利用基因芯片技术检测结核分支杆菌耐利福平分离株rpoB基因突变的研究

Mutation of Rifampicin-resistant Genes in M.tuberculosis with Gene Chip

目的利用基因芯片技术对我国结核分支杆菌耐利福平(RFP)分离株rpoB基因突变进行研究,建立大规模快速检测结核分支杆菌耐药基因型的基因芯片分子药敏试验方法。方法利用寡核苷酸基因芯片技术分析19株结核分支杆菌临床分离株的rpoB基因突变,并经PCR-直接测序法(PCR-DS)验证,以结核分支杆菌H37Rv标准株为对照。结果7株结核分支杆菌药物敏感株芯片检测和PCR-DS分析均未见rpoB突变,12株RFP耐药株中,1株芯片检测和PCR-DS分析均未见rpoB突变;11株rpoB基因有突变,其中5株为531位TCG→TTG或TGG突变,5株为526位CAC→TAC或GAC或CGC突变,531位和526位突变占耐药菌株rpoB突变的83.3%,其余为502、505、506、507、516、518、522、538位突变;芯片检测和PCR-DS分析结果符合率达100%。结论大多数结核分支杆菌耐RFP是由于rpoB基因突变所致,采用基因芯片技术可以快速、准确、大规模地确定结核分支杆菌RFP耐药突变的部位和性质,是一种有前途的结核分支杆菌分子药敏试验方法。

OBJECTIVE To underdstand mutation of rifampicin-resistant genes in Mycobacterium tuberculosis clinical isolates with gene chip,and to develop a new method for detecting drug resistance. METHODS Analyzing the rpoB genes in 19 M. tuberculosis clinical isolates with gene chip and PCR-direct sequencing （PCR-DS）.M. tuberculosis strain H37Rv was used as control. RESULTS Of 7 drug-sensitive isolates,all had no mutations in rpoB sequences. Of 12 rifampicin-resistant isolates, 11 had mutations in rpoB sequences, in which five isolates displayed TCG→TTG or TGG mutations at codon 531, five had CAC→TAC or GAC or CGC mutations at codon 526. The mutations at codon 531 and codon 526 accounted for 83.3％ mutations of rifampicin-resistant selected.The result with gene chip accorded with those of PCR-DS completely. CONCLUSIONS Resistance to rifampicin in most M. tuberculosis isolates is due to the mutations on the genes encoding the RNA polymerase subunit（rpoB）.Mutations detecting with gene chip techniques might become simple, rapid, reliable and potential diagnostic tests for rifampicin-resistance in clinical isolates.