A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus
Identifieur interne : 000304 ( Ncbi/Checkpoint ); précédent : 000303; suivant : 000305A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus
Auteurs : Alejandra Castillo Alvarez [Australie] ; Marion Eg Brunck [Australie] ; Victoria Boyd [Australie] ; Richard Lai [Australie] ; Elena Virtue [Australie] ; Wenbin Chen [Australie] ; Cheryl Bletchly [Australie] ; Hans G. Heine [Australie] ; Ross Barnard [Australie]Source :
- Virology Journal [ 1743-422X ] ; 2008.
Abstract
The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.
Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.
Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.
Url:
DOI: 10.1186/1743-422X-5-77
PubMed: 18613963
PubMed Central: 2483269
Affiliations:
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last="Brunck">Marion Eg Brunck</name><affiliation wicri:level="1"><nlm:aff id="I1">Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author><author><name sortKey="Boyd, Victoria" sort="Boyd, Victoria" uniqKey="Boyd V" first="Victoria" last="Boyd">Victoria Boyd</name><affiliation wicri:level="1"><nlm:aff id="I2">CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic</wicri:regionArea><wicri:noRegion>Vic</wicri:noRegion></affiliation></author><author><name sortKey="Lai, Richard" sort="Lai, Richard" uniqKey="Lai R" first="Richard" last="Lai">Richard Lai</name><affiliation wicri:level="1"><nlm:aff id="I1">Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author><author><name sortKey="Virtue, Elena" sort="Virtue, Elena" uniqKey="Virtue E" first="Elena" last="Virtue">Elena Virtue</name><affiliation wicri:level="1"><nlm:aff id="I2">CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic</wicri:regionArea><wicri:noRegion>Vic</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease, The University of Queensland, St. Lucia, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease, The University of Queensland, St. Lucia, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Wenbin" sort="Chen, Wenbin" uniqKey="Chen W" first="Wenbin" last="Chen">Wenbin Chen</name><affiliation wicri:level="1"><nlm:aff id="I4">Pathology Queensland, Central Laboratory, Herston Hospitals Campus, Herston, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Pathology Queensland, Central Laboratory, Herston Hospitals Campus, Herston, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author><author><name sortKey="Bletchly, Cheryl" sort="Bletchly, Cheryl" uniqKey="Bletchly C" first="Cheryl" last="Bletchly">Cheryl Bletchly</name><affiliation wicri:level="1"><nlm:aff id="I4">Pathology Queensland, Central Laboratory, Herston Hospitals Campus, Herston, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Pathology Queensland, Central Laboratory, Herston Hospitals Campus, Herston, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author><author><name sortKey="Heine, Hans G" sort="Heine, Hans G" uniqKey="Heine H" first="Hans G" last="Heine">Hans G. Heine</name><affiliation wicri:level="1"><nlm:aff id="I2">CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>CSIRO livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Vic</wicri:regionArea><wicri:noRegion>Vic</wicri:noRegion></affiliation></author><author><name sortKey="Barnard, Ross" sort="Barnard, Ross" uniqKey="Barnard R" first="Ross" last="Barnard">Ross Barnard</name><affiliation wicri:level="1"><nlm:aff id="I1">Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Biochip Innovations Pty Ltd., 8 Mile Plains, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="I5">School of Molecular and Microbial Sciences, The University of Queensland, St. Lucia, Queensland, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>School of Molecular and Microbial Sciences, The University of Queensland, St. Lucia, Queensland</wicri:regionArea><wicri:noRegion>Queensland</wicri:noRegion></affiliation></author></analytic><series><title level="j">Virology Journal</title><idno type="eISSN">1743-422X</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.</p></sec><sec><title>Results</title><p>Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.</p></sec><sec><title>Conclusion</title><p>Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Australie</li></country></list><tree><country name="Australie"><noRegion><name sortKey="Alvarez, Alejandra Castillo" sort="Alvarez, Alejandra Castillo" uniqKey="Alvarez A" first="Alejandra Castillo" last="Alvarez">Alejandra Castillo Alvarez</name></noRegion><name sortKey="Barnard, Ross" sort="Barnard, Ross" uniqKey="Barnard R" first="Ross" last="Barnard">Ross Barnard</name><name sortKey="Barnard, Ross" sort="Barnard, Ross" uniqKey="Barnard R" first="Ross" last="Barnard">Ross Barnard</name><name sortKey="Bletchly, Cheryl" sort="Bletchly, Cheryl" uniqKey="Bletchly C" first="Cheryl" last="Bletchly">Cheryl Bletchly</name><name sortKey="Boyd, Victoria" sort="Boyd, Victoria" uniqKey="Boyd V" first="Victoria" last="Boyd">Victoria Boyd</name><name sortKey="Brunck, Marion Eg" sort="Brunck, Marion Eg" uniqKey="Brunck M" first="Marion Eg" last="Brunck">Marion Eg Brunck</name><name sortKey="Chen, Wenbin" sort="Chen, Wenbin" uniqKey="Chen W" first="Wenbin" last="Chen">Wenbin Chen</name><name sortKey="Heine, Hans G" sort="Heine, Hans G" uniqKey="Heine H" first="Hans G" last="Heine">Hans G. Heine</name><name sortKey="Lai, Richard" sort="Lai, Richard" uniqKey="Lai R" first="Richard" last="Lai">Richard Lai</name><name sortKey="Virtue, Elena" sort="Virtue, Elena" uniqKey="Virtue E" first="Elena" last="Virtue">Elena Virtue</name><name sortKey="Virtue, Elena" sort="Virtue, Elena" uniqKey="Virtue E" first="Elena" last="Virtue">Elena Virtue</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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