maeng, J S" uniqKey="Maeng J">J.-S. Maeng</name></author><author><name sortKey="Kim, S J" uniqKey="Kim S">S.J. Kim</name></author><author><name sortKey="Kim, S I" uniqKey="Kim S">S.I. Kim</name></author><author><name sortKey="Kim, B T" uniqKey="Kim B">B.-T. Kim</name></author><author><name sortKey="Lee, M J" uniqKey="Lee M">M.J. Lee</name></author><author><name sortKey="Kim, H G" uniqKey="Kim H">H.G. Kim</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, V M" uniqKey="Corman V">V.M. Corman</name></author><author><name sortKey="Eckerle, I" uniqKey="Eckerle I">I. Eckerle</name></author><author><name sortKey="Bleicker, T" uniqKey="Bleicker T">T. Bleicker</name></author><author><name sortKey="Zaki, A" uniqKey="Zaki A">A. Zaki</name></author><author><name sortKey="Landt, O" uniqKey="Landt O">O. Landt</name></author><author><name sortKey="Eschbach Bludau, M" uniqKey="Eschbach Bludau M">M. Eschbach-Bludau</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S. van Boheemen</name></author><author><name sortKey="Gopal, R" uniqKey="Gopal R">R. Gopal</name></author><author><name sortKey="Ballhause, M" uniqKey="Ballhause M">M. Ballhause</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T.M. Bestebroer</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Muller, M A" uniqKey="Muller M">M.A. Muller</name></author><author><name sortKey="Drexler, J F" uniqKey="Drexler J">J.F. Drexler</name></author><author><name sortKey="Zambon, M" uniqKey="Zambon M">M. Zambon</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A.D. Osterhaus</name></author><author><name sortKey="Fouchier, R M" uniqKey="Fouchier R">R.M. Fouchier</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kumar, S" uniqKey="Kumar S">S. Kumar</name></author><author><name sortKey="Stecher, G" uniqKey="Stecher G">G. Stecher</name></author><author><name sortKey="Tamura, K" uniqKey="Tamura K">K. Tamura</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hiscox, J A" uniqKey="Hiscox J">J.A. Hiscox</name></author><author><name sortKey="Cavanagh, D" uniqKey="Cavanagh D">D. Cavanagh</name></author><author><name sortKey="Britton, P" uniqKey="Britton P">P. Britton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Khorosheva, E M" uniqKey="Khorosheva E">E.M. Khorosheva</name></author><author><name sortKey="Karymov, M A" uniqKey="Karymov M">M.A. Karymov</name></author><author><name sortKey="Selck, D A" uniqKey="Selck D">D.A. Selck</name></author><author><name sortKey="Ismagilov, R F" uniqKey="Ismagilov R">R.F. Ismagilov</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zou, L" uniqKey="Zou L">L. Zou</name></author><author><name sortKey="Ruan, F" uniqKey="Ruan F">F. Ruan</name></author><author><name sortKey="Huang, M" uniqKey="Huang M">M. Huang</name></author><author><name sortKey="Liang, L" uniqKey="Liang L">L. Liang</name></author><author><name sortKey="Huang, H" uniqKey="Huang H">H. Huang</name></author><author><name sortKey="Hong, Z" uniqKey="Hong Z">Z. Hong</name></author><author><name sortKey="Yu, J" uniqKey="Yu J">J. Yu</name></author><author><name sortKey="Kang, M" uniqKey="Kang M">M. Kang</name></author><author><name sortKey="Song, Y" uniqKey="Song Y">Y. Song</name></author><author><name sortKey="Xia, J" uniqKey="Xia J">J. Xia</name></author><author><name sortKey="Guo, Q" uniqKey="Guo Q">Q. Guo</name></author><author><name sortKey="Song, T" uniqKey="Song T">T. Song</name></author><author><name sortKey="He, J" uniqKey="He J">J. He</name></author><author><name sortKey="Yen, H L" uniqKey="Yen H">H.L. Yen</name></author><author><name sortKey="Peiris, M" uniqKey="Peiris M">M. Peiris</name></author><author><name sortKey="Wu, J" uniqKey="Wu J">J. Wu</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Mol Diagn</journal-id><journal-id journal-id-type="iso-abbrev">J Mol Diagn</journal-id><journal-title-group><journal-title>The Journal of Molecular Diagnostics : JMD</journal-title></journal-title-group><issn pub-type="ppub">1525-1578</issn><issn pub-type="epub">1943-7811</issn><publisher><publisher-name>Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32276051</article-id><article-id pub-id-type="pmc">7144851</article-id><article-id pub-id-type="publisher-id">S1525-1578(20)30090-8</article-id><article-id pub-id-type="doi">10.1016/j.jmoldx.2020.03.006</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Park</surname><given-names>Gun-Soo</given-names></name><email>pcdhmk@krict.re.kr</email><email>maengjs@krict.re.kr</email><xref rid="aff1" ref-type="aff">∗</xref><xref rid="aff2" ref-type="aff">†</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Ku</surname><given-names>Keunbon</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Baek</surname><given-names>Seung-Hwa</given-names></name><xref rid="aff1" ref-type="aff">∗</xref><xref rid="aff3" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Kim</surname><given-names>Seong-Jun</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Kim</surname><given-names>Seung Il</given-names></name><xref rid="aff1" ref-type="aff">∗</xref><xref rid="aff4" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Kim</surname><given-names>Bum-Tae</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Maeng</surname><given-names>Jin-Soo</given-names></name><xref rid="aff1" ref-type="aff">∗</xref><xref rid="aff2" ref-type="aff">†</xref></contrib></contrib-group><aff id="aff1"><label>∗</label>Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</aff><aff id="aff2"><label>†</label>Research Group of Food Processing, Korea Food Research Institute, Wanju-gun, Republic of Korea</aff><aff id="aff3"><label>‡</label>Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, Republic of Korea</aff><aff id="aff4"><label>§</label>Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Republic of Korea</aff><author-notes><corresp id="cor1"><label>∗</label>Address correspondence to Gun-Soo Park or Jin-Soo Maeng, Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, 141, Gajeong-ro, Yuseong, Daejeon 34114, Republic of Korea. <email>pcdhmk@krict.re.kr</email><email>maengjs@krict.re.kr</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>7</day><month>4</month><year>2020</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="epub"><day>7</day><month>4</month><year>2020</year></pub-date><history><date date-type="accepted"><day>27</day><month>3</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Association for Molecular Pathology and American Society for Investigative Pathology</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract id="abs0010"><p>Epidemics of coronavirus disease 2019 (COVID-19) now have >100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. Herein, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19, were developed and evaluated. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay so that the tests potentially performed in higher throughput.</p></abstract></article-meta></front><body><p id="p0010">SARS-CoV-2 is the causative viral pathogen of coronavirus disease 2019, of which the outbreaks resulted in 857,641 confirmed cases involving 42,006 deaths over 206 countries, areas, or territories as of April 2, 2020 (<ext-link ext-link-type="uri" xlink:href="https://www.who.int/emergencies/diseases/novel-coronavirus-2019" id="intref0010"><italic>https://www.who.int/emergencies/diseases/novel-coronavirus-2019</italic></ext-link>). As the name suggests, SARS-CoV-2 is closely related to a group of severe acute respiratory syndrome–related coronaviruses, namely subgenus <italic>Sarbecovirus</italic>, showing 96% identity to a bat coronavirus.