Subtype Identification Of Avian Influenza Inactivated Vaccine By Realtime Fluorescence PCR

Immunity with vaccine is an important measure to prevent the outbreak of avian influenza (AI), Chinese government attaches great importance to the quality of AI vaccine. Strengthen the supervision of vaccine quality is an important function of our institute. We assumed national AI cracking project to make research on the establishment of rapid, sensitive and specific method to differentially detect the H5, H9 and H7 subtype AI vaccine. The method will give technical support to quality supervision of vaccine. We plan to detect the AI vaccine using realtime fluorescence PCR.Specific primers and Taqman MGB probes were designed and synthesized according to the conserved region of the H5 AIV hemagglutinin gene to detect the H5 subtype AI inactivated vaccine by realtime fluorescence PCR. It had no cross-reaction with other avian disease vaccines, so the method was specific. Inter-assay and intra-assay showed the method had good reproducibility. We also produced a standard plasmid including HA gene and got a series of gradiently diluted H5 RNA standard by in-vitro transcription. We drew a standard curve using these H5 RNA standards. The standard curve showed the sensitivity was 10 copies/reaction. The linear correlation equation was Ct = -3.347424 ×Log Copy number +35. 885406. If we know the Ct value by fluorescence PCR, then we can get the copy number of genes.50 lots of H5 subtype AI inactivated vaccine were detected by fluorescence PCR and we got the copy number of HA gene. The correlation was also compared between the copy number of HA gene and the HI antibody titers of 50 lots of vaccines. We found there was not correlation. So it was not feasible to evaluate the efficacy of the inactivated vaccine by detecting the number of HA gene by fluorescence PCR. But we can detect the HA gene number and know the AIV (avian influenza virus) number in tissue.It has some significance.Specific primers and Taqman MGB probes were also designed and synthesized according to the conserved region of the H9 AIV hemagglutinin gene. We detected the H9 subtype AI inactivated vaccine using fluorescence PCR. The results showed this method was specific and stable. No cross-reaction was found with other avian disease vaccines.Specific primers and Taqman MGB probes were also designed and synthesized according to the conserved region of the H7 AIV hemagglutinin gene. We detected the H7 subtype...