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

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.