Multiplex real-time reverse transcription polymerase chain reaction for differential detection of H5, N1, and N8 genes of highly pathogenic avian influenza viruses

Rapid and differential diagnosis of highly pathogenic avian influenza virus (HPAIV) subtype H5 is essential for the effective prevention and control of outbreaks caused by this pathogen. In this study, we describe a one-step multiplex real-time reverse transcription polymerase chain reaction (mRRT-PCR), using H5-, N1-, and N8-specific primers and probes, for differential detection of two HPAIVs (H5N1 and H5N8) and other H5-subtype AIVs. Using the mRRT-PCR assay, we were able to detect H5N1, H5N8, and other H5-subtype AIVs in a one-tube reaction, with high specificity; furthermore, using an in silico PCR program, we confirmed that this assay can detect nearly all H5, N1, and N8 genes of AIVs currently available in the Influenza Sequence Database. The limit of detection of the assay was determined to be as low as 100 copies/reaction for each target gene, and was comparable to limits of detection of previously reported mRRT-PCR assays. Thus, the mRRT-PCR assay described here can serve as a rapid and reliable differential diagnostic tool for the monitoring and surveillance of H5N1, H5N8, and other H5-subtype AIVs in countries where these pathogens are problematic.