用DNA芯片检测结核分枝杆菌耐利福平基因突变的初步研究

目的研制一种新型DNA芯片，用于快速检测结核分枝杆菌耐利福平rpoB基因突变。方法根据结核分枝杆菌rpoB基因序列设计探针并制作DNA芯片，PCR掺入法荧光标记根据结核分枝杆菌rpoB基因突变热点的目的片断，与DNA芯片杂交，同时以DNA测序法为对照。结果18株结核分枝杆菌临床分离株中，1株敏感株DNA芯片杂交结果与标准株完全相同；17株耐RFP临床分离株中有17株检测到rpoB基因突变，其中14株单位点突变，1株511和516位双位点突变，与测序结果完全一致。另有两株为517和518、526和517位双位点突变，因为芯片上未点517位突变的探针，所以只检测到518和526位的突变，该突变与测序结果完全一致、结论用DNA芯片可快速、特异地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变，可用于临床耐药性的检测，指导临床用药。

Application of Gene Chip for Detecting Mutations of Rifampin-resistant Genes in Mycobacterium Tuberculosis Strains

Objective To explore the feasibility of detecting the mutations of rpoB gene in rifampin-resistant Mycobacterium tuberculosis strains by gene chip. Methods The DNA chip was designed according to the sequence of Mycobacterium tuberculosis rpoB gene. The DNA fragments which contained hot mutation sites of rpoB gene were labelled with cy3 fluorescence, amplified by PCR technique and hybridized with gene chip. The DNA sequenciug was used as the control. Results Eighteen of the Mycobacterium tuberculosis strains were detected for the mutations of rpoB gene by gene chip. Among them, one sensitive strain was identical with the type strain, and all of 17 strains with rifampin-resistant, rpoB mutations were detected. Of the 17 strains with rpoB mutations, 14 were detected as single point mutation, one was detected as double sites point mutations with the sites of 511 and 516, and two were detected as single point mutation with the sites of 518 and 526, respectively. Because the probe of 517 rifanmpin-resistant did not be dotted on the gene chip, only sites of 518 and 526 mutations were detected, which were consistent with the results of DNA sequencing. Conclusion Using the gene chip could detect mass rpoB gene mutation of rifampin-resistant with higher specialty and sensitivity, which could be used for clinical detection of rifampin-resistant strains and clinical medicine.