Abstract: Combining nested PCR (NP-PCR) with real-time RT-PCR,superimposing advantages of sensitivity and specificity of these two methods,establwhed nested real-time RT-PCR for detecting AIV and subtype H5,which was more sensitive than conventional real-time RT-PCR. Basing on the conserved AIV NP and HA gene downloaded from GenBank,designed nested real-time RT-PCR primers and TaqMan probes for detecting AIV and subtype H5. Target segments recombined plasmids were constructed,and positive controls were prepared with technology of in vitro transcription.Nested real-time RT-PCR was established after reaction mixtures and condition screened out. Test results indicated that this established method can specific detect AIV and H5 subtype,19 of 30 fish aquaculture water samples were presented positive results,multitudinous samples can be detected simultaneously within 5 h. It is a good method for detecting trace AIV from samples of the natural environment,with distinguishing feature of high sensitivity,good specificity,high throughout and rapidity.

Key words: influenza A viruse; general type subtype H5; nested real-time RT-PCR; detection method