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利用分子信标探针熔解曲线技术快速筛查结核分枝杆菌临床分离株中耐乙胺丁醇突变

作　者: NABAGOU Yenhame
导　师: 李庆阁
学　校: 厦门大学
专　业: 生物化学与分子生物学
关键词: 分子信标乙胺丁醇耐药实时PCR
分类号: R450
类　型: 硕士论文
年　份: 2009年
下　载: 93次
引　用: 0次
阅　读: 论文下载

内容摘要

本论文发展了一种利用改良分子信标的熔解曲线分析技术对肺结核的乙胺丁醇耐药和多药耐药突变进行快速筛查的方法。应用该方法完成了84份结核分枝杆菌培养标本和23份痰标本的检测。标本由厦门市疾病防治控制中心、深圳市慢性病防治中心、北京大学医学院提供。结果证明该体系能够检测出单拷贝的结核分枝杆菌基因,并且能识别不同的碱基错配的情况,从而筛选出突变类型。所有野生和突变的检测结果均通过DNA测序验证。本系统对结核分枝杆菌的检测特异性达到100%。如果本方法能够应用于对耐药病人的治疗,将对减少多药耐药的发生提供一定的帮助。

全文目录

ABSTRACT  8
摘要  8-18
CHAPTER ONE:INTRODUCTION AND LITERATURE REVIEW  18-42
  1.1 INTRODUCTION  18-21
  1.2 LITERATURE REVIEW  21-42
    1.2.1 Overview of tuberculosis worldwide  21-22
    1.2.2 Treatment of tuberculosis  22
      1.2.2.1 First line drugs  22
      1.2.2.2 Second line drugs  22
    1.2.3 Evolution of drug resistance in Mycobacterium tuberculosis  22-24
    1.2.4 Clinical circumstance for resistance development  24-25
    1.2.5 Compartmentalization of tuberculosis infection  25-26
    1.2.6 Occurrence of mutation in mycobacterium  26-28
      1.2.6.1 Mutation rate  27
      1.2.6.2 Mutant antagonism  27-28
    1.2.7 Adaptation of mycobacterium  28-29
    1.2.8 Human studies of resistance emergence  29-30
    1.2.9 Multi-drug resistant tuberculosis and global response on TB surveillance  30-33
    1.2.10 Molecular mechanism of EMB action  33-34
    1.2.11 Molecular mechanisms for the emergence of drug resistance  34-36
    1.2.12 Challenge of embB306 as a marker  36-37
    1.2.13 Molecular methods to detect drug resistance  37-41
      1.2.13.1 Current methods used worldwide for ethambutol drug-resistance detection  37-41
    1.2.14 Main Objectives of the study  41-42
CHAPTER TWO:MOLECULAR BEACON PROBE MELTING CURVE ANALYSIS FOR RAPID SCREENING OF ETHAMBUTOL DUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS ISOLATES  42-64
  2.1 Background  42
  2.2 Material and method  42-47
    2.2.1 Principle of methodology  42
    2.2.2 Principle of the original molecular beacon probes  42-43
    2.2.3 Principle of our modified molecular beacon probes melting curve analysis  43-44
    2.2.4 Sample preparation  44-45
      2.2.4.1 Boiling method  44
      2.2.4.2 Bio-v DNA extraction Kit:protocol  44-45
      2.2.4.3 Equipments used during the extraction  45
    2.2.5 Primers and probes  45-46
    2.2.6 Real-time PCR assay  46
    2.2.7 Sensitivity  46
    3.2.8 Specificity  46-47
    2.2.9 Sequencing  47
    2.2.10 Validation of the system  47
  2.3 RESULTS  47-60
    2.3.1 Primers and probe choice  47-48
    2.3.2 Allele discrimination  48-50
    2.3.3 Primer ratio optimization  50-52
    2.3.4 Analytical Sensitivity test  52
    2.3.5 Specificity test  52-53
    2.3.6 Clinical samples tests  53-55
    2.3.7-Sequencing results  55-59
    2.3.8 Drug information  59
    2.3.9 Validation  59-60
  2.4 Discussion  60-63
  2.5 Conclusion  63-64
References  64-69
Acknowledgement  69

利用分子信标探针熔解曲线技术快速筛查结核分枝杆菌临床分离株中耐乙胺丁醇突变

内容摘要

全文目录

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