Rapid and Enclosed System for the Identification of Multi-Drug Resistant TB

简介(申请人提供)：由结核分枝杆菌引起的结核病(TB)是全球第七大死亡原因，在传染病中仅次于人类免疫缺陷病毒(HIV)和获得性免疫缺陷综合征(AIDS)。2004年，估计有890万人患上结核病，170万人死亡。艾滋病毒大流行加剧了这种情况，目前有近1300万人同时感染艾滋病毒和结核病，以及出现耐多药结核病(耐多药结核病)。在美国，每年大约有15,000例新的结核病病例报告，其中大约10%的人对药物有抗药性。结核病感染的快速诊断和耐药性鉴定是疾病控制和治疗的关键。为了实现2010年《健康人》将实验室确诊结核病所需时间减少到48小时的目标，需要进行快速检测以检测结核分枝杆菌或其产品。此外，还需要能够减少检测耐药性的周转时间的快速检测。显然，目前还没有符合目标规格的测试，能够克服限制和应对挑战的新方法将受到欢迎。我们拟议的工作将使用最先进的技术来提供鉴别诊断，以识别结核分枝杆菌感染，并确定它是否存在突变，导致对结核病最常用的抗生素产生耐药性。其目标是在90分钟内实现从样品到结果的检测。在使用自动核酸(NA)提取仪器制备样品后，NA模板将被吸引到在线超快PCR反应中，在该反应中将进行高度多重扩增分析(涵盖25个突变/靶点)。这项来自我们合作者的强大的多重扩增技术克服了传统多重PCR技术发展的障碍。它允许在一次反应中对多个目标进行特定和灵敏的放大。然后，将聚合酶链式反应产物与杂交缓冲液混合，以封闭的形式向下游输送，以执行极其简单的基于微流控的多重检测。PCR产物将通过具有空间可寻址捕获区的线性玻璃毛细管底物传递，捕获区由不同的寡核苷酸捕获探针修饰。直径较小的线性玻璃基板将安装在直径稍大的聚合管中，以提供具有微流控特性的“管中阵列”条件，利用极其简单和廉价的设置进行快速和灵敏的荧光检测。在第一阶段，将开发一个面包板系统，并将利用合作者提供的核酸模板来证明技术可行性。第一阶段的结果将使随后设计和开发一种自动化临床诊断分析方法成为可能，该方法将满足结核病护理以及其他疾病检测方面的关键需求。 结核病每年夺走近200万人的生命，是全球第七大死亡原因，在传染病中仅次于艾滋病毒/艾滋病。更糟糕的是，耐多药结核病(MDR-TB)的出现使治疗变得更加困难和昂贵。在美国，每年大约有15,000例新的结核病病例报告，其中大约10%的人对药物有抗药性。结核病和耐多药结核病的快速和准确诊断工具是疾病控制和治疗的关键，每年可挽救多达625,000人的生命(《自然》S1，46-57；2006)。

DESCRIPTION (provided by applicant): Tuberculosis (TB) caused by Mycobacterium tuberculosis is the seventh leading cause of death worldwide, second only to human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) among infectious diseases. In 2004, an estimated 8.9 million people developed TB and 1.7 million died. This situation is compounded by the HIV pandemic with almost 13 million people currently co-infected with HIV and TB, and the emergence of multidrug resistant TB (MDR-TB). In the Unites States, there are about 15,000 new cases of TB reported each year, and approximately 10% of them have resistance to drugs. Rapid diagnosis of TB infections and identification of drug resistance is key to disease control and treatment. In order to accomplish the Healthy People 2010 goal of reducing the time required for the laboratory confirmation of the diagnosis of tuberculosis to 48 hours, rapid tests to detect Mycobacterium tuberculosis or its products are needed. In addition, rapid tests that can reduce the turnaround-time for detection of drug- resistance are needed as well. It is clear that no test is yet available that meets target specifications, and new methods that can overcome limitations and respond to the challenges posed will be well received. Our proposed work will employ state-of-the-art technology to provide differential diagnostics to identify Mycobacterium tuberculosis infection and determine if it has mutations that cause resistances to the most commonly used antibiotics for TB. The goal is to achieve sample-to-result testing in less than 90 minutes. After sample preparation using automated nucleic acid (NA) extraction instrumentation, the NA template will be drawn into an in-line ultra-fast PCR reaction where a highly multiplexed amplification assay (covering 25 mutation/targets) will take place. This powerful multiplex amplification technology from our collaborator overcomes the obstacles of traditional multiplex PCR assay development. It allows for specific and sensitive amplification of multiple targets in a single reaction. The PCR products, mixed with hybridization buffer, will then be transported downstream in a closed format to perform an extremely simple microfluidic based multiplexed detection. The PCR product will be passed over a linear glass capillary substrate with spatially addressable capture zones that are modified with different oligonucleotide capture probes. The smaller diameter linear glass substrate will be housed in a slightly larger diameter polymeric tubing to provide an "array-in-a tube" conditions with microfluidic characteristics for rapid and sensitive fluorescence detection utilizing an extremely simple and inexpensive setup. In Phase I, a breadboard system will be developed and the technology feasibilities will be demonstrated with the nucleic acid templates provided by collaborators. Phase I results will enable the subsequent design and development of an automated clinical diagnostic assay that will fulfill a crucial need in TB care, as well as the detection of other diseases. Tuberculosis (TB), which claims nearly 2 million lives each year, is the seventh leading cause of death worldwide, second only to HIV/AIDS among infectious diseases. Making the condition worse, the emergence of multidrug resistant TB (MDR-TB) makes the treatment more difficult and costly. In the Unites States, there are about 15,000 new cases of TB reported each year, and approximately 10% of them have resistance to drugs. A quick and accurate diagnostics tool for TB and MDR-TB is key to disease control and treatment and could save up to 625,000 of those lives each year (Nature S1, 46-57; 2006).