DUPLEX REAL-TIME RT-PCR ASSAY FOR DETECTION OF TYPE A AVIAN INFLUENZA VIRUS AND IDENTIFICATION OF A/H5 SUBTYPE
A duplex real-time reverse transcriptase polymerase chain reaction assay (duplex real-time RT- PCR assay) was developed to detect avian influenza type A virus and identify A/H5 subtype. In order to determine analytical sensitivity of the developed method ten-fold dilutions of allantoic fluid containing avian influenza virus isolates (AIV) with the known infectious titer were tested. Sensitivity of the duplex real-time RT- PCR assay was 1.1-3.1 lg EID50/cm3 for M gene and 3-3.4 lg EID50/ cm3 for HA gene. The developed method was used to detect AIV in biomaterial samples from wild birds. The obtained results were confirmed by virus isolation in SPF-chicken embryos. This method can be used to detect AIV in biomaterial samples collected during monitoring.