牛巴贝斯虫巢式PCR检测方法的建立  

Development of a nested PCR assay for detection of Babesia bovis

作　　者：孙明[1,2] 王锦明 刘爱红[1] 刘志杰[1] 聂英[2] 魏华 李玉峰 殷宏 关贵全[1] 罗建勋 SUN Ming;WANG Jin-ming;LIU Ai-hong;LIU Zhi-jie;NIE Ying;WEI Hua;LI Yu-feng;YIN Hong;GUAN Gui-quan;LUO Jian-xun(State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/ Lanzhou Veterinary Research Institute,Chinese A cademy of Agricultural Sciences ,Lanzhou 730046,China;Center for Animal Disease Control of Minqin County,Wuwei 733300,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]民勤县动物疫病预防控制中心,甘肃武威733300 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2019年第2期183-189,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501200);农业科技创新工程项目(ASTIP);国家肉牛牦牛产业技术体系项目(NBCIS;CARS-37)

摘　　要：为建立一种适用于基层兽医实验室特异、敏感、快速检测牛巴贝斯虫感染的方法,以牛巴贝斯虫球状体蛋白4(SBP4)基因为靶基因设计引物,建立检测牛巴贝斯虫的巢式PCR方法。结果表明,该方法可特异性地检测牛巴贝斯虫感染,与14种动物梨形虫无交叉反应。该方法能够检测到1 copy的SBP4基因,其敏感性分别是对应的内、外引物普通PCR的10倍和1 000倍。应用该巢式PCR方法,对田间随机采集的136份野外样品进行检测,检出阳性样品9份,显著高于AbouLaila等报道的方法(5份)。这些数据表明,建立的巢式PCR方法可用于基层兽医实验室敏感、准确地检测牛巴贝斯虫感染。To establish a specific,sensitive and rapid detection method for Babesia bovis that is easy-to-use in practice in local veterinary laboratories,a nested PCR was established using the primers designed targeting SBP4 gene of spherical body protein(SBP) of B.bovis.As a result,this method was specific for B.bovis and no cross-reaction was observed with 14 related piroplasm samples.The analytic sensitivity of the method showed to detect as few as 1 copy of SBP4 gene.When compared with the conventional PCR with both outer primers and inner primers of the nested PCR,the sensitivity increased 1 000 times and 10 times,respectively.Using the developed method,136 field samples were detected,and it gave much higher positives(9 samples) than the method of Abou Laila M et al.(5 samples).In conclusion,the established nested PCR can be implied in practice in local veterinary laboratories due to its satisfactory sensitivity and specificity for the detection of B.bovis.

关 键 词：牛巴贝斯虫 巢式PCR SBP4基因 

分 类 号：S852.723[农业科学—基础兽医学]