结核分枝杆菌耐药基因rpoB.katG.inhA突变快速检测方法研究

Rapid Detection of Rifampin and Isonicazide-Resistant Mycobacterium tuberculosis by Membrane Gene Chip

目的通过PCR及芯片杂交技术平台,建立临床样品中结核分枝杆菌及耐药基因突变的快速诊断新方法。方法根据结核分支杆菌标准株H37Rv序列,我们设计了覆盖rpoB基因81个碱基高变区的5个正常探针和6个可以探测特定突变类型的突变探针,制作膜芯片,并检测临床样品中结核杆菌的基突变情况,以此来判断耐药结果。结果18个利福平培养耐药中的15个样品都在rpoB基因上检出有突变,利福平耐药突变检出率为83.0%(18/15);25个利福平敏感样品rpoB基因上都未检出突变。20个异烟肼培养耐药的样品中有16个在katG或inhA基因上检出有突变,异烟肼耐药突变检出率为80.0%(16/20);25个异烟肼敏感样品katG或inhA基因上都未检出突变。结论用膜芯片检测结核分枝杆菌对利福平和异烟肼的耐药性,具有较高的特异性和敏感性,可用于临床结核杆菌耐药性检测。并具有快速、简便、敏感的特点。

Objective To develop a new membrane gene chip for rapid detection of rpoB, katG and inhA gene mutation in rifampin and/or isonieazide-resistant Mycobacterium tuberculosis clinical isolates. Method According to the sequence of H37Rv strain, we designed 16 oligonueleotide specific probes for the detection of the mutant sequences in rpoB, katG and inhA genes of Mycobacterium tuberculosis. Result Mutations were found in 50 （89.3%） strains of 56 randomly selected rifampin-resistant M.tuberculosis and 24（81.0%）strains of 58 randomly selected isonieazideresistant M. tuberculosis by membrane gene chip. Conclusion Membrane gene chip technology has high sensitivity and specificity for the detection of rifampin and/or isoniazid-resistant M.tuberculosis and may be applied for clinical diagnosis.