等位基因特异多重PCR快速检测结核分枝杆菌异烟肼耐药性研究

Rapid Detection of Isoniazid Resistant Mycobacterium Tuberculosisby Allele Specific Multiplex PCR

了解北京地区异烟肼(INH)耐药结核分枝杆菌(MTB)的分子特点,评价等位基因特异多重PCR在INH耐药性快速检测中的应用价值。选取102株北京地区MTB临床分离株,首先分别采用PCR-RFLP和反向线性杂交技术(RLB)检测耐药相关基因katG和inhA突变,然后运用等位基因特异多重PCR方法同时检测katG和inhA基因突变,并与PCR-RFLP和RLB分别检测的结果进行比较。采用等位基因特异多重PCR检测发现,INH耐药株中60.3%(35/58)存在katG315突变,13.8%(8/58)发生了inhA突变,两者同时突变率为6.9%(4/58),INH敏感株中没有发现katG315突变,而inhA突变率为18.2%(8/44)。上述检测结果与PCR-RFLP和RLB分别检测的结果完全一致,且等于两种方法结果之和,提示等位基因特异多重PCR可完全取代PCR-RFLP和RLB。北京地区INH耐药菌株以katG315 AGC→ACC突变为主;联合检测katG315和inhA基因可提高INH耐药株的检出率。等位基因特异多重PCR操作简便,结果易于判断,可作为临床MTB菌株中INH耐药性快速检测的辅助手段。

To clarify the molecular characteristics of isoniazid （ INH ） resistant Mycobacterium tuberculosis （MTB） in Beijing area and evaluate the application of allele specific multiplex PCR in rapid detection of INH resistant strains. Total 102 MTB strains were selected. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） and reverse line blot assay （RLB） were used to detect INH resistance related genes katG and inhA, respectively; and allele specific multiplex PCR to detect katG and inhA simultaneously. The results obtained from different methods were compared. Thirty-five （60.3%） of 58 INH resistant strains harbored katG315 AGC→ACC, 13.8% （8/58） with inhA mutation, 6.9% （4/58） had both of the mutations. There was no mutation of katG315 in INH sensitive strains, while inhA mutation occurred in 18.2% of them. The results obtained from allele specific multiplex PCR were concordant with PCR-RFLP and RLB, and equivalent to both of them, suggesting that allele specific multiplex PCR can replace PCR-RFLP and RLB. The mutation of katG315 AGC→ACC is prevalent in INH resistant MTB strains in Beijing area. A combined use of both katG315 and inhA can improve the detection rate of INH resistant strains. Allele specific multiplex PCR can be used in clinical laboratories as a fast complement to INH phenotypic methods.