检测结核分枝杆菌rpoB基因突变的研究

目的：建立聚合酶链反应－单链构象多态性（PCR－SSCP）检测结核分枝杆菌rpoB基因突变的方法，并评价共临床应用的价值。方法：依据结核分枝杆菌rpoB基因的耐利福平决定区设计引物，用PCR从临床分离株和直接从痰标本中扩增rpoB基因片段；对扩得的rpoB基因片段做DNA SSCP分析，并随机测定rpoB片段的序列。结果：PCR从所有212株结核分枝杆菌中均扩得230bp片段135份抗酸染色阳性的痰标本中，有113份扩得阳性片段（83．7％），在SSCP分析中，140份经L－J药敏试验检测为利福平耐受的结核分枝杆菌，有130份有rpoB基因突变（符合率为92．9％），72份经L－J药敏试验检测为利福平敏感的菌，有67份的rpoB基因无突变（符合率为93．1％），测序分析发现57份经SSCP检测为突变的rpoB片段均有序列改变，10份经SSCP分析为无突变的有8份无序列改变，SSCP与测序结果的符合率为94．0％，在本研究的菌株中，耐多药结核病（MDR－TB）占76．4％（107／140），有92．5％（99／107）耐多药菌株经SSCP检测有rpoB突变。结论：建立的PCR－SSCP分析方法，是一种较准确和稳定的检测结核分枝杆菌rpoB基因突变的方法，可用于临床快速分析患者结核分枝杆菌对利福平的耐药性，并可作为MDR－TB判断的一个重要指标。

Clinical analysis of rpoB mutations of Mycobacterium tuberculosis by the PCR-SSCP

Objective To establish PCR SSCP for analyzing rpoB mutation of Mycobacterium tuberculosis and to evaluate its use in clinics. Methods A rpoB fragment was amplified from M. tuberculosis strains and sputum specimens by PCR with a pair of primers designed according to the lifampin resistant determinant of rpoB. The fragments of rpoB were analyzed by single stranded conformation polymorphism (SSCP) and some fragments were sequenced. Results A 230 bp fragments of rpoB were amplified from all M. tuberculosis strains and 113 of 135 (83.7%) sputum samples that were positive for acid fast stain, but not from 16 species of nontuberculosis bacteria and sputum from nontuberculosis patients. In SSCP analysis, 130 of 140 (92.9%) strains resistant to lifampin in L J drug susceptibility test were rpoB mutants and 67 of 72 (93.1%) strains susceptible to lifampin were wild type. In sequencing analysis, 57 mutant fragments in SSCP were all found to have sequence changes and 2 of 10 wild types of fragments in SSCP to have sequence changes. Among the lifampin resistant strains, the multi drug resistant (MDR) strains were 76.4%. The MDR strains,92.5%(99/107) rpoB mutation in SSCP. Conclusion This PCR SSCP is an accurate and stable method to identify rpoB showed mutation of M. tuberculosis , and it is useful for quickly identifying the lifampin resistant or the MDR strains in clinics.