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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR <xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Wang, Wei" sort="Wang, Wei" uniqKey="Wang W" first="Wei" last="Wang">Wei Wang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ren, Peijun" sort="Ren, Peijun" uniqKey="Ren P" first="Peijun" last="Ren">Peijun Ren</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mardi, Sek" sort="Mardi, Sek" uniqKey="Mardi S" first="Sek" last="Mardi">Sek Mardi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hou, Lili" sort="Hou, Lili" uniqKey="Hou L" first="Lili" last="Hou">Lili Hou</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Tsai, Cheguo" sort="Tsai, Cheguo" uniqKey="Tsai C" first="Cheguo" last="Tsai">Cheguo Tsai</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok Hung" last="Chan">Kwok Hung Chan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cheng, Peter" sort="Cheng, Peter" uniqKey="Cheng P" first="Peter" last="Cheng">Peter Cheng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sheng, Jun" sort="Sheng, Jun" uniqKey="Sheng J" first="Jun" last="Sheng">Jun Sheng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Buchy, Philippe" sort="Buchy, Philippe" uniqKey="Buchy P" first="Philippe" last="Buchy">Philippe Buchy</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sun, Bing" sort="Sun, Bing" uniqKey="Sun B" first="Bing" last="Sun">Bing Sun</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Toyoda, Tetsuya" sort="Toyoda, Tetsuya" uniqKey="Toyoda T" first="Tetsuya" last="Toyoda">Tetsuya Toyoda</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lim, Wilina" sort="Lim, Wilina" uniqKey="Lim W" first="Wilina" last="Lim">Wilina Lim</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Peiris, J S Malik" sort="Peiris, J S Malik" uniqKey="Peiris J" first="J. S. Malik" last="Peiris">J. S. Malik Peiris</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Zhou, Paul" sort="Zhou, Paul" uniqKey="Zhou P" first="Paul" last="Zhou">Paul Zhou</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Deubel, Vincent" sort="Deubel, Vincent" uniqKey="Deubel V" first="Vincent" last="Deubel">Vincent Deubel</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18971359</idno><idno type="pmc">2620859</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620859</idno><idno type="RBID">PMC:2620859</idno><idno type="doi">10.1128/JCM.01090-08</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000326</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000326</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR <xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Wang, Wei" sort="Wang, Wei" uniqKey="Wang W" first="Wei" last="Wang">Wei Wang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ren, Peijun" sort="Ren, Peijun" uniqKey="Ren P" first="Peijun" last="Ren">Peijun Ren</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Mardi, Sek" sort="Mardi, Sek" uniqKey="Mardi S" first="Sek" last="Mardi">Sek Mardi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hou, Lili" sort="Hou, Lili" uniqKey="Hou L" first="Lili" last="Hou">Lili Hou</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Tsai, Cheguo" sort="Tsai, Cheguo" uniqKey="Tsai C" first="Cheguo" last="Tsai">Cheguo Tsai</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok Hung" last="Chan">Kwok Hung Chan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cheng, Peter" sort="Cheng, Peter" uniqKey="Cheng P" first="Peter" last="Cheng">Peter Cheng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sheng, Jun" sort="Sheng, Jun" uniqKey="Sheng J" first="Jun" last="Sheng">Jun Sheng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Buchy, Philippe" sort="Buchy, Philippe" uniqKey="Buchy P" first="Philippe" last="Buchy">Philippe Buchy</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sun, Bing" sort="Sun, Bing" uniqKey="Sun B" first="Bing" last="Sun">Bing Sun</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Toyoda, Tetsuya" sort="Toyoda, Tetsuya" uniqKey="Toyoda T" first="Tetsuya" last="Toyoda">Tetsuya Toyoda</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lim, Wilina" sort="Lim, Wilina" uniqKey="Lim W" first="Wilina" last="Lim">Wilina Lim</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Peiris, J S Malik" sort="Peiris, J S Malik" uniqKey="Peiris J" first="J. S. Malik" last="Peiris">J. S. Malik Peiris</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Zhou, Paul" sort="Zhou, Paul" uniqKey="Zhou P" first="Paul" last="Zhou">Paul Zhou</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Deubel, Vincent" sort="Deubel, Vincent" uniqKey="Deubel V" first="Vincent" last="Deubel">Vincent Deubel</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Clinical Microbiology</title><idno type="ISSN">0095-1137</idno><idno