摘要
根据GenBank登录的猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)的参考基因序列,设计3对引物分别用于扩增PCV2的ORF2基因、PRV的gE基因、PRRSV的N基因的目的片段,通过优化反应中各个影响因素,建立了PRV、PCV2、PRRSV的多重PCR(mPCR)检测方法。敏感性和特异性的结果表明,该方法对这3种病毒的最低核酸检出量分别为32.5(PRV)、25.2(PCV2)、35.9pg(PRRSV)。该方法对猪流感病毒(SIV)、猪圆环病毒1型(PCV1)、大肠杆菌、猪瘟病毒(CSFV)、猪流行性腹泻病毒(TGE)等病毒的检测结果均为阴性。200份临床样品的多重PCR结果表明,PCV2感染率为80%(160/200),PRV感染率为21%(42/200),PRRSV的感染率为78%(156/200)。200份临床样品主要为PCV2和PRRSV混合感染,阳性率达56.0%(112/200)。该方法的建立对这3种病毒病的早期快速检测和指导临床实践具有十分重要的意义。
According to the gene sequences published in GenBank of porcine pseudorabies virus （PRV）,porcine circovirus type 2 （PCV2） and porcine reproductive and respiratory syndrome virus （PRRSV）, three pairs of specific primers were designed for amplifying the three specific fragments, including the ORF2 of PCV2, the gB gene of PRV and the N gene of PRRSV, respectively. After optimization of annealing temperature and the concentration of the primers,a multiplex PCR assay（mPCR）was established for simultaneous detection of the three viruses. The sensitivity and specificity tests showed that the mPCR was so sensitive that it can detect the pg level,29.0 pg for PCV2,36.8 pg for PRRSV,32.5 pg for PRV. The testing results for classical swine fever virus （CSFV）, swine influenza virus （SIV）, porcine circovirus type 1 （PCV1）, bovine viral diarrhea virus （BVDV） ,Escherichia coli by the mPCR were all negative. 200 clinical samples were detected by the mPCR. The infection rate of PCV2 is 80%（160/200）,PRV 21%（42/200）,PRRSV 78%（156/ 200）. PCV2 was detected from all the samples,among which 3 samples were positive for PPV,68 samples were positive for PRRSV. The multiplex PCR assay established could be used to effectively detect and differentiate PCV2, PRV and PRRSV respective infection or co-infection in clinical samples.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2014年第9期1393-1397,共5页
Chinese Journal of Veterinary Science
基金
沈阳市高新技术产业发展与科技攻关资助项目(F11-112-3-00)