<xref rid="bib1" ref-type="bibr"><sup>1</sup></xref><sup>,</sup><xref rid="bib2" ref-type="bibr"><sup>2</sup></xref></p><p id="p0015">Diagnosis of coronavirus disease 2019 can be done through computed tomographic scan of suspicious patients, and a confirmatory laboratory test is performed using published quantitative RT-PCR (RT-qPCR) methods<xref rid="bib3" ref-type="bibr">3</xref>, <xref rid="bib4" ref-type="bibr">4</xref>, <xref rid="bib5" ref-type="bibr">5</xref>, <xref rid="bib6" ref-type="bibr">6</xref> and recommendations from The World Health Organization (<ext-link ext-link-type="uri" xlink:href="https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance" id="intref0015"><italic>https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance</italic></ext-link>, last accessed April 2, 2020). Although RT-qPCR methods are used as the gold standard for detection of pathogens because of their high sensitivity and specificity, there are still some caveats. Briefly, laboratory-level facilities, with reliable supply of electricity, expensive instruments, and trained personnel, are required to properly perform RT-qPCR tests. These restrictions hinder use of RT-qPCR methods for various point-of-care situations, where pathogen detection might be required.<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref></p><p id="p0020">To overcome such cost restriction of RT-qPCR and still detect pathogens' nucleic acids, isothermal amplification methods have been developed.<xref rid="bib8" ref-type="bibr"><sup>8</sup></xref> Among such methods, the loop-mediated isothermal amplification (LAMP) method has some advantages to be applied for point-of-care testing.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> Well-optimized LAMP assay shows sensitivity comparable to that of PCR, <10 copies per reaction.<xref rid="bib10" ref-type="bibr"><sup>10</sup></xref> Intercalating fluorescent dyes are compatible with LAMP reaction so that amplification can be observed in real-time.<xref rid="bib11" ref-type="bibr"><sup>11</sup></xref> Because amplification efficiency of LAMP reaction is high, changes in reaction mixture components made it possible to detect the result with colorimetric detection methods.<xref rid="bib12" ref-type="bibr">12</xref>, <xref rid="bib13" ref-type="bibr">13</xref>, <xref rid="bib14" ref-type="bibr">14</xref> Moreover, unpurified sample can be directly used for LAMP.<xref rid="bib15" ref-type="bibr">15</xref>, <xref rid="bib16" ref-type="bibr">16</xref>, <xref rid="bib17" ref-type="bibr">17</xref> This indicates that high-throughput test is possible when use of unpurified specimen is combined with noninstrumental (eg, colorimetric) detection.</p><p id="p0025">In this study, one-step reverse transcription LAMP (RT-LAMP) methods to detect SARS-CoV-2 were designed and evaluated. A pair of LAMP primer sets specific to SARS-CoV-2 and accompanying optimized reaction conditions was provided. The leuco crystal violet method was applied to achieve colorimetric detection of LAMP reaction.<xref rid="bib13" ref-type="bibr"><sup>13</sup></xref></p><sec id="sec1"><title>Materials and Methods</title><sec id="sec1.1"><title>Viral RNA Preparation</title><p id="p0030">SARS-CoV-2 viral RNA was prepared, as previously described.<xref rid="bib18" ref-type="bibr"><sup>18</sup></xref> hCoV-229E and hCoV-OC43 viral RNA were isolated from culture media of infected MRC-5 cells (ATCC, Manassas, VA; CCL-171). MERS-CoV RNA was isolated from cell pellet lysate of infected Vero cells (ATCC; CCL-81).</p></sec><sec id="sec1.2"><title>Viral RNA Titration</title><p id="p0035"><italic>In vitro</italic> transcribed standard RNA for SARS-CoV-2 was prepared, as previously described.<xref rid="bib18" ref-type="bibr"><sup>18</sup></xref> To prepare standard RNA for hCoV-229E, first amplicon of PCR (forward primer, 5′-GCTAGTGGATGATCATGCTTTG-3′; reverse primer, 5′-TGGGGCCATAAACTGTTCTATTAC-3′) was cloned to pBluescript II KS (+) plasmid with BamHI and XhoI. Then, <italic>in vitro</italic> transcription template was prepared by restriction enzyme cut with BglI and XhoI and purification after agarose gel electrophoresis. For hCoV-OC43 and MERS-CoV, amplicons of PCR (OC43 forward primer, 5′-AGCAACCAGGCTGATGTCAATACC-3′; OC43 reverse primer, 5′-AGCAGACCTTCCTGAGCCTTCAAT-3′; MERS-CoV, UpE region<xref rid="bib19" ref-type="bibr"><sup>19</sup></xref>) were synthesized and cloned to pBIC-A plasmid (Bioneer, Daejeon, Republic of Korea). <italic>In vitro</italic> transcription template for hCoV-OC43 and MERS-CoV was prepared by restriction enzyme cut with BamHI-XhoI or SspI-XhoI, respectively. <italic>In vitro</italic> transcriptions were done with EZ T7 High Yield In Vitro Transcription kit (Enzynomics, Daejeon, Republic of Korea), per manufacturer's instructions. RNA products were then purified using Agencourt RNAClean XP (Beckman Coulter, Brea, CA). Standard RNA copy numbers were calculated from concentration measured by NanoDrop Lite (Thermo Scientific, Waltham, MA). All restriction enzymes were purchased from Enzynomics.</p><p id="p0040">To evaluate genomic copy number of viral RNAs, dilutions of standard RNAs and viral RNAs in TE buffer (10 mmol/L Tris-Cl, pH 8.0, and 1 mmol/L EDTA) are subjected to one-step RT-qPCR. RT-qPCRs were performed using LightCycler 96 instrument (Roche, Basel, Switzerland) and following reagents: Luna Universal One-Step RT-qPCR Kit [New England Biolabs (NEB), Ipswich, MA] for hCoV-229E and hCoV-OC43, THUNDERBIRD Probe qPCR Master Mix (Toyobo, Osaka, Japan) for MERS-CoV, and Luna Universal Probe One-Step RT-qPCR Kit (NEB) for SARS-CoV-2.</p></sec><sec id="sec1.3"><title>Reverse Transcription</title><p id="p0045">cDNA of SARS-CoV-2 was made using SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, MA) following manufacturer's instructions with modifications. Briefly, 10 pmol of random hexamer was used as reverse transcription primer, and reaction was performed as follows: 20 minutes at 25°C, 30 minutes at 55°C, and 10 minutes at 80°C.</p></sec><sec id="sec1.4"><title>LAMP and RT-LAMP Reaction</title><p id="p0050">LAMP reaction was performed with reaction mixture containing following components: 1.6 μmol/L FIP/BIP primers, 0.2 μmol/L F3/B3 primers, 0.4 μmol/L loop forward/loop backward primers, 1× Isothermal Amplification Buffer II [NEB; 20 mmol/L Tris-HCl, pH 8.8, 10 mmol/L (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 150 mmol/L KCl, 2 mmol/L MgSO<sub>4</sub>, and 0.1% Tween 20], 6 mmol/L MgSO<sub>4</sub> (NEB; final, 8 mmol/L Mg<sup>2+</sup>), 1.4 mmol/L each dNTP (Enzynomics), 0.4 μmol/L SYTO-9 (Invitrogen), and 6 U <italic>Bst</italic> 3.0 DNA polymerase (NEB) in total 15-μL reaction volume. For RT-LAMP, 10 U of SuperScript IV Reverse Transcriptase (Invitrogen) was added. For end-point colorimetric detection of LAMP reaction, tweaked version of 5× stock leuco crystal violet solution<xref rid="bib13" ref-type="bibr"><sup>13</sup></xref> containing 0.5 mmol/L crystal violet (Sigma, St. Louis, MO; C0775), 60 mmol/L sodium sulfite (Sigma; S0505), and 5 mmol/L β-cyclodextrin (Sigma; C4767) was directly added to LAMP reaction mixture to 1× concentration. When using WarmStart Colorimetric LAMP 2X Master Mix (NEB), same concentrations of primers and 0.4 μmol/L SYTO-9 were added. Isothermal incubation and fluorescence signal measurement were performed using LightCycler 96 instrument at 69°C with additional heat inactivation (5 minutes at 95°C) and melting curve analysis steps. Fluorescence signals were measured for every minute during 60 (screening and optimization) or 30 [limit of detection (LoD) and cross-reactivity] minutes of incubation. Any changed conditions are specified for each experiment.