猪水泡病病毒RT-PCR检测方法的建立

Establishment of Rapid Detection of SVDV by RT-PCR

根据基因库中的猪水泡病病毒(SVDV)高度保守的VP1基因的序列,设计了与SVDV互补的2对特异性引物,通过对影响PCR扩增因素的筛选,成功地扩增出251bp和366bp特异性条带,将其克隆入pMD18-T载体中,并进行序列测定,与已发表的SVDV基因比较发现,核苷酸的同源性为100%,证实为SVDV的VP1段基因。通过检测FMDV、VSV、BHK21细胞等,对照的扩增结果均为阴性,验证其特异。对SVDVRNA进行10倍系列稀释后检测,结果SVDVRNA在10-4稀释度时仍能检测到阳性,说明具有较好的敏感性。此项试验能稳定、高效、快速地检测出猪水泡病病毒。

According to swine vesicular disease virus （SVDV） gene published and RT-PCR assay for the diagnosis and characterization of SVDV reported, 2 sets of primers to detect SVDV were designed in the conserved VP1 gene of SVDV which is conserved and specific. The production of the RT-PCR assay is 251bp and 366bp, subsequently cloned into pMD18-T Vector. The nucleotide of production was compared with the corresponding sequences of published sequence of SVDV. The nucleotide homology was 100%. Suggest that it is gene of SVDV. Specificity was tested by that amplification of FMDV,VSV,BHK21 cell was negative. The dilution of 10^-4 SVDV RNA can be detected. Suggest that it has high sensitivity. The RT-PCR method is rapid, sensitive and specific for detecting SVDV.