猪繁殖与呼吸综合征病毒、猪圆环病毒三型、猪流感病毒三重RT-PCR检测方法建立及初步应用

Development and preliminary application of a triplex RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus,porcine circovirus typeⅢ,and swine influenza virus

【背景】猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒三型(porcine circovirus typeⅢ,PCV3)和猪流感病毒(swine influenza virus,SIV)是3种影响猪只健康的重要呼吸道病原,对养猪业造成严重的经济损失,因此需要建立高效快速的检测方法,以了解3种病原在国内的流行情况。【目的】建立能同时检测PRRSV、PCV3和SIV的三重RT-PCR方法,为3种病毒的流行病学调查和疾病监控提供技术支持。【方法】针对猪繁殖与呼吸综合征病毒(PRRSV),猪圆环病毒三型(PCV3),猪流感病毒(SIV)基因序列,分别设计3对特异性引物,扩增PRRSV M和N基因(436 bp)、PCV3 Cap基因(619 bp)和SIV M基因(199 bp)。通过对退火温度和引物浓度优化建立三重RT-PCR检测方法,并对建立的多重检测方法进行特异性、敏感性、重复性试验验证。【结果】建立了能够同时快速检测PRRSV、PCV3、SIV的三重RT-PCR方法,而对猪瘟病毒(classical swine fever virus,CSFV)、猪伪狂犬病病毒(pseudorabies virus,PRV)、猪圆环病毒2型(porcine circovirus typeⅡ,PCV2)、猪细小病毒2型(porcine parvovirusⅡ,PPV2)、猪细小病毒3型(porcine parvovirusⅢ,PPV3)和猪细环病毒1型(torque teno sus virus I,TTSuV1)等6种病原的扩增均为阴性,特异性较好。敏感性结果显示,同时检测PRRSV、PCV3、SIV这3种病原的检测下限为100copies/μL,批间与批内试验结果均一致。用该方法对黑龙江省部分地区猪场的67份临床病料进行检测,结果显示PRRSV阳性率为16.5%,PCV3阳性率为10.5%,SIV阳性率为10.5%,而且存在混合感染。【结论】该方法灵敏度高、特异性强,能够应用于临床样品检测,有效预测病原的流行情况。

[Background]Porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus typeⅢ(PCV3),and swine influenza virus(SIV),the major respiratory pathogens threatening pig health,lead to great economic loss in swine industry.Therefore,it is an urgent task to develop efficient and rapid detection assay for clarifying the prevalence of these pathogens in China.[Objective]We aim to establish a rapid and accurate triplex RT-PCR method for simultaneous detection of PRRSV,PCV3,and SIVand facilitate epidemiological investigation and disease surveillance.[Methods]Three pairs of specific primers were designed according to the gene sequences of PRRSV,PCV3 and SIV and the M and N genes(436 bp)of PRRSV,Cap gene(619 bp)of PCV3,and M gene(199 bp)of SIV were amplified.The annealing temperature and primer concentration were optimized and the specificity,sensitivity and reproducibility of the triplex RT-PCR were tested.[Results]The triplex RT-PCR can rapidly detect PRRSV,PCV3 and SIV,but there was no amplification for classical swine fever virus(CSFV),porcine pseudorabies virus(PRV),porcine circovirus typeⅡ(PCV2),porcine parvovirusⅡ(PPV2),porcine parvovirusⅢ(PPV3)and torque teno sus virus I(TTSuV1).Thus,it was highly specific.The lower limit of detection for PRRSV,PCV3 and SIV was 100 copies/μL and the inter-batch and intra-batch test results were consistent.The method was applied to detect the 67 clinical samples in some farms in Heilongjiang Province.The result indicated that the detection rate of PRRSV,PCV3 and SIV was 16.5%,10.5%and 10.5%respectively,and suggested mixed infection.[Conclusion]The triplex RT-PCR is highly sensitive and specific,which can be used for the detection of clinical samples and prediction of disease prevalence.