Rapid Diagnosis of MDR TB from Sputum using a Small Fully Integrated Nucleic Acid

描述（由申请人提供）：本项目的目标是使用手持式、易于使用、廉价、封闭式单元的完全集成核酸检测设备，在初级卫生保健机构的护理点快速诊断痰液中的耐药结核病。所设想的系统的成本将低10-100倍，并且尺寸将显著小于当前用于护理点核酸测试的基于PCR的完全集成的台式系统。该项目建立在正在进行的工作，旨在开发一个类似的，但更简单的系统，从拭子样本检测单纯疱疹病毒。该测定基于等温指数扩增反应（EXPAR），结合使用侧向流的目视检测。简单而快速的样品制备利用了最初开发用于炭疽孢子机械裂解的小型化珠混合器，其当前版本也能够进行核酸纯化。所提出的概念涉及一种手持式、电池供电的仪器，其上连接有一次性塑料盒。该盒容纳用于样品制备、扩增和检测的腔室、阀门和流体导管，所有这些都在单个封闭系统中。在其最终形式中，测试卡片将包含所有必需的机载试剂。手持式仪器控制测试执行所需的泵送、阀门和等温加热步骤。操作员只需将检测盒连接到仪器上，插入样品，按下按钮即可启动测试，大约30分钟至1小时后即可读取结果。这样的系统将使医疗保健专业人员能够在护理点及时诊断和治疗耐药结核病，从而改善患者的治疗效果并防止进一步传播。本课题的目的一是建立一种简便、快速、有效的痰标本制备方法。在目标2下，我们将建立基于EXPAR的多重等温核酸扩增检测，并结合基于侧向流的检测。本项目的目标3集中于开发检测盒和仪器原型，以执行根据目标1和目标2开发的样品制备、等温扩增和视觉检测方法。目标1-3所述任务的性能验证最初将涉及BSL 2替代物和M的DNA提取物。其次为结核分枝杆菌、耐药分枝杆菌。在培养物中生长并掺入TB阴性痰中的结核分枝杆菌，以及最终已经测试过M.结核病和抗菌药物敏感性使用已建立的方法。该项目提供了一个独特的机会，汇集多学科专业知识，开发下一代核酸检测试剂盒和分析仪。从长远来看，本文开发的测定和装置可以应用于与资源有限环境中的生物威胁检测和传染病诊断相关的病原体谱。该项目的目标是使用手持式、易于使用、价格低廉、封闭式单元的完全集成核酸检测设备，在初级卫生保健设施的护理点快速诊断痰液中的耐药结核病。所设想的系统的成本将低10-100倍，并且尺寸将显著小于当前用于护理点核酸测试的基于PCR的完全集成的台式系统。

DESCRIPTION (provided by applicant): The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing. The project builds upon ongoing work aimed at developing a similar albeit simpler system for detection of Herpes Simplex Virus from swab samples. The assay is based on the isothermal EXponential Amplification Reaction (EXPAR) coupled to visual detection using lateral flow. Simple and rapid sample preparation utilizes a miniaturized bead blender originally development for mechanical lysis of anthrax spores, which in its current version is also capable of nucleic acid purification. The proposed concept involves a handheld, battery operated instrument, to which a disposable plastic cartridge is attached. The cartridge houses chambers, valves, and fluid conduits for sample preparation, amplification and detection, all in a single, closed system. In its final form, the cartridge will contain all necessary reagents on board. The handheld instrument controls the pumping, valving and isothermal heating steps necessary for test execution. The operator simply attaches the cartridge to the instrument, inserts the sample, initiates the test by pushing a button, and after approximately 30 minutes to one hour reads off the result. Such a system will allow health care professionals in a point-of- care setting to diagnose and treat drug resistant tuberculosis in a timely manner, thereby improving patient outcomes and preventing further spread. Aim 1 of this project focuses on establishing a simple, rapid and effective sample preparation method applicable to Mycobacterium tuberculosis in sputum. Under Aim 2, we will establish multiplexed isothermal nucleic acid amplification assays based on EXPAR, coupled with lateral flow based detection. Aim 3 of this project focuses on developing cartridge and instrument prototypes to execute the sample preparation, isothermal amplification, and visual detection methods developed under aims 1 and 2. Performance verification for tasks described under Aims 1-3 will initially involve BSL2 surrogates and DNA extracts of M. tuberculosis, followed by drug susceptible and resistant M. tuberculosis grown in culture and spiked into TB negative sputum, and ultimately clinical sputum samples that have been tested for M. tuberculosis and antimicrobial susceptibility using established methods. This project presents a unique opportunity to bring together multidisciplinary expertise to develop a next generation of assays and analyzers for nucleic acid testing. In the long term, the assays and devices developed herein can be applied to a spectrum of pathogens relevant to biothreat detection and infectious disease diagnosis in resource-limited settings. The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing.