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目的了解苏州市利福平(rifampin,RFP)耐药结核分枝杆菌rpoB基因突变特点,以期为苏州市RFP耐药结核分枝杆菌(mycobacterium tuberculosis,MTB)临床快速诊断提供有价值的分子标记靶点。方法通过裂解MTB抽提基因组DNA,采用PCR扩增和DNA测序方法,分析苏州市结核病患者痰培养阳性并经药敏试验诊断为RFP耐药MTB的rpoB基因RFP耐药决定区(rifampin resistance-determining region,RRDR)和CⅡ区变异谱与变异率。将主要突变形式RRDR-531(TCG→TTG)与传统药敏实验进行诊断学试验,评价此类型突变作为RFP耐药菌株分子标记靶点的临床应用价值。结果 42株RFP耐药MTB的rpoB基因总突变率为90.5%(38/42),在所检测到的突变中,以RRDR-531(TCG→TTG)为主要形式(61.9%)。以此类型的位点突变作为RFP耐药菌株诊断的分子标记,与传统药敏试验比较进行配对2检验,结果表明,与敏感菌株相比,RRDR-531(TCG→TTG)突变在RFP耐药菌株中差异有统计学意义(2=12.51,P<0.01)。结论 rpoB基因RRDR-531(TCG→TTG)突变是苏州市MTB产生RFP耐药性的主要分子机制,其作为RFP耐药MTB诊断的分子标记靶点具有较好的临床应用价值。
Objective To understand the characteristics of rpoB gene mutations in rifampin (RFP) -resistant Mycobacterium tuberculosis in Suzhou City and to provide valuable molecular marker targets for rapid clinical diagnosis of mycobacterium tuberculosis (MTB) in Suzhou city. point. Methods Genomic DNA was extracted by MTB assay. PCR amplification and DNA sequencing were used to analyze the positive sputum cultures of TB patients in Suzhou. The susceptibility test was used to determine the rifampin resistance-determining region (rPBFR) of RFP-resistant MTB region, RRDR) and C Ⅱ region mutation spectrum and mutation rate. The main mutated form of RRDR-531 (TCG → TTG) and traditional susceptibility testing for diagnostic tests to evaluate this type of mutation as RFP-resistant strains of molecular markers of clinical value. Results The total mutation rate of rpoB gene in 42 RFP-resistant MTB strains was 90.5% (38/42). Among the detected mutations, RRDR-531 (TCG → TTG) was the predominant form (61.9%). Compared with the susceptible strains, RRDR-531 (TCG → TTG) mutation was more resistant to RFP than the susceptible strain The difference was statistically significant (2 = 12.51, P <0.01). Conclusion The mutation of rpoB gene RRDR-531 (TCG → TTG) is the main molecular mechanism of MTB-producing RFP resistance in Suzhou city. It has good clinical value as a molecular marker for the diagnosis of RFP-resistant MTB.