Multiplex Reverse Transcriptase-PCR Assay for Typing and Subtyping of Influenza A (H5 & H9) Virus in Iran

Avian influenza virus (AIV) infection is a major cause of bird or human mortality and morbidity, therefore the rapid identification of the virus is of important clinical and epidemiological implication. Methods: A multiplex Reverse Transcriptase PCR (RT-PCR) was optimized for the detection of influenza A virus and the H5 and H9 subtypes. The influenza type A specific primers were directed to the region of the influenza A matrix gene that is conserved among most type A influenza viruses. The H5 and H9 primers were directed to H5 and H9 hemagglutinin (HA) gene regions that are conserved among H5 and H9 subtypes. The selected primer sets were used in the RT-PCR for simultaneous detection of matrix, H5 and H9 responding specific sequences in a multiplex format. Results: Three reaction conditions were optimized which include: i) RT-PCR typing using matrix gene primers for five subtypes of flu A (H1, H3, H5, H7 and H9), ii) RT-PCR subtyping for H5 and H9 subtypes, and iii) multiplex subtyping of H5 and H9. In this study, the multiplex RT-PCR was applied to 147 cloacal and tracheal swabs of clinical poultry cases with similar influenza symptoms. Conclusions: These results suggest that multiplex RT-PCR assay can be a useful test for rapid detection and subtyping of AIV in clinical samples.