M. tuberculosis Fluorescent In-Situ Hybridization

描述（由申请方提供）：结核病-双探针荧光原位杂交（TB-双探针FISH）是一种在单一热固定涂片上检测和区分结核分枝杆菌复合群（MTB）核糖体RNA（rRNA）和分枝杆菌种（rRNA）的方法，该涂片从培养物或直接从标本制备。该检测使用标记有不同荧光染料的MTB和分枝杆菌属特异性探针。该测定法简单、快速且不昂贵（< $5.00/测试和用于过滤器的约$600的一次性费用）。该检测包括五个步骤-预杂交，杂交，洗涤，复染和在荧光显微镜下观察处理后的涂片。结核分枝杆菌和分枝杆菌在特定的双通滤光片下应发出不同颜色的荧光。总测定时间小于1小时。 具体目标：开发一种简单、快速、廉价的双探针FISH检测试剂盒，用于涂片阳性样本中分枝杆菌和MTB的培养确认和/或直接检测。试剂盒应包含所有试剂和质控涂片。结核病是穷人的疾病。世界上超过三分之一的人口（主要在非洲和亚洲）处于危险之中。在这些地区中的一些地区，结核分枝杆菌（MOTT）以外的分枝杆菌的频率可能很高，部分原因是HIV患者的频率很高。有些地区可能没有养殖设施。鉴别结核分枝杆菌的唯一方法是通过抗酸染色涂片的显微镜检查。不幸的是，抗酸涂片缺乏敏感性和特异性。TB-FISH双探针检测可提供与扩增DNA探针检测相当的特异性，以及与AFB血清学阳性相当的灵敏度。在工业化国家，这种测试可能是有用的个人与肺结核，因为他们是高度传染性的，需要立即隔离和抗结核治疗的开始。 第一阶段：（1）双色MTB-FISH检测的开发和优化。(2)专属性研究（3）纯培养物、加标痰液样本和由涂片阳性临床样本制备的一些涂片的检出限研究和可行性研究。(4)公共卫生-结核病实验室、医院实验室主任的反馈，以及潜在临床地点的确定。 第二阶段：在美国建立生产设施，在美国进行临床试验，并获得FDA批准。 第三阶段：美国境内和美国境外的营销。在结核病流行的不发达国家（如肯尼亚）建立制造工厂。

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.