荧光定量PCR与免疫荧光法定量定性检测牛血清中呼肠孤病毒

Establishment of Fluorescent Quantitative PCR and Fluorescent Antibody Test for Reovirus

目的采用TaqMan荧光探针定量PCR技术与免疫荧光技术定量定性检测牛血清中呼肠孤病毒。方法以携带有Reovirus保守系列的重组质粒作为标准品,优化定量PCR体系,建立标准曲线,用Vero细胞培养不同病毒评价方法的特异性;用FITC标记的抗Reovirus病毒蛋白单克隆抗体对感染不同滴度Reovirus的Vero细胞培养物进行直接免疫荧光检测,用Vero细胞培养不同病毒评价方法的特异性。结果 Taq Man Real-time PCR方法检测灵敏度可达1.0×102拷贝/l,线性范围为109～103;免疫荧光法对Reovirus检测具有很高的特异性。结论以TaqMan荧光探针检测Reovirus荧光定量PCR方法,灵敏度高、特异性强,辅以免疫荧光定性检测方法,可以作为牛血清感染Reovirus的定量和定性检测。

Objective To establishment the method of reovirus detection using Taq Man probe fluorescent quantitative PCR and fluorescent antibody.Methods The standard curve with the plasmid taking the conservative sequence of reovirus was established to evaluate its specificity with other virus.,and the Vero cell infected different titer reovirus was inspected with fluorescent antibody to evaluate its specificity with other virus.Results The sensitivity of the Taq Man Real-time PCR assay is up to 1.0×102 copies/μl,linearity range is 109~103,and fluorescent antibody test was very specific.Conclusion The study of the developed methods show a high specificity and sensitivity,the TaqMan Real-time PCR and fluorescent antibody assay could be used for detecting reovirus of fetal bovine serum.