水貂肠炎病毒SYBR GreenⅠ荧光定量PCR检测方法的建立

Development of a SYBR GreenⅠreal-time quantitative PCR assay for the detection of Mink enteritis virus

根据Gen Bank登录的水貂肠炎病毒(MEV)VP2蛋白基因的保守序列设计了1对特异性引物,经PCR扩增出146bp的目的片段,并克隆到pMD 18-T载体上,提取重组质粒经PCR及测序鉴定正确后,以10倍梯度稀释质粒作为阳性标准品,绘制荧光定量标准曲线,以此建立一种基于SYBR Green荧光染料的定量PCR特异性检测水貂肠炎病毒的方法。结果表明:该方法对MEV有很好的特异性,检测灵敏度为34拷贝/μL,且重复性试验变异系数均小于2%。临床检测中,在80份样品中检测到30份阳性样品,高于普通PCR和电镜观察方法的检出率。该方法的建立实现了对MEV临床早期快速诊断和定量分析,为毛皮动物MEV的防控提供了可靠的检测方法。

A quantitative PCR assay was developed for specific and sensitive detection of mink enteritis virus. The primer pair targeting a 146 bp fragment of the conserved VP2 gene was selected based on alignment of viral sequences available at Gen Bank. The analytic sensibility of this assay was 34 copies of plasmid DNA,and it had high repeatability with CV 2%. The assay was evaluated by testing 80 fecal samples collected from suspicious cases. The results showed that 30 samples were found positive for the virus infection,which was higher than that of routine PCR and electron microscopy. Therefore the new assay provides a useful method for the rapid diagnosis of mink enteritis and the prevention and control of the disease.