MOLECULAR SURVEY OF MICROBES IN HUMAN TUBERCULOSIS

说明(改编自申请摘要)：建议为期两年 计划是调查肺肉芽肿的微生物成分 与人类结核病有关，使用最近开发的核糖体RNA (RRNA)基于基因的技术，不需要培养微生物 用于检测和识别它们。虽然结核分枝杆菌 是结核病的主要致病微生物，有可能是其他 生物，可能是土著的，目前还没有被发现，参与或 伴随着疾病的进程。该计划将被实施 与临床医生和结核病实验专家合作，他们将 为分子分析提供材料。从人肺中提取DNA 不同类型的结节肉芽肿(例如，包裹性，增生性， 等)和位置(例如，中央呼吸道近端和远端)将 用聚合酶链式反应扩增所有的 (`普遍性‘)或精选(例如，细菌、古生菌、分枝杆菌、真菌) RRNA基因。混合物种扩增产物将由一个 克隆步骤，通过限制性内切酶分析和唯一类型测序进行排序。 对这些序列的系统发育分析将识别存在于 原来的肉芽肿。荧光原位杂交分析将 用于确认选定的潜在共感染生物体并进行研究 生物体在组织中的分布。据估计，他们可以 在处理和分析大约100个组织样本的过程中 这项为期两年的调查。这一结果将证明该病的单一病因 结核病，或者，或者，牵连到新的潜在参与者。 最后，作为一项次要的努力，当组织可用时，微生物 与人类肺结节病相关的肉芽肿成分将 要下定决心。这一结果可能解决细菌的问题 结节病的病原学，确定致病微生物(S)，如果有，以及 提供基于序列的诊断测试。

DESCRIPTION (adapted from application abstract): This proposed two-year program is to survey the microbial constituents of pulmonary granulomas associated with human tuberculosis, using recently developed, ribosomal RNA (rRNA) gene-based technology that does not require cultivation of microbes for their detection and identification. Although Mycobacterium tuberculosis is the primary causative organism in tuberculosis, it is possible that other organisms, perhaps indigenous and so far undiscovered, participate in or accompany the disease process. The program will be conducted collaboratively with clinicians and tuberculosis experimentalists who will provide materials for molecular analyses. DNA from human pulmonary tubercular granulomas of different types (e.g., encapsulated, proliferative, etc.) and locations (e.g. central airway proximal and distal) will be subjected to polymerase chain reaction using primers that amplify all (`universal`) or selected (e.g., bacterial, archaeal, mycobacterial, fungal) rRNA genes. Mixed-species amplification products will be separated by a cloning step, sorted by restriction analysis and unique types sequenced. Phylogenetic analysis of the sequences will identify organisms present in the original granulomas. Fluorescence in situ hybridization analyses will be used to confirm selected potential co-infecting organisms and to study distributions of organisms in tissues. It is estimated that they can process and analyze approximately 100 tissue samples during the course of the two-year survey. The results will prove the monotypic etiology of tuberculosis, or alternatively, implicate novel potential participants. Finally, as a minor effort and as tissues are available, the microbial constituents of granulomas associated with human pulmonary sarcoidosis will be determined. The results may resolve the question of the bacterial etiology of sarcoidosis, identify the causative organism(s), if any, and provide for a sequence-based diagnostic test.