</p></sec></sec><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>LAMP Primer Design</title><p id="p0055">Five SARS-CoV-2 sequences (MN908947, MN938384, MN988713, MN985325, and MN975262) and seven SARS-CoV sequences (NC_004718, AY613947, AY313906, AY559094, AY502924, AY278491, and AY502927) were aligned by MEGA7 software.<xref rid="bib20" ref-type="bibr"><sup>20</sup></xref> SARS-CoV-2 specific regions for LAMP primer design are manually selected: two regions from <italic>Nsp3</italic> (3055 to 3591 and 6172 to 7273), two regions from <italic>Spike</italic> (21540 to 22549 and 22890 to 23779), and one region from <italic>Orf8</italic> (27824 to 28396). Whole <italic>Nucleocapsid</italic> gene region is also included as <italic>Nucleocapsid</italic> is usual target of molecular diagnosis because of the abundance of its mRNA.<xref rid="bib21" ref-type="bibr"><sup>21</sup></xref> Two to five basic LAMP primer sets are designed and selected with PrimerExplorer V5 (<ext-link ext-link-type="uri" xlink:href="http://primerexplorer.jp/lampv5e/index.html" id="intref0020"><italic>http://primerexplorer.jp/lampv5e/index.html</italic></ext-link>, last accessed February 22, 2020) for each target region. Loop primers are designed by PrimerExplorer V5 or manually selected. Sixteen LAMP primer sets with proper both of loop forward primer and loop backward primer are selected and subjected to further screening.</p></sec><sec id="sec2.2"><title>LAMP Primer Screening</title><p id="p0060">First round of screening was done using WarmStart Colorimetric LAMP 2X Master Mix with cDNA, of which corresponding RNA concentration is 8.3 × 10<sup>4</sup> copies/reaction. Of 16 primer sets, nine were selected by threshold time from the results of 40 minutes’ incubation at 65°C and subjected to further screening.</p><p id="p0065">Second round of screening was done using <italic>Bst</italic> 3.0. The same amount of cDNA as the first round of screening is used. Two primer sets that showed relatively early non-specific amplification are discarded, and the remaining seven primer sets are subjected to further screening.</p><p id="p0070">Third and fourth rounds of screening were done by checking sensitivity to dilutions of cDNA and RNA, respectively. Five of seven primer sets showed specific amplification for at least one replicate of duplicate, with cDNA concentration corresponding to 1.7 × 10<sup>1</sup> copies of input RNA (<xref rid="appsec1" ref-type="sec">Supplemental Figure S1</xref>). Next, sensitivity of the five primer sets to RNA dilutions was evaluated in RT-LAMP using <italic>Bst</italic> 3.0 and SuperScript IV Reverse Transcriptase. Two primer sets, both targeting <italic>Nsp3</italic>, showed best sensitivity that showed specific amplification at 10<sup>−6</sup> dilution of RNA (<xref rid="fig1" ref-type="fig">Figure 1</xref>). Two additional primer sets, one targeting <italic>Spike</italic> and the other targeting <italic>Nucleocapsid</italic>, were added for reaction optimization experiments as they showed fast threshold time for cDNA and to keep ranges of target genes. Primer sequences are represented in <xref rid="tbl1" ref-type="table">Table 1</xref>.<fig id="fig1"><label>Figure 1</label><caption><p>Loop-mediated isothermal amplificationLAMP) primer positions on aligned sequences of SARS-CoV-2 and SARS-CoV. Primer binding sites of Nsp3_1-61 (<bold>A</bold>) and Nsp3_2-24 (<bold>B</bold>) LAMP primer sets are depicted on aligned sequences of five SARS-CoV-2 (from up, MN908947, MN938384, MN988713, MN985325, and MN975262) and seven SARS-CoV (from up, NC_004718, AY613947, AY313906, AY559094, AY502924, AY278491, and AY502927). Conserved sites are toggled at 50% level by MEGA7 software so that SARS-CoV-2 specific residues versus SARS-CoV are background colored. Different color bars are used as follows to distinguish binding sites: F3, blue; F2, red; F1, orange; loop forward (LF), green; loop backward (LB), light green; B1, light orange; B2, brown; and B3, light blue.</p></caption><graphic xlink:href="gr1_lrg"></graphic></fig><table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>LAMP Primers of Which Assays Were Optimized and LoD Evaluated</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Set name</th><th>Primer</th><th>Sequence</th></tr></thead><tbody><tr><td>Nsp3_1-61</td><td>F3</td><td>5′-GGAATTTGGTGCCACTTC-3′</td></tr><tr><td></td><td>B3</td><td>5′-CTATTCACTTCAATAGTCTGAACA-3′</td></tr><tr><td></td><td>FIP</td><td>5′-CTTGTTGACCAACAGTTTGTTGACTTCAACCTGAAGAAGAGCAA-3′</td></tr><tr><td></td><td>BIP</td><td>5′-CGGCAGTGAGGACAATCAGACACTGGTGTAAGTTCCATCTC-3′</td></tr><tr><td></td><td>LF</td><td>5′-ATCATCATCTAACCAATCTTCTTC-3′</td></tr><tr><td></td><td>LB</td><td>5′-TCAAACAATTGTTGAGGTTCAACC-3′</td></tr><tr><td>Nsp3_2-24</td><td>F3</td><td>5′-TGCAACTAATAAAGCCACG-3′</td></tr><tr><td></td><td>B3</td><td>5′-CGTCTTTCTGTATGGTAGGATT-3′</td></tr><tr><td></td><td>FIP</td><td>5′-TCTGACTTCAGTACATCAAACGAATAAATACCTGGTGTATACGTTGTC-3′</td></tr><tr><td></td><td>BIP</td><td>5′-GACGCGCAGGGAATGGATAATTCCACTACTTCTTCAGAGACT-3′</td></tr><tr><td></td><td>LF</td><td>5′-TGTTTCAACTGGTTTTGTGCTCCA-3′</td></tr><tr><td></td><td>LB</td><td>5′-TCTTGCCTGCGAAGATCTAAAAC-3′</td></tr><tr><td>S_1-2-2</td><td>F3</td><td>5′-CTGACAAAGTTTTCAGATCCTCAG-3′</td></tr><tr><td></td><td>B3</td><td>5′-AGTACCAAAAATCCAGCCTCTT-3′</td></tr><tr><td></td><td>FIP</td><td>5′-TCCCAGAGACATGTATAGCATGGAATCAACTCAGGACTTGTTCTTACC-3′</td></tr><tr><td></td><td>BIP</td><td>5′-TGGTACTAAGAGGTTTGATAACCCTGTTAGACTTCTCAGTGGAAGCA-3′</td></tr><tr><td></td><td>LF</td><td>5′-CCAAGTAACATTGGAAAAGAAA-3′</td></tr><tr><td></td><td>LB</td><td>5′-GTCCTACCATTTAATGATGGTGTTT-3′</td></tr><tr><td>N_21</td><td>F3</td><td>5′-GCCAAAAGGCTTCTACGCA-3′</td></tr><tr><td></td><td>B3</td><td>5′-TTGCTCTCAAGCTGGTTCAA-3′</td></tr><tr><td></td><td>FIP</td><td>5′-TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG-3′</td></tr><tr><td></td><td>BIP</td><td>5′-TCTCCTGCTAGAATGGCTGGCATCTGTCAAGCAGCAGCAAAG-3′</td></tr><tr><td></td><td>LF</td><td>5′-TGTTGCGACTACGTGATGAGGA-3′</td></tr><tr><td></td><td>LB</td><td>5′-ATGGCGGTGATGCTGCTCT-3′</td></tr></tbody></table><table-wrap-foot><fn><p>LB, loop backward; LF, loop forward; LAMP, loop-mediated isothermal amplification; LoD, limit of detection.