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目的探讨结核分枝杆菌耐药性与WhIB7,drrA,drrB基因表达的关系。方法通过设计并合成结核分枝杆菌的Whi B7,drrA,drrB基因引物,对耐药的结核分枝杆菌利用逆转录得到三个基因的c DNA。并对c DNA进行PCR扩增,通过优化PCR参数,使PCR方法达到检测c DNA拷贝数的准确性。分析WhIB7,drrA,drrB基因表达在不同类型耐药菌株的表达程度。使用药物刺激观察耐药菌株Whi B7,drrA,drrB基因的变化。结果逆转录荧光定量PCR可以对WhIB7,drrA,drrB基因很好的定量。对不同种耐药菌株进行WhIB7,drrA,drrB基因检测,阳性率分别为:单利福平耐药菌株85.0%(17/20)、35.0%(7/20)、30.0%(6/20),单异烟肼耐药菌株37.5%(12/32)、87.5%(28/32)、81.3%(26/32),利福平和异烟肼均耐药的菌株87.5%(14/16)、75.0%(12/16)、81.3%(13/16),含利福平异烟肼的多重耐药菌株88.2%(30/34)、82.4%(28/34)、85.3%(29/34),药物敏感菌株12.4%(12/97)、8.2%(8/97)、9.3%(9/97)。耐药菌株WhIB7,drrA,drrB基因阳性率高于药物敏感菌株。WhIB7基因与利福平耐药关系紧密,drrA和drrB基因与异烟肼耐药关系紧密。但不同的药物对耐药菌基因表达的诱导能力不同。结论 Whi B7,drrA,drrB基因的逆转录荧光定量PCR方法可以作为耐药结核分枝杆菌筛查的重要参考,为研究结核分枝杆菌日后基因表达与耐药机理研究奠定基础。
Objective To investigate the relationship between the drug resistance of Mycobacterium tuberculosis and the expression of WhIB7, drrA and drrB genes. Methods By designing and synthesizing the primers of Whi B7, drrA and drrB genes of Mycobacterium tuberculosis, the c DNAs of three genes were obtained by reverse transcription of resistant Mycobacterium tuberculosis. And c DNA PCR amplification, PCR parameters to optimize the PCR method to detect c DNA copy number accuracy. The expression of WhIB7, drrA and drrB genes in different types of resistant strains was analyzed. Drug resistance was used to observe the changes of the resistant strains Whi B7, drrA and drrB genes. Results RT-PCR showed that the WhIB7, drrA and drrB genes were well quantitated. The detection rates of WhIB7, drrA and drrB in different kinds of resistant strains were 85.0% (17/20), 35.0% (7/20) and 30.0% (6/20) respectively, 37.5% (12/32), 87.5% (28/32), 81.3% (26/32), 87.5% (14/16) of rifampicin-resistant and isoniazid-resistant isolates of single- , 75.0% (12/16), 81.3% (13/16), 88.2% (30/34), 82.4% (28/34) and 85.3% (29.3%) of rifampin isoniazid- 34), drug-sensitive strains 12.4% (12/97), 8.2% (8/97), 9.3% (9/97). The resistant strains WhIB7, drrA, drrB gene positive rate higher than the drug-sensitive strains. The relationship between WhIB7 gene and rifampicin resistance is closely related, drrA and drrB genes are closely related to isoniazid resistance. However, different drugs have different ability to induce drug-resistant bacterial gene expression. Conclusion The reverse transcription-PCR method of Whi B7, drrA and drrB genes can be used as an important reference for the screening of drug-resistant Mycobacterium tuberculosis, which lays the foundation for the study on the gene expression and drug resistance mechanism of Mycobacterium tuberculosis in the future.