Rapid and sensitive detection of multiple genes from the SARS‐Coronavirus using quantitative RT‐PCR with dual systems
Identifieur interne : 004E43 ( Main/Merge ); précédent : 004E42; suivant : 004E44Rapid and sensitive detection of multiple genes from the SARS‐Coronavirus using quantitative RT‐PCR with dual systems
Auteurs : Jau-Ling Huang [Taïwan] ; Hui-Tsu Lin [Taïwan] ; Yu-Ming Wang [Taïwan] ; Yi-Chien Yeh [Taïwan] ; Konan Peck [Taïwan] ; Bai-Ling Lin [Taïwan] ; Huan-Wun Liu [Taïwan] ; Ann Chen [Taïwan] ; Chang-Shen Lin [Taïwan]Source :
- Journal of Medical Virology [ 0146-6615 ] ; 2005-10.
Descripteurs français
- KwdFr :
- MESH :
- diagnostic : Syndrome respiratoire aigu sévère.
- génétique : Virus du SRAS.
- isolement et purification : Virus du SRAS.
- virologie : Syndrome respiratoire aigu sévère.
- ARN viral, Gènes viraux, Humains, RT-PCR, Sensibilité et spécificité.
English descriptors
- KwdEn :
- MESH :
- chemical : RNA, Viral.
- diagnosis : Severe Acute Respiratory Syndrome.
- genetics : SARS Virus.
- isolation & purification : SARS Virus.
- virology : Severe Acute Respiratory Syndrome.
- Teeft :
- Absolute sensitivity, Academia sinica, Accurate diagnosis, Acute phase, Artus, Assay, Best sensitivity, Bioscience technology, Bniout assays, Cell culturederived viruses, Cell viruses, Chain reaction assay, Chan, Chang jung christian university, Clin microbiol, Clinical application, Clinical samples, Clinical specimens, Coronavirus, Coronaviruses, Defective virions, Disease control, Fields virology, Genes, Viral, Genome, Grant sponsor, Health sciences, Hong kong, Huang, Human coronaviruses, Human sera, Human sera specimens, Humans, Independent experiments, Initial stage, Inoculated vero, Japanese encephalitis virus, Lightcycler, Lippincott williams wilkins, National defense, National health research institutes, Novel coronavirus, Other coronaviruses, Other viruses, Peiris, Plaque assay, Polymerase, Positive results, Preventive medicine, Primer, Primer sets, Prism, Probable sars cases, Probe sets, Quantitative assay, Respiratory syncytial virus, Respiratory syndrome, Reverse Transcriptase Polymerase Chain Reaction, Roche, Roche lightcycler, Roche lightcycler system, Room temperature, Sars, Sars patients, Sars virus, Sars2, Sars2 sets, Sarscov infection, Sensitivity and Specificity, Sequence detection system, Similar sensitivity, Spiked, Syndrome, Taqman, Taqman assay, Taqman probes, Taqman technique, Viral, Viral load, Viral rnas, Virol, Virus, World health organization.
Abstract
The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS‐CoV) in 2003. To detect early SARS‐CoV infection, a one‐step, real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS‐CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture‐derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS‐S, ‐M, and ‐N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture‐derived SARS‐CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS‐N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS‐S and SARS‐M also demonstrated equivalent sensitivity to the commercially available RealArt HPA‐Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS‐CoV; and there was no cross detection with other coronaviruses and human respiratory tract‐associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple‐gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS‐CoV infection. J. Med. Virol. 77:151–158, 2005. © 2005 Wiley‐Liss, Inc.
