Development and Application of Duplex PCR Method for Detection of Ovine and Caprine Theileria and Anaplasma phagocytophilum
宁宇春 1王圆赫 1郭祥杰 1朱宏阳 2田万年1
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作者信息
1. 吉林农业科技学院动物科技学院,吉林 132101
2. 图们市凉水镇畜牧兽医站,图们 133100
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摘要
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875 bp和394 bp.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16 fg/μL和1 fg/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.
Abstract
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％for A.phagocytophilum and 10％for mixed infection.The results showed that the duplex PCR developed here was suitable for quick clinical detection and molecular epidemiological investigation.
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