This technology includes an optimized influenza A/H5 subtyping real-time RT-PCR assay for detection and characterization of Asian lineage A/H5N1 viruses. The highly pathogenic avian influenza (HPAI) A/H5N1 viruses, first isolated in human in 1997, have spread among birds over seventy countries on three continents. In 2014 a novel reassortant HPAI A/H5N8 virus has been detected in poultry in Asia, Europe, As well as Canada and USA. In addition, genetic reassortment of this virus with circulating avian influenza viruses has resulted in the novel viral combinations. Due to the newly emerging novel reasserted A/H5Nx viruses detected in the USA, the assay, has been further updated and optimized by including extra primers and probe into the assay.