Url:
DOI: 10.1002/jmv.20432
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Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Yeh, Yi Hien" sort="Yeh, Yi Hien" uniqKey="Yeh Y" first="Yi-Chien" last="Yeh">Yi-Chien Yeh</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Medical Research, Chang Jung Christian University, Tainan</wicri:regionArea><wicri:noRegion>Tainan</wicri:noRegion></affiliation></author><author><name sortKey="Peck, Konan" sort="Peck, Konan" uniqKey="Peck K" first="Konan" last="Peck">Konan Peck</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biomedical Science, Academia Sinica, 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Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Chang Hen" sort="Lin, Chang Hen" uniqKey="Lin C" first="Chang-Shen" last="Lin">Chang-Shen Lin</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>National Health Research Institutes, Miaoli</wicri:regionArea><wicri:noRegion>Miaoli</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Taïwan</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr" wicri:curation="lc">Taïwan</country><wicri:regionArea>Jau‐Ling Huang, Department of Bioscience Technology, College of Health Sciences, Chang Jung Christian University, 396, Chang Jung Rd., Sec. 1, Kway Jen, Tainan, Taiwan.===Chang‐Shen Lin, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350</wicri:regionArea><wicri:noRegion>Miaoli County 350</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Journal of Medical Virology</title><title level="j" type="alt">JOURNAL OF MEDICAL VIROLOGY</title><idno type="ISSN">0146-6615</idno><idno type="eISSN">1096-9071</idno><imprint><biblScope unit="vol">77</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="151">151</biblScope><biblScope unit="page" to="158">158</biblScope><biblScope unit="page-count">8</biblScope><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2005-10">2005-10</date></imprint><idno type="ISSN">0146-6615</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0146-6615</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Genes, Viral</term><term>Humans</term><term>RNA, Viral</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>SARS Virus (genetics)</term><term>SARS Virus (isolation & purification)</term><term>Sensitivity and Specificity</term><term>Severe Acute Respiratory Syndrome (diagnosis)</term><term>Severe Acute Respiratory Syndrome (virology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral</term><term>Gènes viraux</term><term>Humains</term><term>RT-PCR</term><term>Sensibilité et spécificité</term><term>Syndrome respiratoire aigu sévère (diagnostic)</term><term>Syndrome respiratoire aigu sévère (virologie)</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (isolement et purification)</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>RNA, Viral</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" qualifier="diagnostic" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Absolute sensitivity</term><term>Academia sinica</term><term>Accurate diagnosis</term><term>Acute phase</term><term>Artus</term><term>Assay</term><term>Best sensitivity</term><term>Bioscience technology</term><term>Bniout assays</term><term>Cell culturederived viruses</term><term>Cell viruses</term><term>Chain reaction assay</term><term>Chan</term><term>Chang jung christian university</term><term>Clin microbiol</term><term>Clinical application</term><term>Clinical samples</term><term>Clinical specimens</term><term>Coronavirus</term><term>Coronaviruses</term><term>Defective virions</term><term>Disease control</term><term>Fields virology</term><term>Genes, Viral</term><term>Genome</term><term>Grant sponsor</term><term>Health sciences</term><term>Hong kong</term><term>Huang</term><term>Human coronaviruses</term><term>Human sera</term><term>Human sera specimens</term><term>Humans</term><term>Independent experiments</term><term>Initial stage</term><term>Inoculated vero</term><term>Japanese encephalitis virus</term><term>Lightcycler</term><term>Lippincott williams wilkins</term><term>National defense</term><term>National health research institutes</term><term>Novel coronavirus</term><term>Other coronaviruses</term><term>Other viruses</term><term>Peiris</term><term>Plaque assay</term><term>Polymerase</term><term>Positive results</term><term>Preventive medicine</term><term>Primer</term><term>Primer sets</term><term>Prism</term><term>Probable sars cases</term><term>Probe sets</term><term>Quantitative assay</term><term>Respiratory syncytial virus</term><term>Respiratory syndrome</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Roche</term><term>Roche lightcycler</term><term>Roche lightcycler system</term><term>Room temperature</term><term>Sars</term><term>Sars patients</term><term>Sars virus</term><term>Sars2</term><term>Sars2 sets</term><term>Sarscov infection</term><term>Sensitivity and Specificity</term><term>Sequence detection system</term><term>Similar sensitivity</term><term>Spiked</term><term>Syndrome</term><term>Taqman</term><term>Taqman assay</term><term>Taqman probes</term><term>Taqman