双芽巴贝斯虫TaqMan实时荧光PCR检测方法的建立  

Establishment of a TaqMan real-time PCR for detection of Babesia bigemina

作　　者：刘启生[1] 王振宝 曹雯丽[1] 杜晓杰[1] 哈森 刘绪斌 巴音查汗[1] 

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]伊犁出入境检验检疫局,新疆伊犁835000

出　　处：《中国兽医学报》2014年第5期747-750,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(NSFC);国家自然科学基金资助项目(30960282);新疆联合基金资助项目(U1170301)

摘　　要：根据双芽巴贝斯虫（Babesia bigemina）18SrRNA基因的保守序列,设计特异性引物和TaqMan探针,建立了检测双芽巴贝斯虫的实时荧光PCR方法。该方法灵敏度高,最小检出量为1.1×101 copies/μL的标准质粒,比常规PCR灵敏1 000倍;重复性好,组内和组间重复性试验的变异系数分别为0.21%~1.14%和0.31%~1.50%,均小于2%;特异性强,对牛常见的其他两种血液原虫和健康血液无交叉反应。用建立的实时荧光PCR和常规PCR分别对30份临床样品进行检测,实时荧光PCR和常规PCR的检出率分别为30%和16.7%。结果表明,本研究建立的荧光TaqMan实时荧光PCR方法可以灵敏、准确、快速检测双芽巴贝斯虫感染,将为蜱传牛梨形虫病的流行病学调查和防制提供新的方法。A pair of primers and a TaqMan probe was designed according to the conserved sequence of the 18S rRNA gene of B. bigernina,and a TaqMan real-time PCR method was established for detection of 13. bigemina. The sensitivity of the method was 1.1×10^1 copies/μL which was 1 000 times more than conventional PCR. And the reproducibility tests in inter-assay and intra-assay in- dicated that the coefficients of variation were 0. 21%-1.14% and 0. 31%-1. 50% ,respectively,less than 2%. Moreover,the method was also highly specificity and had no cross-reaction with other bovine piroplasms. A total of 30 clinical samples were tested by the established real-time PCR and the conventional PCR, respectively,the positive rates by the real-time PCR was 30 ~, but 16.7 positive by the conventional PCR. The real-time PCR method for detection of B. bigemina was sensitive,specific and rapid detection technology, which will provided a strong guarantee for the tick-borne bovine piroplasmosis control and epidemiological investigation.

关 键 词：双芽巴贝斯虫 实时荧光PCR 检测 TAQMAN探针 

分 类 号：S858.23[农业科学—临床兽医学]