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="sec2.3"><title>LAMP Reaction Optimization</title><p id="p0075">To optimize RT-LAMP reaction with <italic>Bst</italic> 3.0, first, optimal concentrations of dNTP and Mg<sup>2+</sup> were evaluated for each primer set. Three dNTP- Mg<sup>2+</sup> concentration combinations are tested at 69°C: dNTP (1.4 mmol/L) and Mg<sup>2+</sup> (8 mmol/L), dNTP (1.4 mmol/L) and Mg<sup>2+</sup> (6 mmol/L), and dNTP (1 mmol/L) and Mg<sup>2+</sup> (6 mmol/L). The copy number of RNA template was 1000 copies per reaction. Best concentration combination was selected for each primer set by whether both of duplicates show specific amplification and by threshold time. Average threshold time (Tt_av) and corresponding dNTP and Mg<sup>2+</sup> concentrations are as follows: dNTP (1 mmol/L) and Mg<sup>2+</sup> (6 mmol/L) for Nsp3_1-61 (Tt_av = 11.93), dNTP (1.4 mmol/L) and Mg<sup>2+</sup> (8 mmol/L) for Nsp3_2-24 (Tt_av = 7.74), dNTP (1.4 mmol/L) and Mg<sup>2+</sup> (6 mmol/L) for S_1-2-2 and N_21 (Tt_av = 11.08 and 5.50, respectively).</p><p id="p0080">Next, whether amplification is improved at lower temperature (65°C) was evaluated, because optimal reaction temperature of SuperScript IV Reverse Transcriptase is 50°C to 55°C and the manufacturer recommends 65°C to 72°C for optimal performance of <italic>Bst</italic> 3.0. Notably, Nsp3_1-61 and S_1-2-2 primer sets show improved threshold time (Tt_av = 8.92 and 8.49, respectively).</p></sec><sec id="sec2.4"><title>Assessing LoD and Cross-Reactivity</title><p id="p0085">The LoD of optimized RT-LAMP assays was assessed through 5 to 1000 RNA copies/reaction in triplicate. Among four primer sets subjected to reaction optimization, S_1-2-2 and N_21 sets showed relatively poor sensitivity. For Nsp3_1-61 and Nsp3_2-24 primer sets, LoD was additionally evaluated with twofold SuperScript IV Reverse Transcriptase (20 U/reaction). LoD and Tt_av were improved by increasing reverse transcriptase amount (<xref rid="tbl2" ref-type="table">Table 2</xref>and <xref rid="appsec1" ref-type="sec">Supplemental Figure S2</xref>). As a result, both Nsp3_1-61 and Nsp3_2-24 RT-LAMP assays could detect as low RNA concentration as 100 copies per reaction.<table-wrap position="float" id="tbl2"><label>Table 2</label><caption><p>LoD of RT-LAMP Assays Targeting SARS-CoV-2</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="3">RNA copies</th><th colspan="4">SSIV, 10 U/rxn<hr></hr></th><th colspan="4">SSIV, 20 U/rxn<hr></hr></th></tr><tr><th colspan="2">Nsp3_1-61<hr></hr></th><th colspan="2">Nsp3_2-24<hr></hr></th><th colspan="2">Nsp3_1-61<hr></hr></th><th colspan="2">Nsp3_2-24<hr></hr></th></tr><tr><th>Tt_av</th><th>RPR</th><th>Tt_av</th><th>RPR</th><th>Tt_av</th><th>RPR</th><th>Tt_av</th><th>RPR</th></tr></thead><tbody><tr><td>1000</td><td align="char">13.11</td><td>3:3</td><td align="char">9.31</td><td>3:3</td><td align="char">10.7</td><td>3:3</td><td align="char">7.53</td><td>3:3</td></tr><tr><td>500</td><td align="char">13.47</td><td>3:3</td><td align="char">8.16</td><td>2:3</td><td align="char">10.63</td><td>3:3</td><td align="char">7.59</td><td>3:3</td></tr><tr><td>200</td><td align="char">13.65</td><td>2:3</td><td align="char">11.19</td><td>2:3</td><td align="char">11.34</td><td>2:3</td><td align="char">7.78</td><td>3:3</td></tr><tr><td>100</td><td align="char">14.62</td><td>3:3</td><td></td><td></td><td align="char">12.26</td><td>2:3</td><td align="char">8.47</td><td>2:3</td></tr><tr><td>50</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td align="char">13.88</td><td>1:3</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NTC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table><table-wrap-foot><fn><p>LoD, limit of detection; RPR, ratio of positive replicates; RT-LAMP, reverse transcription loop-mediated isothermal amplification; rxn, reaction, Tt_av, average threshold time.</p></fn></table-wrap-foot></table-wrap></p><p id="p0090">The cross-reactivity of two SARS-CoV-2 RT-LAMP assays targeting <italic>Nsp3</italic> to other human coronaviruses was not found for hCoV-229E, hCoV-OC43, and MERS-CoV (<xref rid="fig2" ref-type="fig">Figure 2</xref>).<fig id="fig2"><label>Figure 2</label><caption><p>Cross-reactivity to other coronaviruses tested for reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting SARS-CoV-2. Real-time amplification fluorescence signal and end-point leuco crystal violet colorimetric detection results of cross-reactivity test for Nsp3_1-61 (<bold>A</bold>) and Nsp3_2-24 (<bold>B</bold>) LAMP primer sets. Reactions are performed with 20 U/reaction of reverse transcriptase and optimized temperature, dNTP concentration, and Mg<sup>2+</sup> concentration for each primer set. RNA copy number of each viral RNA is as follows: hCoV-229E, 1.6 × 10<sup>6</sup>; hCoV-OC43, 1.6 × 10<sup>6</sup>; MERS-CoV, 4.5 × 10<sup>6</sup>; and SARS-CoV-2, 2.5 × 10<sup>3</sup>.</p></caption><graphic xlink:href="gr2_lrg"></graphic></fig></p></sec></sec><sec id="sec3"><title>Discussion</title><p id="p0095">In this study, LAMP primer sets targeting SARS-CoV-2 were designed and screened. Reaction optimization was also performed for selected primer sets. In summary, a pair of RT-LAMP assays for detection of SARS-CoV-2 with LoD of 100 copies per reaction was designed and evaluated. These RT-LAMP assays showed specificity to SARS-CoV-2 versus alphacoronavirus (hCoV-229E), betacoronavirus (hCoV-OC43), and MERS-CoV. Although specificity to SARS-CoV could not be tested because a proper sample was not in hand, specificity of the RT-LAMP assays is easily expectable from the mismatching bases in primer binding sites (<xref rid="fig1" ref-type="fig">Figure 1</xref>). Especially, both of F1/B1 sites of Nsp3_1-61 primer set are in SARS-CoV-2 specific region, of which aligning SARS-CoV sequence does not exist.</p><p id="p0100">About the sensitivity of LAMP assays, note that average threshold time is not well correlated with LoD. In fact, average threshold times of S_1-2-2 and N_21 primer sets for 1000 copies were faster than that of Nsp3_1-61: 7.33 (ratio of positive replicates, 2:3) and 5.06 (ratio of positive replicates, 1:3) minutes, respectively. This disrelation is previously reported.<xref rid="bib22" ref-type="bibr"><sup>22</sup></xref> One peculiar observation is that both S_1-2-2 and N_21 showed good sensitivity to cDNA template (<xref rid="appsec1" ref-type="sec">Supplemental Figure S1</xref>), unlike to RNA. Observed difference of sensitivity to RNA and cDNA seems significant even when accounting for slight amplification that might happened during reverse transcription. The reason may include stochastic nature of forming proper LAMP amplification intermediate-dumbbell structure,<xref rid="bib22" ref-type="bibr"><sup>22</sup></xref> higher stability of DNA/RNA double strand than DNA/DNA double strand, and more.</p><p id="p0105">The LoD of RT-LAMP assays suggested in this study, 100 copies per reaction, may not be enough for sensitive screening of suspicious patients. This relatively high LoD would be from the target sequences used for primer design that are selected by specificity versus SARS-CoV. Indeed, target GC percentage and Tm of primers had to be adjusted to get enough number of starting sets for primer screening during LAMP primer design to give less-optimal primer sets. Therefore, there might be better target sequences for LAMP assay of SARS-CoV-2 in the viewpoint of sensitivity. However, expected high specificity of RT-LAMP assays suggested in this study would be a good feature for a confirmatory test. In addition, considering high viral load of SARS-CoV-2 at early stage after symptom onset<xref rid="bib23" ref-type="bibr"><sup>23</sup></xref> suggested RT-LAMP assays may still be useable for screening tests.