technique</term><term>Viral</term><term>Viral load</term><term>Viral rnas</term><term>Virol</term><term>Virus</term><term>World health organization</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ARN viral</term><term>Gènes viraux</term><term>Humains</term><term>RT-PCR</term><term>Sensibilité et spécificité</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS‐CoV) in 2003. To detect early SARS‐CoV infection, a one‐step, real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS‐CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture‐derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS‐S, ‐M, and ‐N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture‐derived SARS‐CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS‐N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS‐S and SARS‐M also demonstrated equivalent sensitivity to the commercially available RealArt HPA‐Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS‐CoV; and there was no cross detection with other coronaviruses and human respiratory tract‐associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple‐gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS‐CoV infection. J. Med. Virol. 77:151–158, 2005. © 2005 Wiley‐Liss, Inc.</div></front></TEI><double doi="10.1002/jmv.20432"><ISTEX><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Rapid and sensitive detection of multiple genes from the SARS‐Coronavirus using quantitative RT‐PCR with dual systems</title><author><name sortKey="Huang, Jau Ing" sort="Huang, Jau Ing" uniqKey="Huang J" first="Jau-Ling" last="Huang">Jau-Ling Huang</name></author><author><name sortKey="Lin, Hui Su" sort="Lin, Hui Su" uniqKey="Lin H" first="Hui-Tsu" last="Lin">Hui-Tsu Lin</name></author><author><name sortKey="Wang, Yu Ing" sort="Wang, Yu Ing" uniqKey="Wang Y" first="Yu-Ming" last="Wang">Yu-Ming Wang</name></author><author><name sortKey="Yeh, Yi Hien" sort="Yeh, Yi Hien" uniqKey="Yeh Y" first="Yi-Chien" last="Yeh">Yi-Chien Yeh</name></author><author><name sortKey="Peck, Konan" sort="Peck, Konan" uniqKey="Peck K" first="Konan" last="Peck">Konan Peck</name></author><author><name sortKey="Lin, Bai Ing" sort="Lin, Bai Ing" uniqKey="Lin B" first="Bai-Ling" last="Lin">Bai-Ling Lin</name></author><author><name sortKey="Liu, Huan Un" sort="Liu, Huan Un" uniqKey="Liu H" first="Huan-Wun" last="Liu">Huan-Wun Liu</name></author><author><name sortKey="Chen, Ann" sort="Chen, Ann" uniqKey="Chen A" first="Ann" last="Chen">Ann Chen</name></author><author><name sortKey="Lin, Chang Hen" sort="Lin, Chang Hen" uniqKey="Lin C" first="Chang-Shen" last="Lin">Chang-Shen Lin</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:4CE919B653183838A834334ED61DEEE905FCE822</idno><date when="2005" year="2005">2005</date><idno type="doi">10.1002/jmv.20432</idno><idno type="url">https://api.istex.fr/ark:/67375/WNG-ZVNZ6330-B/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000190</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000190</idno><idno type="wicri:Area/Istex/Curation">000190</idno><idno 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Tainan</wicri:regionArea><wicri:noRegion>Tainan</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Taïwan</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr" wicri:curation="lc">Taïwan</country><wicri:regionArea>Jau‐Ling Huang, Department of Bioscience Technology, College of Health Sciences, Chang Jung Christian University, 396, Chang Jung Rd., Sec. 1, Kway Jen, Tainan, Taiwan.===Chang‐Shen Lin, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350</wicri:regionArea><wicri:noRegion>Miaoli County 350</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Hui Su" sort="Lin, Hui Su" uniqKey="Lin H" first="Hui-Tsu" last="Lin">Hui-Tsu Lin</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Wang, Yu Ing" sort="Wang, Yu Ing" uniqKey="Wang Y" first="Yu-Ming" last="Wang">Yu-Ming Wang</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Yeh, Yi Hien" sort="Yeh, Yi Hien" uniqKey="Yeh Y" first="Yi-Chien" last="Yeh">Yi-Chien Yeh</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Medical Research, Chang Jung Christian University, Tainan</wicri:regionArea><wicri:noRegion>Tainan</wicri:noRegion></affiliation></author><author><name sortKey="Peck, Konan" sort="Peck, Konan" uniqKey="Peck K" first="Konan" last="Peck">Konan Peck</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Biomedical Science, Academia Sinica, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Bai Ing" sort="Lin, Bai Ing" uniqKey="Lin B" first="Bai-Ling" last="Lin">Bai-Ling Lin</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Development Center for Biotechnology, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Liu, Huan Un" sort="Liu, Huan Un" uniqKey="Liu H" first="Huan-Wun" last="Liu">Huan-Wun Liu</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Ann" sort="Chen, Ann" uniqKey="Chen A" first="Ann" last="Chen">Ann Chen</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Chang Hen" sort="Lin, Chang Hen" uniqKey="Lin C" first="Chang-Shen" last="Lin">Chang-Shen Lin</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>National Health Research Institutes, Miaoli</wicri:regionArea><wicri:noRegion>Miaoli</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Taïwan</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr" wicri:curation="lc">Taïwan</country><wicri:regionArea>Jau‐Ling Huang, Department of Bioscience Technology, College of Health Sciences, Chang Jung Christian University, 396, Chang Jung Rd., Sec. 1, Kway Jen, Tainan, Taiwan.