利用正交设计优化脑膜炎奈瑟氏菌Taqman-MGB探针检测反应体系

DETECTION OF REACTION SYSTEM BY TAQMAN-MGB PROBE FOR NEISSERIA MENINGITIDES OPTIMIZED WITH ORTHOGONAL DESIGN

[目的]建立Taqman-MGB探针检测脑膜炎奈瑟氏菌的最佳反应体系,探讨影响Taqman-MGB探针检测的多个因素。[方法]利用正交设计,从Buffer、TaqDNA聚合酶、引物、Mg2+、探针、dNTP6种因素对实时荧光PCR体系进行优化,在此基础上,进一步细调,寻求实时荧光PCR反应的最佳反应体系。[结果]正交试验结果表明,最佳反应体系为(50μl):Mg2+7.0mmol/L,引物0.6μmol/L,dNTP300μmol/L,TaqDNA聚合酶2.0U,探针0.1μmol/L,Buffer1.5×。[结论]Mg2+是影响实时荧光PCR的重要因素;所得体系反应稳定,正交法实验设计高效、快捷,科学性强,值得推广。

[ Objective] To establish Taqman-MGB probe to detecting an optimization reaction system of neisseria meningitides. [ Methods] The fluorescence PCR amplification system was optimized by using orthogonal design with six factors （Buffer, Mg^2＋, dNTP, primer, Taq DNA polymerase and Taqman-MGB brobe）. Then the optimization reaction system of realtime fluorescence PCR amplification was explored by further adjustment. [ Results] The results of orthogonal test demonstrated that the optimization reaction system was as follows： 7.0 mmol/L Mg^2＋, 0.6μmoL/L primer, 300μmol/L dNTP, 2.0U Taq DNA polymerase, 0.1μmol/L Taqman-MGB probe, 1.5xBuffer for 50μl reaction system [Conclusion] Mg^2＋ was an important factor to influence the real-time PCR. The reaction system is stability, which demonstrated that orthogonal test design showed to be high performance, shortcut, scientific and worth of generalization.