Real-time RT-PCR assays for type and subtype detection of influenza A and B viruses

Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of 'type,' i.e., influenza virus A and B, and 'subtype,' i.e., H1, H3, and H5, specific, single-step/reaction vessel format, real-time RTPCR assays using total RNA from archived reference strains, shell-vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. 'Type' and 'subtype' specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field-deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.