猪繁殖障碍病毒性疫病六重PCR检测方法的建立及应用

Establishment and Application of a Multiplex PCR Method for Single-step Simultaneous Detection of Six Viral Pathogens of Porcine Reproductive Disorder

参照GenBank中相关的基因序列,设计了6对引物分别用于扩增PRV gE、PPV NS1、PCV ORF2、PRRSV ORF7、CSFVE2、JEVE基因的部分片段。敏感性和特异性试验结果表明,该mPCR对6种病毒的最低核酸检测量分别为PRV 6.6pg、PPV 96pg、PCV 12.9pg、PRRSV 10.5pg、CSFV 51pg、JEV 46pg,对猪流感病毒(SIV)、大肠杆菌(E.coli)的扩增结果均为阴性。103份临床病料检测结果表明,上述6种猪病毒性疫病在贵州省猪群中普遍存在且混合感染情况严重。该六重PCR方法不仅实现了上述6种病毒的同时检测,更实现了DNA病毒及RNA病毒的同步检测,能够对PRV、PPV、PCV、PRRSV、CSFV、JEV单个或混合感染的临床病料进行快速鉴别诊断。

According to the gene sequences in GenBank, six pairs of specific primers were de- signed to amplify part fragments of the specific genes of PRV gE, PPV NS1, PCV ORF2, PRRSV ORFT, CSFV E2 and JEV E, respectively. The results of sensitivity and specificity tests showed that the minimum amounts of nucleic acid detection by the mPCR were as follows： PRV 6.6 pg, PPV 96 pg, PCV2 12.9 pg, PRRSV 10.5 pg, CSFV 51 pg, and JEV 46 pg respectively, and the amplification results of Swine Influenza Virus （SIV） and Escherichia coli （E. coli） were both negative. The results of 103 clinical samples from Guizhou province revealed the six viral diseases exist universally in pig farms of Guizhou province, and the mixed infection was much seri- ous. The Six-plex PCR method could detect the infection of PRV, PPV, PCV, PRRSV, CSFV and JEV simultaneously, could detect DNA and RNA virus synchronously, and could differentiate and diagnose the clinical samples of single infection or mixed infection infected by the six viruses quickly.