分子信标荧光定量PCR检测结核分枝杆菌的反应体系优化方法对比研究  

Comparison of Optimization Design of Molecular Beacon Real-time PCR Reaction System in the Detection of Mycobacterium Tuberculosis

作　　者：陆巧荣[1] 方梅[1] 洪志强[1] 余志祥[1] 胡朝友[1] 

机构地区：[1]昆山市疾病预防控制中心检验科,江苏昆山215300

出　　处：《现代检验医学杂志》2010年第3期27-30,共4页Journal of Modern Laboratory Medicine

基　　金：江苏省卫生厅预防医学科研课题项目（Y2006028）资助.

摘　　要：目的建立分子信标荧光定量PCR检测结核分技杆菌的最佳反应体系，探讨影响分子信标荧光定量PCR扩增结果的多个因素。方法利用单因素法和正交实验法，从MgCl2，引物，分子信标探针，dNTP，Taq DNA聚合酶5种因素对分子信标荧光定量PCR反应体系进行优化，并对这两种方法所得的最优体系进行比较。结果单因素法得到的最优反应体系（25μl）为：4mmol／L MgCl2，上、下游引物各0．3μmol／L，分子信标探针0．1μmol／L，200μmol／L dNTP，2U Taq DNA；正交法得到的最优反应体系（25μl）为：6mmol／L MgCl2，上、下游引物各0．2μmol／L，分子信标探针0．1μmol／L，100μmol／L dNTP，3U Taq DNA聚合酶，正交法得到的最优反应体系的荧光定量PCR扩增曲线形状更好，Q值较小，△Rn较大。结论采用正交法得到的荧光定量PCR反应体系优于单因素法，进行实时荧光定量PCR反应体系优化时，正交实验设计是一条科学、可靠、高效、快捷的途径。Objective To establish an optimization reaction system of molecular beacon real-time PCR in the detection of Mycobacterium tuberculosis,discuss impact factors to results of molecular beacon real-time PCR amplification. Methods The reaction system of molecular beacon real-tlme PCR was optimized respectively by using the single factor and orthogonal test with five factors （MgCl2,primer,molecular beacon probe,dNTP,Taq DNA polymerase）,and compared the single factor with orthogonal test. Results The result of the single factor revealed that the optimization reaction system was follow： 4 mmol/L MgCl2,0.3 μmol/L primer, 0. 1 μmol/L molecular beacon probe, 200μmol/L dNTP, 2U Taq DNA polymerase,total volume was 25 μl. The result of orthogonal test revealed that the optimization reaction system was follow：6 mmol/L MgCl2,0. 2μmol/L primer, 0. 1 μmol/L molecular beacon probe, 100 μmol/L dNTP, 3U Taq DNA polymerase,total volume was 25μl,real-time PCR amplification curve of orthogonal test had lesser Ct and large △Rn. Conclusion The reaction system of molecular beacon real-time PCR was optimal by orthogonal test. So performing optimization trial of real-time PCR reaction system,orthogonal design is scientific,reliable,efficient and shortcut.

关 键 词：结核分枝杆菌 分子信标荧光定量PCR 单因素法 正交法 

分 类 号：R378.911[医药卫生—病原生物学] Q503[医药卫生—基础医学]