type="eISSN">1098-660X</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Clin Microbiol</journal-id><journal-id journal-id-type="publisher-id">jcm</journal-id><journal-title>Journal of Clinical Microbiology</journal-title><issn pub-type="ppub">0095-1137</issn><issn pub-type="epub">1098-660X</issn><publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18971359</article-id><article-id pub-id-type="pmc">2620859</article-id><article-id pub-id-type="publisher-id">1090-08</article-id><article-id pub-id-type="doi">10.1128/JCM.01090-08</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virology</subject></subj-group></article-categories><title-group><article-title>Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR <xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Wei</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ren</surname><given-names>Peijun</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Mardi</surname><given-names>Sek</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Hou</surname><given-names>Lili</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Tsai</surname><given-names>Cheguo</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Kwok Hung</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Sheng</surname><given-names>Jun</given-names></name><xref ref-type="aff" rid="aff1">5</xref></contrib><contrib contrib-type="author"><name><surname>Buchy</surname><given-names>Philippe</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Bing</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Toyoda</surname><given-names>Tetsuya</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lim</surname><given-names>Wilina</given-names></name><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Peiris</surname><given-names>J. S. Malik</given-names></name><xref ref-type="aff" rid="aff1">3</xref><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Deubel</surname><given-names>Vincent</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Shanghai, China,<label>1</label> Institut Pasteur du Cambodge, Phnom Penh, Cambodia,<label>2</label> Department of Microbiology, the University of Hong Kong, Hong Kong,<label>3</label> Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong,<label>4</label> Shanghai Nanxiang Hospital, Shanghai, China,<label>5</label> HKU-Pasteur Research Centre, Hong Kong SAR<label>6</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Unit of Emerging Viruses, Chinese Academy of Sciences, Institut Pasteur of Shanghai, 411 Hefei Road, 200025 Shanghai, People's Republic of China. Phone: 86 21 6384 5146. Fax: 86 21 6384 3571. E-mail: <email>vdeubel@sibs.ac.cn</email></p></fn></author-notes><pub-date pub-type="ppub"><month>1</month><year>2009</year></pub-date><pub-date pub-type="epub"><day>29</day><month>10</month><year>2008</year></pub-date><volume>47</volume><issue>1</issue><fpage>86</fpage><lpage>92</lpage><history><date date-type="received"><day>9</day><month>6</month><year>2008</year></date><date date-type="rev-recd"><day>13</day><month>8</month><year>2008</year></date><date date-type="accepted"><day>13</day><month>10</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2009, American Society for Microbiology</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="zjm00109000086.pdf"></self-uri><abstract><p>Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>Algorithm of the four-tube rRT-PCR assay for the detection of AIV.</p></caption><graphic xlink:href="zjm0010985850001"></graphic></fig><table-wrap position="float" id="t1"><label>TABLE 1.</label><caption><p>Influenza virus and other respiratory viruses used in this study</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Virus<xref ref-type="table-fn" rid="t1fn1"><italic>a</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Reference strain (accession no.)<xref ref-type="table-fn" rid="t1fn2"><italic>b</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Sample<xref ref-type="table-fn" rid="t1fn3"><italic>c</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Source<xref ref-type="table-fn" rid="t1fn4"><italic>d</italic></xref></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">Influenza virus</td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H1N1</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPB890/05</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">HK/948/06</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">PHL</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H2N2</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPU3165/06</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H3N2</td><td colspan="1" rowspan="1" align="left" valign="top">HK/2268/06</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">PHL</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H4N6</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPA2290/07</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H5N1</td><td colspan="1" rowspan="1" align="left" valign="top">Goose/HK/WW119/01</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Phf/HK/FY155/01</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Pigeon/HK/SF216/01</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Chicken/HK/FC2C/02</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Shenzhen/05</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H6N(?)