Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR.
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.
Stone
Belinda
B
Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, P.O. Box 14, Frome Road, South Australia, Rundle Mall, S.A. 5000, Australia.
Burrows
Julie
J
Schepetiuk
Sonia
S
Higgins
Geoff
G
Hampson
Alan
A
Shaw
Robert
R
Kok
TuckWeng
T
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
D012367:RNA, Viral; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins