Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting
We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses. A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuram...
Gespeichert in:
| Veröffentlicht in: | PloS one 2011-06, Vol.6 (6), p.e21446-e21446 |
|---|---|
| Hauptverfasser: | , , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | e21446 |
|---|---|
| container_issue | 6 |
| container_start_page | e21446 |
| container_title | PloS one |
| container_volume | 6 |
| creator | Tong, Steven Y C Dakh, Farshid Hurt, Aeron C Deng, Yi-Mo Freeman, Kevin Fagan, Peter K Barr, Ian G Giffard, Philip M |
| description | We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.
A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was 32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.
The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations. |
| doi_str_mv | 10.1371/journal.pone.0021446 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_plos_</sourceid><recordid>TN_cdi_plos_journals_1304812726</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A476886821</galeid><doaj_id>oai_doaj_org_article_6e5b1f5b85a949688f884df01347d8af</doaj_id><sourcerecordid>A476886821</sourcerecordid><originalsourceid>FETCH-LOGICAL-c691t-3417b6d5c89a74043a516f73b5c3d10398c696489ad3b3a0157cc84ed7d6737c3</originalsourceid><addsrcrecordid>eNqNk92K1DAUx4so7rr6BqIBQfGis0mTJumNMAzqDCyujKvgVUiTtM3SNrNNurg-gw9t5muZkb2QBBKS3_mfj-QkyUsEJwgzdH7txqGX7WTlejOBMEOE0EfJKSpwltIM4scH-5PkmffXEOaYU_o0OckQw4jl-DT5s5Qrq4E2wahgXQ9cBUJjwDxj-U_gvGmD7OytHcBgvPVB9sqAbgxyA9v1rNrR9L8lmJ7P0RcEMggLUN4Bb_u6NaCU3oCVjALLq_TrbAlkr0Fj6yaNgq4dNzpddBPx58mTSrbevNitZ8n3Tx-vZvP04vLzYja9SBUtUEgxQaykOle8kIxAgmWOaMVwmSusEcQFjxwl8VbjEkuIcqYUJ0YzTRlmCp8lr7e6q9Z5sSukFwhDwlHGMhqJxZbQTl6L1WA7OdwJJ63YHLihFnIIVrVGUJOXqMpLnsuCFJTzinOiK4gwYZrLKmp92Hkby85oZfowyPZI9Pimt42o3a3AKMOY8CjwbicwuJvR-CA665VpW9kbN3rBWR6T5Wwd9pt_yIeT21G1jPHHB3TRrVpriilhMQPKMxSpyQNUHNp0VsVPV9l4fmTw_sggMsH8CrUcvReLb8v_Zy9_HLNvD9jGyDY0-5_jj0GyBdXgvB9MdV9jBMW6Z_bVEOueEbueiWavDt_n3mjfJPgv1sQPig</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1304812726</pqid></control><display><type>article</type><title>Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting</title><source>MEDLINE</source><source>DOAJ Directory of Open Access Journals</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Public Library of Science (PLoS)</source><source>PubMed Central</source><source>Free Full-Text Journals in Chemistry</source><creator>Tong, Steven Y C ; Dakh, Farshid ; Hurt, Aeron C ; Deng, Yi-Mo ; Freeman, Kevin ; Fagan, Peter K ; Barr, Ian G ; Giffard, Philip M</creator><contributor>Poon, Leo L. M.</contributor><creatorcontrib>Tong, Steven Y C ; Dakh, Farshid ; Hurt, Aeron C ; Deng, Yi-Mo ; Freeman, Kevin ; Fagan, Peter K ; Barr, Ian G ; Giffard, Philip M ; Poon, Leo L. M.</creatorcontrib><description>We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.
A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.
