M. tuberculosis Fluorescent In-Situ Hybridization

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DESCRIPTION (provided by applicant): Tuberculosis-Dual Probe Fluorescent In-Situ Hybridization (TB-Dual Probe FISH) is a method of detecting and differentiating Mycobacterium tuberculosis complex (MTB) ribosomal RNA (rRNA) and Mycobacterium species (rRNA) on a SINGLE heat fixed smear, prepared either from a culture or directly from a specimen. The assay uses MTB and Mycobacterium genus specific probes labeled with different fluorescent dyes. The assay is simple, rapid and in-expensive (< $5.00/test and a one time expense of approximately $600 for filters). The assay consists of five steps - prehybridization, hybridization, washing, counter staining and viewing the processed smear under a fluorescent microscope. MTB and Mycobacterium shall fluoresce with different colors under specific dual pass filters. The total assay time is less than 1 hour. Specific Aims: Develop a simple, rapid and in-expensive Dual Probe FISH test kit for culture confirmation and/or direct detection of Mycobacterium and MTB in smear positive samples. The kit shall contain all the reagents and control smears. Tuberculosis is the disease of the poor. Over one-third of the world's population (mostly in Africa and Asia) is at risk. In some of these areas, the frequency of Mycobacteria other than MTB (MOTT) maybe high due in part to high frequency of HIV patients. Some areas may not have culturing facilities. The only means of identification of MTB is by microscopic examination of acid-fast stained smears. Unfortunately the acid-fast smear lacks both sensitivity and specificity. The TB-FISH Dual Probe assay may provide the specificity equivalent to amplified DNA probe assays, and sensitivity equivalent to AFB Smear Positives. In the industrialized nations, this test may be useful for individuals with pulmonary tuberculosis since they are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Phase I: (1) Development and optimization of a Dual Color MTB-FISH Assay. (2) Specificity study (3) Limit of Detection Study and Feasibility study on pure cultures, spiked sputum samples, and some smears made from Smear-Positive clinical samples. (4) Feedback from Directors of Public Health-TB Laboratories, Hospital Laboratories, and identification of potential clinical sites. Phase II: Set up manufacturing facilities in the US, perform clinical trials in the US, and get FDA approval. Phase III: Marketing within US and Outside US. Setting up manufacturing plant in an underdeveloped country where TB is endemic, e.g. Kenya.