猪瘟病毒和不同型猪繁殖与呼吸综合征病毒的多重RT-PCR检测方法的建立

为建立能同时检测猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的方法,针对CSFV和PRRSV的基因序列设计3对特异性引物,第1对引物扩增CSFV毒株NS2基因508bp片段,第2对引物扩增PRRSV美洲型经典毒株和变异毒株Nsp2基因338bp/248bp片段,第3对引物扩增PRRSV欧洲型毒株ORF5基因614bp片段。经过反应条件的优化,建立了能同时检测并区分CSFV毒株和PRRSV美洲型经典毒株、变异毒株及欧洲型毒株的多重RT-PCR方法。该方法可以特异扩增CSFV和PRRSV,而与猪口蹄疫病毒(FMDV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV-2)均无交叉反应;对CSFV和PRRSV 4种重组质粒标准品的检出下限均为1.67×103拷贝/μL。对采集的106份临床疑似病料进行检测,结果CSFV和PRRSV变异株混合阳性4份,占3.77%(4/106);CSFV阳性7份,占6.60%(7/106);PRRSV变异株阳性17份,占16.04%(17/106)。结果表明,建立的多重RT-PCR检测方法可以用于CSFV和PRRSV的临床快速鉴别诊断和流行病学调查。

Establishment of a Multiplex RT-PCR for Detection of CSFV and Different Genotype PRRSV

For rapid detection of classical swine fever virus(CSFV) and porcine reproductive and respiratory syndrome virus(PRRSV),three pairs of specific primers were designed,of which one specifically amplified 508bp NS2gene fragment of CSFV,the other specifically amplified 338bp or 248bp Nsp2gene fragment of the classical strain or the highly pathogenic(HP) variant of the North American(NA) genotype PRRSV(NA-PRRSV),and another specifically amplified 614bp ORF5gene fragment of the European(EU) genotype PRRSV(EU-PRRSV).After optimization of the reaction conditions,a multiplex RT-PCR assay was established for simultaneous and differential detection of CSFV,the classical strain and HP variant of NA-PRRSV and the strain of EU-PRRSV.The assay could only react with CSFV and PRRSV,but not with foot-and-mouse disease virus(FMDV),porcine circovirus type 2(PCV-2),porcine pseudorabies virus(PRV) and porcine parvovirus(PPV).The detection limit of the method was as little as 1.67×10 3 copies / μ L of each of the four recombinant plasmid templates.The established assay was successfully used to detect CSFV and PRRSV in 106clinical samples,of which 4samples were positive for CSFV and PRRSV,7samples for CSFV and 17samples for HP-PRRSV.The results indicated that the established multiplex RT-PCR assay was suitable for rapid detection and epidemic surveillance of CSFV and PRRSV.