牛瑟氏泰勒虫二温式PCR快速检测技术的建立及初步应用.pdfVIP

.26. Animal Husbandry Veterinary Medicine 2013 Vol. 45 No.7 牛瑟氏泰勒虫工温式 PCR 快速检测技术的 建立及初步应用 丁晓双，夏晓辉，张晓轩，海旭楠，许应天* (延边大学农学院，吉林延吉 133∞2) 摘要: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凹3 表面蛋白基因序列 (D8斜47) 设计 l 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 l ∞%。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 fglμL，与牛新袍子虫、弓形虫和卵形巳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66.67ì岛。用 同一对引物对28 份临床血液样本分别进行二温式 PCR 和三混式 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。 关键词:牛瑟氏泰勒虫;归3 基因;二温式 PCR 中固分类号: S854.4 文献标志码:A 文章编号: 0529-5130(2013 )07-∞26-04 Establishment of two-temperature PCR for the rapid detection of Theileria sergenti and its preliminary application DING Xiaoshuang , XIA Xiaohui , ZHANG Xiaoxuan , HAI Xunan , XU Yingtian (Agricultural College of Yanbian University , Yanji 133∞2 ， China) Abstract: 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 (D8剁47). 币le 244 bp gene fragment was amplified , and the sequencing analysis showed 1 ∞% of homology with known gene sequence. The two-temperature PCR was established and its highest susceptibility was 171 fglμ1. 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 , re- spectively , the positive coincidence rate was 95% , and the whole coincidence rate was 96. 43 1毛. The results indicated that the two-tempera