牛环形泰勒虫病实时荧光PCR诊断方法的建立及初步应用  

Development and application of Real-time PCR assay for detection of theilera annulatain bovine

作　　者：郭庆勇[1] 王振宝 何晓杰[3] 曹雯丽[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,新疆伊宁835000 [3]伊犁职业技术学院,新疆伊宁835000

出　　处：《中国兽医学报》2013年第9期1369-1372,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(U1170301);新疆自治区国际科技合作计划资助项目(20126008)

摘　　要：参考GenBank中牛环形泰勒虫18SrRNA基因序列,设计合成1对特异性引物和TaqMan探针,通过优化反应参数,建立了检测牛环形泰勒虫的实时荧光PCR方法。研究结果表明,该检测方法最低可检测到10拷贝的核酸,比常规PCR敏感10倍;其组内变异系数<0.85%,组间变异系数<0.56%;对牛巴贝斯虫、双芽巴贝斯虫、伊氏锥虫等无荧光检测信号,与其他蜱传血液原虫无交叉反应;在47份被检样品中,实时荧光PCR和常规PCR的检出率分别为36.17%和25.53%。本研究建立的实时荧光PCR检测方法可用于牛环形泰勒虫病的诊断和分子流行病学调查。To develop a rapid real-time PCR assay for detection of theileriosis in bovine.A pair of primer and TaqMan probe was designed,according to the nuclear DNA region of Theilera annulata containing 18S rRNA in GenBank,and the real-time PCR assay was developed by optimizing the reaction parameter.The results showed that this method could specifically detect 10 copies DNA of T.annulata,which is 10-fold sensitivity more than conventional PCR,the intra-assay was lower than 0.85% and inter-assay was lower than 0.56%.No signal was detected for B.bigemina,B.bovis,T.evansi and no cross-reaction to other tick-borne protozoosis with conventional PCR detection.The 47 blood samples were detected by real-time PCR and conventional PCR,and the positive rate is 36.17% and 25.53%,respectively.It indicates that the real-time PCR assay could be used for the detection of Theilera annulata and molecular epidemiology investigation in bovine.

关 键 词：牛环形泰勒虫病 实时荧光PCR TAQMAN探针 诊断方法 

分 类 号：S852.7[农业科学—基础兽医学]