结核分支杆菌喹诺酮耐药基因的研究

[目的]了解结核分支杆菌耐喹诺酮耐药基因突变情况，建立快速分子药敏实验方法。[方法]通过PCR—SSCP和直接测序技术(PCR～DS)分析结核分支杆菌的gyrA基因突变的情况。[结果]以结核分支杆菌标准菌株H37Rv和卡介苗做对照，30株喹诺酮敏感株的gyrA基因的SSCP图谱均泳动正常，测序分析与对照株相同。45株喹诺酮耐药株中，34株(75．6％)gyrA基因SSCP图谱泳动异常；测序证实10株为90位密码子的突变，24株为94位密码子突变，符合率100％。[结论]gyrA基因突变是结核分支杆菌耐喹诺酮的主要分子机制，PCR—SSCP可能成为测定部分结核分支杆菌耐喹诺酮株的简便、快速的方法．并有望直接用于临床标本喹诺酮敏感性试验。

Studies of Gene of Quinolones- resistant in Mycobacterium Tuberculosis Isolates by PCR-SSCP

ObjectiveTo understand the mutations of gene in M.Tuberculosis quinolones-resistant isolates and to develop a new method for detecting drug resistance.M.tuberculosis gyrA genes were analyzed with PCR-SSCP and PCR-direct sequencing.The standard strain of M. tuberculosis H 37 Rv and BCG were used as control groups, all of 30 quinolones -sensitive isolates had the same gyrA SSCP profiles as the controls. Of 45 quinolones-resistant isolates, 34(75.6%) displayed abnormal gyrA SSCP profiles;DNA direct sequencing of gyrA gene was performed,the mutation at codon 90 of gyrA occurred in 10 quinolones-resistant isolates, the mutation at codon 94 of gyrA occurred in 24 quinolones-resistant isolates.The results were identical completely between PCR-SSCP and DNA sequencing.[Conclusion]The mutations of gyrA were important molecular mechanism in quinolones-resistance of M. tuberculosis. PCR-SSCP might become a simple and rapid diagnostic test for genotypes of M. tuberculosis quinolones -resistance. These methods are expected to be applied to detect drug susceptibility for clinical specimens.