荧光定量PCR检测牛瑟氏泰勒虫方法的建立

Establishment of Real-Time PCR for Detection of Theileria sergenti

本试验旨在建立快速诊断牛瑟氏泰勒虫的荧光定量PCR检测方法。以常规PCR方法扩增牛瑟氏泰勒虫P33基因,将其克隆至载体pMD-18T,然后进行质粒标准品的构建,以此为模板建立牛瑟氏泰勒虫P33基因TaqMan荧光定量PCR和SYBR Green荧光定量PCR检测方法。结果显示:2种方法均能检测到2.26~2.26×10^(8)copies/μL范围,特异性试验中与其他病原体均无特异性扩增曲线,批内和批间变异系数都小于4%。鉴于2种方法均具有较好的敏感性、特异性和重复性,适用于牛瑟氏泰勒虫P33基因的临床实验室鉴别。

This study aimed to establish real-time PCR for rapid detection of Theileria sergenti. The P33 gene was amplified from T. sergenti by PCR, and then inserted into pMD-18 T vector for use as plasmid standard. TaqMan real-time PCR and SYBR Green real-time PCR methods were set up by using the plasmid standard containing T. sergenti P33 gene. The results showed that both TaqMan and SYBR Green real-time PCR methods detected the copy number in the range of 2.26-2.26×10^(8)copies/μL, produced no amplification curve for other pathogens in the specific test, and had intra-assay and the inter-assay coefficients of variation less than 4%. Given their excellent sensitivity, specificity and reproducibility, TaqMan and SYBR Green real-time PCR methods can be applied for clinical laboratory identification of T. sergenti.