<xref ref-type="table-fn" rid="t1fn5"><italic>e</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPC2750/07</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H7N7</td><td colspan="1" rowspan="1" align="left" valign="top">WB/MPB127/05</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H8N3</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MP6684/05</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H9N2</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MP2934/04</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H10N3</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPD351/07</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H11N9</td><td colspan="1" rowspan="1" align="left" valign="top">WB/HK/MPC72/06</td><td colspan="1" rowspan="1" align="center" valign="top">AF</td><td colspan="1" rowspan="1" align="left" valign="top">HKU</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">PIV1</td><td colspan="1" rowspan="1" align="left" valign="top">R3061225006</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">RSV</td><td colspan="1" rowspan="1" align="left" valign="top">R3070118002</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">PIV3</td><td colspan="1" rowspan="1" align="left" valign="top">R3061016010</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">OC43-CoV</td><td colspan="1" rowspan="1" align="left" valign="top">ATCC VR-759 (<ext-link ext-link-type="gen" xlink:href="AY391777">AY391777</ext-link>)</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">CHRC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">229E-CoV</td><td colspan="1" rowspan="1" align="left" valign="top">ATCC VR-740 (<ext-link ext-link-type="gen" xlink:href="EF198679">EF198679</ext-link>)</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">CHRC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">RhV</td><td colspan="1" rowspan="1" align="left" valign="top">R3070719007</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">hMPV</td><td colspan="1" rowspan="1" align="left" valign="top">R3070108003</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">IBV</td><td colspan="1" rowspan="1" align="left" valign="top">R3070315001</td><td colspan="1" rowspan="1" align="center" valign="top">CS</td><td colspan="1" rowspan="1" align="left" valign="top">IPS</td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>RSV, respiratory syncytial virus; PIV, parainfluenza virus; CoV, coronavirus; RhV, rhinovirus; hMPV, human metapneumovirus; IBV, influenza B virus.</p></fn><fn id="t1fn2"><label>b</label><p>Accession numbers are from GenBank.</p></fn><fn id="t1fn3"><label>c</label><p>CS, cell supernatant; AF, allantoic fluid.</p></fn><fn id="t1fn4"><label>d</label><p>HKU, Department of Microbiology, the University of Hong Kong; PHL, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong SAR; IPS, Institut Pasteur of Shanghai, Chinese Academy of Sciences; CHRC, Centre Hospitalier Regional de Caen, Caen, France.</p></fn><fn id="t1fn5"><label>e</label><p>H6N(?), neuraminidase gene not subtyped.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>TABLE 2.</label><caption><p>IAV M and HA gene cDNA clones</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Plasmid</th><th colspan="1" rowspan="1" align="center" valign="bottom">Target</th><th colspan="1" rowspan="1" align="center" valign="bottom">Origin/strain no./yr (antigenic description)</th><th colspan="1" rowspan="1" align="center" valign="bottom">Reference or accession no.</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">pET14b-M1</td><td colspan="1" rowspan="1" align="center" valign="top">M1</td><td colspan="1" rowspan="1" align="left" valign="top">PR/8/34 (H1N1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="C_002016">C_002016</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pPSP-H1<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H1</td><td colspan="1" rowspan="1" align="left" valign="top">PR/8/34 (H1N1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="NC_002017">NC_002017</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pUC-H2<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H2</td><td colspan="1" rowspan="1" align="left" valign="top">Adachi/2/57 (H2N2)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="D13576">D13576</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pME18A-H3<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H3</td><td