47株结核分枝杆菌利福平耐药决定区基因突变特征的研究

Gene mutations analysis for rifampin resistance determining region of 47 mycobacterium tuberculosis strains isolated in Jilin province

目的了解吉林省结核分枝杆菌的利福平耐药决定区(RRDR)基因突变特征。方法结核分枝杆菌(MTB)分离自2008-2009年吉林省临床肺结核病人的痰液标本,培养方法为改良酸性罗氏(L-J)培养基。分离出的抗酸杆菌经对硝基苯甲酸(PNB)培养基初步鉴定后选出结核分枝杆菌复合群并应用比例法进行药敏试验(DST)。根据DST结果,选择22株利福平耐药株、22株利福平敏感株以及3株临界值标本进行聚合酶链式反应(PCR)扩增rpoB中包含RRDR在内的部分基因,阳性DNA片段通过基因测序以及Bioedit软件分析方法比对基因突变情况。结果 22株利福平耐药株中,16株同时对异烟肼耐药;22株利福平敏感株中,1株异烟肼耐药。利福平耐药菌株中,95%(21/22)的菌株在利福平耐药决定区RRDR发生了基因突变,其中第531位密码子突变频率占50%,第526位占27%,第533位占9%,第516位及513位均为4.5%;22株利福平敏感株中,RRDR区全部无基因突变发生;3株临界值标本中,1株在第531位密码子发生了基因突变,1株在533位发生基因突变,最后1株无突变。结论 RRDR区点突变的发生与利福平耐药高度一致;异烟肼耐药与利福平耐药高度相关。

Objective To understand the gene mutations characteristics for rifampin resistance determining region（RRDR） of mycobacterium tuberculosis（MTB）.Methods MTB strains were isolated from sputum samples collected from clinical pulmonary tuberculosis（TB） patients in Jilin province during 2008-2009 by Lwenstein-Jensen（L-J） medium culture.The acid-fast bacilli were then identified by medium contained p-nitrobenzoate（PNB）,then drug susceptibility testing（DST） was performed with proportion method.22 rifampin resistance strains,22 rifampin susceptive strains and 3 cut-off strains were selected based on the DST result and polymerase chain reaction（PCR） was carried out to amplify partial rpoB gene which covered RRDR,then sequence analysis and Bioedit software was used to identify the gene mutations.Results Among 22 rifampin resistance strains,16 were resistant to isoniazid,which was one among the 22 rifampin susceptive strains.95%（21/22）of the rifampin resistance strains were found to have gene mutations in RRDR,among them,the 531 codon mutation percent was 50%,which of the 526 codon was 27%,and which of 533 codon,516 codon and 513 codon was 9%,4.5% and 4.5% respectively;Among the 22 rifampin susceptive strains,no gene mutations were observed in RRDR;Among the three cut-off strains,one had mutation in the 531 codon,the other had mutation in the 533 codon and the last had no mutation observed.Conclusion The occurrence of gene mutation in RRDR is highly consistent with rifampin resistance;Isoniazid resistance is highly correlative with rifampin resistance.