PEDV经典毒株与变异毒株套式RT-PCR鉴别检测方法的建立及应用

Development and application of a nested RT-PCR method for differentiation between variant and classical PEDV strains

我国当前猪流行性腹泻病毒(PEDV)流行毒株与经典毒株相比,在S基因上存在多处变异,其中在58aa处有4个氨基酸QGVN的插入。为快速鉴别经典毒株与变异毒株,本试验根据Gen Bank中当前流行毒株S基因序列设计合成2对特异性扩增引物,在优化RT-PCR反应条件的基础上,建立一套区分PEDV变异毒株与经典毒株的套式RT-PCR检测方法,并完成特异性、敏感性试验及对临床送检样品检测试验。结果表明:该套式PCR检测方法可以分别鉴定PEDV经典毒株和变异毒株,PEDV经典毒株仅在PCR外套产物有749 bp的条带,内套产物没有条带;而PEDV变异毒株在PCR外套产物中有760 bp的条带,而在内套产物中出现510 bp的条带。该检测方法特异性强、灵敏度高、操作简单,为猪流行性腹泻的快速诊断提供了一种较为方便的技术手段。

Compared with classical strains of porcine epidemic diarrhea virus（ PEDV）,the variant strain has multiple mutations in the S gene,with the insertion of four amino acids（ QGVN） at amino acid position 58. For the rapid differentiation of classical and variation strains,two pair of primers were designed based on S gene sequence of PEDV published in Gen Bank and RT-PCR assay was established.The results showed that the method can defferentiate classical and variant PEDV strains. Only the 749 bp band can be detected in classical PEDV strains,while both 760 bp and 510 bp bands appear in the variant PEDV strains. The detection method has a high specificity and sensitivity. This study provides a tool for rapid diagnosis of PEDV infection.