Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay
Identifieur interne : 000F92 ( Ncbi/Merge ); précédent : 000F91; suivant : 000F93Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay
Auteurs : Sisi Luo ; Zhixun Xie ; Jiaoling Huang ; Zhiqin Xie ; Liji Xie ; Minxiu Zhang ; Meng Li ; Sheng Wang ; Dan Li ; Tingting Zeng ; Yanfang Zhang ; Qing Fan ; Xianwen DengSource :
- Frontiers in Microbiology [ 1664-302X ] ; 2019.
Abstract
To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers–nine subtype-specific pairs and one pan-AIV pair–were screened and used to establish the GeXP multiplex RT-PCR assay. A single subtype was detected using the developed GeXP assay; the N1 to N9 AIV subtypes individually generated two target peaks: the NA subtype-specific peak and the general AIV peak. Different concentrations of multiplexed subtypes were tested with this GeXP assay and the peaks of the corresponding NA subtypes were generated, suggesting that this GeXP assay is useful for identifying NA subtypes in mixed samples. Moreover, no peaks were generated for other important avian viruses, indicating negative results and validating the lack of cross-reactions between AIV subtypes and other avian pathogens. RNA templates synthesized through
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DOI: 10.3389/fmicb.2019.01271
PubMed: 31231349
PubMed Central: 6568037
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay</title><author><name sortKey="Luo, Sisi" sort="Luo, Sisi" uniqKey="Luo S" first="Sisi" last="Luo">Sisi Luo</name></author><author><name sortKey="Xie, Zhixun" sort="Xie, Zhixun" uniqKey="Xie Z" first="Zhixun" last="Xie">Zhixun Xie</name></author><author><name sortKey="Huang, Jiaoling" sort="Huang, Jiaoling" uniqKey="Huang J" first="Jiaoling" last="Huang">Jiaoling Huang</name></author><author><name sortKey="Xie, Zhiqin" sort="Xie, Zhiqin" uniqKey="Xie Z" first="Zhiqin" last="Xie">Zhiqin Xie</name></author><author><name sortKey="Xie, Liji" sort="Xie, Liji" uniqKey="Xie L" first="Liji" last="Xie">Liji Xie</name></author><author><name sortKey="Zhang, Minxiu" sort="Zhang, Minxiu" uniqKey="Zhang M" first="Minxiu" last="Zhang">Minxiu Zhang</name></author><author><name sortKey="Li, Meng" sort="Li, Meng" uniqKey="Li M" first="Meng" last="Li">Meng Li</name></author><author><name sortKey="Wang, Sheng" sort="Wang, Sheng" uniqKey="Wang S" first="Sheng" last="Wang">Sheng Wang</name></author><author><name sortKey="Li, Dan" sort="Li, Dan" uniqKey="Li D" first="Dan" last="Li">Dan Li</name></author><author><name sortKey="Zeng, Tingting" sort="Zeng, Tingting" uniqKey="Zeng T" first="Tingting" last="Zeng">Tingting Zeng</name></author><author><name sortKey="Zhang, Yanfang" sort="Zhang, Yanfang" uniqKey="Zhang Y" first="Yanfang" last="Zhang">Yanfang Zhang</name></author><author><name sortKey="Fan, Qing" sort="Fan, Qing" uniqKey="Fan Q" first="Qing" last="Fan">Qing Fan</name></author><author><name sortKey="Deng, Xianwen" sort="Deng, Xianwen" uniqKey="Deng X" first="Xianwen" last="Deng">Xianwen Deng</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31231349</idno><idno type="pmc">6568037</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568037</idno><idno type="RBID">PMC:6568037</idno><idno type="doi">10.3389/fmicb.2019.01271</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000970</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000970</idno><idno type="wicri:Area/Pmc/Curation">000970</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000970</idno><idno type="wicri:Area/Pmc/Checkpoint">000028</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000028</idno><idno type="wicri:Area/Ncbi/Merge">000F92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay</title><author><name sortKey="Luo, Sisi" sort="Luo, Sisi" uniqKey="Luo S" first="Sisi" last="Luo">Sisi Luo</name></author><author><name sortKey="Xie, Zhixun" sort="Xie, Zhixun" uniqKey="Xie Z" first="Zhixun" last="Xie">Zhixun Xie</name></author><author><name sortKey="Huang, Jiaoling" sort="Huang, Jiaoling" uniqKey="Huang J" first="Jiaoling" last="Huang">Jiaoling Huang</name></author><author><name sortKey="Xie, Zhiqin" sort="Xie, Zhiqin" uniqKey="Xie Z" first="Zhiqin" last="Xie">Zhiqin Xie</name></author><author><name sortKey="Xie, Liji" sort="Xie, Liji" uniqKey="Xie L" first="Liji" last="Xie">Liji Xie</name></author><author><name sortKey="Zhang, Minxiu" sort="Zhang, Minxiu" uniqKey="Zhang M" first="Minxiu" last="Zhang">Minxiu Zhang</name></author><author><name sortKey="Li, Meng" sort="Li, Meng" uniqKey="Li M" first="Meng" last="Li">Meng Li</name></author><author><name sortKey="Wang, Sheng" sort="Wang, Sheng" uniqKey="Wang S" first="Sheng" last="Wang">Sheng Wang</name></author><author><name