Fixed E. coli as Low Range Nucleic Acid Controls for M. tuberculosis Testing

描述（由申请人提供）：本项目的主要目的是生产一系列急需的质量控制品，用于分子结核分枝杆菌（M。结核病）诊断测试。良好实验室规范和联邦法规要求进行质量控制，以确保所有医学实验室检测的准确性。具体目标是：1）生产E。大肠杆菌细胞，平行于M.结核分枝杆菌DNA分子检测技术; 2）克隆结核分枝杆菌耐药基因序列;结核病测试，和3）生产和评估原型产品。这项第二阶段的工作将开发一种新的方法来生产克隆的E。大肠杆菌与含有合成M.结核DNA序列用作分子M.肺结核检查这类材料是实施新的快速分子技术的基础设施的必要组成部分。结核病检测有效和准确的诊断，这是这项新技术的承诺，是关键的治疗和预防的主要全球健康威胁的M。结核加强诊断工具和基础设施，改进诊断测试，是NIAID使命的一个重要部分。此外，在“关注结核病”机构的使命中：“NIAID支持强有力的研究组合，以开发新药和诊断方法-"。所产生的质量控制是开发和实施新的分子诊断M。肺结核检查 在研究设计中，使用分子方法来产生含有M.结核病诊断突变体和野生型克隆用用于测试耐药性突变的序列构建。传统上用于固定用于电子显微镜研究的膜的方法适于硬化克隆细胞的膜并使细胞核酸酶失活。改变固定时间、固定剂浓度和固定剂浓度，直到细胞结构硬化到在临床检测过程中其核酸释放与M非常相似的程度。结核杆菌将在临床实验室分子M中对产品进行评价。结核病测试和固定参数调整，以产生最佳用于监测常见分子M的材料。结核病测试

DESCRIPTION (provided by applicant): The BROAD OBJECTIVE of this project is to produce a family of urgently needed quality controls for use in molecular Mycobacterium tuberculosis (M. tuberculosis) diagnostic tests. Quality controls are required by good laboratory practice and federal regulations to assure accuracy of all medical laboratory tests. The SPECIFIC AIMS are to 1) Produce E. Coli cells that parallel the properties of M. tuberculosis in DNA based molecular testing, 2) Clone gene sequences used to identify drug resistance in M. tuberculosis tests, and 3) Produce and evaluate prototype products. This Phase II work will develop a novel method to produce cloned E. coli with toughened, chemically crosslinked membranes containing synthetic M. tuberculosis DNA sequences for use as quality controls in molecular M. tuberculosis tests. Materials of this type are a necessary part of the infrastructure for the implementation of the new and rapid molecular technologies for M. tuberculosis testing. Efficient and accurate diagnoses, which are the promise of this new technology, are key to treatment and prevention of the major global health threat presented by M. tuberculosis. Enhancing the diagnostic tools and infrastructure enabling improved diagnostic testing is an important part of the NIAID mission. Further, in the "FOCUS on TB" agency mission: "NIAID supports a robust portfolio of research to develop new drugs and diagnostics----". The quality controls to be produced are a key element in developing and implementing new molecular diagnostic M. tuberculosis tests. In the RESEARCH DESIGN, molecular methods are used to produce clones that contain DNA sequences utilized in M. tuberculosis diagnosis. Mutant and wild type clones are constructed with sequences used in testing for drug resistance mutations. Methods traditionally used to fix membranes for electron microscopy studies are adapted to harden the membranes of the clone cells and to deactivate cellular nucleases. The fixing time, fixative adjuncts, and fixative concentrations are varied until the cell structure is hardened to a point where its release of nucleic acid during the clinical testing process is very similar to that of M. tuberculosis bacilli. The products will be evaluated in clinical laboratory molecular M. tuberculosis tests and fixing parameters adjusted to produce a material that is optimal for monitoring common molecular M. tuberculosis test.