Rapid detection of the S247N neuraminidase mutation in influenza A(H1N1)pdm09 virus by one-step duplex RT-PCR assay.

Neuraminidase inhibitors, especially oseltamivir, have been used worldwide for the prophylaxis or treatment of pandemic influenza H1N1 2009 (A(H1N1)pdm09) viruses. The most commonly detected oseltamivir-resistant A(H1N1)pdm09 viruses possess the H275Y mutation in neuraminidase (NA). Recently, a novel A(H1N1)pdm09 variant containing the S247N mutation in NA was found mainly in the Asia-Pacific area. This mutation reduces sensitivity to oseltamivir and confers extremely high resistance to oseltamivir in association with the H275Y mutation compared with the resistance caused by the H275Y mutation alone. In this study, a rapid and simple one-step duplex RT-PCR assay for identifying A(H1N1)pdm09 viruses possessing the S247N mutation was developed. This assay is based on an endpoint genotyping analysis method and can use isolates from cell culture supernatants without RNA extraction, similar to the H275Y RT-PCR assay reported previously. The combination of the S247N and H275Y RT-PCR assays is a powerful surveillance method for determining whether A(H1N1)pdm09 viruses with the S247N mutation acquire the H275Y mutation.