西安市耐利福平结核分枝杆菌rpoB基因的突变型分析  

Molecular analysis of rpoB gene mutations of rifampin-resistant Mycobacterium tuberculosisclinical isolates in Xi'an City

作　　者：杜蓬 李峰[3] 董兆麟 陈超 

机构地区：[1]国家微检测系统工程技术研究中心,陕西西安710069 [2]浙江医学高等专科学校基础医学部,浙江杭州310053 [3]西安市结核病医院检验科,陕西西安710061 [4]西北大学生命科学学院,陕西西安710069

出　　处：《西安交通大学学报（医学版）》2012年第1期19-23,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的西安市结核分枝杆菌临床分离株rpoB基因RRDR的基因型分析。方法采用PCR-单链构象多态性(PCR-SSCP)分析32株结核分枝杆菌耐RFP株和10株RFP敏感株的rpoB基因PCR产物,并对8株具有代表性的菌株rpoB基因片段通过DNA测序进行验证。结果 rpoB基因PCR-SSCP分析灵敏度为56.3%(18/32),特异性为80%(8/10)。8株结核分枝杆菌rpoB基因经测序,6株耐RFP菌株中有5株的rpoB基因突变均发生在531或526密码子上。其中有两株在526密码子均发生了双碱基突变;1株PCR-SSCP呈现阴性的耐RFP结核分枝杆菌存在513密码子突变;两株RFP敏感株出现PCR-SSCP假阳性,其rpoB基因均涉及2～3个密码子的突变,其中518密码子突变型AAC→GAC为首次报道。结论 531和526密码子除了单点突变之外,亦出现同密码子双碱基突变型;RFP敏感株多密码子突变型值得关注。Objective To genotype the rifampin(RFP)-resistance-determining region(RRDR) of rpoB gene of clinical Mycobacterium tuberculosis isolates in Xi'an by PCR single-strand conformation polymorphism(PCR-SSCP) combined with DNA sequencing.Methods The rpoB gene of 32 RFP-resistant and 10 RFP-susceptible Mycobacterium tuberculosis strains was analyzed by PCR-SSCP within the 157 bp(Ala500 to Val550) region covers RRDR,and the PCR products of rpoB gene of 8 strains,which have representative patterns of PCR-SSCP combined with drug sensitive testing,were sequenced subsequently.Results The sensitivity and specificity of PCR-SSCP in detected rpoB gene were 56.3%(18/32) and 80%(8/10),respectively.Mutations in codon 531 or 526 were observed in 5 of 6 RFP-resistant isolates by DNA sequencing,and double-base mutations in codon 526 were found in 2 of 6 RFP-resistant isolates.One mutation in codon 513 with false-negative PCR-SSCP result was also unfolded.Two false-positive PCR-SSCP results were revealed among the RFP-susceptible strains with mutations covered Leu511,Asp516 and Asn518,in which new asparagine 518 mutation type was identified.Conclusion The mutations in codon 531 and codon 526 of rpoB gene are still the 'hot spots'.Both point mutant and double mutants in one codon were discovered.Moreover,multiple mutations in codons of RFP-susceptible isolates deserve concern.

关 键 词：结核分枝杆菌 耐利福平 RPOB基因 突变 

分 类 号：R446.5[医药卫生—诊断学]