</p><p id="p0110">In conclusion highly specific RT-LAMP assays for detection of SARS-CoV-2 were developed. The results of these RT-LAMP assays can be detected within 30 minutes after amplification reaction begins. In addition, optimized reaction conditions were provided, to which leuco crystal violet colorimetric detection method is applied that can be used for point-of-care tests.</p></sec></body><back><ref-list id="cebib0010"><title>References</title><ref id="bib1"><label>1</label><element-citation publication-type="journal" id="sref1"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>P.</given-names></name><name><surname>Yang</surname><given-names>X.L.</given-names></name><name><surname>Wang</surname><given-names>X.G.</given-names></name><name><surname>Hu</surname><given-names>B.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Zhang</surname><given-names>W.</given-names></name><name><surname>Si</surname><given-names>H.R.</given-names></name><name><surname>Zhu</surname><given-names>Y.</given-names></name><name><surname>Li</surname><given-names>B.</given-names></name><name><surname>Huang</surname><given-names>C.L.</given-names></name><name><surname>Chen</surname><given-names>H.D.</given-names></name><name><surname>Chen</surname><given-names>J.</given-names></name><name><surname>Luo</surname><given-names>Y.</given-names></name><name><surname>Guo</surname><given-names>H.</given-names></name><name><surname>Jiang</surname><given-names>R.D.</given-names></name><name><surname>Liu</surname><given-names>M.Q.</given-names></name><name><surname>Chen</surname><given-names>Y.</given-names></name><name><surname>Shen</surname><given-names>X.R.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Zheng</surname><given-names>X.S.</given-names></name><name><surname>Zhao</surname><given-names>K.</given-names></name><name><surname>Chen</surname><given-names>Q.J.</given-names></name><name><surname>Deng</surname><given-names>F.</given-names></name><name><surname>Liu</surname><given-names>L.L.</given-names></name><name><surname>Yan</surname><given-names>B.</given-names></name><name><surname>Zhan</surname><given-names>F.X.</given-names></name><name><surname>Wang</surname><given-names>Y.Y.</given-names></name><name><surname>Xiao</surname><given-names>G.F.</given-names></name><name><surname>Shi</surname><given-names>Z.L.</given-names></name></person-group><article-title>A pneumonia outbreak associated with a new coronavirus of probable bat origin</article-title><source>Nature</source><volume>579</volume><year>2020</year><fpage>270</fpage><lpage>273</lpage><pub-id pub-id-type="pmid">32015507</pub-id></element-citation></ref><ref id="bib2"><label>2</label><element-citation publication-type="journal" id="sref2"><person-group person-group-type="author"><name><surname>Wu</surname><given-names>F.</given-names></name><name><surname>Zhao</surname><given-names>S.</given-names></name><name><surname>Yu</surname><given-names>B.</given-names></name><name><surname>Chen</surname><given-names>Y.M.</given-names></name><name><surname>Wang</surname><given-names>W.</given-names></name><name><surname>Song</surname><given-names>Z.G.</given-names></name><name><surname>Hu</surname><given-names>Y.</given-names></name><name><surname>Tao</surname><given-names>Z.W.</given-names></name><name><surname>Tian</surname><given-names>J.H.</given-names></name><name><surname>Pei</surname><given-names>Y.Y.</given-names></name><name><surname>Yuan</surname><given-names>M.L.</given-names></name><name><surname>Zhang</surname><given-names>Y.L.</given-names></name><name><surname>Dai</surname><given-names>F.H.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>Q.M.</given-names></name><name><surname>Zheng</surname><given-names>J.J.</given-names></name><name><surname>Xu</surname><given-names>L.</given-names></name><name><surname>Holmes</surname><given-names>E.C.</given-names></name><name><surname>Zhang</surname><given-names>Y.Z.</given-names></name></person-group><article-title>A new coronavirus associated with human respiratory disease in China</article-title><source>Nature</source><volume>579</volume><year>2020</year><fpage>265</fpage><lpage>269</lpage><pub-id pub-id-type="pmid">32015508</pub-id></element-citation></ref><ref id="bib3"><label>3</label><element-citation publication-type="journal" id="sref3"><person-group person-group-type="author"><name><surname>Zhu</surname><given-names>N.</given-names></name><name><surname>Zhang</surname><given-names>D.</given-names></name><name><surname>Wang</surname><given-names>W.</given-names></name><name><surname>Li</surname><given-names>X.</given-names></name><name><surname>Yang</surname><given-names>B.</given-names></name><name><surname>Song</surname><given-names>J.</given-names></name><name><surname>Zhao</surname><given-names>X.</given-names></name><name><surname>Huang</surname><given-names>B.</given-names></name><name><surname>Shi</surname><given-names>W.</given-names></name><name><surname>Lu</surname><given-names>R.</given-names></name><name><surname>Niu</surname><given-names>P.</given-names></name><name><surname>Zhan</surname><given-names>F.</given-names></name><name><surname>Ma</surname><given-names>X.</given-names></name><name><surname>Wang</surname><given-names>D.</given-names></name><name><surname>Xu</surname><given-names>W.</given-names></name><name><surname>Wu</surname><given-names>G.</given-names></name><name><surname>Gao</surname><given-names>G.F.</given-names></name><name><surname>Tan</surname><given-names>W.</given-names></name><collab>China Novel Coronavirus Investigating and Research Team</collab></person-group><article-title>A novel coronavirus from patients with pneumonia in China, 2019</article-title><source>N Engl J Med</source><volume>382</volume><year>2020</year><fpage>727</fpage><lpage>733</lpage><pub-id pub-id-type="pmid">31978945</pub-id></element-citation></ref><ref id="bib4"><label>4</label><element-citation publication-type="journal" id="sref4"><person-group person-group-type="author"><name><surname>Huang</surname><given-names>C.</given-names></name><name><surname>Wang</surname><given-names>Y.</given-names></name><name><surname>Li</surname><given-names>X.</given-names></name><name><surname>Ren</surname><given-names>L.</given-names></name><name><surname>Zhao</surname><given-names>J.</given-names></name><name><surname>Hu</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Fan</surname><given-names>G.</given-names></name><name><surname>Xu</surname><given-names>J.</given-names></name><name><surname>Gu</surname><given-names>X.</given-names></name><name><surname>Cheng</surname><given-names>Z.</given-names></name><name><surname>Yu</surname><given-names>T.</given-names></name><name><surname>Xia</surname><given-names>J.</given-names></name><name><surname>Wei</surname><given-names>Y.</given-names></name><name><surname>Wu</surname><given-names>W.</given-names></name><name><surname>Xie</surname><given-names>X.