The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0021446</identifier><identifier>PMID: 21731753</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amplification ; Antiviral drugs ; Assaying ; Base Pairing - genetics ; Biology ; Dilution ; Drug Resistance, Viral - drug effects ; Drug Resistance, Viral - genetics ; Exo-a-sialidase ; High resolution ; Humans ; Influenza ; Influenza A ; Influenza A Virus, H1N1 Subtype - drug effects ; Influenza A Virus, H1N1 Subtype - genetics ; Laboratories ; Low cost ; Medicine ; Melting ; Mutants ; Mutation ; Mutation - genetics ; Neuraminidase - genetics ; Nucleic Acid Denaturation - genetics ; Oseltamivir ; Oseltamivir - pharmacology ; Oseltamivir phosphate ; Pandemics ; Polymerase chain reaction ; Reference Standards ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction - methods ; Single-nucleotide polymorphism ; Temperature ; Viruses</subject><ispartof>PloS one, 2011-06, Vol.6 (6), p.e21446-e21446</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Tong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Tong et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c691t-3417b6d5c89a74043a516f73b5c3d10398c696489ad3b3a0157cc84ed7d6737c3</citedby><cites>FETCH-LOGICAL-c691t-3417b6d5c89a74043a516f73b5c3d10398c696489ad3b3a0157cc84ed7d6737c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123348/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123348/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2095,2914,23846,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21731753$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Poon, Leo L. M.</contributor><creatorcontrib>Tong, Steven Y C</creatorcontrib><creatorcontrib>Dakh, Farshid</creatorcontrib><creatorcontrib>Hurt, Aeron C</creatorcontrib><creatorcontrib>Deng, Yi-Mo</creatorcontrib><creatorcontrib>Freeman, Kevin</creatorcontrib><creatorcontrib>Fagan, Peter K</creatorcontrib><creatorcontrib>Barr, Ian G</creatorcontrib><creatorcontrib>Giffard, Philip M</creatorcontrib><title>Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.
A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.
The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.</description><subject>Amplification</subject><subject>Antiviral drugs</subject><subject>Assaying</subject><subject>Base Pairing - genetics</subject><subject>Biology</subject><subject>Dilution</subject><subject>Drug Resistance, Viral - drug effects</subject><subject>Drug Resistance, Viral - genetics</subject><subject>Exo-a-sialidase</subject><subject>High resolution</subject><subject>Humans</subject><subject>Influenza</subject><subject>Influenza A</subject><subject>Influenza A Virus, H1N1 Subtype - drug effects</subject><subject>Influenza A Virus, H1N1 Subtype - genetics</subject><subject>Laboratories</subject><subject>Low cost</subject><subject>Medicine</subject><subject>Melting</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Mutation - genetics</subject><subject>Neuraminidase - genetics</subject><subject>Nucleic Acid Denaturation - genetics</subject><subject>Oseltamivir</subject><subject>Oseltamivir - pharmacology</subject><subject>Oseltamivir phosphate</subject><subject>Pandemics</subject><subject>Polymerase chain reaction</subject><subject>Reference Standards</subject><subject>Reproducibility of Results</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>Single-nucleotide polymorphism</subject><subject>Temperature</subject><subject>Viruses</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk92K1DAUx4so7rr6BqIBQfGis0mTJumNMAzqDCyujKvgVUiTtM3SNrNNurg-gw9t5muZkb2QBBKS3_mfj-QkyUsEJwgzdH7txqGX7WTlejOBMEOE0EfJKSpwltIM4scH-5PkmffXEOaYU_o0OckQw4jl-DT5s5Qrq4E2wahgXQ9cBUJjwDxj-U_gvGmD7OytHcBgvPVB9sqAbgxyA9v1rNrR9L8lmJ7P0RcEMggLUN4Bb_u6NaCU3oCVjALLq_TrbAlkr0Fj6yaNgq4dNzpddBPx58mTSrbevNitZ8n3Tx-vZvP04vLzYja9SBUtUEgxQaykOle8kIxAgmWOaMVwmSusEcQFjxwl8VbjEkuIcqYUJ0YzTRlmCp8lr7e6q9Z5sSukFwhDwlHGMhqJxZbQTl6L1WA7OdwJJ63YHLihFnIIVrVGUJOXqMpLnsuCFJTzinOiK4gwYZrLKmp92Hkby85oZfowyPZI9Pimt42o3a3AKMOY8CjwbicwuJvR-CA665VpW9kbN3rBWR6T5Wwd9pt_yIeT21G1jPHHB3TRrVpriilhMQPKMxSpyQNUHNp0VsVPV9l4fmTw_sggMsH8CrUcvReLb8v_Zy9_HLNvD9jGyDY0-5_jj0GyBdXgvB9MdV9jBMW6Z_bVEOueEbueiWavDt_n3mjfJPgv1sQPig</recordid><startdate>20110624</startdate><enddate>20110624</enddate><creator>Tong, Steven Y C</creator><creator>Dakh, Farshid</creator><creator>Hurt, Aeron C</creator><creator>Deng, Yi-Mo</creator><creator>Freeman, Kevin</creator><creator>Fagan, Peter K</creator><creator>Barr, Ian