羊传染性脓疱病毒SYBR Green Ⅰ实时荧光定量PCR方法的建立  

Establishment of SYBR GreenⅠreal-time fluorescence quantitative PCR for sheep contagious ecthyma virus

作　　者：李旭东[1] 庞方圆 苏日娜[1] 李浩[1] 张岩[1] 申之义[1] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018

出　　处：《中国兽医学报》2015年第9期1441-1445,共5页Chinese Journal of Veterinary Science

摘　　要：根据GenBank已经发表的CEV参考毒株（登录号：AF097215）F1L基因序列,利用Primer Express 3.0软件设计合成1对特异性引物,以pMD19-T-63bp重组阳性质粒作为标准品,建立了羊传染性脓疱病毒SYBR GreenⅠ实时荧光定量PCR检测方法。经过反应条件的优化,建立的羊传染性脓疱病毒SYBR GreenⅠ实时荧光定量PCR的线性关系良好,标准曲线的相关系数R2=0.99,扩增效率E=1.18。该方法敏感性高且特异性强,最低检出下限为65.47拷贝/μL,与羊痘和口蹄疫病毒均不发生交叉反应,重复性好,组内和组间变异系数分别为0.17%~0.59%和0.74%~1.25%。运用该方法对2例羊传染性脓疱病例进行检测,结果均为阳性。因此,可将该方法应用于临床诊断,具有准确、高效、快捷、灵敏的特点。Sheep contagious ecthyma is an acute, contagious disease that is caused by the contagious ecthyma virus （CEV） of sheep and goats. To diagnosing the disease more quickly and accurately, the CEV SYBR Green I real-time fluorescence quantitative PCR was established in this experi- ment. By using recombinant positive plasmid pMD19-T-63 bp as a standard,the method was estab- lished through a pair of specific primers synthesized by using the Primer Express 3.0 based on the FIL gene sequence on CEV reference strains published by GenBank （AF097215）. The result showed that by optimizing reaction conditions, the linear relationship of SYBR Green I real-time fluorescence quantitative PCR was good. The correlation coefficiency was 0. 99 for standard curves, and the amplification efficiency was 1.18. The SYBR Green I real-time fluorescence quanti- tative PCR was proved to have the advantages of high sensibility, strong specificity,and good re- peatability. The minimum detection limit was 65.47 copies/μL. Both CaPV and FMDV were not detected by the SYBR Green I real-time fluorescence quantitative PCR. The coefficient of variation for intra-assay and inter-assay were 0. 17%-0.59% and 0. 74%-1. 25% , respectively. By applying this detection method to two cases of sheep contagious ecthyma,both were tested positive. There- fore, it is feasible to apply this technique to clinical diagnosis for the disease.

关 键 词：羊传染性脓疱病毒 SYBR GreenⅠ 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]