Differential diagnosis of various subtypes of influenza type A virus using RT-PCR-RFLP assay

Influenza type A virus is one of the most important viral agents in human respiratory diseases. The genetic variability of the influenza viruses leads to the incidence of new epidemics worldwide. Hence, there is a growing need for rapid and effective new methods capable of detection and differentiation of influenza virus circulating strains. This study was done to develop a method for rapid differentiation of the subtypes of influenza type A virus. In this experimental study, reverse-transcription and polymerase chain reaction (RT-PCR) were performed using a primer set based on M gene of H1N1, H3N2, H5N1, and H9N2 influenza subtypes. Then the amplified fragments were subjected to digestion using subtype specific restriction endonuclease enzymes. The results of PCR reaction showed that the primer pair of the M gene was specific and capable of amplifying all influenza subtypes understudy. Also, different restriction fragment length polymorphism patterns (RFLP) were generated using enzyme digestion reaction on the amplified segment of M gene. RT-PCR and RFLP analysis of the M gene can be employed as a useful method for differentiating influenza virus subtypes.