广泛耐药结核分枝杆菌筛选及耐药机制初步研究

发布时间：2018-06-09 04:38

本文选题：广泛耐药 + 结核分枝杆菌　；参考：《苏州大学》2010年硕士论文

【摘要】： 目的筛选XDR-TB临床分离株,并从耐药相关基因突变和药物外排泵两方面初步探讨XDR-TB耐药机制,为开发XDR-TB的快速诊断技术和临床治疗方案提供基础理论支持。方法利用罗氏药敏检测技术筛选XDR-TB临床分离株,并检测XDR-TB分离株的最低抑菌浓度(MIC);提取XDR-TB临床分离株基因组DNA,PCR扩增耐药相关基因,测序后分析其突变位点;根据XDR-TB分离株KZN605的15个特有的SNP位点设计引物扩增后测序比对;检测药物外排泵抑制剂利血平,CCCP,异博定对XDR-TB分离株的MIC值的影响,并通过荧光定量PCR检测药物刺激组和未刺激组对编码药物外排泵的相关基因表达量的影响。结果163结核分枝杆菌(MTB)中筛选得到24株XDR-TB (14.7%),随机挑选的10株XDR-TB菌株在rpoB、katG和rpsL均检测到突变,9株分离株在gyrA检测到突变,2株在gyrB检测到突变,6株分离株在rrs检测到突变,未检测到tlyA突变,2株检测到ahpC,3株检测到inhA,4株检测到embB;大部分KZN605株特异的SNP位点在本研究均未检测到;药物外排泵抑制剂利血平导致1株XDR-TB分离株OFLX的MIC值明显下降了16倍,其余药物以及其它分离株的MIC值均无明显变化;在OFLX刺激后编码一种ABC运输蛋白的基因Rv2686大量表达。结论KZN-605的部分SNP位点可能与XDR-TB耐药无关,耐药相关基因突变是XDR-TB分离株耐药的主要机制,Rv2686-Rv2687-Rv2688编码的一种ABC运输蛋白可能部分参与了氟喹诺酮类药物的耐药产生,gyrB基因D500N和rrs基因A1427G是否与耐药性相关还需进一步研究探讨。
[Abstract]:Objective to screen the clinical isolates of XDR-TB and to explore the mechanism of XDR-TB resistance from two aspects: drug resistance related gene mutation and drug efflux pump. Methods the clinical isolates of XDR-TB were screened by Roche's drug sensitivity detection technique, which provided basic theoretical support for the rapid diagnosis and clinical treatment of XDR-TB. The minimal inhibitory concentration (MIC) of XDR-TB isolate was detected, the gene related to drug resistance was amplified by PCR of genomic DNA of XDR-TB clinical isolate, and its mutation site was analyzed after sequencing, and 15 specific SNP loci of XDR-TB isolate KZN605 were designed for sequencing. The effects of reserpine and verapamil on the MIC of XDR-TB isolates were detected. Fluorescence quantitative PCR was used to detect the effect of drug stimulation group and unstimulated group on the expression of genes related to drug efflux pump. Results 24 strains of XDR-TB were screened out of 163 Mycobacterium tuberculosis (MTB), and 10 strains of XDR-TB were randomly selected. 9 strains were detected in gyrA, 2 strains were detected in GyrB, 6 strains were detected in rrs. No tlyA mutation was detected in 2 strains of tlyA mutation and 4 strains of inhAgna were detected in 4 strains of tlyA, most of the specific SNPs of KZN605 strain were not detected in this study, and reserpine, a drug efflux pump inhibitor, significantly decreased the OFLX value of one XDR-TB isolate by 16 times. After OFLX stimulation, Rv2686, a gene encoding an ABC transport protein, was highly expressed. Conclusion some SNP loci of KZN-605 may be independent of XDR-TB resistance. The mutation of drug-resistance-related genes is the main mechanism of drug resistance in XDR-TB isolates. An ABC transport protein encoded by Rv2686-Rv2687-Rv2688 may be partly involved in the production of fluoroquinolones resistance and whether D500N and A1427G of rrs gene are related to drug resistance should be further studied.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.911

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