TUBERCULOSIS--DEVELOPMENT OF DNA BASED DIAGNOSTICS

近年来，结核病在美国急剧死灰复燃。 各州。据估计，已记录了51,700例超额结核病病例 一直到1992年。与结核病发病率的上升相称， 结核分枝杆菌耐药菌株的分离增加 对一种或多种常用抗生素的治疗已经发生。 总而言之，这些疾病趋势代表着一个明确而现实的危险 社会。拟议研究的长期目标是开发基于DNA的 快速识别和药物敏感性的诊断策略 在痰等初级临床样本中检测结核分枝杆菌。 具体地说，研究的目的是， (I)使用自动DNA测序来快速和明确地定义 几个基因强烈突变的类型和频率分布 与抗结核药物耐药有关，包括 利福平、异烟肼、乙硫酰胺、链霉素和环丙沙星。 (2)探索一项名为“按以下方式排序”的新兴技术的能力 “杂交”或“基质测序”，以快速诊断结核病 并确定与初级临床耐药相关的突变 标本和早期阳性培养。 (3)测试使用自动DNA测序策略的可行性 快速诊断结核病和其他分支杆菌引起的感染 通过物种特异性等位基因的特征，而不是结核病 编码热休克蛋白的结构基因(Hsp65)的多态性， 并确定与结核分枝杆菌耐药相关的突变。 初级临床标本和早期阳性培养将用于 忙碌的医院分支杆菌诊断实验室的背景。 拟议研究的成功完成将证明 诊断结核分枝杆菌感染的能力，以及其他 分枝杆菌种类和耐药结核分枝杆菌的鉴定 在不到24小时的时间内从初级标本中分离到菌株。

Tuberculosis has resurged dramatically in recent years in the United States. An estimated 51,700 excess tuberculosis cases have been recorded through 1992. Commensurate with the rise in tuberculosis frequency, an increase in the isolation of Mycobacterium tuberculosis strains resistant to one or more antibiotics commonly used as treatment has occurred. Together, these disease trends represent a clear and present danger to society. The long term goal of the proposed studies is to develop DNA-based diagnostic strategies for rapid identification and drug susceptibility testing of M. tuberculosis in primary clinical samples such as sputum. Specifically, the aims of the research are to, (i) Employ automated DNA sequencing to rapidly and unambiguously define the type and frequency distribution of mutations in several genes strongly associated with resistance to anti-tuberculosis medications, including rifampin, isoniazid, ethionamide, streptomycin, and ciprofloxacin. (ii) Explore the ability of an emerging technology, termed "sequencing by hybridization," or "matrix sequencing," to rapidly diagnose tuberculosis and identify mutations associated with drug resistance in primary clinical specimens and early positive cultures. (iii) Test the feasibility of using automated DNA sequencing strategies to rapidly diagnose tuberculosis and infections caused by mycobacteria other than tuberculosis, by characterization of species-specific allelic polymorphism in the structural gene (hsp65) encoding a heat shock protein, and identify mutations associated with drug resistance in M. tuberculosis. Both primary clinical specimens and early positive cultures will be used in the context of a busy hospital mycobacteriology diagnostic laboratory. Successful accomplishment of the proposed research will demonstrate the capability of diagnosing infection with M. tuberculosis, and other mycobacterial species, and identifying drug resistant M. tuberculosis strains in primary specimens in less than 24-hours.