种公猪精液中CSFV、PCV2和PRRSV多重PCR检测方法的建立及应用  

Establishment and Application of Multiplex PCR Method for Detection of CSFV,PCV2 and PRRV in Semen of Stock Boars

作　　者：杨程 李文军 张雪峰 程宁 刘野 王凯月 邵笑 李双玉 孙英峰 YANG Cheng;LI Wen-jun;ZHANG Xue-feng;CHENG Ning;LIU Ye;WANG Kai-yue;SHAO Xiao;LI Shuang-yu;Sun Ying-feng(College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin,300384,China;Tianjin Nongken Kangjia Ecological Farming Co.,Ltd,Tianjin,300112,China;Jizhou Animal Husbandry and Aquaculture Development Service Centre,Tianjin,301900,China)

机构地区：[1]天津农学院动物科学与动物医学学院,天津300384 [2]天津农垦康嘉生态养殖有限公司,天津300112 [3]天津市蓟州区畜牧水产业发展服务中心,天津301900

出　　处：《动物医学进展》2024年第9期77-80,共4页Progress In Veterinary Medicine

基　　金：天津市科技支撑计划重点项目(22YFZCSN00100);天津市优秀企业科技特派员项目(22YDTPJC00420);天津市优秀农业科技特派员项目(22ZYCGSN00570)。

摘　　要：旨在建立可同时检测猪精液中猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)的多重PCR方法。根据GenBank数据库中的CSFV E2基因、PCV2 ORF2基因及PRRSV ORF5基因,设计3对特异性引物,通过优化反应条件和特异性、敏感性检验,建立可同时检测上述3种病原的多重PCR方法,用该方法对30份精液样品进行检测。结果表明,所建立的方法可同时扩增出734 bp(CSFV)、476 bp(PCV2)、305 bp(PRRSV)的目的条带,且对猪的其他病毒(PRV、PEDV、TEGV)扩增结果呈阴性,多重PCR最低检出限1×10^(7) copies/μL,具有良好的特异性和较高的灵敏度。用单一PCR与多重PCR方法对临床样品进行检测,两种方法的检测结果一致,表明该方法可同时对种公猪精液中CSFV、PCV2和PRRSV的检测。The aim of this study was to establish a multiplex PCR assay for simultaneous detection of classical swine fever virus(CSFV),porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV)in porcine semen.According to the CSFV E2 gene,PCV2 ORF2 gene and PRRSV ORF5 gene in GenBank database,three pairs of specific primers were designed respectively,and a multiplex PCR method was established to detect the above three pathogens at the same time through optimization of reaction conditions and specificity and sensitivity tests.The method was used to detect 30 semen samples.The results showed that the established method could simultaneously amplify 734bp(CSFV),476bp(PCV2),and 305bp(PRRSV)target bands,and the amplification results of other porcine viruses(PRV,PEDV,TEGV)were negative.The minimum detection limit of multiplex PCR was 1×10^(7) copies/μL,which showed good specificity and high sensitivity.Single PCR and multiplex PCR were used to detect clinical samples,and the results of the two methods were consistent,indicating that the multiplex PCR method can simultaneously detect classical swine fever virus,porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus in the semen of stock boars.

关 键 词：种公猪精液 猪瘟病毒 猪繁殖与呼吸综合征病毒 猪圆环病毒2型 多重PCR 

分 类 号：S857.29[农业科学—临床兽医学]