G</creator><creator>Giffard, Philip M</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20110624</creationdate><title>Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting</title><author>Tong, Steven Y C ; Dakh, Farshid ; Hurt, Aeron C ; Deng, Yi-Mo ; Freeman, Kevin ; Fagan, Peter K ; Barr, Ian G ; Giffard, Philip M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c691t-3417b6d5c89a74043a516f73b5c3d10398c696489ad3b3a0157cc84ed7d6737c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Amplification</topic><topic>Antiviral drugs</topic><topic>Assaying</topic><topic>Base Pairing - genetics</topic><topic>Biology</topic><topic>Dilution</topic><topic>Drug Resistance, Viral - drug effects</topic><topic>Drug Resistance, Viral - genetics</topic><topic>Exo-a-sialidase</topic><topic>High resolution</topic><topic>Humans</topic><topic>Influenza</topic><topic>Influenza A</topic><topic>Influenza A Virus, H1N1 Subtype - drug effects</topic><topic>Influenza A Virus, H1N1 Subtype - genetics</topic><topic>Laboratories</topic><topic>Low cost</topic><topic>Medicine</topic><topic>Melting</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Mutation - genetics</topic><topic>Neuraminidase - genetics</topic><topic>Nucleic Acid Denaturation - genetics</topic><topic>Oseltamivir</topic><topic>Oseltamivir - pharmacology</topic><topic>Oseltamivir phosphate</topic><topic>Pandemics</topic><topic>Polymerase chain reaction</topic><topic>Reference Standards</topic><topic>Reproducibility of Results</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - methods</topic><topic>Single-nucleotide polymorphism</topic><topic>Temperature</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tong, Steven Y C</creatorcontrib><creatorcontrib>Dakh, Farshid</creatorcontrib><creatorcontrib>Hurt, Aeron C</creatorcontrib><creatorcontrib>Deng, Yi-Mo</creatorcontrib><creatorcontrib>Freeman, Kevin</creatorcontrib><creatorcontrib>Fagan, Peter K</creatorcontrib><creatorcontrib>Barr, Ian G</creatorcontrib><creatorcontrib>Giffard, Philip M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection (ProQuest)</collection><collection>Natural Science Collection (ProQuest)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tong, Steven Y C</au><au>Dakh, Farshid</au><au>Hurt, Aeron C</au><au>Deng, Yi-Mo</au><au>Freeman, Kevin</au><au>Fagan, Peter K</au><au>Barr, Ian G</au><au>Giffard, Philip M</au><au>Poon, Leo L. M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2011-06-24</date><risdate>2011</risdate><volume>6</volume><issue>6</issue><spage>e21446</spage><epage>e21446</epage><pages>e21446-e21446</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.
A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.
The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>21731753</pmid><doi>10.1371/journal.pone.0021446</doi><tpages>e21446</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2011-06, Vol.6 (6), p.e21446-e21446 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1304812726 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amplification Antiviral drugs Assaying Base Pairing - genetics Biology Dilution Drug Resistance, Viral - drug effects Drug Resistance, Viral - genetics Exo-a-sialidase High resolution Humans Influenza Influenza A Influenza A Virus, H1N1 Subtype - drug effects Influenza A Virus, H1N1 Subtype - genetics Laboratories Low cost Medicine Melting Mutants Mutation Mutation - genetics Neuraminidase - genetics Nucleic Acid Denaturation - genetics Oseltamivir Oseltamivir - pharmacology Oseltamivir phosphate Pandemics Polymerase chain reaction Reference Standards Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction - methods Single-nucleotide polymorphism Temperature Viruses |
| title | Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-24T01%3A05%3A07IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Rapid%20detection%20of%20the%20H275Y%20oseltamivir%20resistance%20mutation%20in%20influenza%20A/H1N1%202009%20by%20single%20base%20pair%20RT-PCR%20and%20high-resolution%20melting&rft.jtitle=PloS%20one&rft.au=Tong,%20Steven%20Y%20C&rft.date=2011-06-24&rft.volume=6&rft.issue=6&rft.spage=e21446&rft.epage=e21446&rft.pages=e21446-e21446&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0021446&rft_dat=%3Cgale_plos_%3EA476886821%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1304812726&rft_id=info:pmid/21731753&rft_galeid=A476886821&rft_doaj_id=oai_doaj_org_article_6e5b1f5b85a949688f884df01347d8af&rfr_iscdi=true |