PCR—SSCP快速检测结核分枝杆菌耐利福平rpoB基因的研究

目的探讨PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因作为新的分子药敏试验方法的价值。方法应用PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因核心区的突变，同时对片段长度为215bp的PCR扩增产物进行测序分析。受试菌为23株利福平敏感结核分枝杆菌以72．35株利福平抗性结核分枝杆菌。结果23株利福平敏感结核分枝杆菌分别用PCR—SSCP和PCR扩增片段测序均没有检测到巾0B碱基突变。而在35株利福平抗性菌株中，PCR—SSCP检测到31株与H37Rv标准株不同的带谱，PCR—SSCP检测基因突变的敏感度和特异度分别为88．6％（31／35）和100％（23／23）。对35株利福平抗性菌株PCR扩增产物进行测序分析，在其中32株中检出了基因突变。测序分析检测基因突变的敏感度和特异度分别为91．4％（32／35）和100％（23／23）。卡方检验，PCR—SSCP和PCR扩增片段测序两种方法的敏感度之间没有显著性差异（P〉0．05）。若以DNA测序为标准，则PCR—SSCP检测基因突变的准确度、敏感度和特异度分别为93．1％（54／58），96．9％（31／32）和100％（23／23）。结论PCR—SSCP检测结核分枝杆菌耐利福平rpoB基因突变可用于利福平药物敏感度的快速测定。

Evaluation and Comparison Research on PCR-SSCP Method and DNA Sequencing for Rapid Detection of rpoB Mutations in Rifampin-Resistant Mycobacterium Tubeculosis Isolates

Objective To evaluate the significance of PCR-SSCP method in drug susceptibility analysis of M. tuberculosis to rifampin. Methods The rpoB gene of known 23 strains of rifampin-sensitive and 35 strains of rifampin-resistant clinical isolates of M. tuberculosis were analyzed by PCR-SSCP and DNA sequencing. Results 23 strains of rifampin-sensitive strains had the same agarose gel images of PCR-SSCP with M. tuberculosis H37Rv,while 31 of 35 rifampin-resistant strains showed different gel images to that of control ,the sensitivity of PCR-SSCP was 88.6% （31/35）,the speciality was 100% （23/23）. No mutation was found in 23 refampin-sensitive strains by DNA sequencing,while 32 of 35 rifampin-resistant strainshad multations in resistant determination region of ropB gene,the sensitivity of DNA sequencing was 91.4%（32/ 35） ,the speciality was 100% （23/23）. There was no significant difference between PCR-SSCP and DNA sequencing（P〉 0. 05）. The accordance rate of DNA sequencing and PCR-SSCP was 93.1% （54/58）. Compared with DNA sequencing,the sensitivity of PCR-SSCP was 96.9% （31/32） and the specialty was 100% （23/23）. Conclusion PCR-SSCP is effective and feasible in detection of rifampin resistant strains.