title: Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR.
abstract: A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.
authors.affiliation: Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, P.O. Box 14, Frome Road, South Australia, Rundle Mall, S.A. 5000, Australia.
authors.forename: Belinda
authors.initials: B
authors.lastname: Stone
authors.forename: Julie
authors.initials: J
authors.lastname: Burrows
authors.forename: Sonia
authors.initials: S
authors.lastname: Schepetiuk
authors.forename: Geoff
authors.initials: G
authors.lastname: Higgins
authors.forename: Alan
authors.initials: A
authors.lastname: Hampson
authors.forename: Robert
authors.initials: R
authors.lastname: Shaw
authors.forename: TuckWeng
authors.initials: T
authors.lastname: Kok
mesh_terms: D001483:Base Sequence; D005814:Genes, Viral; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D012367:RNA, Viral; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins