猪瘟疫苗病毒荧光定量PCR检测方法的建立

【目的】建立一种检测猪瘟疫苗病毒(HCLV)含量的荧光定量PCR方法。【方法】用RT-PCR方法扩增HCLV基因200bp片段,构建含有该基因片段的重组质粒,对此重组质粒进行系列稀释后作为SYBR GreenⅠ荧光定量PCR模板,绘制定量检测HCLV的标准曲线。【结果】在(6.4×107~6.4×102)copies/μL模板浓度范围内,荧光定量PCR的扩增效率为92.2%,标准曲线的决定系数为0.9997。该方法的精确灵敏度为640copies/μL,重复性试验的变异系数小于2%,对牛病毒性腹泻病毒、猪繁殖与呼吸综合征病毒等的检测结果均为阴性。临床检测结果显示,不同商品猪瘟疫苗之间的病毒载量存在明显差异。【结论】建立了1种具有良好特异性与敏感性的HCLV荧光定量PCR检测方法。

Development of a fluorescence quantitative PCR assay for hog cholera lapinized virus

Objective]In order to develop a fluorescence quantitative (FQ-) PCR method to quantitatively determine hog cholera lapinized virus (HCLV).Method]A fragment of 200 base pair specific to the HCLV genome was amplified by RT-PCR and a recombinant plasmid with the gene fragment was constructed.The recombinant plasmid was diluted serially as templates for SYBR Green Ⅰ FQ-PCR to draw a standard curve for quantitative detection of HCLV.Result]During the templates concentration of (6.4× 107 6.4× 102) copies/μL,the amplification efficacy was 92.2％,the coefficient of determination for the developed standard curve was 0.9997.The accurate sensitivity of the generated assay was 640 copies/μL,and its coefficient of variation was less than 2 ％ in the reproducible assays.The detection results of bovine viral diarrhea virus,porcine reproductive and respiratory syndrome virus and other epidemic swine RNA viruses were all negative.The clinical detection results indicated that there were obvious differences in the virus load of several different commercial classical swine fever vaccines.Conclusion]A FQ-PCR assay for HCLV with good specificity and sensitivity was developed.