用DNA芯片检测结核分枝杆菌耐乙胺丁醇基因突变的研究

Rapid detection of embB mutation in ethambutol-resistant Mycobacterium tuberculosis strains by using gene chip

目的利用DNA芯片快速检测结核分枝杆菌耐乙胺丁醇embB基因突变。方法根据结核分枝杆菌embB基因序列设计探针并制作DNA芯片,根据结核从分枝杆菌embB基因突变的热点片段设计引物,该片段用TAMRA荧光标记PCR扩增增后与DNA芯片杂交,同时以PCR—SSCP和DNA测序法为对照。结果120株结核分枝杆菌临床分离株中,35株敏感株DNA芯片杂交结果与标准株完全相同；85株耐乙胺丁醇临床分离株中有50株检测到embB基因突变[58．8％(50／85)],均为306位密码子突变,该突变与PCR-SSCP和测序结果完全一致；85株耐药株中后有35株未检测到突变,占41．2％(35／85)。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乙胺丁醇分离株的embB基因突变,可协助临床医生制定合理的治疗方案,特别是复治患者,可帮助选择有效药物。

Objective To explore the feasibility of detecting the mutations of ernbB genes in ethambutol-resistant Mycobacterium tuberculosis by gene chip. Methods Probe was designed according to the sequence of chip. The DNA fragment which contains hot mutation sites of ernbB gene was labelled with TAMARA and amplified by PCR technique, then it was hybridized with gene chip. PCR-SSCP and DNA sequence was used as the control. Result Among 120 clinical isolates, 50 strains were detected to harbor the mutations of ernbB gene in 85 ethambutol-resistant Mycobacterium tuberculosis strains by gene chip. 35 sensitive strains were identical with these reference strains. Among fifty strains of ethambutol-resistant M. tuberculosis, 25 strains were ATG→GTG mutations, 15 strains were ATG→ATA mutations, 7 strains were ATG→ATC mutations, 2 strains were ATG→ ATT mutations, and 1 strain was ATG→CTG mutation, which were consistent with those of PCR-SSCP and DNA sequencing; and 35 strains in 85 ethambutol-resistant Mycobacterium tuberculosis strains didn＇t detect to have mutations by gene chip, which was 41.2%. Conclusion It showed that chip could detect mass ernbB gene mutations in ethambutol-resistant M. tuberculosis and could be used to for clinical detection of ethambutol-resistant strains.