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一种便携的、易于使用的、集成的核酸检测设备，在没有用户干预的情况下执行样本制备、等温DNA扩增和基于横向流动的检测，从而在低资源、高负担的国家实现在护理点对肺结核的诊断，以比目前低得多的成本提供1.5小时的样本输入到回答，以显著低于目前的成本 提供完全集成的台式系统。该项目建立在同一调查团队和机构参与的持续努力的基础上，并得到新的合作伙伴的加强。到目前为止，我们已经开发了一种用于痰液化和消毒、病原体裂解和DNA提取的新的、快速的样品制备工艺，并利用临床痰标本建立了该工艺的合适性能。我们已经证明，我们的系统可以适应不同的等温放大方法与侧向流动检测相结合，并开发了一种新的母液混合试剂稳定工艺。展望未来，我们将利用等温交叉启动扩增(CPA)，临床验证用于结核病诊断，它与侧向流动相结合，并包括内部扩增对照。我们设计并制作了一个样机，集成了色谱柱和仪器，并证明了样品制备和扩增/检测子单元的功能。在提议的项目开始时，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。拟议的项目将使我们能够进一步开发和验证该系统，并获得监管部门的批准，以IVD的形式在市场上发行。在目标1中，我们将优化与样品前处理、DNA提取、CPA和侧向流动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，完善车载试剂存储，采用横向流动条带读取器，并确保整体系统的健壮性。在目标2中，我们将改进可制造性设计，并进行工业设计和人因工程。在最终确定药盒和仪器的设计后，我们将根据国际标准化组织13485标准扩大制造规模，以生产足够数量的药盒和仪器来执行目标3，这需要对系统进行分析和临床验证。这三个目标最终将是申请CE-IVD监管批准，以便进入市场。在目标4中，我们将开发一个对接站，可以连接四个便携式仪器单元，作为对基本系统的升级。这个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，实现外部质量保证和电子结果传输，这是在患者附近资源较少的情况下传播结核病诊断的两个重要操作方面。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，这被确定为全球遏制结核病计划的一项关键目标。从长远来看，该系统可以应用于与资源有限的环境中的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.