</given-names></name><name><surname>Yin</surname><given-names>W.</given-names></name><name><surname>Li</surname><given-names>H.</given-names></name><name><surname>Liu</surname><given-names>M.</given-names></name><name><surname>Xiao</surname><given-names>Y.</given-names></name><name><surname>Gao</surname><given-names>H.</given-names></name><name><surname>Guo</surname><given-names>L.</given-names></name><name><surname>Xie</surname><given-names>J.</given-names></name><name><surname>Wang</surname><given-names>G.</given-names></name><name><surname>Jiang</surname><given-names>R.</given-names></name><name><surname>Gao</surname><given-names>Z.</given-names></name><name><surname>Jin</surname><given-names>Q.</given-names></name><name><surname>Wang</surname><given-names>J.</given-names></name><name><surname>Cao</surname><given-names>B.</given-names></name></person-group><article-title>Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China</article-title><source>Lancet</source><volume>395</volume><year>2020</year><fpage>497</fpage><lpage>506</lpage><pub-id pub-id-type="pmid">31986264</pub-id></element-citation></ref><ref id="bib5"><label>5</label><element-citation publication-type="journal" id="sref5"><person-group person-group-type="author"><name><surname>Corman</surname><given-names>V.M.</given-names></name><name><surname>Landt</surname><given-names>O.</given-names></name><name><surname>Kaiser</surname><given-names>M.</given-names></name><name><surname>Molenkamp</surname><given-names>R.</given-names></name><name><surname>Meijer</surname><given-names>A.</given-names></name><name><surname>Chu</surname><given-names>D.K.</given-names></name><name><surname>Bleicker</surname><given-names>T.</given-names></name><name><surname>Brunink</surname><given-names>S.</given-names></name><name><surname>Schneider</surname><given-names>J.</given-names></name><name><surname>Schmidt</surname><given-names>M.L.</given-names></name><name><surname>Mulders</surname><given-names>D.G.</given-names></name><name><surname>Haagmans</surname><given-names>B.L.</given-names></name><name><surname>van der Veer</surname><given-names>B.</given-names></name><name><surname>van den Brink</surname><given-names>S.</given-names></name><name><surname>Wijsman</surname><given-names>L.</given-names></name><name><surname>Goderski</surname><given-names>G.</given-names></name><name><surname>Romette</surname><given-names>J.L.</given-names></name><name><surname>Ellis</surname><given-names>J.</given-names></name><name><surname>Zambon</surname><given-names>M.</given-names></name><name><surname>Peiris</surname><given-names>M.</given-names></name><name><surname>Goossens</surname><given-names>H.</given-names></name><name><surname>Reusken</surname><given-names>C.</given-names></name><name><surname>Koopmans</surname><given-names>M.P.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name></person-group><article-title>Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR</article-title><source>Euro Surveill</source><volume>25</volume><year>2020</year><fpage>2000045</fpage></element-citation></ref><ref id="bib6"><label>6</label><element-citation publication-type="journal" id="sref6"><person-group person-group-type="author"><name><surname>Chu</surname><given-names>D.K.W.</given-names></name><name><surname>Pan</surname><given-names>Y.</given-names></name><name><surname>Cheng</surname><given-names>S.M.S.</given-names></name><name><surname>Hui</surname><given-names>K.P.Y.</given-names></name><name><surname>Krishnan</surname><given-names>P.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Ng</surname><given-names>D.Y.M.</given-names></name><name><surname>Wan</surname><given-names>C.K.C.</given-names></name><name><surname>Yang</surname><given-names>P.</given-names></name><name><surname>Wang</surname><given-names>Q.</given-names></name><name><surname>Peiris</surname><given-names>M.</given-names></name><name><surname>Poon</surname><given-names>L.L.M.</given-names></name></person-group><article-title>Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia</article-title><source>Clin Chem</source><volume>66</volume><year>2020</year><fpage>549</fpage><lpage>555</lpage><pub-id pub-id-type="pmid">32031583</pub-id></element-citation></ref><ref id="bib7"><label>7</label><element-citation publication-type="journal" id="sref7"><person-group person-group-type="author"><name><surname>Nayak</surname><given-names>S.</given-names></name><name><surname>Blumenfeld</surname><given-names>N.R.</given-names></name><name><surname>Laksanasopin</surname><given-names>T.</given-names></name><name><surname>Sia</surname><given-names>S.K.</given-names></name></person-group><article-title>Point-of-care diagnostics: recent developments in a connected age</article-title><source>Anal Chem</source><volume>89</volume><year>2017</year><fpage>102</fpage><lpage>123</lpage><pub-id pub-id-type="pmid">27958710</pub-id></element-citation></ref><ref id="bib8"><label>8</label><element-citation publication-type="journal" id="sref8"><person-group person-group-type="author"><name><surname>Niemz</surname><given-names>A.</given-names></name><name><surname>Ferguson</surname><given-names>T.M.</given-names></name><name><surname>Boyle</surname><given-names>D.S.</given-names></name></person-group><article-title>Point-of-care nucleic acid testing for infectious diseases</article-title><source>Trends Biotechnol</source><volume>29</volume><year>2011</year><fpage>240</fpage><lpage>250</lpage><pub-id pub-id-type="pmid">21377748</pub-id></element-citation></ref><ref id="bib9"><label>9</label><element-citation publication-type="journal" id="sref9"><person-group person-group-type="author"><name><surname>Notomi</surname><given-names>T.</given-names></name><name><surname>Okayama</surname><given-names>H.</given-names></name><name><surname>Masubuchi</surname><given-names>H.</given-names></name><name><surname>Yonekawa</surname><given-names>T.</given-names></name><name><surname>Watanabe</surname><given-names>K.</given-names></name><name><surname>Amino</surname><given-names>N.</given-names></name><name><surname>Hase</surname><given-names>T.</given-names></name></person-group><article-title>Loop-mediated isothermal amplification of DNA</article-title><source>Nucleic Acids Res</source><volume>28</volume><year>2000</year><fpage>E63</fpage><pub-id pub-id-type="pmid">10871386</pub-id></element-citation></ref><ref id="bib10"><label>10</label><element-citation publication-type="journal" id="sref10"><person-group person-group-type="author"><name><surname>Shirato</surname><given-names>K.</given-names></name><name><surname>Yano</surname><given-names>T.</given-names></name><name><surname>Senba</surname><given-names>S.</given-names></name><name><surname>Akachi</surname><given-names>S.</given-names></name><name><surname>Kobayashi</surname><given-names>T.</given-names></name><name><surname>Nishinaka</surname><given-names>T.</given-names></name><name><surname>Notomi</surname><given-names>T.</given-names></name><name><surname>Matsuyama</surname><given-names>S.</given-names></name></person-group><article-title>Detection of middle east respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)</article-title><source>Virol J</source><volume>11</volume><year>2014</year><fpage>139</fpage><pub-id pub-id-type="pmid">25103205</pub-id></element-citation></ref><ref id="bib11"><label>11</label><element-citation publication-type="journal" id="sref11"><person-group person-group-type="author"><name><surname>Oscorbin</surname><given-names>I.P.