羊泰勒虫与嗜吞噬细胞无浆体双重PCR检测方法的建立

Development and Application of Duplex PCR Method for Detection of Ovine and Caprine Theileria and Anaplasma phagocytophilum

为建立一种能快速对羊泰勒虫(ovine and caprine theileria)和嗜吞噬细胞无浆体(A.phagocytoph-ilum)同时进行检测的双重PCR方法。根据GenBank已报道的羊泰勒虫MPSP基因和羊嗜吞噬细胞无浆体16S rRNA基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。双重PCR可特异扩增出羊泰勒虫和嗜吞噬细胞无浆体目的条带,片段大小分别为875bp和394bp。该方法具有较好的特异性,对羊泰勒虫和嗜吞噬细胞无浆体的最低检出浓度为16fg/μL和1fg/μL。对采集的60份血液样本进行双重PCR检测,羊泰勒虫阳性率为46.67%(28/60),嗜吞噬细胞无浆体阳性率为13.33%(8/60),混合感染率为10%(6/60)。结果表明,双重PCR方法可用于羊泰勒虫和嗜吞噬细胞无浆体的快速诊断和流行病学调查。

In order to develop a fast duplex PCR method to simultaneously detect ovine and caprine Theileria and Anaplasma phagocytophilum,specific primers were designed and used for amplification of MPSP of Ovine and caprine and 16S rRNA of A.phagocytophilum.The amplified DNA fragments were 875 bp for ovine and caprine and 394 bp for A.phagocytophilum.The detection limit was 16 fg/µL for ovine and caprine and 1 fg/µL for A.phagocytophilum,respectively.Clinical samples from Hunchun in Jilin province were tested by this duplex PCR and the detection rates were 46.67%for ovine and caprine,13.33%forA.phagocytophilumand 10%for mixed infection.The results showed that the duplex PCR developed here was suitable for quick clinical detection and molecular epidemiological investigation.