Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays.
Identifieur interne : 000288 ( Hal/Checkpoint ); précédent : 000287; suivant : 000289Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays.
Auteurs : Leo L M. Poon [République populaire de Chine] ; K. H. Chan [République populaire de Chine] ; G. J. Smith [République populaire de Chine] ; C. S. W. Leung [République populaire de Chine] ; Y. Guan [République populaire de Chine] ; K. Y. Yuen [République populaire de Chine] ; J. S. M. Peiris [République populaire de Chine]Source :
- Clinical Chemistry [ 0009-9147 ] ; 2009-08.
Abstract
BACKGROUND: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed. METHODS: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses. RESULTS: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses. CONCLUSIONS: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.
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DOI: 10.1373/clinchem.2009.130229
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<front><div type="abstract" xml:lang="en"> <p>BACKGROUND: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed. METHODS: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses. RESULTS: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses. CONCLUSIONS: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.</p>
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<seriesStmt> <idno type="stamp" n="RIIP">Institut Pasteur RIIP (Réseau International)</idno>
<idno type="stamp" n="RIIP_HONGKONG" corresp="RIIP">Centre de Recherche Université de Hong-Kong-Pasteur</idno>
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<notesStmt> <note type="audience" n="1">Not set</note>
<note type="popular" n="0">No</note>
<note type="peer" n="1">Yes</note>
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<sourceDesc> <biblStruct> <analytic> <title xml:lang="en">Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays.</title>
<author role="crp"> <persName> <forename type="first">Leo L M</forename>
<surname>Poon</surname>
</persName>
<email type="md5">12dd69b4fc6098669afe833ac40b281b</email>
<email type="domain">hkucc.hku.hk</email>
<idno type="halauthorid">603530</idno>
<affiliation ref="#struct-136023"></affiliation>
</author>
<author role="aut"> <persName> <forename type="first">K. H.</forename>
<surname>Chan</surname>
</persName>
<idno type="halauthorid">603628</idno>
<affiliation ref="#struct-160704"></affiliation>
</author>
<author role="aut"> <persName> <forename type="first">G. J.</forename>
<surname>Smith</surname>
</persName>
<idno type="halauthorid">603629</idno>
<affiliation ref="#struct-136023"></affiliation>
</author>
<author role="aut"> <persName> <forename type="first">C. S. W.</forename>
<surname>Leung</surname>
</persName>
<idno type="halauthorid">603630</idno>
<affiliation ref="#struct-136023"></affiliation>
</author>
<author role="aut"> <persName> <forename type="first">Y.</forename>
<surname>Guan</surname>
</persName>
<idno type="halauthorid">283241</idno>
<affiliation ref="#struct-136023"></affiliation>
</author>
<author role="aut"> <persName> <forename type="first">K. Y.</forename>
<surname>Yuen</surname>
</persName>
<idno type="halauthorid">603631</idno>
<affiliation ref="#struct-136023"></affiliation>
</author>
<author role="crp"> <persName> <forename type="first">J. S. M.</forename>
<surname>Peiris</surname>
</persName>
<email type="md5">cc6ecab6abc0bb33758973d51e488f7b</email>
<email type="domain">hkucc.hku.hk</email>
<idno type="halauthorid">603528</idno>
<affiliation ref="#struct-136023"></affiliation>
<affiliation ref="#struct-55925"></affiliation>
</author>
</analytic>
<monogr> <idno type="halJournalId" status="VALID">11766</idno>
<idno type="issn">0009-9147</idno>
<idno type="eissn">1530-8561</idno>
<title level="j">Clinical Chemistry</title>
<imprint> <publisher>American Association for Clinical Chemistry</publisher>
<biblScope unit="volume">55</biblScope>
<biblScope unit="issue">8</biblScope>
<biblScope unit="pp">1555-8</biblScope>
<date type="datePub">2009-08</date>
<date type="dateEpub">2009-05-13</date>
</imprint>
</monogr>
<idno type="doi">10.1373/clinchem.2009.130229</idno>
<idno type="pubmed">19439731</idno>
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<profileDesc> <langUsage> <language ident="en">English</language>
</langUsage>
<textClass> <classCode scheme="mesh">Animals</classCode>
<classCode scheme="mesh">Base Sequence</classCode>
<classCode scheme="mesh">Orthomyxoviridae Infections</classCode>
<classCode scheme="mesh">RNA, Viral</classCode>
<classCode scheme="mesh">Reverse Transcriptase Polymerase Chain Reaction</classCode>
<classCode scheme="mesh">Sensitivity and Specificity</classCode>
<classCode scheme="mesh">Swine</classCode>
<classCode scheme="mesh">Time Factors</classCode>
<classCode scheme="mesh">DNA, Viral</classCode>
<classCode scheme="mesh">Humans</classCode>
<classCode scheme="mesh">Influenza A Virus, H1N1 Subtype</classCode>
<classCode scheme="mesh">Influenza A Virus, H2N2 Subtype</classCode>
<classCode scheme="mesh">Influenza A Virus, H3N2 Subtype</classCode>
<classCode scheme="mesh">Influenza A Virus, H5N1 Subtype</classCode>
<classCode scheme="mesh">Influenza, Human</classCode>
<classCode scheme="mesh">Molecular Diagnostic Techniques</classCode>
<classCode scheme="halDomain" n="sdv.mp.vir">Life Sciences [q-bio]/Microbiology and Parasitology/Virology</classCode>
<classCode scheme="halTypology" n="ART">Journal articles</classCode>
</textClass>
<abstract xml:lang="en"> <p>BACKGROUND: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed. METHODS: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses. RESULTS: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses. CONCLUSIONS: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.</p>
</abstract>
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