Analytical and clinical validation of novel real-time reverse transcriptase–polymerase chain reaction assays for the clinical detection of swine-origin H1N1 influenza viruses

During the early stages of the 2009/2010 swine-origin H1N1 influenza A (S-OIV H1N1 FluA) outbreak, the development and validation of sensitive and specific detection methods were a priority for rapid and accurate diagnosis. Between May and June 2009, 2 real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) assays targeting the hemagglutinin and neuraminidase genes of the S-OIV H1N1 FluA virus were developed. These assays are highly specific, showing no cross-reactivity against a panel of respiratory viruses and can differentiate S-OIV H1N1 from seasonal FluA viruses. Analytical sensitivities of the 2 assays were found to be 10−1 tissue culture infectious dose, 50%/ml. Clinical testing showed 99.2% sensitivity and 94.6–98.1% specificity. A large prospective analysis showed that 94.8–95.5% of S-OIV positive specimens were negative by seasonal H1/H3 subtyping. The large-scale validation data presented in this report indicate that these novel assays provide an accurate and efficient method for the rapid detection of S-OIV H1N1 FluA viruses.