Multiplex TB-M.avium complex Fluorescent in Situ hybridization Assay, direct from

描述（由申请方提供）：TB-MAC FISH检测试剂盒是一种荧光原位杂交检测试剂盒，可检测和区分培养物和痰液中的结核分枝杆菌复合群（MTB）和鸟分枝杆菌复合群（MAC）。该测定使用分别靶向MTB和MAC核糖体RNA（rRNA）的荧光标记MTB和MAC特异性探针。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，总时间不超过2小时。在美国，非M。结核分枝杆菌（NTM）是HIV患者的重要病因，在某些地区，NTM分离株的数量超过MTB。肺部疾病是最常见的NTM局部表现，通常与MAC相关。肺部疾病患者具有高度传染性，需要立即隔离并开始抗结核治疗。因此，所有涂片阳性患者都被隔离，直到确诊。这是非常昂贵的。目前还没有直接从痰液中检测MAC的方法。检测MAC的标准方法是通过培养Nalc/NaOH处理的痰液，然后使用Gen-Probe AccuProbe测定、自制扩增测定或生化方法进行培养确认。这可能需要3天到2周的时间。因此，如果有一种方法可以确定哪些涂片阳性患者在同一天是MTB和/或MAC阳性，这将降低患者管理成本。TB-MAC FISH测试可能是一个答案。具体目的：研制一种简便、快速、廉价的结核分枝杆菌MAC荧光原位杂交检测试剂盒，用于直接检测培养物和痰液中的结核分枝杆菌和MAC。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)TBMAC FISH测定在室温下的性能优化。(4)制定质量控制程序，以确保正确阅读载玻片，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-MAC 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。公共卫生相关性：在美国，除M.结核病（NTM）是重要的疾病原因，NTM分离株的数量超过了M。一些地区的肺结核。肺部疾病是最常见的非结核性脑膜炎的局部表现，常与结核性脑膜炎有关。鸟复合体（MAC）。肺部疾病患者具有高度传染性，需要立即隔离并开始抗结核治疗。因此，所有涂片阳性患者都被隔离，直到确诊。这是非常昂贵的。目前还没有直接从痰液中检测MAC的方法。检测MAC的标准方法是通过培养Nalc/NaoH处理的痰液，然后使用Gen-Probe AccuProbe测定、自制扩增测定或生物化学方法进行培养确认。这可能需要3天到2周的时间。因此，如果有一种方法可以确定哪些涂片阳性患者在同一天是MTB和/或NTM阳性，这将降低患者管理成本。因此，具有以下属性的TB-MAC FISH检测试剂盒将非常有用。1.可直接在痰液或培养物上进行。2.具有扩增检测的特异性，灵敏度等同于或优于抗酸染色。3.不需要昂贵的设备，除了显微镜。4.从收到痰液样本到得到结果大约需要2小时。

DESCRIPTION (provided by applicant): TB-MAC FISH assay is a fluorescent in-situ hybridization assay that detects and differentiates Mycobacterium tuberculosis complex (MTB) and Mycobacterium avium complex (MAC) in culture and sputum. This assay uses fluorescent labeled MTB and MAC specific probes targeted to MTB and MAC ribosomal RNA (rRNA) respectively. 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 2 hours from receipt of sputum. In the US, non-M. tuberculosis mycobacteria (NTMs) are important cause of disease in HIV patients, with the number of NTM isolates exceeding those of MTB in some areas. Pulmonary disease, the most commonly reported localized manifestation of NTMs, is often associated with the MAC. Patients with pulmonary disease are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Therefore, all smear-positive patients are kept in isolation until confirmed. This is very costly. Currently there are no tests to detect MAC directly from sputum. The standard method of detecting MAC is by culturing Nalc/NaOH processed sputum followed by culture confirmation, using Gen-Probe AccuProbe assay, home-brew amplified assays or biochemical methods. This can take anywhere from 3 days to 2 weeks. Thus, if there was a way of determining which of the smear-positive patients were MTB and/or MAC positive on the same day, this would reduce the patient management cost. The TB-MAC FISH test may be an answer. Specific Aims: Develop a simple, rapid and in-expensive TB-MAC FISH test kit for direct detection of MTB and MAC in culture and 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 TBMAC 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, assay sensitivity and specificity in pure cultures and spiked sputum samples. (5) Performance of TB-MAC 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. PUBLIC HEALTH RELEVANCE: In the US, Mycobacteria other than M. tuberculosis (NTMs) are important cause of disease, with the number of NTMs isolates exceeding those of M. tuberculosis in some areas. Pulmonary disease, the most commonly reported localized manifestation of NTMs, is often associated with the M. avium complex (MAC). Patients with pulmonary disease are highly infectious and require immediate isolation and initiation of antituberculosis therapy. Therefore, all smear-positive patients are kept in isolation until confirmed. This is very costly. Currently there are no tests to detect MAC directly from sputum. The standard method of detecting MAC is by culturing Nalc/NaoH processed sputum followed by culture confirmation, using Gen-Probe AccuProbe assay, home-brew amplified assays or biochemical methods. This can take anywhere from 3 days to 2 weeks. Thus, if there was a way of determining which of the smear-positive patients were MTB and/or NTMs positive on the same day, this would reduce the patient management cost. Therefore, a test like TB-MAC FISH assay with the following attributes would be very useful. 1. Can be performed directly on sputum or culture. 2. Has specificity of amplified assays and sensitivity equivalent or better than acid-fast staining. 3. Does not require expensive equipment other than a microscope. 4. From time of receipt of sputum sample to result is about 2 hours.