Abstract: According to the conservative region of H5 AIV HA gene,the primers were devised and synthesized. A serial 10 fold dilutions positive plasmid was prepared and used for standard. The standard curve revealed the linear relationship between Ct (cycle threshold) and template concentration with a good correlation (R²＝0.995). The RRT-PCR method for the detection of AIV was highly specific and sensitive,and it could be used for rapid quantitative detection of AIV H5 subtype. No cross-reaction was detected against other avian disease viruses. Sensitivity was 8 copies of AIV genome. The total meet rate with traditional virus isolation method was more than 90.0% in detecting 178 clinical cloacal swab samples. It showed that a real-time RT-PCR method for detection of H5 subtype avian influenza virus had been established in this study,which had properties such as high speed,specific,sensitive,cheap,high-throughput,and good biological safety as well as displaying good prospects in the rapid screening of clinical samples and epidemiological monitoring of avian influenza virus H5 subtype.

Key words: AIV H5 subtype; real-time RT-PCR; development; validation