JEV、PRRSV、PPV及PRV多重PCR检测方法的建立及应用  

Establishment and Application of Multiplex PCR Assay for JEV,PRRSV,PPVandPRV'

作　　者：李斌[1] 赵硕 周英宁[1] 赵武[1] 蒋家霞 何颖[1] 郭旋 卢冰霞[1] 林昌华 秦毅斌[1] 段群棚[1] 全琛宇 许心婷 陈婷婷 许艺兰 陈忠伟[1] 张宁 LI Bin;ZHAO Shuo;ZHOU Ying-ning;ZHAO Wu;JIANG Jia-xia;HE Ying;GUO Xuan;LU Bing-xia;LIN Chang-hua;QIN Yi-bin;DUAN Qun-peng;QUAN Chen-yu;XU Xin-ting;CHEN Ting-ting;XU Yi-lan;CHEN Zhong-wei;ZHANG Ning(Guangri Veterinary Research Institute/key Laboratory of Veterinary Biotechnology of Guangxi/Key Laboratory of China(Guangri)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Nanning,Guangxi,530o01,China;Guangzi Nongken Yongrin Animal Husbandry Group Xinring Co.,Ltd,Liuzhou,Guangxi,545000,China;Guangri Nongken Yongxin Animal Husbandry Group Jinguang Co.,Ltd,Nanning,Guangxi,530042,China;Guangzi Nongken Yongxin Animal Husbandry Group Xijiang Co.,Ltd,Guigang,Guangxi,537100,China;Guangxi Guiken Animal Husbandry Co.,Ltd,Nanning,Guangxi,530022,China)

机构地区：[1]广西兽医研究所/广西兽医生物技术重点实验室/农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西南宁530001 [2]广西农垦永新畜牧集团新兴有限公司,广西柳州545000 [3]广西农垦永新畜牧集团金光有限公司,广西南宁530042 [4]广西农垦永新畜牧集团西江有限公司,广西贵港537100 [5]广西桂垦牧业有限公司,广西南宁530022

出　　处：《动物医学进展》2023年第3期48-53,共6页Progress In Veterinary Medicine

基　　金：广西创新驱动发展专项资金项目(桂科AA18118051);广西基本科研业务费专项(桂科专项21-3);柳州市科技计划项目(2020NACB0804);南宁市科学研究与技术开发计划项目(20202090)。

摘　　要：为了建立一种能快速鉴别猪日本脑炎病毒(JEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪伪狂犬病病毒(PRV)的准确、高效的多重常规PCR检测方法,针对JEV、PRRSV、PPV、PRV基因保守区域合成特异性引物,优化后获得最优反应条件,对该方法特异性、敏感性、重复性进行了测定,并应用该方法对临床样品进行初步应用。结果显示,该方法对JEV、PRRSV、PPV、PRV可进行特异性扩增,对混合阳性质粒检测下限达2×10^(6)copies/μL,对猪圆环病毒2型、猪瘟病毒、猪流行性腹泻病毒、猪丁型冠状病毒、猪传染性胃肠炎病毒等相关病毒均无扩增,且重复性良好。用该方法检测51份2021年采集的广西区内组织样品,检出JEV、PRRSV、PPV、PRV阳性率分别为7.84%、50.98%、5.88%、23.53%,且存在多重混合感染的情况。所建立的多重PCR方法具有良好的特异性、敏感性及重复性,可应用于临床常见猪繁殖障碍性病毒病的快速诊断和监测预警,为猪繁殖障碍性病毒病提供了诊断技术支持。This study was to establish a rapid and accurate efficient routine multiplex PCR detection method for four common clinical porcine reproductive disorder viruses:Japanese encephalitis virus(JEV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine parvovirus(PPV)and porcine pseudorabies virus(PRV).Four pairs of specific primers were synthesized for genome sequencing of JEV,PRRSV,PPV and PRV.The reaction conditions of this multiplex PCR were obtained after optimization,and specificity,sensitivity,reproducibility of multiplex PCR were determination.The method was subsequently applied to clinical samples for preliminary application.Results showed that the multiplex PCR could simultaneously detecting JEV,PRRSV,PPV and PRV,and the limit was 2×10^(6)copies/μL.Epidemic diarrhea virus,porcine delta-coronavirus,and porcine transmissible gastroenteritis virus were not amplified by this method.The method was used to detect 51 porcine clinical tissue samples in Guxnagxi and the positive rates of JEV,PRRSV,PPV and PRV were 7.84%,50.98%,5.88%and 23.53%,respectively.There were multiple mixed infections.The multiplex PCR method established in this study has good specificity,sensitivity and repeatability which can be applied to the rapid diagnosis,monitoring and early warning of common clinical porcine reproductive dysfunction viral diseases,and provides a diagnostic detection technology support for porcine reproductive dysfunction viral diseases.

关 键 词：日本脑炎病毒 猪繁殖与呼吸综合征病毒 猪细小病毒 猪伪狂犬病病毒 多重PCR 

分 类 号：S852.65[农业科学—基础兽医学]