sortKey="Li, Dan" sort="Li, Dan" uniqKey="Li D" first="Dan" last="Li">Dan Li</name></author><author><name sortKey="Zeng, Tingting" sort="Zeng, Tingting" uniqKey="Zeng T" first="Tingting" last="Zeng">Tingting Zeng</name></author><author><name sortKey="Zhang, Yanfang" sort="Zhang, Yanfang" uniqKey="Zhang Y" first="Yanfang" last="Zhang">Yanfang Zhang</name></author><author><name sortKey="Fan, Qing" sort="Fan, Qing" uniqKey="Fan Q" first="Qing" last="Fan">Qing Fan</name></author><author><name sortKey="Deng, Xianwen" sort="Deng, Xianwen" uniqKey="Deng X" first="Xianwen" last="Deng">Xianwen Deng</name></author></analytic><series><title level="j">Frontiers in Microbiology</title><idno type="eISSN">1664-302X</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers–nine subtype-specific pairs and one pan-AIV pair–were screened and used to establish the GeXP multiplex RT-PCR assay. A single subtype was detected using the developed GeXP assay; the N1 to N9 AIV subtypes individually generated two target peaks: the NA subtype-specific peak and the general AIV peak. Different concentrations of multiplexed subtypes were tested with this GeXP assay and the peaks of the corresponding NA subtypes were generated, suggesting that this GeXP assay is useful for identifying NA subtypes in mixed samples. Moreover, no peaks were generated for other important avian viruses, indicating negative results and validating the lack of cross-reactions between AIV subtypes and other avian pathogens. RNA templates synthesized through <italic>in vitro</italic> transcription were used to analyze the sensitivity of the assay; the limit of detection was 100 copies per reaction mixture. The results obtained from clinical samples using this GeXP method were consistent with the results of the neuraminidase inhibition (NI) test, and the ability of the GeXP assay to identify mixed infections was superior to amplicon sequencing of isolated viruses. In conclusion, this GeXP assay is proposed as a specific, sensitive, rapid, high-throughput, and versatile diagnostic tool for nine NA subtypes of AIV.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Yuan, H" uniqKey="Yuan H">H. 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Xie</name></author><author><name sortKey="Deng, X" uniqKey="Deng X">X. Deng</name></author><author><name sortKey="Xie, Z" uniqKey="Xie Z">Z. Xie</name></author><author><name sortKey="Luo, S" uniqKey="Luo S">S. Luo</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Microbiol</journal-id><journal-id journal-id-type="iso-abbrev">Front Microbiol</journal-id><journal-id journal-id-type="publisher-id">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type="epub">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31231349</article-id><article-id pub-id-type="pmc">6568037</article-id><article-id pub-id-type="doi">10.3389/fmicb.2019.01271</article-id><article-categories><subj-group subj-group-type="heading"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Luo</surname><given-names>Sisi</given-names></name><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/695330/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Zhixun</given-names></name><xref ref-type="corresp" rid="c001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/333891/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Jiaoling</given-names></name></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Zhiqin</given-names></name></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Liji</given-names></name></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Minxiu</given-names></name><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/377468/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Meng</given-names></name></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Sheng</given-names></name></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Dan</given-names></name></contrib><contrib contrib-type="author"><name><surname>Zeng</surname><given-names>Tingting</given-names></name></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Yanfang</given-names></name></contrib><contrib contrib-type="author"><name><surname>Fan</surname><given-names>Qing</given-names></name></contrib><contrib contrib-type="author"><name><surname>Deng</surname><given-names>Xianwen</given-names></name></contrib></contrib-group><aff><sup>1</sup><institution>Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute</institution>,<addr-line>Nanning</addr-line>,<country>China</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: Juan-Carlos Saiz, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain</p></fn><fn fn-type="edited-by"><p>Reviewed by: Xiaorong Peng, Zhejiang