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保健设施的护理点使用一种手持、易于使用、价格低廉的封闭式完全集成的核酸检测设备来快速诊断痰中的耐药结核病。设想的系统的成本将比目前用于护理点核酸测试的基于PCR的全集成台式系统低10-100倍，并且尺寸将显著小于当前的系统。该项目建立在正在进行的旨在开发一种类似但更简单的系统的工作基础上，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放大反应(EXPAR)与使用侧向流动的视觉检测相结合的。简单而快速的样品制备利用了最初为机械裂解炭疽孢子而开发的微型珠子搅拌器，在其当前版本中，它也能够提纯核酸。拟议的概念涉及一种手持、由电池供电的仪器，其上连接着一个一次性塑料墨盒。色谱柱容纳了用于样品制备、放大和检测的腔室、阀门和流体管道，所有这些都在一个封闭的系统中。在其最终形式中，试剂盒将包含机上所有必要的试剂。手持仪器控制测试执行所需的泵送、阀门和等温加热步骤。操作员只需将试剂盒连接到仪器上，插入样本，按下按钮启动测试，大约30分钟到1小时后就会读出结果。这样的系统将使医疗保健专业人员能够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。本项目的目标一是建立一种简便、快速、有效的痰中结核分枝杆菌标本制备方法。在目标2下，我们将建立基于EXPAR的多重等温核酸扩增分析，并结合基于横向流动的检测。该项目的目标3侧重于开发试剂盒和仪器原型，以执行根据AIMS 1和2开发的样品制备、等温扩增和目视检测方法。AIMS 1-3中描述的任务的性能验证最初将涉及BSL2代用品和结核分枝杆菌DNA提取物，然后是在培养物中培养并添加到结核病阴性痰中的敏感和耐药结核分枝杆菌，最后是使用既定方法进行结核分枝杆菌和抗菌药敏感性检测的临床痰样本。该项目提供了一个独特的机会，可以将多学科的专业知识结合在一起，开发用于核酸测试的下一代分析和分析器。从长远来看，这里开发的分析和设备可以应用于与资源有限的环境中的生物安全检测和传染病诊断相关的病原体谱。该项目的目标是在初级卫生保健设施的护理点使用一种手持、易于使用、价格低廉的封闭式完全集成的核酸检测设备，促进从痰中快速诊断耐药结核病。设想的系统的成本将比目前用于护理点核酸测试的基于PCR的全集成台式系统低10-100倍，并且尺寸将显著小于当前的系统。

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.