A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

Alvarez, Alejandra Castillo, Brunck, Mario E. G., Boyd, Victoria, Lai, Richard, Virtue, Elena, Chen, Wenbin, Bletchly, Cheryl, Heine, Hans G. and Barnard, Ross (2008) A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus. Virology Journal, 5 77: 1-11.


Author Alvarez, Alejandra Castillo
Brunck, Mario E. G.
Boyd, Victoria
Lai, Richard
Virtue, Elena
Chen, Wenbin
Bletchly, Cheryl
Heine, Hans G.
Barnard, Ross
Title A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus
Journal name Virology Journal   Check publisher's open access policy
ISSN 1743-422X
Publication date 2008-07-09
Sub-type Article (original research)
DOI 10.1186/1743-422X-5-77
Volume 5
Issue 77
Start page 1
End page 11
Total pages 11
Place of publication London, United Kingdom
Publisher BioMed
Language eng
Subject 1108 Medical Microbiology
Formatted abstract Background: 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.
Results:
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.
Conclusion: 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.
Keyword Influenza A virus
Neuraminidase genes
PCR
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemistry and Molecular Biosciences
 
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Created: Thu, 03 Sep 2009, 09:40:07 EST by Mr Andrew Martlew on behalf of Faculty of Science