CSFV和PRRSV二联RT-PCR检测方法的建立

摘要

参照GenBank中猪瘟病毒(CSFV)和猪繁殖与呼吸障碍综合征病毒(PRRSV)基因保守序列,设计2对特异}l物,通过对最佳反应条件的优化,建立了CSFV和PRRSV的二联RT-PCR检测方法,该方法可同时扩增得到2条与试验设计相符的443 bp(CSFV)和246 bp(PRRSV)特异性条带.应用该二联RT-PCR方法检测猪细小病毒(PPV)、猪伪狂犬病毒(PRV)、牛病毒性腹泻病毒(BVDV)结果均为阴性,证明该方法有良好的特异性.将已知阳性病毒PCR模板最高稀释倍数作为PCR的敏感度,结果表明该二联RT-PCR体系扩增的敏感度为10-3,可对CSFV和PRRSV单个或混合感染的临床样品进行快速鉴别诊断.%According to the gene sequences in GenBank of swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus(PRRSV), two pairs of specific primers were designed for amplifying the two specific fragments of CSFV and PRRSV. After optimization of annealing temperature and primers concentrations, a double RT- PCR was established for simultaneous detection of the two viruses, and two specific bands of CSFV 443 bp and PRRSV 246 bp were detected. Porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and bovine viral diarrhea virus (BVDV) were detected by the double RT- PCR, and the results were all negative, showing that this method has good specificity. The maximum dilution of template of known viruses for positive PCR was defined as the sensitivity of PCR, and the results showed that the sensitivity of double RT- PCR was 10-3. Positive samples were detected through double PCR, and the results showed that the method could be used to effectively detect and differentiate CSFV and PRRSV single or co-infection infected in clinical samples.