ASFV和PRRSV多重TaqMan荧光定量PCR检测方法的建立与初步应用  

Establishment and preliminary application of multiple TaqMan real-time PCR detection methods for ASFV and PRRSV

作　　者：赵硕 陈忠伟 肖婷 何颖 张宁 覃国喜 卢冰霞 段振华 秦毅斌 张胜斌 段群棚 赵武 韦明宇 ZHAO Shuo;CHEN Zhong-wei;XIAO Ting;HE Ying;ZHANG Ning;QIN Guo-xi;LU Bing-xia;DUAN Zhen-hua;QIN Yi-bin;ZHANG Sheng-bin;DUAN Qun-peng;ZHAO Wu*;WEI Ming-yu(Key Laboratory of Veterinary Biotechnology of Guangxi/Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China/Guangxi Veterinary Research Institute,Nanning 530001,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;Guangxi Nongken Yongxin Animal Husbandry Group Jinguang Co.,Ltd,Nanning 530042,China;Yulin Animal Disease Prevention and Control Center,Yulin 537000,China;Guangxi Nongken Yongxin Animal Husbandry Group Xijiang Co.,Ltd,Guigang 537100,China;Guangxi Nongken Yongxin Animal Husbandry Group Co.,Ltd,Nanning 530022,China)

机构地区：[1]广西壮族自治区兽医研究所,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]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [3]广西农垦永新畜牧集团金光有限公司,广西南宁530042 [4]玉林市动物疫病预防控制中心,广西玉林537000 [5]广西农垦永新畜牧集团西江有限公司,广西贵港537100 [6]广西农垦永新畜牧集团有限公司,广西南宁530022

出　　处：《中国兽医科学》2023年第6期671-678,共8页Chinese Veterinary Science

基　　金：广西基本科研业务费专项(桂科专项22-4,21-3);南宁市科学研究与技术开发计划项目(20222043);玉林市科学研究与技术开发计划项目(玉市科20220515,20220516);贵港市科学研究与技术开发计划项目(桂科计2117003,202203011);南宁市乡塘区科学研究与技术开发计划项目(2020021605)。

摘　　要：为了建立一种可同时精准、快捷鉴别检测非洲猪瘟病毒(ASFV)和猪繁殖与呼吸综合征病毒(PRRSV)的方法,本研究选择猪源β-actin为内参基因,根据ASFV的P72基因及PRRSV的ORF6基因设计特异性引物及TaqMan探针,通过优化反应条件,建立了ASFV和PRRSV多重TaqMan荧光定量PCR检测方法,对该方法的特异性、敏感性、重复性进行验证,并初步应用于临床样品的检测。结果显示,该方法可在45 min内完成对ASFV、PRRSV及猪源β-actin基因的特异性扩增;对ASFV、PRRSV及猪源β-actin标准品模板的最低检测拷贝数分别为7.87×101 copies/μL、1.19×102 copies/μL和5.99×103 copies/μL,同一模板的3次重复试验的组内及组间变异系数均小于2%;用该方法检测92份临床样品,未检出ASFV,检出19份PRRSV阳性样品。结果表明,本研究建立了一种可快速鉴别检测ASFV及PRRSV的多重荧光定量PCR方法,较普通PCR敏感性高103倍,可应用于ASFV和PRRSV的快速鉴别诊断及流行病学调查。The purpose of the study was to establish a rapid detection method for African swine fever virus(ASFV)and porcine reproductive and respiratory syndrome virus(PRRSV).In this study,primers and probes were designed and synthesized based on the conserved gene sequences of P72 of ASFV and ORF6 of PRRSV.β-actin gene was selected as internal reference gene,and a multiple TaqMan real-time quantitative PCR detection method wase stablished.The reaction conditions were optimized and the specificity experiments,sensitivity experiments and repeatability experiments were verified.And clinical samples were used for preliminary application by this method.The results showed that the method could specifically amplify P72 gene of ASFV and ORF6 gene of PRRSV within 45 min.The lower limit were 7.87×101 copies/μL(ASFV),1.19×102 copies/μL(PRRSV)and 5.99×103 copies/μL(β-actin)respectively.The repeatability test showed that the inter-assay and intra-assay coefficient of variation were both less than 2%.Using this method to detect 92 clinical pig tissue samples,ASFV wasn’t detected while 19 PRRSV were detected.The multiple TaqMan real-time quantitative PCR detection method for ASFV and PRRSV was established,which was 103 times more sensitive than ordinary PCR.The method could be used for the application for rapid detection and epidemiological survey of ASFV and PRRSV.

关 键 词：非洲猪瘟病毒 猪繁殖与呼吸综合征病毒 多重TaqMan荧光定量PCR 

分 类 号：S852.659.1[农业科学—基础兽医学]