===Chang‐Shen Lin, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350</wicri:regionArea><wicri:noRegion>Miaoli County 350</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Journal of Medical Virology</title><title level="j" type="alt">JOURNAL OF MEDICAL VIROLOGY</title><idno type="ISSN">0146-6615</idno><idno type="eISSN">1096-9071</idno><imprint><biblScope unit="vol">77</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="151">151</biblScope><biblScope unit="page" to="158">158</biblScope><biblScope unit="page-count">8</biblScope><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2005-10">2005-10</date></imprint><idno type="ISSN">0146-6615</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0146-6615</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Absolute sensitivity</term><term>Academia sinica</term><term>Accurate diagnosis</term><term>Acute phase</term><term>Artus</term><term>Assay</term><term>Best sensitivity</term><term>Bioscience technology</term><term>Bniout assays</term><term>Cell culturederived viruses</term><term>Cell viruses</term><term>Chain reaction assay</term><term>Chan</term><term>Chang jung christian university</term><term>Clin microbiol</term><term>Clinical application</term><term>Clinical samples</term><term>Clinical specimens</term><term>Coronavirus</term><term>Coronaviruses</term><term>Defective virions</term><term>Disease control</term><term>Fields virology</term><term>Genome</term><term>Grant sponsor</term><term>Health sciences</term><term>Hong kong</term><term>Huang</term><term>Human coronaviruses</term><term>Human sera</term><term>Human sera specimens</term><term>Independent experiments</term><term>Initial stage</term><term>Inoculated vero</term><term>Japanese encephalitis virus</term><term>Lightcycler</term><term>Lippincott williams wilkins</term><term>National defense</term><term>National health research institutes</term><term>Novel coronavirus</term><term>Other coronaviruses</term><term>Other viruses</term><term>Peiris</term><term>Plaque assay</term><term>Polymerase</term><term>Positive results</term><term>Preventive medicine</term><term>Primer</term><term>Primer sets</term><term>Prism</term><term>Probable sars cases</term><term>Probe sets</term><term>Quantitative assay</term><term>Respiratory syncytial virus</term><term>Respiratory syndrome</term><term>Roche</term><term>Roche lightcycler</term><term>Roche lightcycler system</term><term>Room temperature</term><term>Sars</term><term>Sars patients</term><term>Sars virus</term><term>Sars2</term><term>Sars2 sets</term><term>Sarscov infection</term><term>Sequence detection system</term><term>Similar sensitivity</term><term>Spiked</term><term>Syndrome</term><term>Taqman</term><term>Taqman assay</term><term>Taqman probes</term><term>Taqman technique</term><term>Viral</term><term>Viral load</term><term>Viral rnas</term><term>Virol</term><term>Virus</term><term>World health organization</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS‐CoV) in 2003. To detect early SARS‐CoV infection, a one‐step, real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS‐CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture‐derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS‐S, ‐M, and ‐N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture‐derived SARS‐CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS‐N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS‐S and SARS‐M also demonstrated equivalent sensitivity to the commercially available RealArt HPA‐Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS‐CoV; and there was no cross detection with other coronaviruses and human respiratory tract‐associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple‐gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS‐CoV infection. J. Med. Virol. 77:151–158, 2005. © 2005 Wiley‐Liss, Inc.</div></front></TEI></ISTEX><PubMed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems.