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

描述(由申请人提供)：该项目的广泛目标是产生一系列急需的质量控制，用于分子结核分枝杆菌(结核分枝杆菌)诊断试验。良好的实验室实践和联邦法规要求质量控制，以确保所有医学实验室测试的准确性。其具体目标是：1)在基于DNA的分子检测中生产与结核分枝杆菌特性相似的大肠杆菌；2)在结核分枝杆菌检测中克隆用于鉴定耐药性的基因序列；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，用含有合成结核分枝杆菌DNA序列的增韧、化学交联膜来生产克隆大肠杆菌，用作分子结核分枝杆菌检测的质量控制。这类材料是实施用于结核分枝杆菌检测的新的快速分子技术的基础设施的必要组成部分。高效和准确的诊断是这项新技术的希望所在，它是治疗和预防结核分枝杆菌构成的主要全球健康威胁的关键。加强诊断工具和基础设施以改进诊断测试是NIAID任务的重要组成部分。此外，在“关注结核病”机构的使命中：“NIAID支持一个强有力的研究组合，以开发新药和诊断-”。将产生的质量控制是开发和实施新的分子诊断结核分枝杆菌检测的关键要素。 在研究设计中，分子方法被用来产生包含用于结核分枝杆菌诊断的DNA序列的克隆。用用于检测耐药突变的序列构建突变型和野生型克隆。传统上用于电子显微镜研究的固定膜的方法适用于使克隆细胞的膜变硬并使细胞核酸酶失活。固定时间、固定剂附件和固定剂浓度都是不同的，直到细胞结构硬化到在临床测试过程中其释放的核酸与结核分枝杆菌非常相似。这些产品将在临床实验室中进行分子结核分枝杆菌检测，并调整固定参数，以生产出最适合监测常见分子结核分枝杆菌检测的材料。

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.