羊泰勒虫荧光定量PCR检测方法的建立

Establishment of real-time PCR assay for detection of Theileria sp

为建立一种敏感、快速的羊泰勒虫检测方法,本研究根据GenBank中登录的羊泰勒虫18S rRNA保守区设计1对特异引物,经各反应条件的优化,建立了羊泰勒虫荧光定量PCR检测方法。结果显示该方法可以特异地检测羊泰勒虫,而对卵形巴贝斯虫、双芽巴贝斯虫、羊巴贝斯虫和瑟氏泰勒虫检测均为阴性;该方法的灵敏度可达2.08×10~1拷贝/μL,比普通PCR灵敏度高100倍。组内及组间重复试验变异系数均小于5%。对50份临床样品进行检测,荧光定量PCR和常规PCR的阳性检出率分别为48%和38%。本实验建立的荧光定量PCR检测方法适用于羊泰勒虫定量分析和分子流行病学调查。

A real-time PCR assay was established for the detection of Theileria sp with a pair of specific primers based on the Theileria sp 18S rRNA gene. The results showed that the assay was specific for detecting Theileria sp, but not for B.ovata, B.bigemina, B.ovis and T.sergenti. The sensitivity of the method was 2.08×10^1 copies/μL which was 100 times more than convention PCR. The coefficients of variations were less than 5% for both intra-assay and inter-assay. Among 50 clinical samples, the positive rate was 48% detected by real-time PCR and 38% by convention PCR. The results showed that the real-time PCR assay could be used for detection and epidemiology investigation of Theileriosis in goats and sheep.