colspan="1" rowspan="1" align="left" valign="top">Aichi/2/68 (H3N2)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="J02090">J02090</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pGMT-aH5<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Ck/Henan/13/04 (H5N1 clade 8)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="AY950233">AY950233</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pGMT-hH5</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Vietnam/CL36/04 (H5N1 clade 1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="DQ497724">DQ497724</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCMV/R-H5HA(C05)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Cambodia/05 (H5N1 clade 1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="EF473073">EF473073</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCDNA-H5HA(C06)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Cambodia/06 (H5N1 clade 1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="QO321176">QO321176</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCDNA-H5HA(SH)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Shanghai/06 (H5N1 clade 2.3.2)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="AB462295">AB462295</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCMV/R-H5HA(SZ)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Shenzhen/05 (H5N1 subclade 2.3.1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="EF541394">EF541394</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCMV/R-H5HA(Indo)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">Indonesia/1/05 (H5N1 clade 2.1.3)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="EF137706">EF137706</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pCMV/R-H5HA(HK)</td><td colspan="1" rowspan="1" align="center" valign="top">H5<xref ref-type="table-fn" rid="t2fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">HongKong/156/97 (H5N1 clade 0)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="AF036356">AF036356</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pUC-H7<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H7</td><td colspan="1" rowspan="1" align="left" valign="top">FPV/Rostock/34 (H7N1)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="M24457">M24457</ext-link></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">pT-H9<xref ref-type="table-fn" rid="t2fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">H9</td><td colspan="1" rowspan="1" align="left" valign="top">Ck/Shanghai/F/98 (H9N2)</td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="gen" xlink:href="AY743216">AY743216</ext-link></td></tr></tbody></table><table-wrap-foot><fn id="t2fn1"><label>a</label><p>HA clones used for RNA transcription and for RNA sensitivity tests (see Table <xref ref-type="table" rid="t4">4</xref>).</p></fn><fn id="t2fn2"><label>b</label><p>The nomenclature follows that established by the WHO/OIE/FAO H5N1 Evolution Working Group (<xref ref-type="bibr" rid="r52">52</xref>).</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t3"><label>TABLE 3.</label><caption><p>Sequences of primers and probes used in rRT-PCR</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Target</th><th colspan="1" rowspan="1" align="center" valign="bottom">Oligonucleotide<xref ref-type="table-fn" rid="t3fn1"><italic>a</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Sequence 5′→3′<xref ref-type="table-fn" rid="t3fn2"><italic>b</italic></xref></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">M</td><td colspan="1" rowspan="1" align="center" valign="top">M1-f-245</td><td colspan="1" rowspan="1" align="left" valign="top">CCAGTGAGCGAGGACTGCAG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">M1-r-332</td><td colspan="1" rowspan="1" align="left" valign="top">GGATCCCCATTCCCATTGAG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H1</td><td colspan="1" rowspan="1" align="center" valign="top">c2h1-f-553</td><td colspan="1" rowspan="1" align="left" valign="top">GGTTTGTTTGGAGCCATTGC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">c2h1-r-662</td><td colspan="1" rowspan="1" align="left" valign="top">TCCGCAGCATAGCCAGATC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">c2h1-p-592</td><td colspan="1" rowspan="1" align="left" valign="top">Texas Red-TGGACTGGAATGATAGATG-BHQ2</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H2</td><td colspan="1" rowspan="1" align="center" valign="top">h2-f-350</td><td colspan="1" rowspan="1" align="left" valign="top">AACATCTCCTCAGCAGCGTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h2-r-467</td><td colspan="1" rowspan="1" align="left" valign="top">GATGGATTACCAGACACCGC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h2-p-445</td><td