University, China; Huaguang Lu, Pennsylvania State University, United States</p></fn><corresp id="c001">*Correspondence: Zhixun Xie, <email>xiezhixun@126.com</email></corresp><fn fn-type="other" id="fn002"><p>This article was submitted to Virology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type="epub"><day>07</day><month>6</month><year>2019</year></pub-date><pub-date pub-type="collection"><year>2019</year></pub-date><volume>10</volume><elocation-id>1271</elocation-id><history><date date-type="received"><day>28</day><month>2</month><year>2019</year></date><date date-type="accepted"><day>22</day><month>5</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2019 Luo, Xie, Huang, Xie, Xie, Zhang, Li, Wang, Li, Zeng, Zhang, Fan and Deng.</copyright-statement><copyright-year>2019</copyright-year><copyright-holder>Luo, Xie, Huang, Xie, Xie, Zhang, Li, Wang, Li, Zeng, Zhang, Fan and Deng</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>To date, nine neuraminidase (NA) subtypes of avian influenza virus (AIV) have been identified in poultry and wild birds. Rapid and effective methods for differentiating these nine NA subtypes are needed. We developed and validated a rapid, sensitive, and robust method utilizing a GeXP analyzer-based multiplex RT-PCR assay and capillary electrophoresis for the simultaneous differentiation of the N1 to N9 subtypes in a single-tube reaction. Ten pairs of primers–nine subtype-specific pairs and one pan-AIV pair–were screened and used to establish the GeXP multiplex RT-PCR assay. A single subtype was detected using the developed GeXP assay; the N1 to N9 AIV subtypes individually generated two target peaks: the NA subtype-specific peak and the general AIV peak. Different concentrations of multiplexed subtypes were tested with this GeXP assay and the peaks of the corresponding NA subtypes were generated, suggesting that this GeXP assay is useful for identifying NA subtypes in mixed samples. Moreover, no peaks were generated for other important avian viruses, indicating negative results and validating the lack of cross-reactions between AIV subtypes and other avian pathogens. RNA templates synthesized through <italic>in vitro</italic> transcription were used to analyze the sensitivity of the assay; the limit of detection was 100 copies per reaction mixture. The results obtained from clinical samples using this GeXP method were consistent with the results of the neuraminidase inhibition (NI) test, and the ability of the GeXP assay to identify mixed infections was superior to amplicon sequencing of isolated viruses. In conclusion, this GeXP assay is proposed as a specific, sensitive, rapid, high-throughput, and versatile diagnostic tool for nine NA subtypes of AIV.</p></abstract><kwd-group><kwd>avian influenza virus</kwd><kwd>neuraminidase</kwd><kwd>GeXP analyzer</kwd><kwd>multiplex PCR</kwd><kwd>differentiation diagnosis</kwd></kwd-group><counts><fig-count count="2"></fig-count><table-count count="3"></table-count><equation-count count="0"></equation-count><ref-count count="26"></ref-count><page-count count="10"></page-count><word-count count="0"></word-count></counts></article-meta></front></pmc><affiliations><list></list><tree><noCountry><name sortKey="Deng, Xianwen" sort="Deng, Xianwen" uniqKey="Deng X" first="Xianwen" last="Deng">Xianwen Deng</name><name sortKey="Fan, Qing" sort="Fan, Qing" uniqKey="Fan Q" first="Qing" last="Fan">Qing Fan</name><name sortKey="Huang, Jiaoling" sort="Huang, Jiaoling" uniqKey="Huang J" first="Jiaoling" last="Huang">Jiaoling Huang</name><name sortKey="Li, Dan" sort="Li, Dan" uniqKey="Li D" first="Dan" last="Li">Dan Li</name><name sortKey="Li, Meng" sort="Li, Meng" uniqKey="Li M" first="Meng" last="Li">Meng Li</name><name sortKey="Luo, Sisi" sort="Luo, Sisi" uniqKey="Luo S" first="Sisi" last="Luo">Sisi Luo</name><name sortKey="Wang, Sheng" sort="Wang, Sheng" uniqKey="Wang S" first="Sheng" last="Wang">Sheng Wang</name><name sortKey="Xie, Liji" sort="Xie, Liji" uniqKey="Xie L" first="Liji" last="Xie">Liji Xie</name><name sortKey="Xie, Zhiqin" sort="Xie, Zhiqin" uniqKey="Xie Z" first="Zhiqin" last="Xie">Zhiqin Xie</name><name sortKey="Xie, Zhixun" sort="Xie, Zhixun" uniqKey="Xie Z" first="Zhixun" last="Xie">Zhixun Xie</name><name sortKey="Zeng, Tingting" sort="Zeng, Tingting" uniqKey="Zeng T" first="Tingting" last="Zeng">Tingting Zeng</name><name sortKey="Zhang, Minxiu" sort="Zhang, Minxiu" uniqKey="Zhang M" first="Minxiu" last="Zhang">Minxiu Zhang</name><name sortKey="Zhang, Yanfang" sort="Zhang, Yanfang" uniqKey="Zhang Y" first="Yanfang" last="Zhang">Yanfang Zhang</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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