Rapid quantitative detection of H5 subtype avian influenza inactivated vaccine by real-time fluorescent PCR and exploration of vaccine efficacy by in vitro test
Objective To establish a specific, sensitive, rapid method to detect virus gene of H5 sub- type inactivated vaccine and to analyze the correlation between the gene copy number and the HI antibody titer. Methods Specific primers and TaqMan MGB(minor groove binder) probes were designed with Primer Express 2.0 software according to the conserved region of the H5 subtype AIV hemagglutinin (HA) gene to make research of real-time fluorescent PCR in the quantitative detection of H5 subtype avian influenza inactivated vaccine. A se- ries of gradient diluted H5 RNA standard was prepared by in vitro transcription to draw a standard curve. Fifty batches of H5 subtype avian influenza inactivated vaccine were detected by fluorescence PCR and the copy number of HA gene of each vaccine was obtained. The correlation was analyzed between the copy number of HA gene and the HI antibody titers of 50 batches of vaccines. Results The standard curve showed the sensitivity was 10 copies/ reaction. The correlation coefficient of standard curve was 0.998003. The result showed that the method was spe- cific and reproducible. No cross-reaction was discovered with other avian disease vaccines. There was no correla- tion between the copy number of HA gene and the HI antibody titers of the vaccine. Conclusion Real-time fluo- rescent PCR provided a specific, sensitive and rapid method for the quantitative detection of virus gene of H5 sub- type avian influenza inactivated vaccine.