结核分枝杆菌利福平耐药基因rpoB突变特征初步分析

目的 阐明结核分枝杆菌利福平耐药株rpoB基因突变特点。方法 对110株结核分枝杆菌临床分离株包括rpoB基因利福平耐药决定区（RRDR）81个碱基在内的286bp片段进行聚合酶链反应．单链构象多态性（PCR．SSCP）分析，并对经检测显示有突变的结核分枝杆菌rpoB基因此扩增区域进行序列测定。其中利福平耐药株73株，非利福平耐药株11株，敏感株26株。结果 PCR—SSCP检测显示利福平耐药株中有47株有突变存在，经直接测序分析其中76．6％的菌株存在rpoB基因的单位点突变，主要集中在531位（61．1％）和526位（25．0％）；联合突变发生率为23．4％。此外，经PCR—SSCP检测，11株非利福平耐药株中2株有突变存在，26株敏感株中1株有突变存在。经直接测序分析突变涉及位点为511位、526位和535位。结论 rpoB基因耐药决定区发生突变是结核分枝杆菌利福平耐药的主要原因，其中531位丝氨酸和526位组氨酸是最常见的突变位点。

Study on the characteristics of mutation on Mycobacterium tuberculosis rifampicin-resistance gene

Objective To elucidate the characters of rpoB mutation in rifampin-resistant clinical isolates of Mycobacterium tuberculosis.Methods 286 bp DNA fragment of rpoB gene including 81 bp code region (rifampin resistance deteremination region,RRDR)was analyzed by PCR-single-strand conformation polymorphism(SSCP).The 286 bp DNA fragment of each strain which had been proved to have mutation by PCR-SSCP was then sequenced.110 strains of M.tuberculosis,including 73 rifampin- resistant strains,11 rifampin-susceptible drug-resistant strains and 26 drug-susceptible strains were studied. Results 47 rifampin-resistant strains were detected to have mutations by PCR-SSCP method.76.6% rifampin-resistant strains showed that rpoB gene was carrying single point mutation analyzed by direct sequencing technique,which mainly located at 531-Ser(61.1%) and 526-His (25.0%).The combined mutation rate was 23.4 %.In addition,2 rifampin-susceptible drug-resistant strains and 1 drug-susceptible strain were mutated,detected by PCR-SSCP method.Sequencing results showed that the mutations located at 511-Leu,526-His and 535-Pro.Conclusion Mutations in the 81 bp RRDR of rpoB were the main reasons of M.tuberculosis resistant to rifampin.531-Ser and 526-His were the most common positions of mutations.