痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。

Detection of multidrug-resistant Mycobacterium tuberculosis by sputum smears

Objective To investigate the possibility of using DNA extracts from patients sputum smears to detect the multidrug-resistant Mycobacterium tuberculosis (MDRTB).Methods Totally 18 isolates of M. tuberculosis were collected from three hospitals and their resistance to rifampin were detected. After PCR the rpoB gene from cultured strains DNA and DNA extracts from stained slides,identification of mutant rpoB gene was performed by hybridization using line probe assay and DNA sequencing.Results Among the 18 isolates, 12 were resistant to rifampin. In 12 resistant strains,analysis of the rpoB gene mutant revealed that DNA samples from the cultures were 100% (12/12),whereas that from sputums were 91.7% (11/12). Sequence analysis of the rpoB mutants DNA extracts rifampin-resistant M. tuberculosis carried altered codons within the core region of the rpoB gene.Conclusion Molecular diagnosis method of the rpoB gene mutant in M. tuberculosis DNA directly extracted from stained sputum slides could provide simple method for the clinical laboratory.