A multiplex reverse transcription-PCR assay for the detection of influenza A virus and differentiation of the H1, H3, H5 and H9 subtypes.

A multiplex reverse transcription-PCR (mRT-PCR) assay was developed for the rapid detection of influenza A viruses. The assay simultaneously differentiated H1, H3, H5 and H9 hemagglutinin subtypes in a single reaction mixture. Five sets of specific primers targeted to the M, H1, H3, H5 and H9 genes were used in this assay. The amplified products were visualized by agarose gel electrophoresis. The sizes of the PCR amplified fragments were 612 bp for H1, 187 bp for H3, 338 bp for H5, 289 bp for H9 and 239 bp for M. The detection limit of the viral RNA template was 1 ng for the H1, H3 and H5 subtypes and 0.1 ng for the H9 subtype. Nonspecific product bands from RNAs of other viral pathogens were not amplified. The sensitivity analysis demonstrated that the mRT-PCR assay is as sensitive as conventional RT-PCR and 10 times less sensitive than SYBR Green real-time RT-PCR. In conclusion, the mRT-PCR assay developed in this study was able to type influenza A viruses and simultaneously differentiate H1, H3, H5 and H9 subtypes in both human and avian clinical specimens, and thus, the mRT-PCR assay could be a rapid, convenient and relatively inexpensive molecular diagnostic tool for large-scale screening of clinical samples.