A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses
Identifieur interne : 001017 ( Main/Exploration ); précédent : 001016; suivant : 001018A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses
Auteurs : Zhenzhou Wan [République populaire de Chine] ; Ya An Zhang ; Zhixiang He ; Jia Liu ; Ke Lan ; Yihong Hu ; Chiyu ZhangSource :
- International Journal of Molecular Sciences [ 1422-0067 ] ; 2016.
Abstract
Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 102 copies for HCoV-OC43, and 3 × 101 copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.
Url:
DOI: 10.3390/ijms17111880
PubMed: 27886052
PubMed Central: 5133880
Affiliations:
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Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Hu, Yihong" sort="Hu, Yihong" uniqKey="Hu Y" first="Yihong" last="Hu">Yihong Hu</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Zhang, Chiyu" sort="Zhang, Chiyu" uniqKey="Zhang C" first="Chiyu" last="Zhang">Chiyu Zhang</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27886052</idno><idno type="pmc">5133880</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133880</idno><idno type="RBID">PMC:5133880</idno><idno type="doi">10.3390/ijms17111880</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000896</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000896</idno><idno type="wicri:Area/Pmc/Curation">000896</idno><idno 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Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijms-17-01880">Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou 225300, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou 225300</wicri:regionArea><wicri:noRegion>Taizhou 225300</wicri:noRegion></affiliation></author><author><name sortKey="Zhang, Ya An" sort="Zhang, Ya An" uniqKey="Zhang Y" first="Ya An" last="Zhang">Ya An Zhang</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="He, Zhixiang" sort="He, Zhixiang" uniqKey="He Z" first="Zhixiang" last="He">Zhixiang He</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Liu, Jia" sort="Liu, Jia" uniqKey="Liu J" first="Jia" last="Liu">Jia Liu</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Lan, Ke" sort="Lan, Ke" uniqKey="Lan K" first="Ke" last="Lan">Ke Lan</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Hu, Yihong" sort="Hu, Yihong" uniqKey="Hu Y" first="Yihong" last="Hu">Yihong Hu</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author><author><name sortKey="Zhang, Chiyu" sort="Zhang, Chiyu" uniqKey="Zhang C" first="Chiyu" last="Zhang">Chiyu Zhang</name><affiliation><nlm:aff id="af1-ijms-17-01880">Pathogen Diagnostic Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China;<email>wanlv@126.com</email> (Z.W.);<email>13901433616@139.com</email> (Y.Z.);<email>kevin2003xzyz@163.com</email> (Z.H.);<email>liujia02@sibs.ac.cn</email> (J.L.);<email>lanke@sibs.ac.cn</email> (K.L.)</nlm:aff></affiliation></author></analytic><series><title level="j">International Journal of Molecular Sciences</title><idno type="eISSN">1422-0067</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10<sup>2</sup> copies for HCoV-OC43, and 3 × 10<sup>1</sup> copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. 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Kibbe</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="He, Zhixiang" sort="He, Zhixiang" uniqKey="He Z" first="Zhixiang" last="He">Zhixiang He</name><name sortKey="Hu, Yihong" sort="Hu, Yihong" uniqKey="Hu Y" first="Yihong" last="Hu">Yihong Hu</name><name sortKey="Lan, Ke" sort="Lan, Ke" uniqKey="Lan K" first="Ke" last="Lan">Ke Lan</name><name sortKey="Liu, Jia" sort="Liu, Jia" uniqKey="Liu J" first="Jia" last="Liu">Jia Liu</name><name sortKey="Zhang, Chiyu" sort="Zhang, Chiyu" uniqKey="Zhang C" first="Chiyu" last="Zhang">Chiyu Zhang</name><name sortKey="Zhang, Ya An" sort="Zhang, Ya An" uniqKey="Zhang Y" first="Ya An" last="Zhang">Ya An Zhang</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Wan, Zhenzhou" sort="Wan, Zhenzhou" uniqKey="Wan Z" first="Zhenzhou" last="Wan">Zhenzhou Wan</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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