基因芯片快速检测结核分枝杆菌katG基因突变及其与异烟肼耐药相关性

摘要

The objective was to determine the correlation between the mutation at S315 site in katG gene of M. tuberculosis and isoniazid (INH) resistance, and to develop a genetic microarray for rapid and convenient detection of the S315 mutation of katG gene. The INH resistance of 123 M. tuberculosis isolates was determined by using proportional dilution method.The frequency and types of S315 mutation in katG gene of the isolates were detected by PCR and sequencing. A group of Cy3 fluorescent labeling probes were designed and a genetic microarray for detecting S315 mutation in katG gene was developed base on the PCR and sequencing data. The result of microarray detection was compared to that of PCR and sequencing. Of the 123 M. tuberculosis isolates, 39.0％ (48/123) strains were INH-sensitive while 61.0％ (75/123) strains were INH-resistant. All the isolates and M. tuberculosis strain H37Rv presented amplification product of katG gene. In the 75 INH-resistant isolates,69.3％ (52/75) strains presented the S315 mutation in katG gene including S315N (AGC→AAC) in 25 strains, S315T (AGC →ACC) in 12 strains, S315I (AGC→ATC) in 7 strains, S315T (AGC→CGC) in 3 strains, S315R (AGC→AGA) in 3 strains and S315G (AGC→GGC) strains. The detection results of the generated microarray for S315 mutation rate and its mutational types in the katG genes of 123 M. tuberculosis isolates were absolutely same as those of PCR and sequencing. It is evident that there is a close correlation between S315 mutantion in katG gene of M. tuberculosis and INH resistance, and the genetic microarray prepared in this study could be used for rapid, convenient and accurate detection of the S315 mutation and mutational types with high sensitivity and specificity.%目的 了解结核分枝杆菌katG基因S315突变与异烟肼(isoniazid,INH)耐药相关性,建立快速简便的katG基因S315突变基因芯片检测方法.方法 采用二倍稀释法检测123株结核分枝杆菌临床分离菌株对INH的耐药性.PCR及测序确定上述结核分枝杆菌katG基因S315位点突变率及突变类型.根据PCR及测序结果,设计Cy3荧光标记探针并制备katG基因S315位点突变检测基因芯片.基因芯片检测结果与PCR及测序结果进行对比分析.结果 123株结核分枝杆菌临床菌株中,39.0%(48/123)菌株对INH敏感,61.0%(75/123)菌株对INH耐药.结核分枝杆菌H37Rv株及所有临床菌株均能扩增出katG基因片段.75株INH耐药菌株中,69.3%(52/75)菌株katG基因出现S315位突变,其中25株突变类型为S315N(AGC→AAC)、12株为S315T(AGC→ACC)、7株为S315I(AGC→ATC)、各有3株分别为S315T(AGC→CGC)和S315R(AGC→AGA)、2株为S315G(AGC→GGC).所制备的基因芯片对123株结核分枝杆菌katG基因S315野生型或突变型检测结果与PCR及测序结果完全一致.结论 结核分枝杆菌katG基因S315突变与INH耐药密切相关.本研究中制备的基因芯片可快速、简便、准确、敏感和特异地检测结核分枝杆菌katG基因S315突变及其类型.