TUBERCULOSIS--DEVELOPMENT OF DNA BASED DIAGNOSTICS

近年来，结核病在美国急剧死灰复燃。 States.据估计， 直到1992年。与结核病发病率的上升相对应， 结核分枝杆菌耐药菌株的分离增加 一种或多种常用的抗生素作为治疗已经发生。 这些疾病趋势共同构成了一个明确和现实的危险， 社会拟议研究的长期目标是开发基于DNA的 快速鉴定和药物敏感性的诊断策略 测试M.结核病的主要临床样品，如痰。 具体而言，研究的目的是， (i)采用自动化DNA测序快速明确地定义 几个基因突变的类型和频率分布强烈 与抗结核药物耐药性有关，包括 利福平、异烟肼、乙硫异烟胺、链霉素和环丙沙星。 (ii)探索一种新兴技术的能力，称为“测序 杂交”或“矩阵测序”来快速诊断结核病 并在初级临床中识别与耐药性相关的突变 标本和早期阳性培养物。 (iii)测试使用自动化DNA测序策略的可行性， 快速诊断结核病和其他分枝杆菌引起的感染 通过物种特异性等位基因的特征， 编码热休克蛋白的结构基因（hsp 65）的多态性， 并鉴定与M耐药性相关的突变。结核 主要临床标本和早期阳性培养物将用于 在一个繁忙的忙碌医院分枝杆菌诊断实验室的背景下。 成功完成拟议的研究将表明， 诊断M.结核病及其他 分枝杆菌菌种，并鉴定耐药分枝杆菌。结核 在不到24小时的时间内，

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.