【作者】 陆军；

【导师】 叶松； 李朝品；

【作者基本信息】 安徽理工大学 ， 病原生物学， 2006， 硕士

【摘要】 目的 研究淮南矿区尘肺结核患者感染的结核分枝杆菌L型katG、rpoB、rpsL基因的突变特点及规律,探讨结核分枝杆菌L型耐药性的产生与其相关耐药基因突变之间的内在联系,以及聚合酶链反应.单链构象多态性分析(PCR-SSCP)方法的临床应用价值。 方法 选择典型的尘肺结核患者,采集痰标本进行结核分枝杆菌L型培养和鉴定;提取结核分枝杆菌L型菌株的DNA,应用PCR技术扩增katG、rpoB、rpsL基因,并对分离的L型菌株进行回复试验及药物敏感性检测;通过单链构象多态性分析法(SSCP)比较结核分枝杆菌L型菌株与细菌型菌株的katG、rpoB、rpsL耐药基因的差异,分析其突变规律。 结果 研究组 52例结核分枝杆菌L型感染的尘肺结核患者中,采用PCR-SSCP方法检测katG、rpoB和rpsL基因,检出率分别为57.70%(30/52)、65.38%(32/52)和40.38%(21/52)。回复试验后,52株结核分枝杆菌L型中有41株回复为结核分枝杆菌细菌型,回复率为78.85%(41/52),回复后耐药基因的检测结果与回复前相同。采用常规药敏试验(AST)法检测异烟肼(INH)、利福平(RFP)和链霉素(SM)的耐药,检出率分别为76.92%(40/52)、90.38%(47/52)和50.00%(26/52),但是耐药株中有10株katG基因检测阴性,15株rpoB基因检测阴性,5株rpsL基因检测阴性。其中多耐药株40例(76.92%),包括同时耐三种药的21例(40.38%),耐两种药的19例(36.54%),单耐药株12例(23.08%),耐药率100%。PCR-SSCP方法检测出3个基因联合突变的有15株,与AST法的符合率为71.43%(15/21),检出2个基因突变的有16株,符合率为84.21%(16/19),检出1个基因突变的有6株,符合率为50.00%(6/12)。 对照组 28例结核分枝杆菌细菌型感染的尘肺结核患者中,采用PCR-SSCP方法检测katG、rpoB和rpsL基因,检出率分别为42.86%(12/28)、42.86%(12/28)和32.14%(9/28)。采用AST法检测INH、RFP和SM的耐药,检出率分别为64.23%(18/28)、60.71%(17/28)和53.57%(15/28),在耐药株中有6株katG基因检测阴性,5株rpoB基因检测阴性,6株rpsL基因检测阴性。其中多耐药株20例(71.43%),包括同时耐三种药的12例(42.86%),耐两种药的8例(28.57%),单耐药株6例(21.43%),敏感株2例,耐药率92.086%。 结核分枝杆菌L型与细菌型采用PCR-SSCP方法检测的结果分别做χ~2检验,发现katG、rpoB和rpsL基因检测二者之间均无显著差异(P>0.05);采用AST更多还原

【Abstract】 Objective To study the features and regularities of mutation of katG, rpoB and rpsL drug-resistant genetic mutation in Mycobacterium tuberculosis L-forms among coal worker’s pneumoconiosis patients complicated with tuberculosis in Huainan mining district, approach internal relationship between drug resistances and drug-resistant related genes, and to explore the value of clinical application by PCR-SSCP. Methods Sputum specimens of Mycobacterium tuberculosis L-forms were selected among typical pneumoconiosis patients complicated with tuberculosis, and cultivated, identified and extracted DNA. Then the PCR technique was amplified the mutational genes of katG, rpoB and rpsL. We must carry out the reversion test and traditional antimicrobial susceptibility test (AST). Finally we compared the gene mutational rates of strains of L-forms with those of popular types by PCR-SSCP, and the results were compared in order to analysis the mutational regularity.Results In research group, of total 52 Mycobacterium tuberculosis L-forms cases, PCR-SSCP assay can be used to detect the genes of katG, rpoB and rpsL, the rate of drug-resistance were respectively 57.70%(30/52), 65.38%(32/52) and 40.38%(21/52). Furthermore, the rate of reversion was 78.85%(41/52) and the result of drug-resistant genes was invariable. At the same time, we detected INH, RFP and SM by AST, the results were 76.92%(40/52), 90.38%(47/52) and 50.00%(26/52). Nevertheless, there were 10 strains of the negative of katG, 15 strains of the negative of rpoB and 10 strains of the negative of rpsL among persisters. Multi-drug resistant strains were 40(76.92%), including 21 strains (40.38%) resisting to 3 kinds of drugs, 19(36.54%) to 2 kinds of drugs and 12(23.08%) to single drug by AST. But there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.41% and 50.00%.While in contrast group, 28 general bacteric cases was found in sputum Mycobacterium tuberculosis culture positive, the results of katG, rpoB and rpsL by PCR-SSCP assay were 42.86%(12/28), 42.86%(12/28) and 32.14%(9/280), but those of INH, RFP and SM by AST were 64.23%(18/28), 60.71%(17/28) and 53.57%(15/28). Among multi-drug resistant strains were 20(71.43%), including 12 strains (42.86%) resisting to 3 kinds of drugs, 8 strains (28.57%) to 2 kinds of drugs and 6(21.43%) tosingle drug, but have 2 kinds of sensitive strains. Nevertheless, there were 6 strains of the negative of katG, 5 strains of the negative of rpoB and 6 strains of the negative of rpsL among persisters. Multi-drug resistant strains were 20(71.43%), including 12 strains (42.86%) resisting to 3 kinds of drugs, 18(28.57%) to 2 kinds of drugs and 6(21.43%) to single drug by AST.The difference of katG was not quite notable 0^=1.6062, P>0.05) at both L-forms and bacteric cases by PCR-SSCP assay, as well as rpoB and rpsL. By AST, we can find that RFP was quite notable (/2= 10.0137, P<0.005) , INH and SM were no. Conclusion The infectious rate of Mycobacterium tuberculosis L-forms in cases of pneumoconiosis complicated with tuberculosis is much high, and the status of drug resistance is pretty bad. PCR-SSCP technique has higher sensibility and specificity to detect to the genes of katG, rpoB and rpsL, to have advantages at earlier diagnosis and guidance of clinical medications. The generations of drug-resistance of L-forms were concerned not only with absence of cell wall, but also with the mutations of drug-resistant related genes.更多还原