colspan="1" rowspan="1" align="left" valign="top">TET-CGCGATCGATGGACACAGCATACAACAACTGGAGGATCGCG-BHQ1</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H3</td><td colspan="1" rowspan="1" align="center" valign="top">h3-f-1098</td><td colspan="1" rowspan="1" align="left" valign="top">GGTTGGGAGGGAATGGTAGAC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h3-r-1199</td><td colspan="1" rowspan="1" align="left" valign="top">GTCGATTGCTGCTTGAGTGC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h3-p-1170</td><td colspan="1" rowspan="1" align="left" valign="top">FAM-CTGCTGCTTGTCCTGTGCCCTCAG-BHQ1</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H5</td><td colspan="1" rowspan="1" align="center" valign="top">h5c-f-729</td><td colspan="1" rowspan="1" align="left" valign="top">AGATCCAAAGTAAACGGGCAAA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h5c-r-808</td><td colspan="1" rowspan="1" align="left" valign="top">TCGAAGTTGATTGCATCATTCG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">ch5a-f-244</td><td colspan="1" rowspan="1" align="left" valign="top">GGAAACCCAATGTGTGACGAA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">ch5a-r-318</td><td colspan="1" rowspan="1" align="left" valign="top">ATTGGCTGGATTGGCCTTCT</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H7</td><td colspan="1" rowspan="1" align="center" valign="top">c2h7-f-520</td><td colspan="1" rowspan="1" align="left" valign="top">CTGAGCAGCGGCTACAAAGA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">c2h7-r-609</td><td colspan="1" rowspan="1" align="left" valign="top">GAAGACAAGGCCCATTGCAA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">c2h7-p-542</td><td colspan="1" rowspan="1" align="left" valign="top">FAM-TGATACTTTGGTTTAGCTTC-BHQ1</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H9</td><td colspan="1" rowspan="1" align="center" valign="top">h9-f-169</td><td colspan="1" rowspan="1" align="left" valign="top">ATGTCCCTGTGACACATGCTAA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h9-r-281</td><td colspan="1" rowspan="1" align="left" valign="top">CAGTCCTTCAATGGTACAGG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top">h-9p-239</td><td colspan="1" rowspan="1" align="left" valign="top">Texas Red-TGT CCC AGG CTT GTT GCA CAC AGC-BHQ2</td></tr></tbody></table><table-wrap-foot><fn id="t3fn1"><label>a</label><p>Forward (f) and reverse (r) primers and probes (p) are indicated, followed by the position of the 5′ nucleotide in the M or H gene coding sense sequence.</p></fn><fn id="t3fn2"><label>b</label><p>FAM, 6-carboxyfluorescein.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t4"><label>TABLE 4.</label><caption><p>Comparison of <italic>C<sub>T</sub></italic> values of monoplex-specific and multiplex real-time PCR for HA RNA transcripts</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="center" valign="middle">HA gene source</th><th colspan="1" rowspan="2" align="center" valign="middle">Origin (antigen description)</th><th colspan="1" rowspan="2" align="center" valign="middle">No. of RNA copies in reaction<xref ref-type="table-fn" rid="t4fn1"><italic>a</italic></xref></th><th colspan="2" rowspan="1" align="center" valign="bottom"><italic>C<sub>T</sub></italic> value for<xref ref-type="table-fn" rid="t4fn3"><italic>c</italic></xref>:
<hr></hr></th></tr><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Monoplex rRT-PCR<xref ref-type="table-fn" rid="t4fn2"><italic>b</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Multiplex rRT-PCR</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">H1</td><td colspan="1" rowspan="1" align="left" valign="top">A/PR/8/34 (H1N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">16.24</td><td colspan="1" rowspan="1" align="char" char="." valign="top">21.5</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">17.2</td><td colspan="1" rowspan="1" align="char" char="." valign="top">25.3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">18.2</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30.9</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">21.08</td><td colspan="1" rowspan="1" align="char" char="." valign="top">31.1</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="char" char="." valign="top">23.0</td><td colspan="1" rowspan="1" align="char" char="." valign="top">34.7</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.53</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H2</td><td colspan="1" rowspan="1" align="left" valign="top">A/Adachi/2/57 (H2N2)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">19.2</td><td colspan="1" rowspan="1" align="char" char="." valign="top">22.78</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">23.3</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.19</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">27.3</td><td