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
发布时间：2018-07-15 16:26
【摘要】：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。
[Abstract]:Compared with the classical strains of porcine epidemic diarrhea virus (PEDV) in China, there are many mutations in S gene, among which four amino acids QGVN are inserted at 58aa. In order to quickly identify the classical and variant strains, two pairs of specific primers were designed and synthesized according to the S gene sequence of the current prevalent strain in GenBank. Based on the optimization of the conditions of RT-PCR reaction, two pairs of primers were designed and synthesized. A nested RT-PCR method was established to distinguish PEDV variant strains from classical strains, and the specificity, sensitivity and clinical samples were tested. The results showed that the classical PEDV strain and the mutant strain PEDV could be identified by nested PCR, only 749 BP bands were found in the PCR coat product, and there were 760 BP bands in the PCR coat product of PEDV mutant strain, and there were 749 BP bands in the PCR coat product, and 760 BP band in the PCR coat product of the PEDV mutant strain. A 510 BP band was found in the inner product. This method has the advantages of high specificity, high sensitivity and simple operation, which provides a more convenient technique for the rapid diagnosis of swine epidemic diarrhea.
【作者单位】： 华南农业大学兽医学院;广东省动物源性人兽共患病预防与控制重点实验室;
【基金】：广东省生猪产业体系创新团队项目
【分类号】：S852.651
[Abstract]:Compared with the classical strains of porcine epidemic diarrhea virus (PEDV) in China, there are many mutations in S gene, among which four amino acids QGVN are inserted at 58aa. In order to quickly identify the classical and variant strains, two pairs of specific primers were designed and synthesized according to the S gene sequence of the current prevalent strain in GenBank. Based on the optimization of the conditions of RT-PCR reaction, two pairs of primers were designed and synthesized. A nested RT-PCR method was established to distinguish PEDV variant strains from classical strains, and the specificity, sensitivity and clinical samples were tested. The results showed that the classical PEDV strain and the mutant strain PEDV could be identified by nested PCR, only 749 BP bands were found in the PCR coat product, and there were 760 BP bands in the PCR coat product of PEDV mutant strain, and there were 749 BP bands in the PCR coat product, and 760 BP band in the PCR coat product of the PEDV mutant strain. A 510 BP band was found in the inner product. This method has the advantages of high specificity, high sensitivity and simple operation, which provides a more convenient technique for the rapid diagnosis of swine epidemic diarrhea.
【作者单位】： 华南农业大学兽医学院;广东省动物源性人兽共患病预防与控制重点实验室;
【基金】：广东省生猪产业体系创新团队项目
【分类号】：S852.651
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