实时荧光PCR检测牛边缘无浆体方法的建立及应用  

Detection and identification of Anaplasma marginale by real-time PCR

作　　者：王贵强[1] 李树清[2] 张子群 陈志飞[2] 由轩 王巧全[2] 杜凯[1] 姚宝安[1] 

机构地区：[1]华中农业大学,湖北武汉430070 [2]上海出入境检验检疫局,上海200135 [3]黑龙江省出入境检验检疫局,黑龙江哈尔滨150036

出　　处：《中国预防兽医学报》2007年第8期634-636,654,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家标准专项上海地方实施项目(04DZ05017);国家质检总局课题(2005IK179)

摘　　要：根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血(奶牛和肉牛),其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。A real-time PCR method was developed for detecting A.marginale using primers and TaqMan-MGB Probe designed for major surface protein 4 gene. This method could specifically detect as low as 200 fg DNA of A.marginale. No product was detected for A.centrale, A.ovis, B.bovis, B.bigemina, B.motasi, T.hirci, E.wenyonii, orientalis sp. Nov, T.gondii, T.evansi. Among 180 samples from Jiangsu and Heilongjiang, 8.9 % were detected positive, which is consistent with the results of nested PCR. The results showed that the real-time PCR method was highly specific ＆ sensitive for diagnosis of Anaplasmosis.

关 键 词：牛边缘无浆体 实时荧光PCR TAQMAN-MGB探针 检测 

分 类 号：S852.7[农业科学—基础兽医学] S854.44[农业科学—兽医学]