【作者】 赛文莉；

【导师】 叶松；

【作者基本信息】 安徽理工大学 ， 病原生物学， 2008， 硕士

【摘要】 结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的一种慢性传染性疾病,是目前世界上最普遍和最严重的人类传染病之一,也是全世界由单一致病菌致死最多的疾病之一。据世界卫生组织报告显示,约三分之一的人口感染了结核分枝杆菌,估计每年有800～1 000万新发病例。从1953年～1985年,我国结核病发病率呈下降趋势,但上世纪90年代,由于耐药性结核分枝杆菌的出现与传播、艾滋病的流行以及人口的频繁流动等原因,结核病疫情呈现出全球性的回升。在抗结核病治疗药物中,RFP是治疗结核病最有效的一线药物之一,但其耐药率也非常高。有报告称,约90%的结核分枝杆菌耐RFP菌株同时也对INH耐药,因而,单独进行RFP敏感性分析也可以作为耐多药结核分枝杆菌的筛选指标。有关RFP的耐药机制及其耐药性的快速检测也是近年来结核病防治工作中的研究热点。【目的】本研究旨在使用结核分枝杆菌RFP耐药性的噬菌体生物扩增法(Phage Amplified Biologically Assay,PhaB)快速检测方法,探讨和评价此方法在实际检测临床标本应用中的价值;同时对结核分枝杆菌RFP耐药菌株的RFP耐药相关基因rpoB的突变进行研究,对RFP耐药机制进行初步探索,为以后进行RFP耐药机制研究提供相关实验基础。【方法】实验第一部分,用抗酸染色法和酸性L-J培养基培养法检测临床痰标本中的结核分枝杆菌,并对分离出来的菌株做菌型鉴定,获得结核分枝杆菌临床分离株。实验第二部分,以绝对浓度法对结核分枝杆菌临床分离株进行RFP耐药性检测,筛选出对RFP耐药的临床分离株。实验第三部分,应用PhaB法,对108例结核分枝杆菌临床分离株进行RFP耐药性的测定,并将检测结果与常规绝对浓度法结果进行比较,探讨和评价PhaB法的应用价值。实验第四部分,将结核分枝杆菌RFP耐药相关基因rpoB进行PCR扩增后对全基因进行测序,对rpoB基因突变进行研究。【结果】对108株结核分枝杆菌临床分离株进行RFP耐药性测定,常规绝对浓度药敏法检测结果为敏感26株,耐药82株;PhaB法检测结果为敏感24株,耐药84株,与绝对浓度法检测结果符合率为92.59%。如果以绝对浓度法检测RFP耐药性结果为评判标准,则PhaB法测定RFP耐药性的敏感性、特异性、阳性预测值、阴性预测值和准确性分别为96.34%、77.78%、94.05%、87.50%和92.59%。且在实际操作过程中,PhaB法检测RFP耐药性仅需要2～3天,与常规绝对浓度药敏法符合率高且实验结果重复性好,操作者的安全也有保障。对43株RFP耐药株进行rpoB基因测序,结果42株的rpoB基因存在各种不同形式的突变,突变率达到97.61%。在所有突变形式中,除已经见报道的突变形式外,还发现了1个新的突变位点,新突变的形成原因还有待进一步的研究。【结论】PhaB法检测结核分枝杆菌RFP耐药性具有快速、操作简便、安全性高、与常规药敏方法符合率较高等特点。且不需要特殊仪器设备,实验成本低廉,可以作为RFP耐药性快速检测的筛选方法。在我国这样的结核病耐药率较高的发展中国家和其它经济落后的贫困国家和地区,更适宜推广使用。本研究也证明,RFP耐药相关基因rpoB的突变是结核分枝杆菌对RFP产生耐药的主要原因,更重要的是在对rpoB基因序列检测过程中,我们发现了1种新的突变位点,且此突变位点是在突变核心区域之外,该结果为进一步从分子水平研究RFP耐药的理论研究以及研制RFP耐药检测基因芯片具有重要的参考价值。更多还原

