Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China

Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China.