A modified strategy of multiplex RT-PCR for simultaneous detection of H5, H7, and H9 subtypes of avian influenza virus based on common forward oligo

Abstract Avian influenza virus subtypes H5, H7, and H9 related epidemics are accountable for huge losses to the poultry and avian industry in Pakistan. Well-timed and accurate diagnosis of subtype(s) concomitant with a specific epidemic allows a margin of prophylaxis to farmers through requisite vaccination. The current study was designed to develop and validate a rapid, accurate, and efficient diagnostic technique based on a modified strategy in reverse transcription PCR (RT-PCR) for simultaneous detection of avian influenza virus subtypes H5, H7, and H9. Instead of 3 pairs of primers, a single common forward sense primer and 3 negative sense primers specific for 3 subtypes were used. Genotyping on 2% agarose gel sorted out specific products of desired sizes for individual and mixture samples of three subtypes.