A multiplex real-time RT-PCR method for detecting H5, H7 and H9 subtype avian influenza viruses in field and clinical samples

•A quadruplex real-time RT-PCR method for detecting H5, H7 and H9 subtype AIVs was developed with high sensitivity and specificity.•The clinical performance of the method was evaluated by various specimens from poultries and humans.•The multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis. In recent years, H5 and H7 subtypes of highly pathogenic avian influenza viruses (HPAIVs) have been identified in poultry worldwide, resulting in large economic losses to poultry production. Furthermore, H9N2 low pathogenic AIVs are reported to provide internal genes for generating novel reassortant AIVs, leading to potential pandemic risks. To establish an accurate, sensitive and convenient diagnostic method for H5, H7 and H9 subtype AIVs in Eurasian lineage, four groups of specific primers and probes were designed based on the conserved fragments of M, H5, H7 and H9 genes, and a multiplex real-time RT-PCR (RRT-PCR) method was established. High sensitivity was achieved for the multiplex RRT-PCR approach, with a detection limit of 1−10 copies (plasmid DNA) per reaction. The specificity of the method was evaluated using diverse subtypes of AIVs and other avian respiratory viruses isolated in eastern China over the last 9 years. Compared with virus isolation, a higher consistency was achieved when assessing 135 field samples and 126 clinical samples. The results showed that the multiplex RRT-PCR method is a fast, convenient and practical method for AIV clinical detection and epidemiological analysis.