牛巴贝斯虫实时荧光PCR检测方法的建立  

Development of real-time PCR assay for detection of Babesia bovis

作　　者：刘启生[1] 王振宝 王真[1] 哈森 张玉婷[1] 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：《中国预防兽医学报》2013年第1期58-61,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30960282);国家自然科学基金会(NSFC)-新疆联合基金(U1170301)

摘　　要：为建立牛巴贝斯虫(B.bovis)的TaqMan实时荧光PCR检测方法,本研究根据GenBank中B.bovis的18S rRNA基因保守序列,设计引物和TaqMan探针,通过优化反应体系,建立检测B.bovis的实时荧光PCR方法。试验结果表明:实时荧光PCR对靶基因的最低检测值为1.31×101copies/μL,比常规PCR的敏感性高1 000倍;而且与牛的其他血液原虫无交叉反应;组内及组间重复性试验的变异系数均小于3%,具有良好的重复性;在23份被检样品中,实时荧光PCR和常规PCR的检出率分别为52.17%和30.43%。该检测方法的建立为B.bovis的检测提供了一种快速、敏感、特异的技术手段。To detect the Babesia bovis, a real-time PCR method was established with a pair of primers and a TaqMan probe designed according to the conserved sequence of 18S rRNA genes in GenBank. The results showed that： the limit detection of real-time PCR was about 1.31 × 101 copies/μL for the target gene which was 1,000 times higher sensitivity than conventional PCR, and no cross-reaction with other piroplasmasida in cattle. The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than 3%. A total of 23 clinical samples were tested by the real-time PCR comparing with conventional PCR, and positive rates were 52.17% （12/23） and 30.43% （7/23）, respectively. The establishment of the real-time PCR method for detection of bovine Babesia was sensitive, specific and rapid detection technology, which is provided a assay for the tick-borne bovine piroplasmosis detection.

关 键 词：牛巴贝斯虫 实时荧光PCR TAQMAN探针 检测 

分 类 号：S852.72[农业科学—基础兽医学]