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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV</title>
<author><name sortKey="Kim, Jin Hwa" sort="Kim, Jin Hwa" uniqKey="Kim J" first="Jin Hwa" last="Kim">Jin Hwa Kim</name>
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<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kang, Minhee" sort="Kang, Minhee" uniqKey="Kang M" first="Minhee" last="Kang">Minhee Kang</name>
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<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Department of Medical Device Management and Research, SAIHST,</institution>
<institution>Sungkyunkwan University,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Park, Eunkyoung" sort="Park, Eunkyoung" uniqKey="Park E" first="Eunkyoung" last="Park">Eunkyoung Park</name>
<affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Department of Medical Device Management and Research, SAIHST,</institution>
<institution>Sungkyunkwan University,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chung, Doo Ryeon" sort="Chung, Doo Ryeon" uniqKey="Chung D" first="Doo Ryeon" last="Chung">Doo Ryeon Chung</name>
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<institution-id institution-id-type="ISNI">0000 0001 0640 5613</institution-id>
<institution>Center for Infection Prevention and Control,</institution>
<institution>Samsung Medical Center,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff4">Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Division of Infectious Diseases, Department of Internal Medicine, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Jiyeon" sort="Kim, Jiyeon" uniqKey="Kim J" first="Jiyeon" last="Kim">Jiyeon Kim</name>
<affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.31501.36</institution-id>
<institution-id institution-id-type="ISNI">0000 0004 0470 5905</institution-id>
<institution>Department of Microbiology and Immunology,</institution>
<institution>Seoul National University College of Medicine,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.412484.f</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0302 820X</institution-id>
<institution>Institute of Endemic Diseases,</institution>
<institution>Seoul National University Medical Research Center,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hwang, Eung Soo" sort="Hwang, Eung Soo" uniqKey="Hwang E" first="Eung Soo" last="Hwang">Eung Soo Hwang</name>
<affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.31501.36</institution-id>
<institution-id institution-id-type="ISNI">0000 0004 0470 5905</institution-id>
<institution>Department of Microbiology and Immunology,</institution>
<institution>Seoul National University College of Medicine,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.412484.f</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0302 820X</institution-id>
<institution>Institute of Endemic Diseases,</institution>
<institution>Seoul National University Medical Research Center,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">32226589</idno>
<idno type="pmc">7097549</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097549</idno>
<idno type="RBID">PMC:7097549</idno>
<idno type="doi">10.1007/s13206-019-3404-3</idno>
<date when="2019">2019</date>
<idno type="wicri:Area/Pmc/Corpus">000327</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000327</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV</title>
<author><name sortKey="Kim, Jin Hwa" sort="Kim, Jin Hwa" uniqKey="Kim J" first="Jin Hwa" last="Kim">Jin Hwa Kim</name>
<affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kang, Minhee" sort="Kang, Minhee" uniqKey="Kang M" first="Minhee" last="Kang">Minhee Kang</name>
<affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Department of Medical Device Management and Research, SAIHST,</institution>
<institution>Sungkyunkwan University,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Park, Eunkyoung" sort="Park, Eunkyoung" uniqKey="Park E" first="Eunkyoung" last="Park">Eunkyoung Park</name>
<affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Department of Medical Device Management and Research, SAIHST,</institution>
<institution>Sungkyunkwan University,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Chung, Doo Ryeon" sort="Chung, Doo Ryeon" uniqKey="Chung D" first="Doo Ryeon" last="Chung">Doo Ryeon Chung</name>
<affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.414964.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0640 5613</institution-id>
<institution>Center for Infection Prevention and Control,</institution>
<institution>Samsung Medical Center,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff4">Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Division of Infectious Diseases, Department of Internal Medicine, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Jiyeon" sort="Kim, Jiyeon" uniqKey="Kim J" first="Jiyeon" last="Kim">Jiyeon Kim</name>
<affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.31501.36</institution-id>
<institution-id institution-id-type="ISNI">0000 0004 0470 5905</institution-id>
<institution>Department of Microbiology and Immunology,</institution>
<institution>Seoul National University College of Medicine,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.412484.f</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0302 820X</institution-id>
<institution>Institute of Endemic Diseases,</institution>
<institution>Seoul National University Medical Research Center,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hwang, Eung Soo" sort="Hwang, Eung Soo" uniqKey="Hwang E" first="Eung Soo" last="Hwang">Eung Soo Hwang</name>
