等位特异PCR检测结核分支杆菌耐异烟肼基因katG突变研究

Establishment of an AS-PCR for Detection of katG Gene Mutation Associated with Mycobacterium tuberculosis Resistance to Isoniazid.

目的 建立等位特异多聚酶链反应检测结核分支杆菌耐异烟肼基因katG突变的快速敏感检测方法。方法 利用在PCR时 ,3′端的碱基如果与模板DNA不配对 ,PCR扩增阴性的原理 ,根据我们对katG基因突变特点的研究结果 ,建立了AS -PCR检测katG基因突变的方法。结果 AS -PCR检测结核菌耐异烟肼相关基因katG的敏感性为85 %,特异性达 90 %。 15株经测序证实有katG突变的菌株 ,AS -PCR检测均为阳性。二者的符合率为 10 0 %。与PCR-SSCP检测的总符合率为 88%,阳性符合率 81.0 %,阴性结果的符合率为 93.1%。检测试验可在数小时内完成。结论 AS -PCR方法是检测结核菌耐异烟肼基因突变的敏感方法 ,可为临床医师提供判断结核菌对异烟肼是否敏感的依据。

Objective To establish an allele specific polymerase chain reaction(AS-PCR) for rapid and early detection of Mycobacterium tuberculosis katG gene mutation. Methods Primers containing bases matching the mutated gene was used in PCRs. Results The sensitivity and the specificity of the AS-PCR we developed was 85% and 90% respectively. Mutations detected by our methods were confirmed by direct sequencing of katG genes. There was no significant difference between the results detected by PCR-SSCP,MIC determination and AS-PCR. Conclusion AS-PCR can be applied for rapid and early detection of katG mutations which are useful for the physicians to evaluate the strains'susceptibility to isoniazid.