结核分枝杆菌临床分离株基因分型与耐药性的检测

发布时间：2018-05-29 08:16

本文选题：结核分枝杆菌 + RD105基因缺失　；参考：《吉林大学》2015年硕士论文

【摘要】：目的了解22株结核分枝杆菌临床分离株的基因型分布情况，探索适合结核分枝杆菌北京基因型鉴定的方法。探讨结核分枝杆菌北京基因型菌株与耐药是否相关。分析结核分枝杆菌耐药性与耐药基因之间的联系。方法对收集的吉林省22株结核分枝杆菌临床分离株进行RD105缺失基因检测法基因分型研究。应用绝对浓度法药物敏感试验方法检测22株结核分枝杆菌对异烟肼、利福平、链霉素、卡那霉素、氧氟沙星及对氨基水杨酸六种抗结核药物的耐药性，最后对药敏结果和基因分型结果进行分析。对于耐药菌株将其katG基因、rpoB基因、rpsl基因、rrs基因、gyrA基因及thyA基因进行测序，检测其耐药基因是否存在突变并且确定突变的类型。结果22株结核分枝杆菌临床分离株中有10株耐药菌株，占45.5%。对利福平耐药的有6株，占27.3%；对异烟肼耐药的有4株，占18.0%；对链霉素耐药的有2株，占9%；对卡那霉素耐药的有4株，占18.0%；对氧氟沙星耐药的有3株，占13.6%，对对氨基水杨酸耐药的有一株，占4.5%，北京基因型菌株占77.3%(17/22)。17株北京家族结核分枝杆菌临床分离株中52.9%（9/17）表现对六种抗结核药物敏感，47.1%（8/17）表现为耐药；非北京家族菌株中60%（3/5）表现为敏感，40%（2/5）表现为耐药。经检验，北京家族菌株与非北京家族菌株耐药率差异无统计学意义。4株异烟肼耐药株中，有1株未发生katG基因突变，其它3株katG基因发生突变，均发生在315位点，由AGC（Ser）突变成ACC（Thr）。6株利福平耐药株中，有4株rpoB基因发生531位点突变，由TCG（Ser）突变成TTG（Leu）。2株rpoB基因发生526位点突变，由CAC（His）突变成GAC（Asp）。2株链霉素耐药株rpsl基因均发生43位点突变，由AAG（Lys）突变成AGG（Arg）。4株卡那霉素耐药株中，有1株rrs基因未发生突变，其它3株rrs基因均发生11位点突变，由GGT（Gly）突变成GTT（Val）。3株氧氟沙星耐药株gyrA基因均发生94位点突变，GAC（Asp）突变成GCC（Gly）。1株对氨基水杨酸耐药株thyA基因在355位点C碱基缺失，由CTG（Leu）突变成GTC（Val）。结论RD105缺失基因检测法是鉴定MTB北京基因型的简便、有效的方法；北京基因型菌株为所研究的结核分枝杆菌株中的优势菌株；北京基因型菌株与非北京基因型菌株两者耐药率的差别无统计学意义。结核分枝杆菌对抗结核药物耐药与相关耐药基因突变密切相关。
[Abstract]:Objective to investigate the genotypic distribution of 22 clinical isolates of Mycobacterium tuberculosis and to explore a suitable method for identification of mycobacterium tuberculosis in Beijing. To investigate whether the Beijing genotype of Mycobacterium tuberculosis is related to drug resistance. To analyze the relationship between drug resistance and drug resistance genes of Mycobacterium tuberculosis. Methods 22 clinical isolates of Mycobacterium tuberculosis collected from Jilin Province were genotyped by RD105 deletion method. The drug resistance of 22 strains of Mycobacterium tuberculosis to isoniazid, rifampicin, streptomycin, kanamycin, ofloxacin and p-aminosalicylic acid was detected by absolute concentration method. Finally, the results of drug sensitivity and genotyping were analyzed. The katG gene, rpsl gene, rrs gene, gyrA gene and thyA gene were sequenced to detect whether the drug resistance gene had mutation and determine the type of mutation. Results among the 22 clinical isolates of Mycobacterium tuberculosis, 10 strains were drug-resistant, accounting for 45.5%. Six strains were resistant to rifampicin, accounting for 27.3um; four strains were resistant to isoniazid, accounting for 18.0%; two strains were resistant to streptomycin (9 strains); four strains were resistant to kanamycin (18.0%); three strains were resistant to ofloxacin, One strain was resistant to aminosalicylic acid, accounting for 4.5%, and the Beijing genotype strain accounted for 77.3% 22 / 22 .17 strains of Mycobacterium tuberculosis in the family of Beijing. 52.9% of the 17 strains showed resistance to the six anti-tuberculosis drugs (47.1% / 87%). 60 / 5 of the non-Beijing strains showed resistance to drugs. The results showed that there was no significant difference in drug resistance rate between Beijing family strains and non-Beijing family strains. Of the 4 isoniazid resistant strains, 1 strain had no katG gene mutation, while the other 3 strains had katG gene mutation, all of which occurred at 315 locus. Among the rifampicin resistant strains, there were 531 mutation in rpoB gene, 526 site mutation in rpoB gene in TTG(Leu).2 strain, 43 locus mutation in rpsl gene of streptomycin resistant strain GAC(Asp).2. The rrs gene was not mutated in one of the kanamycin resistant strains of AGG(Arg).4, and 11 loci were found in all the other three strains of rrs gene. The 94 locus mutation of the gyrA gene of GTT(Val).3 strain was mutated to the deletion of thyA gene at 355 locus from GCC(Gly).1 strain. Conclusion the detection of RD105 deletion gene is a simple and effective method to identify the Beijing genotype of MTB. There was no significant difference in drug resistance between Beijing genotype strains and non-Beijing genotype strains. Anti-TB drug resistance of Mycobacterium tuberculosis is closely related to the mutation of related drug resistance genes.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

【参考文献】