牛瑟氏泰勒虫与牛卵形巴贝斯虫多重PCR检测方法的建立

Establishment of multiplex PCR for detection of bovine Theileria sergentiand Babesia ovata

根据GenBank上登录的牛瑟氏泰勒虫内转录间隔子基因(ITS基因)和牛卵形巴贝斯虫CCTη基因序列,设计合成了2对特异性引物。通过对反应条件进行优化,建立了同时检测牛瑟氏泰勒虫和牛卵形巴贝斯虫的多重PCR方法。结果显示,用该多重PCR方法可同时扩增出2条与试验设计相符的1 020bp(牛瑟氏泰勒虫)和537bp(牛卵形巴贝斯虫)的特异性条带,对牛瑟氏泰勒虫和牛卵形巴贝斯虫的最低检出限分别为10pg/μL和1pg/μL。用该方法对采自吉林省珲春市的23份临床样本进行检测,结果牛瑟氏泰勒虫的阳性率为69%,牛卵形巴贝斯虫的阳性率为52%,混合感染率为52%。表明,建立的多重PCR方法可用于临床诊断。

For simultaneous detection of Theileria sergenti and Babesia ovata, a multiplex PCR was es- tablished using 2 pairs of specific primers designed on the basis of ITS gene of Theileria sergenti and CCTr/ gene of Babesia ovata. Reactionconditions of the multiplex PCR were optimized. Two targeting fragments, 1 020 bp（for T. sergenti） and 537 bp for （B. ovata） ,were amplified. The detection limit was 10 pg/μL for T. sergenti and 1 pg/μL for B. ovata, respectively. Twenty-three clinical samples from Hunchun in Jilin province were detected by the multiplex PCR and the detection rate was 69% for T. sergenti;52% for B. ovata and 52% for mixed infection. This method could be useful in clinic diagnoses.