【作者】 施开创； 陈进喜； 屈素洁； 许瑞胜； 熊毅； 刘棋； 陈汉忠； 李刚；

【Author】 Shi-kaichuang~(1,2),Chen-jinxi~3,Qu-sujie~2,Xu-ruisheng~3, Xiong-yi~2,Liu-qi~2,Chen-hanzhong~3,Li-gang~1 (1.Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China; 2.Guangxi Center for Animal Disease Control and Prevention,Nanning 530001,China; College of Animal Science and Technology,Guangxi University,Nanning 530005,China)

【机构】 中国农业科学院北京畜牧兽医研究所； 广西动物疫病预防控制中心； 广西大学动物科学技术学院；

【摘要】 针对猪脑心肌炎病毒(Encephalomyocarditis virus,EMCV)VP1基因序列设计并合成一对引物,构建含有该引物扩增序列的重组质粒作为阳性标准品,建立了检测EMCV核酸的SYBR Green I real-time PCR方法。该方法线性关系好,标准曲线的相关系数达到0.991;敏感性高,初始模板的检出下限为1×10~1拷贝/μL,比常规PCR方法高100倍;特异性强,与其他猪源病毒如FMDV、PRRSV、CSFV、PRV均不发生交叉反应;重复性好,组内与组间的变异系数均小于3%。应用所建立的方法检测17份临床可疑病例的心、脑组织样品,样品1份为阳性。结果表明,本研究建立的real-timePCR检测方法灵敏度高、特异性强、重复性好,可以用于EMCV的病原检测及定量分析。更多还原

【Abstract】 A pair of primers was designed according to the sequence of the VP1 gene of porcine encephalomyocarditis virus(EMCV) and a recombinant plasmid containing the target gene was constructed as a standard control.Then,a Real-time PCR assay based on SYBR Green I for detection of EMCV was established.The assay had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.991.Also,it was highly sensitive and had a detection limit of 1×10~1 copies/μL of initial templates,which was 100 times more sensitive than the conventional PCR.Moreover,it was highly specific and had no cross-reaction with other porcine viruses including FMDV, PRRSV,CSFV and PRV.Finally,it was highly reproducible and had a coefficient of variations less than 3 percent for both intra-and inter-assay.Seventeen heart and brain samples from suspicious cases in field were detected by the Real-time PCR,and one was positive for EMCV.The high correlativity,sensitivity,specificity and reproducibility of this assay,together with its relatively rapid and simple procedure,indicates that the SYBR Green I Realtime PCR can be used as an effective assay for detection of nucleic acids and quantification of viral loads of EMCV.更多还原