猪源盖塔病毒TaqMan实时荧光定量PCR检测方法的建立  

Development of TaqMan Real-time Quantitative PCR for Detection of Porcine Gatah Virus

作　　者：王娟 王帅勇 虞凌雪[1] 张毅峰 严杰聪 王曼茱 周艳君[1] 单同领[1] 童武[1] 郑浩[1] 童光志[1] 于海[1,2] WANG Juan;WANG Shuaiyong;YU Lingxue;ZHANG Yifeng;YAN Jiecong;WANG Manzhu;ZHOU Yanjun;SHAN Tongling;TONG Wu;ZHENG Hao;TONG Guangzhi;YU Hai(Shanghai Veterinary Research Institute,CAAS,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]中国农业科学院上海兽医研究所,上海200241 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2023年第3期120-126,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(31970175);中国农业科学院创新工程“猪病毒荏繁殖障碍疫病防控技术团队”项目。

摘　　要：猪盖塔病毒感染是由盖塔病毒(GETV)引起母猪妊娠初期胎儿死亡、初生仔猪发病的一种繁殖障碍性疾病。猪是自然界中GETV的主要扩增宿主,可以产生足够高的病毒血症滴度来感染叮咬的蚊子并维持GETV的传播周期,所以定期对猪群进行GETV监测对于预防潜在的GETV暴发具有重要意义。本研究通过分析GETV E2蛋白基因特征,设计特异性引物和探针,条件优化后建立检测GETV感染的TaqMan实时荧光定量PCR方法。结果表明:以阳性质粒为标准品建立的标准曲线在1×10^(2)~1×10^(7)copies/μL内呈现良好的线性关系,扩增效率为1.61;该检测方法的最低检出限低至3.16 TCID50/mL,远远低于普通PCR的检测限3.16×10^(3)TCID50/mL;组内、组间重复性试验的变异系数均小于2%,具有良好的重复性和稳定性。本研究建立的方法能够快速有效的检测和定量GETV,为猪群中GETV监测提供可靠的检测方法。Porcine Getah virus(GETV)infection can cause reproductive disorder including fetal death in the early pregnancy of sows and disease in newborn piglets.Pigs develop high GETV viremia level and mosquitoes biting maintains the transmission cycle in nature.Therefore,it is of great significance to monitor GETV transmission in pigs on a regular basis to prevent potential outbreaks.In this study,specific primers and probes were designed by Oligo7 software targeting the GETV E2 protein gene after genetic analysis and comparison.A TaqMan real-time fluorescent quantitative PCR method for detecting GETV infection was developed and conditions were optimized.The results showed that the good linear relationship was present when Ct values of the standard positive plasmids existed in the range of 1×10^(2)~1×10^(7)copies/μL,and the amplification efficiency was 1.61.The detection limit of this method was as low as 3.16 TCID50/mL,which was far lower than the limit of 3.16×10^(3)TCID50/mL of ordinary PCR.Both the intra-assay and inter-assay coefficients of variation(CV)of Ct values were less than 2%,which indicated good repeatability and stability.The TaqMan real-time PCR method developed in this study was able to quickly and effectively detect and quantify GETV,providing a reliable monitoring tool for pig herds.

关 键 词：猪源盖塔病毒 TaqMan实时荧光定量PCR方法 检测 

分 类 号：S858.28[农业科学—临床兽医学]