TB- FISH Assay for Direct Detection of M. tuberculosis in unprocessed sputum

TB-FISH 检测直接检测未处理痰中的结核分枝杆菌

• 批准号: 7327538
• 负责人: JYOTSNA S SHAH
• 金额: $ 16.1万
• 依托单位: ID FISH TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7327538
• 关键词: Acid Fast Bacillae Staining Method; Adult; Antitubercular Agents; Area; Biological Assay; Centrifugation; Clinical; Clinical Trials; Complex; Country; Detection; Development; Diagnosis; Diagnostic Procedure; Disease; Ensure; Equipment; Fluorescent Dyes; Fluorescent in Situ Hybridization; Gold; Hour; Incubators; Individual; Label; Lung diseases; Marketing; Microscope; Microscopic; Mycobacterium tuberculosis; Performance; Phase; Preparation; Procedures; Process; Pulmonary Tuberculosis; Quality Control; Reading; Reagent; Reporting; Resources; Ribosomal RNA; Sampling; Sensitivity and Specificity; Slide; Specificity; Sputum; Staining method; Stains; Standards of Weights and Measures; Temperature; Testing; Time; Title; Tuberculosis; United States Food and Drug Administration; Week; base; clinical research site; cost; day; fluorescence microscope; manufacturing facility; protocol development; sample fixation

DESCRIPTION (provided by applicant): Tuberculosis is a disease of the poor. Between 80-90% of active tuberculosis (TB) is due to pulmonary tuberculosis. Individuals with pulmonary disease are highly contagious. Therefore, in order to control spread of TB, diagnostic methods that are sensitive, rapid and affordable in resource-poor countries are required. Some areas may not have culturing facilities. Often, the only means of identification of M. tuberculosis complex (MTB) is by microscopic examination of acid-fast stained unprocessed sputum smears. Unfortunately the acid- fast smear prepared form unprocessed sputum lacks both sensitivity and specificity. TB-FISH (Fluorescent In Situ Hybridization) assay for direct detection of MTB in unprocessed sputum, may be one answer. The TB- FISH test detects MTB ribosomal RNA. The test can be performed at room temperature, and does not require centrifugation or incubators. The assay uses MTB specific probes labeled with fluorescent dyes. The assay is simple, rapid and in-expensive (~$5.00/test). The assay consists of six steps: smear preparation and fixation, pretreatment with a proprietary reagent; hybridization, washing, counterstaining and viewing the processed smear under a fluorescent microscope. The total time is less than 1.5 h from receipt of sputum. Specific Aims: Develop a simple, rapid and in-expensive TB-FISH test kit for direct detection of MTB in unprocessed sputum. The kit shall contain all the reagents and control smears. Phase I: (1) Development of protocols. (2) Preparation of slides for development of test. (3) Optimization of TB- FISH Assay for performance at room temperature. (4) Develop Quality Control procedures to ensure correct reading of slides and determination of the limit of detection and assay sensitivity and specificity in pure cultures and spiked sputum samples. (5) Performance of TB-FISH assay in clinical samples. (6) Identification of potential Clinical Sites in the US. (7) Analyzing and compiling Report Phase II: Set up manufacturing facilities in the US and perform clinical trials in the US. Phase III: Get FDA approval. Marketing within US and Overseas. Tuberculosis caused by Mycobacterium tuberculosis complex (MTB), is the second leading killer of adults in the world, with 8.8 million new cases diagnosed every year. Culture is the gold standard. Unfortunately it can take anywhere from one week to 2 months. Amplified assays have reduced the time to detection and confirmation to less than 2 days. They are especially useful in individuals with pulmonary tuberculosis, since they are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Unfortunately, most of the TB endemic world is resource poor countries. Because of the cost of the amplified assays, they are of little or no use in the TB endemic world. Some don't even have culturing facilities. The only means of diagnosis is based on acid-fast staining of direct sputum smears that lacks sensitivity and specificity. Therefore, a test like TB-FISH assay with the following attributes would be very useful. 1. Can be performed directly on unprocessed sputum at room temperature. 2. Has specificity of amplified assays and sensitivity superior to direct acid-fast staining. 3. Does not require expensive equipment other than a microscope. 4. Cost less than <$5.00 5. From time of receipt of sputum sample to result is less than 2 hours.

描述（由申请人提供）：结核病是穷人的疾病。80-90%的活动性结核病（TB）是由于肺结核。患有肺部疾病的人具有高度传染性。因此，为了控制结核病的传播，需要在资源贫乏的国家提供敏感、快速和负担得起的诊断方法。有些地区可能没有养殖设施。通常，识别M的唯一手段。结核复合菌（MTB）是通过抗酸染色的未处理痰涂片的显微镜检查。不幸的是，从未经处理的痰液中制备的抗酸涂片既缺乏敏感性又缺乏特异性. TB-FISH（荧光原位杂交）检测直接检测未处理的痰中的MTB，可能是一个答案。TB-FISH检测检测结核分枝杆菌核糖体RNA。该试验可在室温下进行，不需要离心或孵育器。该测定使用荧光染料标记的MTB特异性探针。该检测方法简单、快速且价格低廉（约5.00美元/次）。该检测包括六个步骤：涂片制备和固定，用专有试剂预处理;杂交，洗涤，复染和在荧光显微镜下观察处理后的涂片。从收到痰液起，总时间少于1.5小时。具体目的：开发一种简单、快速、廉价的TB-FISH检测试剂盒，用于直接检测未处理痰液中的结核分枝杆菌。试剂盒应包含所有试剂和质控涂片。第一阶段：（1）制定方案。(2)制备用于试验开发的载玻片。(3)TB-FISH测定在室温下的性能优化。(4)制定质量控制程序，以确保载玻片的正确阅读，并确定纯培养物和加标痰液样本的检测限、测定灵敏度和特异性。(5)TB-FISH测定在临床样品中的性能。(6)确定美国的潜在临床研究中心。(7)分析和编写报告第二阶段：在美国建立生产设施，并在美国进行临床试验。第三阶段：获得FDA批准。在美国和海外销售。 由结核分枝杆菌复合体（MTB）引起的结核病是世界上第二大成人杀手，每年诊断出880万新病例。文化是黄金标准。不幸的是，它可能需要一周到两个月的时间。扩增检测已将检测和确认时间缩短至不到2天。它们对肺结核患者特别有用，因为它们具有高度传染性，需要立即隔离并开始抗结核治疗。不幸的是，结核病流行的世界大多数是资源贫乏的国家。由于扩增检测的成本，它们在结核病流行的世界中几乎没有用处。有些甚至没有养殖设施。唯一的诊断方法是直接痰涂片抗酸染色，缺乏敏感性和特异性。因此，具有以下属性的TB-FISH检测试剂盒将非常有用。1.可在室温下直接对未处理的痰液进行。2.具有扩增试验的特异性和上级优于直接抗酸染色的灵敏度。3.不需要昂贵的设备，除了显微镜。4.价格低于5.00元。从收到痰液样本到得出结果的时间不超过2小时。