猪流行性腹泻病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of SYBR GreenⅠfluorescence quantitative RT-PCR method for detection of porcine epidemic diarrhea virus

作　　者：李飞[1] 曾喻兵 江朝源 张儒博 彭珂楠 陈斌[2] 朱玲[1,3] 徐志文[1,3] 程顺财 LI Fei;ZENG Yu-bing;JIANG Chao-yuan;ZHANG Ru-bo;PENG Ke-nan;CHEN Bin;ZHU Ling;XU Zhi-wen;CHENG Shun-cai(Animal Biotechnology Center,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Animal Disease Prevention and Control Center,Chengdu 610041,China;Sichuan Key Laboratory of Animal Diseases and Human Health,Sichuan Agricultural University,Chengdu 611130,China;Agricultural Comprehensive Service Center of Lede Town,Rong County,Zigong 643100,China)

机构地区：[1]四川农业大学动物生物技术中心,四川成都611130 [2]四川省动物疫病预防控制中心,四川成都610041 [3]四川农业大学动物疫病与人类健康四川省重点实验室,四川成都611130 [4]荣县乐德镇农业综合服务中心,四川自贡643100

出　　处：《中国兽医科学》2021年第11期1355-1360,共6页Chinese Veterinary Science

基　　金：四川省农业厅农业产业技术体系项目(CARS-SVDIP);四川省重点研发计划项目(2020YFN0009)。

摘　　要：为开发一种快速、准确、灵敏及定量检测猪流行性腹泻病毒(PEDV)的实时荧光定量PCR方法,根据GenBank中登录的S基因高度保守核苷酸序列,设计1对PEDV S基因的特异性引物,建立以SYBR GreenⅠ为染料的荧光定量RT-PCR检测方法,对疑似PEDV感染的临床样品进行检测并与普通RT-PCR结果进行比较。结果显示,所建立的SYBR GreenⅠ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R^(2)=1,其扩增效率E=2.03,熔解曲线为尖锐单峰;不与猪传染性胃肠炎病毒、猪细小病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪丁型冠状病毒和猪轮状病毒发生交叉反应,具有较强的特异性;最低检出浓度(1×10^(1)copies/μL)比普通RT-PCR方法灵敏100倍;批内批间重复性试验的变异系数均小于2%,重复性和稳定性好;将36份临床样品检测结果进行比较,与普通RT-PCR的总符合率为88.89%。结果表明,建立的实时荧光定量RT-PCR检测方法特异性强、重复性好、灵敏度高,对PEDV的快速及定量检测具有重要意义。To develop a real-time fluorescent quantitative PCR method for rapid,accurate,sensitive and quantitative detection of porcine epidemic diarrhea virus(PEDV),according to the highly conserved nucleotide sequence of S gene reported by Gen Bank,a pair of PEDV S gene specific primers were designed,and a fluorescent quantitative RT-PCR detection method using SYBR GreenⅠas the dye was established.The clinical samples suspected of PEDV infection were tested and compared with the results of ordinary RT-PCR.The results showed that the established standard curve of the SYBR GreenⅠfluorescence quantitative RT-PCR method had a good linear relationship.The linear correlation coefficient R^(2)=1,its amplification efficiency E=2.03,and the melting curve was a sharp single peak.The amplification of transmissible gastroenteritis virus,porcine parvovirus,classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine deltacoronavirus and porcine rotavirus was negative and had strong specificity.The lowest detection concentration of 1×10^(1) copies/μL was100 times more sensitive than that of the ordinary RT-PCR method.The coefficient of variation of intra-and inter-assay repeatability test were both less than 2%,with good repeatability and stability.Comparing the test results of 36 clinical samples,the total coincidence rate with ordinary RT-PCR was 88.89%.The results show that the established real-time fluorescent quantitative RT-PCR detection method has strong specificity,good reproducibility,and high sensitivity,which is of great significance for the rapid and quantitative detection of PEDV.

关 键 词：猪流行性腹泻病毒 S基因 荧光定量RT-PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]