应用pcr-sscp技术检测结核分枝杆菌耐链霉素相关基因rpsl和rrs突变

目的 应用聚合酶链反应-单链构象多态性(pcr-sscp)技术,建立快速检测结核分枝杆菌链霉素耐药相关基因rpsl和rrs突变的新方法.方法 采用常规药敏试验检测112株结核分枝杆菌临床分离株对链霉素的耐药性,建立pcr-sscp法检测耐药相关基因rpsl和rrs的突变.采用pcr直接测序技术(pcr-ds)确定rpsl和rrs基因突变情况并与pcr-sscp法检测结果进行比较.结果 112株结核分枝杆菌临床分离株均携带rpsl和rrs基因,52株(46.4％)对链霉素敏感,其中有1株敏感菌株rrs基因pcr-sscp条带异常,pcr-sscp方法的特异性为98.1％ (51/52).60株(53.6％)结核分枝杆菌对链霉素耐药,用pcr-sscp方法分别检出46株(76.6％) rpsl基因和11株(18.3％)rrs基因条带异常,1株同时出现rpsl和rrs基因条带异常,pcr-sscp检测的灵敏度为93.3％ (56/60).结论 pcr-sscp方法检测结核分枝杆菌耐药相关基因rpsl和rrs突变的特异性和敏感性高,临床上值得推广应用.abstract：objective to establish a novel rapid detection method based on pcr-single-strand-conformational polymorphism (pcr-sscp) to determine mutation of streptomycin-resistance associated rpsl and rrs genes in isolates of mycobacterium tuberculosis (mtb).methods streptomycin-resistance of 112 mtb isolates was detected using the routine drug susceptibility test,and a special pcr-sscp assay was established.the mutations of rpsl and rrs genes in streptomycin-resistant mtb isolates were detected by pcr-sscp and pcr direct sequencing (pcr-ds) ; the results from two techniques were compared.results all isolates had both rpsl and rrs genes.fifty-two isolates (46.4％) were streptomycin susceptible,in which only 1 isolate showed abnormal pcr-sscp fragments from rrs gene,and the specificity of pcr-sscp was 98.1％ (51/52).sixty isolates (53.6％) were streptomycin-resistant,in which 46 (76.6％) and 11 ( 18.3％ ) isolates presented the abnormal pcr-sscp fragments of rpsl and rrs gene,respectively.one streptomycin-resistant isolate showed abnormal pcr-sscp fragments from both rpsl and rrs genes.the sensitivity of pcr-sscp was 93.3％ (56/60).conclusion the pcr-sscp that established in this study is a specific and sensitive method for rapid detection of the streptomycin-resistance associated mutations in rpsl and rrs genes of mtb.

detection of streptomycin-resistance associated rpsl and rrs gene mutations in mycobacterium tuberculosis by pcr-single-strand-conformational polymorphism