</given-names></name><name><surname>Belousova</surname><given-names>E.A.</given-names></name><name><surname>Zakabunin</surname><given-names>A.I.</given-names></name><name><surname>Boyarskikh</surname><given-names>U.A.</given-names></name><name><surname>Filipenko</surname><given-names>M.L.</given-names></name></person-group><article-title>Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP)</article-title><source>Biotechniques</source><volume>61</volume><year>2016</year><fpage>20</fpage><lpage>25</lpage><pub-id pub-id-type="pmid">27401670</pub-id></element-citation></ref><ref id="bib12"><label>12</label><element-citation publication-type="journal" id="sref12"><person-group person-group-type="author"><name><surname>Goto</surname><given-names>M.</given-names></name><name><surname>Honda</surname><given-names>E.</given-names></name><name><surname>Ogura</surname><given-names>A.</given-names></name><name><surname>Nomoto</surname><given-names>A.</given-names></name><name><surname>Hanaki</surname><given-names>K.</given-names></name></person-group><article-title>Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue</article-title><source>Biotechniques</source><volume>46</volume><year>2009</year><fpage>167</fpage><lpage>172</lpage><pub-id pub-id-type="pmid">19317660</pub-id></element-citation></ref><ref id="bib13"><label>13</label><element-citation publication-type="journal" id="sref13"><person-group person-group-type="author"><name><surname>Miyamoto</surname><given-names>S.</given-names></name><name><surname>Sano</surname><given-names>S.</given-names></name><name><surname>Takahashi</surname><given-names>K.</given-names></name><name><surname>Jikihara</surname><given-names>T.</given-names></name></person-group><article-title>Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes</article-title><source>Anal Biochem</source><volume>473</volume><year>2015</year><fpage>28</fpage><lpage>33</lpage><pub-id pub-id-type="pmid">25575759</pub-id></element-citation></ref><ref id="bib14"><label>14</label><element-citation publication-type="journal" id="sref14"><person-group person-group-type="author"><name><surname>Tanner</surname><given-names>N.A.</given-names></name><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Evans</surname><given-names>T.C.</given-names><suffix>Jr.</suffix></name></person-group><article-title>Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes</article-title><source>Biotechniques</source><volume>58</volume><year>2015</year><fpage>59</fpage><lpage>68</lpage><pub-id pub-id-type="pmid">25652028</pub-id></element-citation></ref><ref id="bib15"><label>15</label><element-citation publication-type="journal" id="sref15"><person-group person-group-type="author"><name><surname>Nie</surname><given-names>K.</given-names></name><name><surname>Qi</surname><given-names>S.X.</given-names></name><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Luo</surname><given-names>L.</given-names></name><name><surname>Xie</surname><given-names>Y.</given-names></name><name><surname>Yang</surname><given-names>M.J.</given-names></name><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Shen</surname><given-names>H.</given-names></name><name><surname>Li</surname><given-names>Q.</given-names></name><name><surname>Ma</surname><given-names>X.J.</given-names></name></person-group><article-title>Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens</article-title><source>PLoS One</source><volume>7</volume><year>2012</year><fpage>e52486</fpage><pub-id pub-id-type="pmid">23272248</pub-id></element-citation></ref><ref id="bib16"><label>16</label><element-citation publication-type="journal" id="sref16"><person-group person-group-type="author"><name><surname>Nyan</surname><given-names>D.C.</given-names></name><name><surname>Ulitzky</surname><given-names>L.E.</given-names></name><name><surname>Cehan</surname><given-names>N.</given-names></name><name><surname>Williamson</surname><given-names>P.</given-names></name><name><surname>Winkelman</surname><given-names>V.</given-names></name><name><surname>Rios</surname><given-names>M.</given-names></name><name><surname>Taylor</surname><given-names>D.R.</given-names></name></person-group><article-title>Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay</article-title><source>Clin Infect Dis</source><volume>59</volume><year>2014</year><fpage>16</fpage><lpage>23</lpage><pub-id pub-id-type="pmid">24704724</pub-id></element-citation></ref><ref id="bib17"><label>17</label><element-citation publication-type="journal" id="sref17"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>D.</given-names></name><name><surname>Shin</surname><given-names>Y.</given-names></name><name><surname>Chung</surname><given-names>S.</given-names></name><name><surname>Hwang</surname><given-names>K.S.</given-names></name><name><surname>Yoon</surname><given-names>D.S.</given-names></name><name><surname>Lee</surname><given-names>J.H.</given-names></name></person-group><article-title>Simple and highly sensitive molecular diagnosis of Zika virus by lateral flow assays</article-title><source>Anal Chem</source><volume>88</volume><year>2016</year><fpage>12272</fpage><lpage>12278</lpage><pub-id pub-id-type="pmid">28193014</pub-id></element-citation></ref><ref id="bib18"><label>18</label><element-citation publication-type="journal" id="sref18"><person-group person-group-type="author"><name><surname>Jung</surname><given-names>Y.J.</given-names></name><name><surname>Park</surname><given-names>G.-S.</given-names></name><name><surname>Moon</surname><given-names>J.H.</given-names></name><name><surname>Ku</surname><given-names>K.</given-names></name><name><surname>Beak</surname><given-names>S.-H.</given-names></name><name><surname>Kim</surname><given-names>S.</given-names></name><name><surname>Park</surname><given-names>E.C.</given-names></name><name><surname>Park</surname><given-names>D.</given-names></name><name><surname>Lee</surname><given-names>J.-H.</given-names></name><name><surname>Byeon</surname><given-names>C.W.</given-names></name><name><surname>Lee</surname><given-names>J.J.</given-names></name><name><surname>Maeng</surname><given-names>J.-S.</given-names></name><name><surname>Kim</surname><given-names>S.J.</given-names></name><name><surname>Kim</surname><given-names>S.I.</given-names></name><name><surname>Kim</surname><given-names>B.-T.</given-names></name><name><surname>Lee</surname><given-names>M.J.</given-names></name><name><surname>Kim</surname><given-names>H.G.</given-names></name></person-group><article-title>Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2</article-title><source>BioRxiv</source><year>2020</year></element-citation></ref><ref id="bib19"><label>19</label><element-citation publication-type="journal" id="sref19"><person-group person-group-type="author"><name><surname>Corman</surname><given-names>V.M.</given-names></name><name><surname>Eckerle</surname><given-names>I.</given-names></name><name><surname>Bleicker</surname><given-names>T.</given-names></name><name><surname>Zaki</surname><given-names>A.