猪流行性腹泻病毒微芯片荧光定量RT-qPCR检测方法的建立  

Establishment of Microchip RT-qPCR Method for Detection of Porcine Epidemic Diarrhea Virus

作　　者：曹洪志[1] 王新杰 高姗姗 孙晓明 邓飞[2] 石艳萍 陈弟诗[2] 陈昌英 李平顺 CAO Hong-zhi;WANG Xin-jie;GAO Shan-shan;SUN Xiao-ming;DENG Fei;SHI Yan-ping;CHEN Di-shi;CHEN Chang-ying;LI Ping-shun(Yibin Vocational and Technical College,Yibin,Sichuan,644100,China;Sichuan Animal Disease Prevention and Control Center,Chengdu,Sichuan,610041,China;Beijing Yisen Biotechnology co.,Ltd,Beijing,100176,China;Chengdu Zhengda agriculture and animal husbandry food Co.,LTD,Chengdu,Sichuan,611134,China)

机构地区：[1]宜宾职业技术学院,四川宜宾644100 [2]四川省动物疫病预防控制中心,四川成都610041 [3]北京亿森宝生物科技有限公司,北京100176 [4]成都正大农牧食品有限公司,四川成都611134

出　　处：《动物医学进展》2024年第9期45-48,共4页Progress In Veterinary Medicine

基　　金：四川省科技计划重点研发项目(2023YFN0021);宜宾职业技术学院科技创新团队项目(ybzy21cxtd-08);宜宾职业技术学院自然科学研究项目(22ZRZD-09)。

摘　　要：为了建立简便快速的猪流行性腹泻病毒(PEDV)荧光定量RT-qPCR检测方法,根据GenBank数据库公布的猪流行性腹泻病毒囊膜蛋白E基因,设计特异性引物和探针结合微芯片技术,建立微芯片荧光定量RT-qPCR检测方法。该方法除对PEDV检测为阳性外,对与其同源性较近的致病病毒的检测均为阴性;最低检测限为1 copies/μL,常规荧光RT-qPCR检测限为10 copies/μL。批内、批间重复性试验表明,Ct值变异系数均在2%以内,可对PEDV临床样本进行检测。建立的检测方法特异性好、敏感性高、检测时间短且重复性好,适用于临床检测。In order to establish a fluorescent quantitative reverse transcription-polymerase chain reaction(RT-qPCR)assay for porcine epidemic diarrhea virus(PEDV)with easy operation and rapidity,specific primers and probes were designed based on the published PEDV vesicular membrane protein E gene in the GenBank database combined with the microchip technology,and a microchip RT-qPCR assay was established.The method was positive for PEDV and negative for other disease-causing viruses with close homology,and the lowest detection limit was 1 copies/μL,while the detection limit of conventional RT-qPCR was 10 copies/μL.The intra-and inter-batch reproducibility experiments showed that the coefficient of variation of Ct value was within 2%,and the method was able to detect clinical-positive samples of PEDV.A novel microchip RT-qPCR assay of PEDV with good specificity,high sensitivity,short detection time and good reproducibility was established,which is suitable for clinical testing.

关 键 词：猪流行性腹泻病毒 微芯片 实时荧光定量逆转录-聚合酶链反应 囊膜蛋白 

分 类 号：S852.2[农业科学—基础兽医学]