牛流行热病毒SYBR GreenⅠ荧光定量PCR检测方法的建立及应用

The establishment and application of the SYBR Green Ⅰ real-time PCR for the detection of bovine ephemeral fever virus

为建立牛流行热病毒(BEFV)的荧光定量PCR检测方法,本研究根据GenBank中已登录的BEFV的G基因保守序列设计了1对特异性引物。结果显示,建立的标准曲线线性关系较好,其相关系数为0.996 3。该方法仅对BEFV检测为阳性,表明其特异性强。该方法的检测下限约为4.0×10^1 copies/μL,比普通PCR敏感性高10倍。重复性试验结果显示,该方法的组内和组间变异系数均小于0.76%。利用本方法分别对45份牛病料样品进行检测,结果检出了11份阳性样品,表明所建立的荧光定量PCR方法准确可靠,可以用于BEFV的快速诊断和定量分析。

In order to establish a real-time PCR detection method for bovine ephemeral fever virus,a pair of specific primers were designed and synthesized according to the conservative sequence of G gene in the published sequence of BEFV.The method achieved a good linear relationship with a correlation of 0.996 3.This method had no cross-reaction with the other viruses and had high specificity.And the detection sensitivity was up to 4.0×10^1 copies/μL,which is 10 times higher than the conventional PCR method.The results of repetitive experiments showed that the method had a good repeatability with coefficients of variation of intra-assay and inter-assay less than 0.76%.Using this method,the samples of 45 diseased cattle were detected,and 11 positive samples were determined.The above-mentioned results showed the detection method is accurate and reliable,and will provide technical support for the early rapid diagnosis and quantitative analysis of BEFV.