【Abstract】 Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB),which is one of the most epidemic human diseases.It is also one of the diseases caused the largest number of death that caused by s single pathogen. The World Health Organization estimates that one-third of the world’s population is infected with Mycobacterium tuberculosis which results in 80～100 million new cases ever year.Between 1953 and 1985,the number of tuberculosis cases reported declined in our country,however,a reverse in this downward trend was not prominent in 1990s and became upwards in same areas.Because the drug-resistance Mycobacterium tuberculosis come forth and spread abroad,AIDS prevail and all so,Tuberculosis epidemics situation become serious again in the world.Rifampin(RFP) is one of the most effective one-line drugs that treat tuberculosis.The quick veracious ways that detect the RFP resistance in Mycobacterium tuberculosis is very important to Tuberculosis treating and control.The precise molecular mechanism of RFP-resistance is unclear;to detect the mutation of rpoB is very significative in theory and application. Since RFP is vital in the treatment of tuberculosis,so studies on the mechanism of resistance to RFP and the rapid detection methods are the focuses in the fields of tuberculosis control.【Objective】To evaluate the method of Phage Amplified Biologically Assay(PhaB) in the rapid detection of Rifampin(RFP) resistance.Study the mutation of rpoB gene. Explore the mechanism of RFP-resistance in Mycobacterium tuberculosis.Then,to get more knowledge about the rpoB gene mutations and provide some scientific information to study the molecule mechanism of resistance to Rifampin in Mycobacterium tuberculosis,the rpoB gene sequence of Mycobacterium tuberculosis clinical isolates was analyzed by direct sequencing of PCR products.【Method】The first part,detecting sputum specimens of Mycobacterium tuberculosis of pulmonary tuberculosis patients by Ziehl-Neelsen staining and acidity Lowenstein-Jensen medium.To identify the clinical isolates,in order to obtain the Mycobacterium tuberculosis.The second part,detect the resistance of Rifampin in Mycobacterium tuberculosis by improvement Lowenstein-Jensen culture.To obtain the Rifampin drug strains of clinical isolates Mycobacterium tuberculosis.The third part,detecting RFP resistance in 108 clinical isolates of Mycobacterium tuberculosis and compare it with the result of Lowenstein-Jensen culture method.The forth part,to amplify the rpoB genes of 42 RFP-resistance strains by PCR and sequence rpoB genes of RFP-resistance strains.【Results】All of 108 strains of Mycobacterium tuberculosis,26strains were RFP-susceptible and 82 strains were RFP-resistible detected by PhaB while 24 strains were RFP-susceptible and 84 strains were RFP-resistible by absolute concentration method.21 of 108 strains were RFP-susceptible and 79 strains were RFP-resistible with two methods.The concordant isolates of determination of RFP resistance were 100 strains in two methods and the concordance rate was 92.59%.There were 8 strains in discordant isolates.If the results of absolute concentration method is the golden standard,the sensitivity,specificity,positive and negative predictive value and accuracy,as well as accuracy of PhaB assay was 96.34%,77.78%,94.05%,87.50% and 92.59%respectively.The result of the sequences of the RFP-resistance strains indicates that the mutations of rpoB are detected in 42 of 43;the ratio of mutations is 97.67%.There is 1 new style of mutation formed is founded.【Conclusions】The result of this study indicates that the PhaB assay can be used for rapid method to detect drug susceptibility of RFP in clinical isolates of MTB because it is simplicity,rapidity,low-cost and only for 3 days.The PhaB assay is especially suitable for being popularized and applied in these developing countries and poor areas that the rate of drug-resistant TB is high.The study also shows that the rpoB mutations are the main reason that causes RFP-resistance in Mycobacterium tuberculosis.Further more 1 new style of mutation is founded.It is very valuable for the further research of RFP-resistance and for development of the drug-resistance detecting gene-chip.更多还原