DHPLC法和测序法检测结核分枝杆菌临床分离株链霉素的耐药性  

Detection of streptomycin resistance in mycobacterium tuberculosis clinical isolates as determined by denaturing HPLC analysis and DNA sequencing

作　　者：孙芳[1] 赵玉玲 王子玲[1] 苏振修[1] 赫金平[1] 石瑞如[1] 张国龙[1] 

机构地区：[1]河南省胸科医院呼吸内科,郑州450008 [2]河南CDC结核病预防控制所

出　　处：《医药论坛杂志》2011年第5期15-19,22,共6页Journal of Medical Forum

基　　金：河南省医学科技攻关重点项目(200702015)

摘　　要：目的我国是WHO认定的结核病高发区域之一。链霉素在我国已经使用50年以上,现在在结核病治疗中仍广泛使用。本工作采用变性高效液相色谱(DHPLC)法和测序法来分析结核分枝杆菌链霉素耐药基因rpsL和rrs基因突变,从而检测是否对链霉素耐药。方法215株结核分枝杆菌临床分离株(经常规方法证实,115株为链霉素耐药,100株为敏感),测定链霉素最小抑菌浓度(MIC),同时采用DHPLC和测序法检测rpsL和rrs基因突变。结果85.2%(98/115)的链霉素耐药株携有rpsL或/和rrs基因突变,其中rpsL基因突变占大多数(76.5%,88/115)。对98株带有基因突变的菌株的分析表明,rpsL,rrs基因的突变类型与MIC之间未见密切关系。100株敏感株未检出基因突变。DHPLC法与测序法结果完全一致。结论本工作建立的DHPLC法可以认为是结核分枝杆菌链霉素耐药分析的一种有效的工具。Objective China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis（M.tuberculosis） infection.Streptomycin has been deployed in China for 50 years and still widely used nowadays in tuberculosis treatment.The present study was to determine rpsL and rrs gene mutation by denaturing high-performance liquid chromatography（DHPLC） and DNA sequencing.Methods Two hundred and fifteen M.tuberculosis clinical isolates（115 shown to be streptomycin-resistant and 100 susceptible by a routine proportional method） were tested to determine the streptomycin MIC and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutation.Results 85.2%（98/115） streptomycin-resistant isolates harbored rpsL or rrs mutation while rpsL mutation（76.5%,88/115） dominated.MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance.No mutation was found in all the susceptible isolates.DHPLC results were completely consistent to that of sequencing.Conclusions The DHPLC method devised in this study can be regarded as a useful and powerful tool for streptomycin resistance detection in Mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 链霉素耐药 DHPLC RPSL rrs MIC 

分 类 号：R378.91[医药卫生—病原生物学]