猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of Real-time qPCR Method for Detection of PEDV

作　　者：杨峰[1] 杨猛超 周宏超[1] 许信刚[1] 张为民[1] 张琪[1] YANG Feng;YANG Meng-chao;ZHOU Hong-chao;XU Xin-gang;ZHANG Wei-min;ZHANG Qi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China;Shaanxi Supply and Marketing Fudi Animal Husbandry Limited Liability Company,Zhouzhi,Shaanxi,710400,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]陕西供销福地牧业有限责任公司,陕西周至710400

出　　处：《动物医学进展》2018年第7期1-5,共5页Progress In Veterinary Medicine

基　　金：陕西省农业科技创新与攻关项目(2016NY-095);西安市农业科技创新计划研发类项目(2017050NC/NY010-2);杨凌示范区产学研用协同创新重大项目(2017CXY-17);杨凌示范区农业科技示范推广能力提升项目(TS-2016-12)

摘　　要：为建立一种特异、灵敏、量化、快速的猪流行性腹泻病毒(PEDV)检测方法,设计1对PEDV N基因的特异性引物,建立Eva Green染料的实时荧光定量RT-PCR的检测方法,并对疑似PEDV感染的临床样品进行检测。结果显示,所建立的实时荧光定量RT-PCR方法的标准曲线具有良好的线性关系,线性相关系数R2=0.999;不与猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)发生交叉反应,具有较强的特异性;灵敏度比常规RT-PCR方法高100倍;重复性试验的变异系数均小于5%,重复性良好。对43份临床样品进行检测,结果比普通PCR多检出2份阳性。结果表明,建立的实时荧光定量RT-PCR检测方法在特异性、灵敏度和重复性方面都很好,可用于临床PEDV检测。To establish a quick,specific,sensitive and quantitative detection method of porcine epidemic diarrhea virus(PEDV),a pair of specific primers were designed on the basis of conservative sequence of PEDV N gene.Eva Green RT-qPCR was established,and its specificity,sensitivity,repeatability were determined.The results showed that the standard curve of RT-qPCR revealed a good linear relationship with the correlation coefficient R 2=0.999,high specificity that no cross-reactions with TGEV,CSFV and PRRSV,high sensitivity that 100 times more sensitive than the conventional RT-PCR method,and well repeatability with the coefficient of variation less than 5%.This method had high specificity,good sensitivity and good repeatability,and can be used for clinical detection of PEDV.

关 键 词：猪流行性腹泻病毒 实时荧光定量RT-PCR 检测 

分 类 号：S852.659.6[农业科学—基础兽医学] S854.44[农业科学—兽医学]