JEV、PRRSV、PPV及PRV多重PCR检测方法的建立及应用

Establishment and Application of Multiplex PCR Assay for JEV,PRRSV,PPVandPRV'

为了建立一种能快速鉴别猪日本脑炎病毒(JEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪伪狂犬病病毒(PRV)的准确、高效的多重常规PCR检测方法,针对JEV、PRRSV、PPV、PRV基因保守区域合成特异性引物,优化后获得最优反应条件,对该方法特异性、敏感性、重复性进行了测定,并应用该方法对临床样品进行初步应用。结果显示,该方法对JEV、PRRSV、PPV、PRV可进行特异性扩增,对混合阳性质粒检测下限达2×10^(6)copies/μL,对猪圆环病毒2型、猪瘟病毒、猪流行性腹泻病毒、猪丁型冠状病毒、猪传染性胃肠炎病毒等相关病毒均无扩增,且重复性良好。用该方法检测51份2021年采集的广西区内组织样品,检出JEV、PRRSV、PPV、PRV阳性率分别为7.84%、50.98%、5.88%、23.53%,且存在多重混合感染的情况。所建立的多重PCR方法具有良好的特异性、敏感性及重复性,可应用于临床常见猪繁殖障碍性病毒病的快速诊断和监测预警,为猪繁殖障碍性病毒病提供了诊断技术支持。

This study was to establish a rapid and accurate efficient routine multiplex PCR detection method for four common clinical porcine reproductive disorder viruses:Japanese encephalitis virus(JEV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine parvovirus(PPV)and porcine pseudorabies virus(PRV).Four pairs of specific primers were synthesized for genome sequencing of JEV,PRRSV,PPV and PRV.The reaction conditions of this multiplex PCR were obtained after optimization,and specificity,sensitivity,reproducibility of multiplex PCR were determination.The method was subsequently applied to clinical samples for preliminary application.Results showed that the multiplex PCR could simultaneously detecting JEV,PRRSV,PPV and PRV,and the limit was 2×10^(6)copies/μL.Epidemic diarrhea virus,porcine delta-coronavirus,and porcine transmissible gastroenteritis virus were not amplified by this method.The method was used to detect 51 porcine clinical tissue samples in Guxnagxi and the positive rates of JEV,PRRSV,PPV and PRV were 7.84%,50.98%,5.88%and 23.53%,respectively.There were multiple mixed infections.The multiplex PCR method established in this study has good specificity,sensitivity and repeatability which can be applied to the rapid diagnosis,monitoring and early warning of common clinical porcine reproductive dysfunction viral diseases,and provides a diagnostic detection technology support for porcine reproductive dysfunction viral diseases.