快速检测猪瘟兔化弱毒疫苗株的TaqMan荧光定量RT-PCR方法的建立

Development of a TaqMan fluorogenic quantitative RT-PCR assay for rapid quantification of hog cholera lapinized virus

在猪瘟病毒兔化弱毒疫苗株(HCLV)的5′端非编码区设计一对引物和一条TaqMan探针,通过优化反应条件,成功建立了特异性检测HCLV的荧光定量RT-PCR方法.结果表明:该方法检测的最低拷贝数为45拷贝/μL,灵敏度比普通PCR方法高104倍,在较广的范围内(4.5×101～4.5×106拷贝/μL)有良好的线性关系(r=0.994);分别以乙型脑炎病毒、猪繁殖与呼吸综合征病毒、副猪嗜血杆菌、牛病毒性腹泻/黏膜病病毒作为模板进行TaqManRT-PCR扩增,未出现阳性信号;4个不同浓度标准品组内试验变异系数为1.90%～5.82%,组间试验变异系数为4.02%～5.69%;HCLV3个cDNA样本组内试验变异系数为3.72%～4.93%;组间试验变异系数为2.99%～4.02%.该方法具有很好的灵敏性、特异性及稳定性,能够快速准确定量检测HCLV,为HCLV疫苗的研制、猪瘟病毒分子生物学等方面研究提供了一种快捷有效的工具.

A pair of primers and an internal TaqMan fluorogenic probe spanning the 5′ non-coding region （5′NCR） of hog cholera lapinized virus（HCLV） are designed and a fluorogenie quantitative RT-PCR assay for rapid detection of HCLV is established through optimizating the reaction conditions. The results show that the sensitivity of this method is 45 copies/μL of virus RNA, which is 104 times higher than that of regular PCR, and the assay has good linear relationship in a wide range of 4.5×10^1-4.5×10^6 copies/μL（r=0.994）. Taking encephalitis virus（JEV）, porcine reproductive and respiratory syndrome virus（PRRSV）, haemophilus parasuis（Hps） and bovine viral diarrhea-mucosal disease virus （BVDV） as templates, the TaqMan RT-PCR amplification is conducted and there are no positive signals in the reaction. The coefficient of variation （CV） of 4 different concentration of positive plasmids is 1.90%-5.82% and 4.02%-5.69% in intra-assay and in inter-assay respectively, while the CV of 3 HCLV cDNA samples is 3.72%-4.93% and 2.99%-4.02% in intra-assay and in inter-assay respectively. The method established in this paper has the advantages of high sensibility, specificity and good stability, which can detect HCLV rapidly and accurately and provides a simple and effective tool for the development of HCLV vaccine and the molecular biological studies on classical swine fver virus（CSFV）.