colspan="1" rowspan="1" align="char" char="." valign="top">28.88</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">31.8</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30.06</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="char" char="." valign="top">33.7</td><td colspan="1" rowspan="1" align="char" char="." valign="top">32.05</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="char" char="." valign="top">34.9</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H3</td><td colspan="1" rowspan="1" align="left" valign="top">A/Aichi/2/68 (H3N2)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">13.56</td><td colspan="1" rowspan="1" align="char" char="." valign="top">12.96</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">16.95</td><td colspan="1" rowspan="1" align="char" char="." valign="top">17.02</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">20.71</td><td colspan="1" rowspan="1" align="char" char="." valign="top">20.60</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">23.14</td><td colspan="1" rowspan="1" align="char" char="." valign="top">22.40</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.82</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.84</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="char" char="." valign="top">31.23</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H5<xref ref-type="table-fn" rid="t4fn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="left" valign="top">A/Chicken/Henan/13/2004 (H5N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">11.24</td><td colspan="1" rowspan="1" align="char" char="." valign="top">8.299</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">14.29</td><td colspan="1" rowspan="1" align="char" char="." valign="top">11.04</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">17.06</td><td colspan="1" rowspan="1" align="char" char="." valign="top">14.22</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">17.80</td><td colspan="1" rowspan="1" align="char" char="." valign="top">14.50</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="char" char="." valign="top">20.04</td><td colspan="1" rowspan="1" align="char" char="." valign="top">16.21</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H7</td><td colspan="1" rowspan="1" align="left" valign="top">A/FPV/Rostock/34 (H7N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">19.12</td><td colspan="1" rowspan="1" align="char" char="." valign="top">20.70</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">23.06</td><td colspan="1" rowspan="1" align="char" char="." valign="top">24.28</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">27.69</td><td colspan="1" rowspan="1" align="char" char="." valign="top">29.08</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">32.45</td><td colspan="1" rowspan="1" align="char" char="." valign="top">32.63</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="char" char="." valign="top">35.55</td><td colspan="1" rowspan="1" align="char" char="." valign="top">36.63</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">H9</td><td colspan="1" rowspan="1" align="left" valign="top">A/Chicken/Shanghai/F/98 (H9N2)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">100,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">22.11</td><td colspan="1" rowspan="1" align="char" char="." valign="top">22.24</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">26.58</td><td colspan="1" rowspan="1" align="char" char="." valign="top">25.02</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">1,000</td><td colspan="1" rowspan="1" align="char" char="." valign="top">27.69</td><td colspan="1" rowspan="1" align="char" char="." valign="top">28.54</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">100</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30.34</td><td colspan="1" rowspan="1" align="char" char="." valign="top">35.21</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="char" char="." valign="top">10</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td><td colspan="1" rowspan="1" align="center" valign="top">NEG</td></tr></tbody></table><table-wrap-foot><fn id="t4fn1"><label>a</label><p>A 10-fold dilution series of each HA RNA transcript is used. The copy number in each reaction is shown with the mean relative <italic>C<sub>T</sub></italic> value.</p></fn><fn id="t4fn2"><label>b</label><p>Monoplex rRT-PCR was performed for H5 RNA titration using either primers h5c-f-729 and h5c-r-808 or primers ch5a-f-244 and ch5a-r-318, indicated in Table <xref ref-type="table" rid="t3">3</xref>, and showed similar results.</p></fn><fn id="t4fn3"><label>c</label><p>NEG, negative.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t5"><label>TABLE 5.