CaO-heated DNA amplification obviates electricity requirement at point of care

CaO 加热 DNA 扩增消除了护理点的电力需求

• 批准号: 8227819
• 负责人: Paul D. LaBarre
• 金额: $ 69.28万
• 依托单位: PATH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-19 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8227819
• 关键词: Adoption; Area; Biological Assay; Blinded; Blood specimen; Buffers; Calcium oxide; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Chemistry; Classification; Clinical; Cold Chains; Communicable Diseases; Containment; Cytolysis; DNA amplification; Data; Development; Diagnosis; Diagnostic tests; Drug Formulations; Electricity; Engineering; Environment; Eye; Fluorescence; Genomics; HIV; HIV Seropositivity; HIV-1; Heating; Hour; Incubators; Label; Laboratories; Lead; Liquid substance; Malaria; Maps; Mediating; Medical; Methods; Modification; Nucleic Acid Amplification Tests; Observer Variation; Particle Size; Performance; Population; Preparation; Process; Property; Qualifying; Ramp; Reaction; Reagent; Reporting; Reproducibility; Research; Research Infrastructure; Resources; Sampling; Sensitivity and Specificity; Source; Specific qualifier value; Staging; System; Technology; Temperature; Testing; Text; Thermal Conductivity; Time; Training; Tube; Tuberculosis; Validation; Variant; Water; Whole Blood; Work; communicable disease diagnosis; cost; design; disorder prevention; energy density; evidence base; flexibility; innovation; instrument; instrumentation; melting; novel; novel diagnostics; pathogen; phase change; point of care; prototype; stability testing

DESCRIPTION (provided by applicant): Simple, easy-to-use, low-cost nucleic acid amplification tests (NAATs) are not available for diagnosis of infectious diseases in low-resource settings (LRS). Challenges to easy adoption or modification of existing NAAT technologies for LRS use include time-consuming sample preparation; cold chain requirements for reagent storage; and lack of instrumentation, electricity, and training at the point of care. Unfortunately, there are many infectious diseases rampant in LRS where the lack of appropriate NAATs is a critical barrier to timely diagnosis and treatment. PATH proposes a collaborative effort with the CDC that simultaneously merges scientific and technological capabilities with this clinical need to develop a calcium oxide (CaO)-heated DNA amplification kit that obviates the requirements for electricity and instrumentation at the point of care. PATH has demonstrated the use of CaO for heat and a proprietary engineered phase change material to stabilize an assay mixture within a narrow temperature range suitable for isothermal amplification. The CDC has developed a novel combination of lysis buffer; loop mediated isothermal amplification (LAMP) reaction and fluorescently-labeled primers to enable a simplified, extraction-free and lysis-free workflow. These recent innovations can be combined with a simple two-chamber, high-containment reaction tube prefilled with lyophilized LAMP mixture to create the first electricity-free, instrument-free, and easy-to-use, low-cost NAAT kit. We envision multiple NAAT kits customized and validated for specific strains of infectious diseases such as tuberculosis, malaria, and HIV that will advance evidence-based medical practice at the point of care in LRS. We will develop, integrate, and validate components of this NAAT kit with the sensitivity of PCR, the simplicity of a strip test, and sufficient stability for storage out of the cold chain for long periods of time. Aims 1 and 2 below describe development of the kit components. Aim 3 describes the integration of these components into a stand-alone kit designed for low-infrastructure use. Finally, Aim 4 proposes the laboratory validation of this kit using HIV-1, a highly relevant pathogen afflicting many populations in LRS. "Aim 1: Specify and optimize the properties of the electricity-free incubator active materials using the existing incubator prototype. "Aim 2: Optimize CDC's innovations to LAMP HIV-1 assay, establish dry reagent formulations, and define sample. "Aim 3: Design and fabricate optimized kit hardware and consumables: the isothermal incubator and reaction containment tube. "Aim 4: Validate the electricity-free and instrument-free assay kit created in Aims 1-3. PUBLIC HEALTH RELEVANCE: We propose to integrate two innovations to create and validate the first electricity-free, instrument-free, easy-to-use, low-cost, nucleic acid amplification test kit for diagnosis of infectious diseases in low-resource settings. PATH has demonstrated the use of calcium oxide and a proprietary engineered phase change material to stabilize the temperature of an assay mixture within a narrow range suitable for isothermal amplification. The Centers for Disease Control and Prevention have developed a novel combination of lysis buffer, loop mediated isothermal amplification (LAMP) reaction and fluorescently-labeled primers to enable a simplified, extraction-free and lysis-free workflow. These two technologies will combine with a simple, two-chamber, disposable, high containment reaction tube prefilled with lyophilized LAMP mixture.

描述（由申请人提供）：简单、易于使用、低成本的核酸扩增测试（NAAT）不适用于资源匮乏环境（LRS）中传染病的诊断。轻松采用或修改现有 NAAT 技术以供 LRS 使用所面临的挑战包括耗时的样品制备；试剂储存的冷链要求；护理点缺乏仪器、电力和培训。不幸的是，LRS 中存在许多猖獗的传染病，缺乏适当的 NAAT 是及时诊断和治疗的关键障碍。 PATH 提议与 CDC 合作，同时将科学技术能力与临床需求相结合，开发氧化钙 (CaO) 加热的 DNA 扩增套件，从而消除护理点对电力和仪器的需求。 PATH 已证明使用 CaO 加热和专有的工程相变材料可在适合等温扩增的狭窄温度范围内稳定测定混合物。 CDC 开发了一种新型裂解缓冲液组合；环介导等温扩增 (LAMP) 反应和荧光标记引物可实现简化、免提取和免裂解的工作流程。这些最新的创新可以与预填充冻干 LAMP 混合物的简单的两室、高密封反应管相结合，创建第一个无电、无需仪器、易于使用、低成本的 NAAT 套件。我们设想针对结核病、疟疾和艾滋病毒等特定传染病菌株定制和验证多种 NAAT 试剂盒，这将推动 LRS 护理点的循证医疗实践。我们将开发、集成和验证该 NAAT 试剂盒的组件，使其具有 PCR 的灵敏度、条带测试的简单性以及在冷链外长期储存的足够稳定性。下面的目标 1 和 2 描述了套件组件的开发。目标 3 描述了将这些组件集成到专为低基础设施使用而设计的独立套件中。最后，目标 4 建议使用 HIV-1（一种影响 LRS 中许多人群的高度相关病原体）对该试剂盒进行实验室验证。 “目标 1：使用现有培养箱原型指定和优化无电培养箱活性材料的特性。”目标 2：优化 CDC 在 LAMP HIV-1 检测方面的创新，建立干试剂配方，并定义样品。 “目标 3：设计和制造优化的试剂盒硬件和耗材：等温培养箱和反应容器。”目标 4：验证目标 1-3 中创建的无电和无仪器检测试剂盒。 公共健康相关性：我们建议整合两项创新，创建并验证第一个无电、无仪器、易于使用、低成本的核酸扩增检测试剂盒，用于在资源匮乏的环境中诊断传染病。 PATH 已证明使用氧化钙和专有的工程相变材料可以将测定混合物的温度稳定在适合等温扩增的狭窄范围内。美国疾病控制和预防中心开发了一种新型裂解缓冲液、环介导等温扩增 (LAMP) 反应和荧光标记引物组合，可实现简化、免提取和免裂解的工作流程。这两种技术将与预填充有冻干 LAMP 混合物的简单、两室、一次性、高密封性反应管相结合。