作　　者： (蔡骁垚）; (魏晓锋）; (熊炜）; (陈懿斐）; (林颖峥）; (张泉）; (陈鸿军）;

机构地区： 中国农业科学院上海兽医研究所,上海200241 扬州大学兽医学院,扬州225009

出　　处： 《中国实验动物学报》 2017年第4期433-437,共5页

摘　　要： 目的建立一种能在临床上快速、准确地检测大鼠疑似泰勒氏病毒(Theiler’s-like virus of rats,TLV)的方法,采用TaqMan探针荧光定量聚合酶链式反应(qPCR)技术,特异性针对TLV病毒核酸进行检测。方法通过基因合成序列作为质粒标准品的模板,同时选择特异性的序列在3622~3729 nt处,设计一对引物和TaqMan性探针,优化反应体系及条件,进行qPCR扩增,从而建立TLV TaqMan探针qPCR方法,并对其灵敏度、稳定性和特异性进行评价。结果建立的TLV qPCR检测方法,标准曲线线性关系良好,R^2值可达到0.99,灵敏性最低能够检测到10个拷贝数/μL,对比普通PCR方法,高出其100倍;对其他常见大鼠病毒均无非特异反应;重复性良好,批内和批间变异系数均小于1%。结论利用TaqMan探针建立快速检测TLV的荧光定量PCR方法,该方法具有操作简便、灵敏度高、特异性好等特点。 Objective To establish an accurate TaqMan probe real-time quantitative PCR（ qPCR） method for detection of Theiler＇s-like virus of rats（ TLV）. Methods Primers and TaqMan probes specific to 3622 - 3729 nt region were designed according to the whole genomic sequence of TLV representative strain. Using a synthesized plasmid as DNA standard template,the stability,specificity,and sensitivity of the qPCR method were determined. Results In the standard curve,R^2 value was 0. 99 with a high specificity. The sensitivity of the real-time PCR was less than 10 copies/μL,which was 100 times higher than the ordinary PCR method. No cross reactions appeared to the other rat viruses. Conclusions The TaqMan probe qPCR method established in this study has advantages such as simple to use,high sensitivity and specificity.

关 键 词： 大鼠 疑似泰勒氏病毒 荧光定量