</title><author><name sortKey="Huang, Jau Ling" sort="Huang, Jau Ling" uniqKey="Huang J" first="Jau-Ling" last="Huang">Jau-Ling Huang</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan. JauLingH@mail.cju.edu.tw</nlm:affiliation><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Hui Tsu" sort="Lin, Hui Tsu" uniqKey="Lin H" first="Hui-Tsu" last="Lin">Hui-Tsu Lin</name></author><author><name sortKey="Wang, Yu Ming" sort="Wang, Yu Ming" uniqKey="Wang Y" first="Yu-Ming" last="Wang">Yu-Ming Wang</name></author><author><name sortKey="Yeh, Yi Chien" sort="Yeh, Yi Chien" uniqKey="Yeh Y" first="Yi-Chien" last="Yeh">Yi-Chien Yeh</name></author><author><name sortKey="Peck, Konan" sort="Peck, Konan" uniqKey="Peck K" first="Konan" last="Peck">Konan Peck</name></author><author><name sortKey="Lin, Bai Ling" sort="Lin, Bai Ling" uniqKey="Lin B" first="Bai-Ling" last="Lin">Bai-Ling Lin</name></author><author><name sortKey="Liu, Huan Wun" sort="Liu, Huan Wun" uniqKey="Liu H" first="Huan-Wun" last="Liu">Huan-Wun Liu</name></author><author><name sortKey="Chen, Ann" sort="Chen, Ann" uniqKey="Chen A" first="Ann" last="Chen">Ann Chen</name></author><author><name sortKey="Lin, Chang Shen" sort="Lin, Chang Shen" uniqKey="Lin C" first="Chang-Shen" last="Lin">Chang-Shen Lin</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:16121372</idno><idno type="pmid">16121372</idno><idno type="doi">10.1002/jmv.20432</idno><idno type="wicri:Area/PubMed/Corpus">002571</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002571</idno><idno type="wicri:Area/PubMed/Curation">002571</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002571</idno><idno type="wicri:Area/PubMed/Checkpoint">002553</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002553</idno><idno type="wicri:Area/Ncbi/Merge">001132</idno><idno type="wicri:Area/Ncbi/Curation">001132</idno><idno type="wicri:Area/Ncbi/Checkpoint">001132</idno><idno type="wicri:doubleKey">0146-6615:2005:Huang J:rapid:and:sensitive</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems.</title><author><name sortKey="Huang, Jau Ling" sort="Huang, Jau Ling" uniqKey="Huang J" first="Jau-Ling" last="Huang">Jau-Ling Huang</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan. JauLingH@mail.cju.edu.tw</nlm:affiliation><country xml:lang="fr">Taïwan</country><wicri:regionArea>Institute of Preventive Medicine, National Defense Medical Center, Taipei</wicri:regionArea><wicri:noRegion>Taipei</wicri:noRegion></affiliation></author><author><name sortKey="Lin, Hui Tsu" sort="Lin, Hui Tsu" uniqKey="Lin H" first="Hui-Tsu" last="Lin">Hui-Tsu Lin</name></author><author><name sortKey="Wang, Yu Ming" sort="Wang, Yu Ming" uniqKey="Wang Y" first="Yu-Ming" last="Wang">Yu-Ming Wang</name></author><author><name sortKey="Yeh, Yi Chien" sort="Yeh, Yi Chien" uniqKey="Yeh Y" first="Yi-Chien" last="Yeh">Yi-Chien Yeh</name></author><author><name sortKey="Peck, Konan" sort="Peck, Konan" uniqKey="Peck K" first="Konan" last="Peck">Konan Peck</name></author><author><name sortKey="Lin, Bai Ling" sort="Lin, Bai Ling" uniqKey="Lin B" first="Bai-Ling" last="Lin">Bai-Ling Lin</name></author><author><name sortKey="Liu, Huan Wun" sort="Liu, Huan Wun" uniqKey="Liu H" first="Huan-Wun" last="Liu">Huan-Wun Liu</name></author><author><name sortKey="Chen, Ann" sort="Chen, Ann" uniqKey="Chen A" first="Ann" last="Chen">Ann Chen</name></author><author><name sortKey="Lin, Chang Shen" sort="Lin, Chang Shen" uniqKey="Lin C" first="Chang-Shen" last="Lin">Chang-Shen Lin</name></author></analytic><series><title level="j">Journal of medical virology</title><idno type="ISSN">0146-6615</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Genes, Viral</term><term>Humans</term><term>RNA, Viral</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>SARS Virus (genetics)</term><term>SARS Virus (isolation & purification)</term><term>Sensitivity and Specificity</term><term>Severe Acute Respiratory Syndrome (diagnosis)</term><term>Severe Acute Respiratory Syndrome (virology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral</term><term>Gènes viraux</term><term>Humains</term><term>RT-PCR</term><term>Sensibilité et spécificité</term><term>Syndrome respiratoire aigu sévère (diagnostic)</term><term>Syndrome respiratoire aigu sévère (virologie)</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (isolement et purification)</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>RNA, Viral</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" qualifier="diagnostic" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Genes, Viral</term><term>Humans</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Sensitivity and Specificity</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ARN viral</term><term>Gènes viraux</term><term>Humains</term><term>RT-PCR</term><term>Sensibilité et spécificité</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS-CoV) in 2003. To detect early SARS-CoV infection, a one-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture-derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS-S, -M, and -N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture-derived SARS-CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS-N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS-S and SARS-M also demonstrated equivalent sensitivity to the commercially available RealArt HPA-Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS-CoV; and there was no cross detection with other coronaviruses and human respiratory tract-associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple-gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS-CoV infection.</div></front></TEI></PubMed></double></record>Pour manipuler ce document sous Unix (Dilib)
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