Single-step real-time reverse transcription-polymerase chain reaction for simultaneous detection of H5N1 and H5N8 highly pathogenic avian influenza viruses

For efficient monitoring of Highly Pathogenic Avian Influenza Viruses (HPAIVs) in countries where various subtype H5 HPAIVs co-circulate in avian populations, a Simultaneous Differential Detection Method for major and minor subtypes of HPAIVs is needed. In this study, we developed a single-step Real-time Reverse Transcription-Polymerase Chain Reaction (sRRT-PCR) assay for the simultaneous detection of HPAIVM, H5, Nl and A/5 genes. The M gene primer set in the assay detected all subtypes of AIV including representative H5N1 andH5N8 strains from Korea as reported in earlier studies and the H5, Nl and A/5 gene primer sets were able to specifically detect H5, Nl and N8 subtypes of HP AIV, respectively. The detection limits for the M, H5, and Nl or 7V5 genes were 0.6, 0.7 and 0.9 EID₅₀ for H5N1 HP AIV and 1.0, 0.8 and 1.3 EID₅₀ for H5N8 HP AIV, respectively. The assay was sufficiently sensitive for monitoring and surveillance of HPAIVs and can be used in the differential diagnosis of H5N1, H5N8 and other subtypes of AIV in countries where subtype H5 HP AIV outbreaks are occurring.