羊痘病毒实时荧光定量TaqMan PCR检测方法的建立

参照羊痘病毒(CaPV)P32的基因序列,设计合成了2套引物和1条探针,建立了实时荧光定量PCR技术,对细胞培养物、皮肤丘疹、痂皮等组织病料中的GPV进行了特异性检测和敏感性试验。结果显示,用300 nmol／L引物浓度和200 nmol／L探针浓度,获得的Cr值较小,而△Rn最大;可检测到相当于0．1 TCID50的病毒DNA;制作的标准曲线中各浓度范围内有极好的线性关系且线性范围宽,相关系数为0．999 5以上;组内和组间试验重复性的变异系数分别为2．3％和3．4％;与常规的PCR相比较,该方法具有快速、特异、敏感、可定量,可同时检测大量样品等优点。表明。荧光TaqMan PCR是一种检测CaPV的良好方法,可对组织病料中低含量的CaPV或持续带毒宿主进行准确检测。

Development of a real-time fluorescent TaqMan-quantitative PCR assay for detection of capripoxvirus

A real-time fluorescent TaqMan-quantitative PCR assay using 2 pairs of primers and a probe designed and synthesized according to the P32 gene of the virus genome was developed for the specific detection of capripox virus in epithelial suspensions and cell culture preparations. The real-time fluorescent PCR assay detected specifically CaPV virus in samples with greater sensitivity than the conventional PCR procedure. The fluorescent PCR assay was fast, and could quantitatively assess the virus amounts and could handle more samples and/or replicates of samples in a single assay than the conventional PCR procedure. The results showed that this assay is a valuable complementary tool to the routine diagnostic procedures for the detection of capripox virus infection.