羊泰勒虫套式PCR检测方法的建立

Development of a nested PCR assay for detection of ovine and caprine Theileria

为了建立一种特异、敏感、快速检测羊泰勒虫方法,以羊泰勒虫主要表面蛋白(MPSP)基因设计引物,建立羊泰勒虫套式PCR检测方法。结果表明,该方法只能扩增出羊泰勒虫,而对照组羊巴贝斯虫、羊无浆体和弓形虫基因组DNA均未扩出目的条带。该方法敏感性是普通PCR的1 000倍,其最低检测到1拷贝数的MPSP基因。对采自吉林珲春地区30份羊血液样本检测发现,套式PCR阳性率为46.6%(14/30),高于普通PCR(40.00%,12/30)和血液涂片染色(23.33%,7/30),三者阳性符合率为100%。结果表明,所建立的套式PCR可用于羊泰勒虫病的诊断和流行病学调查。

To establish a rapid,specific and sensitive method for detection of ovine and caprine Theileria,a nested PCR was established u­sing the primers designed to target the major piroplasm surface protein(MPSP)gene of ovine and caprine theileriosis.As a result,this meth­od was specific in detecting ovine and caprine theileriosis,but not so testing the samples of Babesia sp,A.ovis and Toxoplasma gondii.The sensitivity of the method was one copy of the MPSP gene minimally,and was 1000 times the sensitivity of the convention PCR.Among 30clinical samples detected by the nested PCR,the positive rate was 46.67%(14/30),much higher than tested by the convention PCR(40.00%,12/30)and by microscopic examination(23.33%,7/30).And the coincidence rate between the three methods was 100%.Thepresent results indicated that the nested PCR assay was useful and effective in detection and epidemiology investigation of theileriosis in goatsand sheep.