牛瑟氏泰勒虫二温式PCR快速检测技术的建立及初步应用

Establishment of two-temperature PCR for the rapid detection of Theileria sergenti and its preliminary application

为建立一种牛瑟氏泰勒虫快速检测技术,根据GenBank上已发表的牛瑟氏泰勒虫P23表面蛋白基因序列(D84447)设计1对特异性引物,扩增出大小为244 bp的基因片段,经克隆、测序分析,与已知基因序列同源性为100%。用该对引物建立的牛瑟氏泰勒虫二温式PCR能检测出的最高敏感度为171 fg/μL,与牛新孢子虫、弓形虫和卵形巴贝斯虫不产生交叉反应,对48份临床血液样本进行检测,阳性检出率为66.67%。用同一对引物对28份临床血液样本分别进行二温式PCR和三温式PCR检测,阳性符合率为95%,总符合率为96.43%。结果表明,该方法具有快速、敏感、特异等优点,可用于牛瑟氏泰勒虫病的快速临床诊断及流行病学调查。

To establish a rapid method to detect Theileria sergenti,a pair of primers was designed according to the gene encoding P23 major surface protein of T.sergenti（D84447）.The 244 bp gene fragment was amplified,and the sequencing analysis showed 100% of homology with known gene sequence.The two-temperature PCR was established and its highest susceptibility was 171 fg/μL.The method showed no cross reaction with bovine Neospora caninum,Toxoplasma gondii and Babesia ovata.In 48 tested clinical blood samples,the positive rate was 66.67%.When 28 clinical blood samples were detected using the same pair of primers by two-temperature and three-temperature PCR,respectively,the positive coincidence rate was 95%,and the whole coincidence rate was 96.43%.The results indicated that the two-tempera-ture PCR could be used in the rapid clinical diagnosis and epidemiological investigation in T.sergenti infection.