Multiplex reverse transcription-PCR assay for detection of type A influenza virus plus differentiation of avian H7 and H9 hemagglutinin subtypes in Iran

ABSTRACT

Avian influenza virus [AIV] infection is a major cause of bird and human morbidity and mortality. We aimed to evaluate a specific and sensitive multiplex RT-PCR that can simultaneously detect influenza type A viruses and differentiate the two most important subtypes of avian influenza viruses H7 and H9 subtypes. A multiplex reverse transcriptase-polymerase chain reaction [mRT-PCR] was developed and optimized for the detection of type A influenza virus. Simultaneously avian H7 and H9 hemagglutinin subtypes was differentiated. Three sets of specific oligonucleotide primers were used in this test for type A influenza virus, H7 and H9 heamagglutinin subtypes. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 313 bp for H7 and 428 bp for H9 hemagglutinin subtypes, and 101 bp for type A influenza virus. The common set of primers for type A influenza virus were able to amplify a 101 bp DNA band for any of the other subtypes of influenza A virus The mRT-PCR assay developed in this study was found to be sensitive and specific. No specific amplification bands of the same sizes [313 and 428 bp] could be amplified for RNA of other influenza hemagglutinin subtypes, nor specific amplification bands of type A influenza [101 bp] for Influenza B, C, or other viral or bacterial pathogens tested in this study