结核分枝杆菌乙胺丁醇耐药性与分子特征的相关性研究

发布时间：2018-06-21 06:48

本文选题：结核分枝杆菌 + 乙胺丁醇　；参考：《南华大学》2015年硕士论文

【摘要】：目的比较3种以细菌培养为基础的药物敏感试验方法检测结核分枝杆菌对乙胺丁醇的药物敏感性;分析结核分枝杆菌emb CAB基因突变特征及其与乙胺丁醇耐药的关联性、emb CAB突变与乙胺丁醇耐药水平间的关联性,为建立结核分枝杆菌乙胺丁醇耐药的分子检测新方法提供依据。方法(1)采用罗氏培养基比例法(L-J法)、Bactec MGIT 960系统检测法(960法)和微孔板阿尔玛蓝显色法(MABA)同步对126株结核分枝杆菌临床分离株进行乙胺丁醇药物敏感性试验,比较分析3种方法的药敏试验结果;(2)通过溴化十六烷基三甲铵(CTAB)法提取结核分枝杆菌全基因组脱氧核糖核酸(DNA),采用聚合酶链反应(PCR)扩增目的基因片段,基因测序技术分别对126株试验菌株的emb CAB基因进行测序,分析突变高发位点与乙胺丁醇耐药的相关性;(3)用微孔板Alamar Blue显色法(MABA法)检测结核分枝杆菌对乙胺丁醇最低抑菌浓度(MIC)值,分析emb CAB基因突变与EMB耐药水平的相关性。结果(1)三种方法总体一致率为75.4%。若以L-J法测定结果为判断标准,则960法和MABA法的敏感度、特异度和一致率分别为62.8%(49/78)、100.0%(48/48)、77.0%(97/126)和82.1%(64/78)、97.9%(47/48)、88.1%(111/126);若以960法测定结果为判断标准,则L-J法和MABA法的敏感度、特异度和一致率分别为100%(49/49)、62.3%(48/77)、77.0%(97/126)和98.0%(48/49)、77.9%(60/77)、85.7%(108/126),若以MABA法测定耐药结果为判断标准,则L-J法和960法的敏感度、特异度和一致率分别为98.5%(64/65)、77.0%(47/61)、88.1%(111/126)和73.8%(48/65)、98.4%(60/61)、85.7%(108/126)。MABA法与L-J法、960法均有较好一致性而L-J法与960法一致性一般。三种方法的不一致性主要表现在MIC值4.0μg/ml-16.0μg/ml的药物浓度范围。(2)在126株结核分枝杆菌中有78株在emb CAB基因有突变发生,突变率为61.9%,其中,75株菌发生emb B突变,emb B306位点突变率最高,16株emb A及其上游区域突变,2株emb C有突变。emb B306-497突变检验结核分枝杆菌乙胺丁醇耐药的灵敏度、特异度和一致率分别为79.5%、72.9%和77.0%。(3)14株MIC5.0μg/ml的菌株中6株发生emb B单突变,菌株MIC值随emb AB联合突变率提高而升高。结论(1)三种方法对EMB耐药检测一致性良好,MABA法更适合临床及科研推广。(2)emb B、emb A上游非编码区突变与EMB耐药有相关性,emb C突变与EMB耐药无相关性,emb B306-497更适合作为EMB耐药检测的分子标志。联合突变比单突变耐药谱更广泛,突变率与耐药种数呈正相关,而与北京家族无相关性。(3)结核分枝杆菌emb AB联合突变能提升其对EMB耐药水平,低水平EMB耐药与耐药基因突变相关性差,可能存在其他耐药机制,有待进一步研究。
[Abstract]:Objective to compare the susceptibility of Mycobacterium tuberculosis to ethambutanol by three drug sensitivity tests based on bacterial culture. To analyze the relationship between emb cab gene mutation and ethambutanol resistance of Mycobacterium tuberculosis and its relationship with the level of ethambutanol resistance, and to provide the basis for establishing a new molecular method for the detection of ethambutanol resistance of Mycobacterium tuberculosis. Methods 1) the susceptibility of 126 clinical isolates of Mycobacterium tuberculosis to ethambutanol was tested by the method of L-J method and microplate Alma-blue method, and the method of Bactec MGIT 960 system detection was used to detect the drug sensitivity of 126 clinical isolates of Mycobacterium tuberculosis. To compare and analyze the results of drug sensitivity test of three methods, we extracted the whole genome DNA of Mycobacterium tuberculosis by cetyltrimethylammonium bromide (CTAB) method, and amplified the target gene fragment by polymerase chain reaction (PCR). The emb cab genes of 126 strains were sequenced by gene sequencing technique. To analyze the correlation between mutation high frequency and ethambutanol resistance. (3) to detect the minimum inhibitory concentration of Mycobacterium tuberculosis to ethambutanol by Alamar Blue method, and to analyze the correlation between the mutation of emb cab gene and the level of EMB resistance. Results the overall consistency rate of the three methods was 75.4%. If the results of the L-J method were taken as the criterion, the sensitivity, specificity and consistency of 960 and MABA methods were 62.80.49 / 78 / 100.048 / 48 and 77.097 / 126, respectively, and 82.1 / 4888.88 / 97 / 126, respectively. If the results of the 960 method were used as the criterion, then the sensitivity of the L-J method and the MABA method, The specificity and the consistency rate were 100 / 49 / 49 / 62.3and 48 / 77 / 77.70 / 97 / 126, respectively, and 98.0 / 48 / 49 / 77.90 / 77 / 85.70.The sensitivity of the L-J method and the 960 method was determined by using the MABA method to determine the results of drug resistance. The specificity and the consistency rate were 98.5 / 64 / 65 / 77.0 / 47 / 61 / 88 / 11 / 126 and 73.8% / 65 / 65 respectively. There was good consistency between L-J method and L-J method 960 method, and 85.70% 108126U% MABA method and 960% L-J method respectively. The inconsistency of the three methods was mainly manifested in the drug concentration range of 4.0 渭 g/ml-16.0 渭 g/ml.) of 126 strains of Mycobacterium tuberculosis, 78 strains had mutations in emb cab gene. The mutation rate was 61.9, of which 75 strains had the highest mutation rate of emb B mutation, 16 strains of emb A and 2 strains of upstream region mutation, emb C had mutation .emb B306-497 mutation to test the susceptibility of Mycobacterium tuberculosis to ethambutanol resistance. The specific and consistent rates were 72.9% and 77.0%, respectively. Six of the 314 strains of MIC5.0 渭 g/ml had single emb B mutation, and the MIC value increased with the increase of co-mutation rate of emb AB. Conclusion (1) the three methods have good consistency in the detection of EMB resistance. MABA method is more suitable for clinical and scientific research. There is a correlation between the mutation of the upstream non-coding region and the resistance of EMB. The mutation of amb C and the resistance of EMB B306-497 are more suitable to be used as EMB resistance. A molecular marker for drug testing. The mutation rate was positively correlated with the number of drug resistant species, but not with the Beijing family. The combined mutation of Mycobacterium tuberculosis emb AB could improve the drug resistance level of Mycobacterium tuberculosis. The relationship between low level EMB resistance and drug resistance gene mutation is poor, and there may be other drug resistance mechanisms, which need to be further studied.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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