耐利福平结核分枝杆菌rpoB基因序列研究

Study on rpoB genes of rifampin-resistant Mycobacterium tuberculosis

目的研究杭州地区结核分枝杆菌(Mycobacteriumtuberculosis)对利福平(RFP)的耐受与rpoB基因突变的关系。方法随机挑选了90例结核杆菌感染的病例,选择了利福平作为主要的药物进行药敏实验,PCR扩增39株耐RFP结核分支杆菌的rpoB基因片段(580bp);测定扩增片段的序列,并与美国国立生物信息中心(www.ncbi.nlm.nih.gov/nucleotide)检索获得结核杆菌野生株(利福平敏感株)序列(登陆号:AJ749948)作对比分析。结果结果显示PCR结核测序方法得到了39例结核杆菌耐药株,51例敏感株,与药敏实验的方法检测的结果完全一致。测定的11株临床分离的耐药株中,526位或531位有突变,其中526位有突变的有7株,占耐药测定株63.6%,531位突变的有4株,占耐药测定株36.4%,未见两位点同时突变。检测的11株敏感株,未见526位或531位有突变。结论杭州地区结核分支杆菌耐利福平的发生与rpoB基因的526位或531位突变密切相关,与国外的报告基本相似。但526位突变占耐药测定株高达63.6%,如此高比例目前国内外未见相似报道。PCR扩增和产物测序将是临床检测结核分支杆菌耐利福平和耐多药的一种迅速、准确的方法。

Objective To investigate the mutations of rpoB genes interrelated rifampin （RFP） resistance of Mycobacterium tuberculosis（Mt） in Hangzhou area. Methods 90 strains of Mt from the patients were chosen randomly, rpoB genes were performed by PCR amplification and direct sequencing, and contrasted the rpoB DNA sequences of the strains with the rpoB sequences of Mt wild-type strain （RFP sensitive）. Results The results indicated that there were 39 strains of RFP-resistant Mt and 51 strains of RFP-sensitive Mt. Furthermore, the results from PCR and sequencing were the same to the results from drug susceptibility testing. The rate of RFP-resistant Mt was 43%. The substitution at single codon in 526（63.6%） or 531（36.4 %） was found in all 11 strains of RFP-resistant. 11 sensitive strains of RFP were not detected the mutation in 531 or 526 points. Conclusions The results reveal that there is a strong correlation between resistance to rifampin and rpoB mutations of M. tuberculosis, but the report about the substitution at single codon mutation in 526 （63.6%） have not found in published reports, the sequencing analysis of the rpoB fragment amplified by PCR may accurately identify the rifampin-resistant or multiple resistant strains of M. tuberculosis.