</given-names></name><name><surname>Landt</surname><given-names>O.</given-names></name><name><surname>Eschbach-Bludau</surname><given-names>M.</given-names></name><name><surname>van Boheemen</surname><given-names>S.</given-names></name><name><surname>Gopal</surname><given-names>R.</given-names></name><name><surname>Ballhause</surname><given-names>M.</given-names></name><name><surname>Bestebroer</surname><given-names>T.M.</given-names></name><name><surname>Muth</surname><given-names>D.</given-names></name><name><surname>Muller</surname><given-names>M.A.</given-names></name><name><surname>Drexler</surname><given-names>J.F.</given-names></name><name><surname>Zambon</surname><given-names>M.</given-names></name><name><surname>Osterhaus</surname><given-names>A.D.</given-names></name><name><surname>Fouchier</surname><given-names>R.M.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name></person-group><article-title>Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction</article-title><source>Euro Surveill</source><volume>17</volume><year>2012</year><fpage>20285</fpage><pub-id pub-id-type="pmid">23041020</pub-id></element-citation></ref><ref id="bib20"><label>20</label><element-citation publication-type="journal" id="sref20"><person-group person-group-type="author"><name><surname>Kumar</surname><given-names>S.</given-names></name><name><surname>Stecher</surname><given-names>G.</given-names></name><name><surname>Tamura</surname><given-names>K.</given-names></name></person-group><article-title>MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets</article-title><source>Mol Biol Evol</source><volume>33</volume><year>2016</year><fpage>1870</fpage><lpage>1874</lpage><pub-id pub-id-type="pmid">27004904</pub-id></element-citation></ref><ref id="bib21"><label>21</label><element-citation publication-type="journal" id="sref21"><person-group person-group-type="author"><name><surname>Hiscox</surname><given-names>J.A.</given-names></name><name><surname>Cavanagh</surname><given-names>D.</given-names></name><name><surname>Britton</surname><given-names>P.</given-names></name></person-group><article-title>Quantification of individual subgenomic mRNA species during replication of the coronavirus transmissible gastroenteritis virus</article-title><source>Virus Res</source><volume>36</volume><year>1995</year><fpage>119</fpage><lpage>130</lpage><pub-id pub-id-type="pmid">7653093</pub-id></element-citation></ref><ref id="bib22"><label>22</label><element-citation publication-type="journal" id="sref22"><person-group person-group-type="author"><name><surname>Khorosheva</surname><given-names>E.M.</given-names></name><name><surname>Karymov</surname><given-names>M.A.</given-names></name><name><surname>Selck</surname><given-names>D.A.</given-names></name><name><surname>Ismagilov</surname><given-names>R.F.</given-names></name></person-group><article-title>Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP</article-title><source>Nucleic Acids Res</source><volume>44</volume><year>2016</year><fpage>e10</fpage><pub-id pub-id-type="pmid">26358811</pub-id></element-citation></ref><ref id="bib23"><label>23</label><element-citation publication-type="journal" id="sref23"><person-group person-group-type="author"><name><surname>Zou</surname><given-names>L.</given-names></name><name><surname>Ruan</surname><given-names>F.</given-names></name><name><surname>Huang</surname><given-names>M.</given-names></name><name><surname>Liang</surname><given-names>L.</given-names></name><name><surname>Huang</surname><given-names>H.</given-names></name><name><surname>Hong</surname><given-names>Z.</given-names></name><name><surname>Yu</surname><given-names>J.</given-names></name><name><surname>Kang</surname><given-names>M.</given-names></name><name><surname>Song</surname><given-names>Y.</given-names></name><name><surname>Xia</surname><given-names>J.</given-names></name><name><surname>Guo</surname><given-names>Q.</given-names></name><name><surname>Song</surname><given-names>T.</given-names></name><name><surname>He</surname><given-names>J.</given-names></name><name><surname>Yen</surname><given-names>H.L.</given-names></name><name><surname>Peiris</surname><given-names>M.</given-names></name><name><surname>Wu</surname><given-names>J.</given-names></name></person-group><article-title>SARS-CoV-2 viral load in upper respiratory specimens of infected patients</article-title><source>N Engl J Med</source><volume>382</volume><year>2020</year><fpage>1177</fpage><lpage>1179</lpage><pub-id pub-id-type="pmid">32074444</pub-id></element-citation></ref></ref-list><sec id="appsec1" sec-type="supplementary-material"><title>Supplemental Data</title><p id="p0120"><fig id="dfig1" position="anchor"><label>Supplemental Figure S1</label><caption><p>Sensitivity of SARS-CoV-2 loop-mediated isothermal amplification (LAMP) assays to cDNA dilutions. Real-time amplification fluorescence signal (<bold>A</bold>) and end-point leuco crystal violet (LCV) colorimetric detection results (<bold>B</bold>) of four SARS-CoV-2 LAMP primer sets that were subjected to reaction optimization. Color of amplification signal curve and LCV detection label for each cDNA dilution are matched. Designated copy number is corresponding reverse transcription input RNA copy number. Some low fluorescence of amplification signal plateaus is due to baseline correction process of LightCycler 96 software. NTC, XXX.</p></caption><graphic xlink:href="figs1_lrg"></graphic></fig><fig id="dfig2" position="anchor"><label>Supplemental Figure S2</label><caption><p>Colorimetric detection of SARS-CoV-2 reverse transcription loop-mediated isothermal amplification limit of detection (LoD) tests for Nsp3_1-61 and Nsp3_2-24. Leuco crystal violet colorimetric detection results of LoD tests for Nsp3_1-16 and Nsp3_2-24. A 20 U/reaction of reverse transcriptase was used. See main text for detailed reaction conditions. NTC, XXX.</p></caption><graphic xlink:href="figs2_lrg"></graphic></fig></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We thank Korea Centers for Disease Control and Prevention for kind and rapid sharing of isolated strain of SARS-CoV-2.</p></ack><fn-group><fn id="d32e840"><p id="ntpara0010">Supported by the <funding-source id="gs1">National Research Council of Science and Technology</funding-source> grant by the <funding-source id="gs2">Ministry of Science</funding-source> and ICT grant CRC-16-01-KRICT (J.-S.M).</p></fn><fn id="d32e852"><p id="ntpara0015">Disclosures: None declared.</p></fn><fn id="appsec2" fn-type="supplementary-material"><p id="p0125">Supplemental material for this article can be found at <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1016/j.jmoldx.2020.03.006" id="intref0025"><italic>https://doi.org/10.1016/j.jmoldx.2020.03.006</italic></ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001203 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 001203 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= SrasV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:7144851
|texte= Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting SARS-CoV-2
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:32276051" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a SrasV1
| This area was generated with Dilib version V0.6.33. |  |