CSFV、PRRSV、PEDV和PRV四重荧光定量PCR方法的建立及应用  

Establishment and application of a quadruple real-time PCR assay for CSFV,PRRSV,PEDV and PRV

作　　者：王利丽[1,2] 路超 李富强[1,2] 鄢明华 任卫科[1,2] 江珊 池晶晶[1,2] 张莉 李程[1,2] 李秀丽 WANG Li-li;LU Chao;LI Fu-qiang;YAN Ming-hua;REN Wei-ke;JIANG Shan;CHI Jing-jing;ZHANG Li;LI Cheng;LI Xiu-li(Animal Husbandry and Veterinary Research Institute,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China;Tianjin Observation and Experiment Site of National Animal Health,Tianjin 300381,China)

机构地区：[1]天津市农业科学院畜牧兽医研究所,天津300381 [2]国家动物疫病天津观测实验点,天津300381

出　　处：《中国兽医科学》2022年第10期1259-1267,共9页Chinese Veterinary Science

基　　金：中央级公益性科研院所基本科研业务费院级统筹项目:动物疫病数据中心;天津市科技计划项目(19YFZCSN00570);天津市重大推广项目(ITTPRS2021003)

摘　　要：为了快捷地进行规模化猪场疫病风险评估,建立了一种同时检测猪场环境中猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪流行性腹泻病毒(PEDV)和猪伪狂犬病病毒(PRV)的荧光定量PCR方法。根据CSFV 5′UTR基因、PRRSV Nsp2基因、PEDV M基因和PRV g E基因保守区,设计特异性引物和探针,通过优化PCR反应条件,建立了四重荧光定量PCR方法。结果显示,该方法可特异性扩增CSFV、PRRSV、PEDV和PRV,与TGEV、RV、PCV2、PPV、PRV Bartha-K61、JEV、E.coli、S.suis 2等病原体无交叉反应,四种病毒的最低检出值为4.62×10^(1)copies/μL,批内及批间变异系数为0.003~0.016。与国标方法相比,两种方法阳性符合率为83.33%~100.00%(182份组织样品)。运用该方法检测猪场采集的434份肛拭子、鼻拭子及环境样品,肛拭子和环境样品中PEDV检出率较高,分别为16.53%和11.90%;环境样品中PRV检出率较高,为8.28%。综上结果,此方法为规模化猪场进行疫病风险评估提供了一种有效的技术手段。In order to quickly assess the disease risk caused by four viruses including classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine epidemic diarrhea virus(PEDV)and porcine pseudorabies virus(PRV)on large-scale pig farms,a quadruple real-time PCR assay for CSFV,PRRSV,PEDV and PRV was established.We designed and synthesized the specific primers based on the conserved region of CSFV 5′UTR gene,PRRSV Nsp2 gene,PEDV M gene and PRV g E gene respectively and optimized the PCR reaction conditions.The assay could specifically detected CSFV,PRRSV,PEDV and PRV,meanwhile the detection of TGEV,RV,PCV2,PPV,PRV Bartha-K61,JEV,E.coli,S.suis 2 were negative.The detection limit was 4.62×10^(1)copies/μL for four viruses.The variation coefficient of the intraand inter-assay ranged from 0.003—0.016.The comparison results between using the national standard methods and this method showed that the positive coincidence rate was 83.33%—100.00%(182 tissue samples).Using this method to detect the anal swabs,nasal swabs and environmental samples of pig farms(434 samples),the positive rate of PEDV in anal swabs and environmental samples was high(16.53%and 11.90%,respectively),and the positive rate of PRV in environmental samples was high(8.28%).In summary,the established method in this study could provide an effective technical way for virus epidemic risk assessment in large-scale pig farms.

关 键 词：猪瘟病毒 猪繁殖与呼吸综合征病毒 猪流行性腹泻病毒 猪伪狂犬病病毒 荧光定量PCR方法 疫病风险评估 

分 类 号：S852.65[农业科学—基础兽医学]