發(fā)布時(shí)間：2018-06-21 06:48

  本文選題：結(jié)核分枝桿菌 + 乙胺丁醇��； 參考：《南華大學(xué)》2015年碩士論文

【摘要】：目的比較3種以細(xì)菌培養(yǎng)為基礎(chǔ)的藥物敏感試驗(yàn)方法檢測(cè)結(jié)核分枝桿菌對(duì)乙胺丁醇的藥物敏感性;分析結(jié)核分枝桿菌emb CAB基因突變特征及其與乙胺丁醇耐藥的關(guān)聯(lián)性、emb CAB突變與乙胺丁醇耐藥水平間的關(guān)聯(lián)性,為建立結(jié)核分枝桿菌乙胺丁醇耐藥的分子檢測(cè)新方法提供依據(jù)。方法(1)采用羅氏培養(yǎng)基比例法(L-J法)、Bactec MGIT 960系統(tǒng)檢測(cè)法(960法)和微孔板阿爾瑪藍(lán)顯色法(MABA)同步對(duì)126株結(jié)核分枝桿菌臨床分離株進(jìn)行乙胺丁醇藥物敏感性試驗(yàn),比較分析3種方法的藥敏試驗(yàn)結(jié)果;(2)通過(guò)溴化十六烷基三甲銨(CTAB)法提取結(jié)核分枝桿菌全基因組脫氧核糖核酸(DNA),采用聚合酶鏈反應(yīng)(PCR)擴(kuò)增目的基因片段,基因測(cè)序技術(shù)分別對(duì)126株試驗(yàn)菌株的emb CAB基因進(jìn)行測(cè)序,分析突變高發(fā)位點(diǎn)與乙胺丁醇耐藥的相關(guān)性;(3)用微孔板Alamar Blue顯色法(MABA法)檢測(cè)結(jié)核分枝桿菌對(duì)乙胺丁醇最低抑菌濃度(MIC)值,分析emb CAB基因突變與EMB耐藥水平的相關(guān)性。結(jié)果(1)三種方法總體一致率為75.4%。若以L-J法測(cè)定結(jié)果為判斷標(biāo)準(zhǔn),則960法和MABA法的敏感度、特異度和一致率分別為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法測(cè)定結(jié)果為判斷標(biāo)準(zhǔn),則L-J法和MABA法的敏感度、特異度和一致率分別為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法測(cè)定耐藥結(jié)果為判斷標(biāo)準(zhǔn),則L-J法和960法的敏感度、特異度和一致率分別為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法與L-J法、960法均有較好一致性而L-J法與960法一致性一般。三種方法的不一致性主要表現(xiàn)在MIC值4.0μg/ml-16.0μg/ml的藥物濃度范圍。(2)在126株結(jié)核分枝桿菌中有78株在emb CAB基因有突變發(fā)生,突變率為61.9%,其中,75株菌發(fā)生emb B突變,emb B306位點(diǎn)突變率最高,16株emb A及其上游區(qū)域突變,2株emb C有突變。emb B306-497突變檢驗(yàn)結(jié)核分枝桿菌乙胺丁醇耐藥的靈敏度、特異度和一致率分別為79.5%、72.9%和77.0%。(3)14株MIC5.0μg/ml的菌株中6株發(fā)生emb B單突變,菌株MIC值隨emb AB聯(lián)合突變率提高而升高。結(jié)論(1)三種方法對(duì)EMB耐藥檢測(cè)一致性良好,MABA法更適合臨床及科研推廣。(2)emb B、emb A上游非編碼區(qū)突變與EMB耐藥有相關(guān)性,emb C突變與EMB耐藥無(wú)相關(guān)性,emb B306-497更適合作為EMB耐藥檢測(cè)的分子標(biāo)志。聯(lián)合突變比單突變耐藥譜更廣泛,突變率與耐藥種數(shù)呈正相關(guān),而與北京家族無(wú)相關(guān)性。(3)結(jié)核分枝桿菌emb AB聯(lián)合突變能提升其對(duì)EMB耐藥水平,低水平EMB耐藥與耐藥基因突變相關(guān)性差,可能存在其他耐藥機(jī)制,有待進(jìn)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5

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