Integrated Cost-Effective Point of Care Nucleic Acid Testing for TB diagnosis

描述（申请人提供）：该项目的目标是通过开发便携式、易于使用的集成核酸检测设备，在没有用户干预的情况下执行样本制备、等温DNA扩增和基于侧流的检测，在< 1.5小时的样本输入到应答输出内提供结果，以比目前低得多的成本 提供完全集成的台式系统。该项目是在现有工作的基础上开展的，这些工作涉及同一组调查人员和机构，并得到新伙伴的加强。到目前为止，我们已经开发出一种新的，快速的样本制备过程中的痰液液化和消毒，病原体裂解，DNA提取，并已建立了适当的性能，该过程中使用临床痰样本。我们已经证明，我们的系统可以适应不同的等温扩增方法耦合到侧流检测，并开发了一种新的主混合试剂稳定化的过程。展望未来，我们将利用等温交叉引发扩增（CPA），临床验证的结核病诊断，这是耦合到横向流动，并包括内部扩增控制。我们已经设计并建立了一个原型集成盒和仪器，我们已经证明，样品制备和扩增/检测亚单位的功能。在项目开始时，我们的目标是在集成的alpha原型系统中演示完全自动化的流程执行。拟议项目将使我们能够进一步开发和验证该系统，并获得作为IVD上市的监管批准。在目标1中，我们将优化与样品预处理、DNA提取、CPA和侧流检测相关的关键系统组件。我们将提高主混合试剂的热稳定性，完善机载试剂储存，整合侧流式试纸条阅读器，并确保整个系统的稳健性。在目标2中，我们将完善可制造性设计，并执行工业设计和人因工程。在完成测试卡片和仪器的设计后，我们将根据ISO 13485扩大生产规模，以生产足够数量的测试卡片和仪器来执行目标3，这需要对系统进行分析和临床确认。这三个目标将最终申请CE-IVD监管批准，以进入市场。在目标4中，我们将开发一个可连接四个便携式仪器单元的对接，作为对基本系统的升级。该对接站将提供增强的质量控制、更好的用户界面以及无线连接，从而实现外部质量保证和电子结果传输，这是在资源匮乏的患者附近环境中传播结核病诊断的两个重要业务方面。该项目提供了一个独特的机会，使所有结核病病例能够得到早期诊断，这被确定为遏制结核病全球计划的一个关键目标。从长远来看，该系统可应用于资源有限环境中与生物威胁检测和传染病诊断相关的其他病原体。

DESCRIPTION (provided by applicant): The goal of this project is to enable diagnosis of pulmonary tuberculosis at the point of care in low resource high burden countries by developing a portable, easy to use, integrated nucleic acid testing device that executes sample preparation, isothermal DNA amplification and lateral flow based detection without user intervention, providing a result in < 1.5 h sample-in-to-answer-out, at a significantly lower cost than currently available fully integrated bench-top systems. The project builds upon ongoing efforts that involve the same team of investigators and institutions, and is strengthened by new partners. To date, we have developed a novel, rapid sample preparation process for sputum liquefaction and disinfection, pathogen lysis, and DNA extraction, and have established suitable performance of this process using clinical sputum samples. We have demonstrated that our system can accommodate different isothermal amplification methods coupled to lateral flow detection, and have developed a novel process for master-mix reagent stabilization. Going forward we will utilize an isothermal cross-priming amplification (CPA), clinically validated for TB diagnosis, which is coupled to lateral flow, and includes an internal amplification control. We have designed and built a prototype integrated cartridge and instrument, and we have demonstrated that the sample preparation and amplification/detection subunits are functional. By the beginning of the proposed project, we aim to demonstrate fully automated process execution in the integrated alpha prototype system. The proposed project will enable us to further develop and validate the system, and to obtain regulatory approval for market release as an IVD. In Aim 1, we will optimize key system components related to sample pre-processing, DNA extraction, CPA, and lateral flow detection. We will improve the thermal stability of master-mix reagents, refine on-board reagent storage, incorporate a lateral flow strip reader, and ensure overall system robustness. In Aim 2, we will refine the design for manufacturability, and perform industrial design and human factors engineering. After finalizing the design of the cartridge and instrument, we will scale up manufacturing under ISO 13485 to produce a sufficient number of cartridges and instruments to execute Aim 3, which entails analytical and clinical validation of the system. These three aims will culminate in application for CE-IVD regulatory approval, to enable market entry. In Aim 4, we will develop a docking station to which four of the portable instrument units can be attached, as an upgrade to the basic system. This docking station will provide enhanced quality control, a better user interface, plus wireless connectivity, enabling external quality assurance and electronic result transmission, two important operational aspects of disseminated TB diagnostics in low resource near patient settings. This project presents a unique opportunity to enable early diagnosis of all TB cases, which was identified as a key objective in the Global Plan to Stop TB. In the long term, the system can be applied to other pathogens relevant to bio-threat detection and infectious disease diagnosis in resource-limited settings.