<affiliation><nlm:aff id="Aff6"><institution-wrap><institution-id institution-id-type="GRID">grid.31501.36</institution-id>
<institution-id institution-id-type="ISNI">0000 0004 0470 5905</institution-id>
<institution>Department of Microbiology and Immunology,</institution>
<institution>Seoul National University College of Medicine,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="Aff7"><institution-wrap><institution-id institution-id-type="GRID">grid.412484.f</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0302 820X</institution-id>
<institution>Institute of Endemic Diseases,</institution>
<institution>Seoul National University Medical Research Center,</institution>
</institution-wrap>
Seoul, South Korea</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Biochip Journal</title>
<idno type="ISSN">1976-0280</idno>
<idno type="eISSN">2092-7843</idno>
<imprint><date when="2019">2019</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
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</teiHeader>
<front><div type="abstract" xml:lang="en"><p>The current diagnosis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) relies on laboratory-based tests since its clinical features are nonspecific, unlike other respiratory pathogens. Therefore, the development of a rapid and simple method for on-site detection of SARS-CoV is crucial for the identification and prevention of future SARS outbreaks. In this study, a simple colorimetric and multiplex loop-mediated isothermal amplification (LAMP) assay was developed to rapid screening of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). It can be visually detected based on color change and monitored in real-time with fluorescent signals. The performance of this assay, based on six primers targeting open reading frame (ORF1b) and nucleocapsid (N) genes located in different regions of the SARS-CoV, was compared with real-time RT-PCR assay using various concentrations of target genes. The detection limit of the LAMP assay was comparable to that of real-time RT-PCR assay and therefore a few target RNA to 10<sup>4</sup>
-10<sup>5</sup>
copies could be detected within a short period of time (20–25 min). In addition, we established a multiplex real-time LAMP assay to simultaneously detect two target regions within the SARS-CoV genome. Two target sequences were amplified by specific primers in the same reaction tube and revealed that it was able to detect down to 10<sup>5</sup>
copies. The standard curve had a linear relationship with similar amplification efficiencies. The LAMP assay results in shorter “sample-to-answer” time than conventional PCR method. Therefore, it is suitable not only for diagnosis of clinical test, but also for surveillance of SARS virus in developing countries.</p>
<sec><title>Electronic Supplementary Material</title>
<p>Supplementary material is available for this article at 10.1007/s13206-019-3404-3 and is accessible for authorized users.</p>
</sec>
</div>
</front>
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</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Biochip J</journal-id>
<journal-id journal-id-type="iso-abbrev">Biochip J</journal-id>
<journal-title-group><journal-title>Biochip Journal</journal-title>
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<issn pub-type="ppub">1976-0280</issn>
<issn pub-type="epub">2092-7843</issn>
<publisher><publisher-name>The Korean BioChip Society (KBCS)</publisher-name>
<publisher-loc>Seoul</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">32226589</article-id>
<article-id pub-id-type="pmc">7097549</article-id>
<article-id pub-id-type="publisher-id">3404</article-id>
<article-id pub-id-type="doi">10.1007/s13206-019-3404-3</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject>
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</article-categories>
<title-group><article-title>A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV</article-title>
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<xref ref-type="aff" rid="Aff1">1</xref>
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<contrib contrib-type="author" corresp="yes" equal-contrib="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-0330-7828</contrib-id>
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<contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0001-9267-101X</contrib-id>
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<aff id="Aff1"><label>1</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</aff>
<aff id="Aff2"><label>2</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Department of Medical Device Management and Research, SAIHST,</institution>
<institution>Sungkyunkwan University,</institution>
</institution-wrap>
Seoul, Republic of Korea</aff>
<aff id="Aff3"><label>3</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.414964.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0640 5613</institution-id>
<institution>Center for Infection Prevention and Control,</institution>
<institution>Samsung Medical Center,</institution>
</institution-wrap>
Seoul, Republic of Korea</aff>
<aff id="Aff4"><label>4</label>
Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea</aff>
<aff id="Aff5"><label>5</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.264381.a</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 2181 989X</institution-id>
<institution>Division of Infectious Diseases, Department of Internal Medicine, Samsung Medical Center,</institution>
<institution>Sungkyunkwan University School of Medicine,</institution>
</institution-wrap>
Seoul, Republic of Korea</aff>
