210株结核分枝杆菌利福平耐药基因rpoB突变特征研究

目的了解结核分枝杆菌利福平耐药株rpoB基因突变特点。方法对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的片段进行PCR-SSCP分析,并对经检测显示有突变的结核分枝杆菌的rpoB基因此扩增区域进行序列测定。结果共对210株结核分枝杆菌临床分离菌株进行了研究,其中利福平耐药株110株,非利福平耐药株40株,敏感株60株。PCR-SSCP检测显示110株利福平耐药株中有75株有突变存在,经测序分析,77.3%(58/75)的菌株发生rpoB基因的单位点突变,主要集中在456位60.3%(35/58)和451位25.9%(15/58);22.7%(25/110)的菌株发生了联合突变。此外,经PCR-SSCP检测,40株非利福平耐药株中有3株发生突变,60株敏感株中有2株发生突变。经直接测序分析突变涉及位点为436位、451位和460位。结论结核分枝杆菌利福平耐药的主要原因是rpoB基因耐药决定区发生突变,其中456位丝氨酸和451位组氨酸是最常见的突变位点。

Study of the mutation characteristics of rifampicin-resistance gene (rpoB) in 210 clinical isolates of M. tuberculosis

To find out the mutation characteristics of ropB gene in rifampicin-resistant strains of Mycobacterium tuberculosis. 286bp DNA fragment of rpoB gene including 81bp code region (rifampicin resistance deteremination region, RRDR) was analyzed with PCR-SSCP. Then the 286bp DNA fragment of each strain which had been proved to have mutation by PCR-SSCP was sequenced. 210 strains of Mycobacterium tuberculosis, including 110 rifampicin-resistant strains, 40 rifarmpicin-susceptible drug-resistant strains and 60 drug-susceptible strains were studied. 75 rifampicin-resistant strains were detected to have mutations by PCR-SSCP. By means of direct sequencing analysis, the results showed that 77.3% (58/75) rifarmpicin-resistant strains had single point mutation in rpoB gene, which mainly located at 456-Ser (60.3%) and 451-His (25.9%), and combinative mutation rate was 22.7%. In addition, 3 rifampicin-susceptible drug-resistant strains and 2 drug-susceptible strains had mutations detected by PCR-SSCP method. Sequencing results showed that the mutations located at 436-Leu, 451-His and 460-Pro. The main reason of Mycobacterium tuberculosis resistant to rifampicin is mutations in the 81bp RRDR of rpoB gene. 456-Ser and 451-His are the most common mutation positions in rpoB gene.