</label><caption><p>Comparison of monoplex RT-PCR and multiplex rRT-PCR sensitivities<xref ref-type="table-fn" rid="t5fn1"><italic>a</italic></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Reaction</th><th colspan="1" rowspan="1" align="center" valign="bottom">Gene</th><th colspan="1" rowspan="1" align="center" valign="bottom">Virus</th><th colspan="1" rowspan="1" align="center" valign="bottom"><italic>C<sub>T</sub></italic> limit value</th><th colspan="1" rowspan="1" align="center" valign="bottom">Limit of TCID<sub>50</sub> detection</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="left" valign="top">M1</td><td colspan="1" rowspan="1" align="left" valign="top">Hong Kong/948/06 (H1N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30.7</td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td></tr><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="left" valign="top">M1</td><td colspan="1" rowspan="1" align="left" valign="top">Hong Kong/2268/06 (H3N2)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">30.7</td><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td></tr><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">1</td><td colspan="1" rowspan="1" align="left" valign="top">M1</td><td colspan="1" rowspan="1" align="left" valign="top">Shenzhen/05 (H5N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">29.9</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.1</td></tr><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">2</td><td colspan="1" rowspan="1" align="left" valign="top">H5 HA</td><td colspan="1" rowspan="1" align="left" valign="top">Shenzhen/05 (H5N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">25.6</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.01</td></tr><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">3</td><td colspan="1" rowspan="1" align="left" valign="top">H1 HA</td><td colspan="1" rowspan="1" align="left" valign="top">Hong Kong/948/06 (H1N1)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">28.5</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.1</td></tr><tr><td colspan="1" rowspan="1" align="char" char="." valign="top">3</td><td colspan="1" rowspan="1" align="left" valign="top">H3 HA</td><td colspan="1" rowspan="1" align="left" valign="top">Hong Kong/2268/06 (H3N2)</td><td colspan="1" rowspan="1" align="char" char="." valign="top">32.3</td><td colspan="1" rowspan="1" align="char" char="." valign="top">0.1</td></tr></tbody></table><table-wrap-foot><fn id="t5fn1"><label>a</label><p>Assessments of sensitivity levels of monoplex RT-PCR (using the M gene) and multiplex rRT-PCR (using the HA gene) were made with IAV RNA extracted from titrated virus stocks (TCID<sub>50</sub>).</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t6"><label>TABLE 6.</label><caption><p>Comparison of detection of rRT-PCR and RT-PCR assays<xref ref-type="table-fn" rid="t6fn1"><italic>a</italic></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="center" valign="middle">rRT-PCR result</th><th colspan="2" rowspan="1" align="center" valign="bottom">No. (%) of samples with indicated RT-PCR result
<hr></hr></th><th colspan="1" rowspan="2" align="center" valign="middle">Total no. (%) of samples</th></tr><tr><th colspan="1" rowspan="1" align="center" valign="bottom">+</th><th colspan="1" rowspan="1" align="center" valign="bottom">−</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="center" valign="top">+</td><td colspan="1" rowspan="1" align="center" valign="top">36 (17.8)<xref ref-type="table-fn" rid="t6fn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">3 (31.5)<xref ref-type="table-fn" rid="t6fn3"><italic>c</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">39 (19.3)</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top">−</td><td colspan="1" rowspan="1" align="center" valign="top">2 (1)<xref ref-type="table-fn" rid="t6fn4"><italic>d</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="top">161 (79.7)</td><td colspan="1" rowspan="1" align="center" valign="top">163 (80.1)</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top">Total</td><td colspan="1" rowspan="1" align="center" valign="top">38 (18.8)</td><td colspan="1" rowspan="1" align="center" valign="top">164 (80)</td><td colspan="1" rowspan="1" align="center" valign="top">202 (100)</td></tr></tbody></table><table-wrap-foot><fn id="t6fn1"><label>a</label><p>Detection by rRT-PCR was compared with that by RT-PCR using influenza viruses in NPS samples, by reaction 1.</p></fn><fn id="t6fn2"><label>b</label><p>Two specimens were positive for subtype H1, and 34 specimens were positive for subtype H3 in reaction 3.</p></fn><fn id="t6fn3"><label>c</label><p>One specimen was positive for subtype H3 in reaction 3.</p></fn><fn id="t6fn4"><label>d</label><p>Two specimens were positive for subtype H3 in reaction 3.</p></fn></table-wrap-foot></table-wrap></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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