猪传染性胃肠炎病毒TaqMan实时荧光定量PCR检测方法的建立与应用  

Establishment and application of TaqMan real-time quantitative PCR for detection of porcine transmissible gastroenteritis virus

作　　者：张羽欣 王树茂 段宏勇 赵款 董世山[1] 高飞[2,3] 李丽薇 ZHANG Yuxin;WANG Shumao;DUAN Hongyong;ZHAO Kuan;DONG Shishan;GAO Fei;LI Liwei(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Shanghai Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Shanghai 200241,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]中国农业科学院上海兽医研究所,上海200241 [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》2024年第4期479-484,共6页Chinese Veterinary Science

基　　金：上海市科技兴农项目(2022-02-08-00-12-F01162);国家重点研发计划项目(2022YFD1800305,2022YFD1800804,2021YFD1801401);中国农业科学院科技创新工程项目(CAAS-CSLPDCP-202301)。

摘　　要：本研究针对猪传染性胃肠炎病毒(TGEV)的N基因保守序列设计特异性引物和探针,构建重组质粒,建立针对TGEV N基因的Taq Man荧光定量PCR检测方法,用于临床快速诊断和实验室检测。结果显示,该方法特异性好,与其他具有相似临床症状的猪源病毒相比,不存在交叉反应,Ct值在1×10^(9)~1×10^(1)copies/μL的范围内具有很好的线性关系,R^(2)=0.99。该方法最低检测限为10 copies/μL,标准曲线为y=-3.0587x+37.7,斜率为-3.0587。该方法重复性好、敏感度高、特异性强,适用于临床样品中TGEV抗原核酸的检测和实验室病毒生长特性的研究,为准确定量及诊断TGEV提供了有效的试验手段和检测工具。In this study,we designed highly specific primers and probes for the N gene fragment of porcine transmissible gastroenteritis virus(TGEV),constructed the recombinant plasmid,and estab-lished the TaqMan fluorescence quantitative PCR detection method for the rapid clinical diagnosis and laboratory testing.The results showed that the method had good specificity.Compared with other porcine viruses,there was no cross-reaction between viruses with similar clinical symptoms.The C val-ues displayed good linear relationship in the range of 1×10^(9)~1×10^(1)copies/μL,R^(2)=0.99.The lowest detection limit of this method was 10 copies/μL.The standard curve was y=-3.0587x+37.7,and the slope was-3.0587.The method has good repeatability,high sensibility,and strong specificity.The method is suitable for the detection of TGEV antigen nucleic acid in clinical samples and the study of the growth characteristics of laboratory viruses.It provides an effective test and detection tool for the accurate quantification and diagnosis of TGEV.

关 键 词：猪传染性胃肠炎病毒 N基因 荧光定量PCR TAQMAN 

分 类 号：S852.659.6[农业科学—基础兽医学]