| Marek's disease(MD)is a highly contagious,malignant T-lymphomatosis of chickens.The etiological agent is Marek's disease virus(MDV) and belong to the Alpha herpesvirinae.The MDV group consists of three closely related viruses with many antigens in common,and their division into individual serotypes,namely serotype 1,2,and 3.Today MD is the only one in all tumorous disease that can be prevented by vaccine. HVT vaccine, its representative poisonous Fc-126, takes one kind of freeze-drying vaccine, from formulating until now it widely be used in our country and played the vital role in preventting and controlling MD.The turkey herpes virus can send CEF to pathological changes, The potency examination of HVT vaccine usually takes the plaque technology. The result of measuring poisonous not only receives intrinsic factor like different chicken embryo, extraneous virus and so on;but also external factor like nourishing cream pH value, covering agar-agar quantity, viral diluenand so on; The experimental operation is tedious and the cycle is too long.Moreover,the HVT vaccine can not be massly detected. Therefore, how fast,accurately effective determined the live virus's content in the HVT vaccine is the key link of this vaccine potency examination, is also the one of important reference norms wich success prevents MDThis research is for the purpose which establishes corresponding relationships between Viral content and plaque-forming unit (PFU)counting in the turkey herpes virus vaccine,which determines the live virus's content.First,published sequences of the selected genes(SORF1 for HVT)were obtained from GenBank. A pair of primers and a internal dual-labeled fluorogenic probes were designed and synthesized.The fragments were cloned into T vector and transformed into DH5a.The positive recombinant plasmids were used as standard quantitative template. Carries on FQ-PCR increasing, obtains the specification curve.This method examination's sensitivity can reach 3.97 copies/μL. No positive was observed, taking newcastle disease virus(NDV), avian influenza virus (H9), infectious pancreatic necrosis virus (IBDV), avian leukosis viruses (ALV), avian encephalomyetitis virus (AEV), avian infectious bronchitis virus (AIBV) and double distilled water as the template. The coefficient of variation (CV) of HVT DNA samples is 0.91%～3.25% intra-assay and 1.79%～4.92% inter-assay. The methods have the character of high isomerism, repeatability and sensitivity.The detection can be rapidly completed in 1.5-2.0 hours.Second,9 different batchs of half-finished product vaccine and 51 different batchs of finished product vaccine were quantified by HVT FQ-PCR method.Therefore,41 different batch of finished product vaccine which were 100,000 times diluted determine PFU with plaque assay. The test results indicated that the number of feather copies, the number of PFU and its contains the number of viral granule have a batter stability. Each batch of vaccine viral granule numbers ware situated in a stable order of magnitude. The statistical analysis, one PFU least contains 104 copy virus.Eeach batch of vaccine's PFU numbers ware far beyond the country standard. The corresponding relationships were established between the FQ-PCR method and plaque counting initially.This corresponding relationships may apply to determine the half-finished product HVT number of feather copies,the configuration and examination of finished product vaccine;Researching on well-regulated multiplication in vitro,this provides valid guarantee and measure for vaccinal quality.These tools were used to accurately dynamic determine the role that vaccine plays in prevention,and offer feasible approach to study mechanism of immunity.Meanwhile, this method will have certain instruction function on improving the technique of HVT vaccine procedure... |
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