CSFV,SIV,PCV2,PRV和PRRSV多重PCR检测方法的建立及初步应用

参照GenBank中的猪瘟病毒(CSFV)、猪流感病毒(SIV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合病毒(PRRSV)基因序列,设计了5对引物分别用于扩增CSFVE2、SIV、PCV2ORF2、PRVgB、PRRSVN基因的目的片段。通过对反应条件进行优化,建立了检测CSFV、SIV、PCV2、PRV和PRRSV的多重PCR(mPCR)诊断方法。敏感性和特异性结果显示,该mPCR对5种病毒的最低核酸检测量为10pg。而对大肠杆菌、葡萄球菌、链球菌、猪细小病毒、鸡传染性喉气管炎病毒、鸡新城疫病毒、猪传染性胃肠炎病毒的多重PCR的扩增结果均为阴性。临床样品的多重PCR检测结果表明,建立的多重PCR方法能够对SIV、CSFV、PCV2、PRV、PRRSV单个或混合感染的临床样品进行快速鉴别诊断。

Establishment and Application of a Multiplex PCR Assay for Simultaneous Detection of CSFV,SIV,PCV2,PRV and PRRSV

According to the gene sequences in GenBank of classical swine fever virus （CSFV）,swine influenza virus （SIV）,porcine circovirus type 2 （PCV2）,porcine pseudorabies virus （PRV）,porcine reproductive and respiratory comprehensive virus （PRRSV） gene sequences,designed 5 pairs of primers were used to amplify CSFV E 2,SIV,PCV 2 ORF 2,PRV gB,PRRSV N gene.Reaction conditions were optimized by the establishment of a detection CSFV,SIV,PCV2,PRV and PRRSV multiple PCR （mPCR） diagnosis.Sensitivity and specificity results showed that the mPCR minimum of 5 of the virus nucleic acid detection volume were 10 pg.Against E.coli,staphylococcus,streptococcus,porcine parvovirus,infectious laryngotracheitis virus,newcastle disease virus,porcine transmissible gastroenteritis virus,multiple PCR amplification results were negative.Clinical samples of multiple PCR results showed that the multiplex PCR method capable of SIV,CSFV,PCV2,PRV,PRRSV infection of single or mixed clinical samples for rapid diagnosis.