荧光定量PCR检测牛瑟氏泰勒虫方法的建立  

Establishment of Real-Time PCR for Detection of Theileria sergenti

作　　者：王轶男[1] 胡诗悦 陈洪涛 金春梅[1] 金一[1] 于龙政[1] WANG Yi-nan;HU Shi-yue;CHEN Hong-tao;JIN Chun-mei;JIN Yi;YU Long-zheng(Agricultural College,Yanbian University,Yanji 133002,China;Changchun Animal Disease Prevention and Control Centre,Changchun 130000,China;Government of Quangou Town,Xintai City,Shandong Province,Xintai 271207,China)

机构地区：[1]延边大学农学院,吉林延吉133002 [2]长春市动物疫病预防控制中心,吉林长春130000 [3]山东省新泰市泉沟镇人民政府,山东新泰271207

出　　处：《中国兽医杂志》2021年第11期33-37,共5页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(32060807);吉林省教育厅项目(JJKH20200522KJ);高等学校学科创新引智计划资助(D20034)。

摘　　要：本试验旨在建立快速诊断牛瑟氏泰勒虫的荧光定量PCR检测方法。以常规PCR方法扩增牛瑟氏泰勒虫P33基因,将其克隆至载体pMD-18T,然后进行质粒标准品的构建,以此为模板建立牛瑟氏泰勒虫P33基因TaqMan荧光定量PCR和SYBR Green荧光定量PCR检测方法。结果显示:2种方法均能检测到2.26~2.26×10^(8)copies/μL范围,特异性试验中与其他病原体均无特异性扩增曲线,批内和批间变异系数都小于4%。鉴于2种方法均具有较好的敏感性、特异性和重复性,适用于牛瑟氏泰勒虫P33基因的临床实验室鉴别。This study aimed to establish real-time PCR for rapid detection of Theileria sergenti. The P33 gene was amplified from T. sergenti by PCR, and then inserted into pMD-18 T vector for use as plasmid standard. TaqMan real-time PCR and SYBR Green real-time PCR methods were set up by using the plasmid standard containing T. sergenti P33 gene. The results showed that both TaqMan and SYBR Green real-time PCR methods detected the copy number in the range of 2.26-2.26×10^(8)copies/μL, produced no amplification curve for other pathogens in the specific test, and had intra-assay and the inter-assay coefficients of variation less than 4%. Given their excellent sensitivity, specificity and reproducibility, TaqMan and SYBR Green real-time PCR methods can be applied for clinical laboratory identification of T. sergenti.

关 键 词：牛瑟氏泰勒虫 荧光定量PCR P33基因 检测方法 

分 类 号：S852.7[农业科学—基础兽医学]