山羊地方性鼻内肿瘤病毒TaqMan荧光定量RT-PCR检测方法的建立  

Development of a TaqMan Real-time RT-PCR Assay for detection of Enzootic Nasal Tumor Virus in Goats

作　　者：黎家明 郑烁东 罗世诚 张浩权 颜广智 陈盛楠 刘明杰 黄良宗 LI Jiaming;ZHENG Shuodong;LUO Shicheng;ZHANG Haoquan;YAN Guangzhi;CHEN Shengnan;LIU Mingjie;HUANG Liangzong(School of life science and engineering,Foshan University,Foshan Guangdong 528000;Guangdong Findergene Biotechnology Co.,Ltd.,Foshan Guangdong 528000)

机构地区：[1]佛山科学技术学院生命科学与工程学院,广东佛山528000 [2]广东方道基因生物科技有限公司,广东佛山528000

出　　处：《广东畜牧兽医科技》2023年第1期24-28,62,共6页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省现代草牧业(羊)产业技术体系(2019KJ127);广东省企业科技特派员项目(kh21368)。

摘　　要：为建立山羊地方性鼻内肿瘤病毒(Enzootic nasal tumor virus of goats,ENTV-2)快速检测方法,根据ENTV-2 env基因保守区域设计引物和TaqMan探针,通过优化反应条件,建立TaqMan荧光定量RT-PCR检测方法,并对其特异性、灵敏性、重复性与临床检测效果进行验证。结果显示,该方法建立的标准曲线在重组质粒浓度为1.019×10^(9)—1.019×10^(2)copies/μL时,相关系数R^(2)为0.998,Ct值和重组质粒浓度有良好的线性关系;与相关的其他5种羊常见病原无交叉反应,最低检测限度为10.19 copies/μL,组间和组内的变异系数均在1.5%内。利用本研究建立的方法与常规RT-PCR对20份临床样品进行对比检测,两种方法的ENTV-2阳性检出率分别为60%(12/20)和30%(6/20),两种方法的总符合率为70%(14/20)。结果表明,本研究建立的方法特异性强、灵敏度高、重复性好,为ENTV-2的诊断和流行病学调查提供技术手段。In order to establish a rapid detection method for Enzootic nasal tumor virus of goats(ENTV-2),primers and TaqMan probes were designed according to the conserved region of ENTV-2 env gene.By optimizing the reaction conditions,the ENTV-2 TaqMan fluorescence quantitative RT-PCR detection method was established,and its specificity,sensitivity,repeatability and clinical detection effect were verified.The results showed that the standard curve established by this method had a correlation coefficient(R^(2))of 0.998 when the concentration of recombinant plasmid was 1.019×10^(9)—1.019×10^(2)copies/μL,and the Ct value had a good linear relationship with the concentration of recombinant plasmid.This approach was specific for ENTV-2,and there was no cross-reaction observed against other five kinds of pathogens related to goats.The minimum detection limit was 10.19 copies/μL,and the coefficient of variation intra-assays and inter-assays was within 0.46%and 1.5%.It was more sensitive than the conventional RT-PCR and gave higher ENTV-2 positive detection rate(60%,12/20)than the conventional RT-PCR(30%,6/20)from clinical samples,the total coincidence rate of the two methods was 70%(14/20).These data indicated that the TaqMan-based realtime RT-PCR assay established here was an effective method with high sensitivity,specificity and reproducibility for faster and more accurate detection and quantification of ENTV-2.

关 键 词：山羊地方性鼻内肿瘤病毒 TAQMAN 荧光定量RT-PCR 

分 类 号：S856.3[农业科学—临床兽医学]