<aff id="Aff6"><label>6</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.31501.36</institution-id>
<institution-id institution-id-type="ISNI">0000 0004 0470 5905</institution-id>
<institution>Department of Microbiology and Immunology,</institution>
<institution>Seoul National University College of Medicine,</institution>
</institution-wrap>
Seoul, South Korea</aff>
<aff id="Aff7"><label>7</label>
<institution-wrap><institution-id institution-id-type="GRID">grid.412484.f</institution-id>
<institution-id institution-id-type="ISNI">0000 0001 0302 820X</institution-id>
<institution>Institute of Endemic Diseases,</institution>
<institution>Seoul National University Medical Research Center,</institution>
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Seoul, South Korea</aff>
</contrib-group>
<pub-date pub-type="epub"><day>11</day>
<month>11</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="ppub"><year>2019</year>
</pub-date>
<volume>13</volume>
<issue>4</issue>
<fpage>341</fpage>
<lpage>351</lpage>
<history><date date-type="received"><day>15</day>
<month>7</month>
<year>2019</year>
</date>
<date date-type="accepted"><day>23</day>
<month>8</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>© The Korean BioChip Society and Springer 2019</copyright-statement>
<license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p>
</license>
</permissions>
<abstract id="Abs1"><p>The current diagnosis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) relies on laboratory-based tests since its clinical features are nonspecific, unlike other respiratory pathogens. Therefore, the development of a rapid and simple method for on-site detection of SARS-CoV is crucial for the identification and prevention of future SARS outbreaks. In this study, a simple colorimetric and multiplex loop-mediated isothermal amplification (LAMP) assay was developed to rapid screening of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). It can be visually detected based on color change and monitored in real-time with fluorescent signals. The performance of this assay, based on six primers targeting open reading frame (ORF1b) and nucleocapsid (N) genes located in different regions of the SARS-CoV, was compared with real-time RT-PCR assay using various concentrations of target genes. The detection limit of the LAMP assay was comparable to that of real-time RT-PCR assay and therefore a few target RNA to 10<sup>4</sup>
-10<sup>5</sup>
copies could be detected within a short period of time (20–25 min). In addition, we established a multiplex real-time LAMP assay to simultaneously detect two target regions within the SARS-CoV genome. Two target sequences were amplified by specific primers in the same reaction tube and revealed that it was able to detect down to 10<sup>5</sup>
copies. The standard curve had a linear relationship with similar amplification efficiencies. The LAMP assay results in shorter “sample-to-answer” time than conventional PCR method. Therefore, it is suitable not only for diagnosis of clinical test, but also for surveillance of SARS virus in developing countries.</p>
<sec><title>Electronic Supplementary Material</title>
<p>Supplementary material is available for this article at 10.1007/s13206-019-3404-3 and is accessible for authorized users.</p>
</sec>
</abstract>
<kwd-group xml:lang="en"><title>Keywords</title>
<kwd>SARS-CoV</kwd>
<kwd>Loop-mediated isothermal amplification</kwd>
<kwd>Colorimetric detection</kwd>
<kwd>Point-of-care test</kwd>
</kwd-group>
<custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name>
<meta-value>© The Korean BioChip Society and Springer-Verlag GmbH Germany, part of Springer Nature 2019</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec sec-type="supplementary-material"><title>Electronic supplementary material</title>
<sec id="Sec1"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="13206_2019_3404_MOESM1_ESM.pdf"><caption><p><bold>Supplementary Table 1</bold>
. The target sequences for amplification.</p>
<p><bold>Supplementary Figure 1S</bold>
. Color change in LAMP Assay. Primer set II (six primers) for N genes was used for amplification. A,
Before amplification; B, After amplification. An orange color indicates a positive and a neutral pink color indicates a negative reaction. C, Gel electrophoresis of LAMP products (POS: positive; NEG: negative; C: control).</p>
<p><bold>Supplementary Figure 2S</bold>
. LAMP primer set optimization with two LAMP primer set. A, four primers (F3, B3, FIP, BIP); and B, six primers (added Loop F and Loop B primers to A primer set). Detection of LAMP products by (a) naked eye with color change and (b) agarose gel electrophoresis (Top: negative control (non-template control, NTC); Bottom: positive control with 50 ng of genomic DNA). An orange color indicates a positive and a neutral pink color indicates a negative reaction. (b) Agarose electrophoresis results of LAMP assay (temperature gradient from 50°C (lane 2) to 72°C (lane 12), 1kb DNA ladder; lane 1). Optimal temperature was determined to be 65°C for both targets.</p>
</caption>
</media>
</supplementary-material>
</p>
</sec>
</sec>
</body>
<back><fn-group><fn><p><bold>Conflict of Interests</bold>
The authors declare no competing financial interests.</p>
</fn>
<fn><p>These authors contrilbuted equally.</p>
</fn>
</fn-group>
<ack><title>Acknowledgements</title>
<p>This research was supported by the government-wide R&D Fund for the research of infectious diseases in Republic of Korea (HG18C 0062), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science (2016M3 A9B6919187, 2016M3A9B6919189).</p>
</ack>
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