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

描述（由申请人提供）：该项目的目标是促进在初级卫生保健设施的医疗点使用手持式、易于使用、价格低廉、封闭单元的全集成核酸检测设备从痰液中快速诊断耐药结核病。设想的系统的成本将降低10-100倍，尺寸将大大小于目前基于pcr的全集成台式系统，用于护理点核酸检测。该项目建立在正在进行的工作的基础上，旨在开发一种类似但更简单的系统，用于从拭子样本中检测单纯疱疹病毒。该分析是基于等温指数扩增反应（EXPAR）与使用横向流动的视觉检测相结合。简单快速的样品制备利用最初为炭疽孢子的机械裂解而开发的微型头部搅拌器，其目前的版本也能够进行核酸纯化。所提出的概念包括一个手持的、电池供电的仪器，上面附有一个一次性塑料药筒。药筒内装有室、阀门和流体管道，用于样品制备、放大和检测，所有这些都在一个单独的封闭系统中。在其最终形式中，墨盒将包含机上所有必要的试剂。手持式仪器控制泵送，阀门和等温加热步骤所需的测试执行。操作人员只需将墨盒连接到仪器上，插入样品，通过按按钮启动测试，并在大约30分钟至1小时后读取结果。这样一个系统将使卫生保健专业人员在护理点环境中及时诊断和治疗耐药结核病，从而改善患者的预后并防止进一步传播。本课题的目的1是建立一种适用于痰液中结核分枝杆菌的简单、快速、有效的样品制备方法。在目标2中，我们将建立基于EXPAR的多路等温核酸扩增检测，并结合基于侧流的检测。该项目的目标3侧重于开发墨盒和仪器原型，以执行目标1和目标2中开发的样品制备、等温扩增和视觉检测方法。目标1-3所述任务的性能验证将首先涉及结核分枝杆菌的BSL2替代品和DNA提取物，然后是在培养物中生长的药敏和耐药结核分枝杆菌，并加入到结核阴性痰中，最后是使用既定方法检测结核分枝杆菌和抗菌药物敏感性的临床痰样本。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方法和分析仪。从长远来看，在资源有限的环境中，本文开发的检测方法和设备可应用于与生物威胁检测和传染病诊断相关的一系列病原体。该项目的目标是促进在初级卫生保健设施的医疗点使用手持式、易于使用、价格低廉、全封闭单元的全面综合核酸检测装置从痰液中快速诊断耐药结核病。设想的系统的成本将降低10-100倍，尺寸将大大小于目前基于pcr的全集成台式系统，用于护理点核酸检测。

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.