应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型，评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系，比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中，78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸，6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T，14株（14．3％）未发现突变；69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致，RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明，应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性，反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。

Detection of Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates Using Four Molecular Methods in China

To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuber-culosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL re-sulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequenc-ing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.