Switching gears for an influenza pandemic: validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus.
Rapid methods for the detection and confirmatory identification of pandemic influenza A virus (also known as pandemic [H1N1] 2009) are of utmost importance. In this study, a conventional reverse transcriptase PCR (RT-PCR) assay for the detection of influenza A virus and the hemagglutinin of swine lineage H1 (swH1) was designed, optimized, and validated. Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal, or throat swab specimens collected early in the outbreak (127 negative specimens, 66 specimens with pandemic [H1N1] 2009 influenza virus, 3 specimens with seasonal [H1N1] influenza A virus, and 2 specimens with seasonal [H3N2] influenza A virus). The performance characteristics of the duplex RT-PCR assay were assessed and compared to those of various detection methods: a monoplex RT-PCR assay at the National Microbiology Laboratory, a real-time RT-PCR assay using a Centers for Disease Control and Prevention protocol, an in-house multiplex RT-PCR assay (targeting influenza A virus, influenza B virus, and respiratory syncytial virus), and a rapid antigen test (the Binax Now Influenza A & B assay). The sensitivity of the duplex RT-PCR assay for influenza A virus detection was 97.2%, whereas the sensitivities were 74.6%, 71.8%, 47.8%, and 12.7% for the other four assays, respectively. The duplex RT-PCR assay was also able to identify swH1 in 94% of the cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmatory identification. Only a limited number of specimens that contained influenza A virus had amounts of virus that fell below the limit of detection of the assay with the swH1 primers. Overall, the duplex RT-PCR assay is a reliable method for the simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus and would be particularly attractive to laboratories without real-time RT-PCR capabilities.
Division of Microbiology, Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, 5788 University Avenue, Room 315, MacKenzie Building, Halifax, Nova Scotia B3H 1V8, Canada. jason.leblanc@cdha.nshealth.ca
Jason J
JJ
LeBlanc
Yan
Y
Li
Nathalie
N
Bastien
Kevin R
KR
Forward
Ross J
RJ
Davidson
Todd F
TF
Hatchette
D017931:DNA Primers / Q000235:genetics; D004196:Disease Outbreaks; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012621:Seasons; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral