A Reaction- Diffusion-Based Approach for Nucleic Acid Quantification

基于反应扩散的核酸定量方法

• 批准号: 9912154
• 负责人: Changchun Liu
• 金额: $ 36.85万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-11-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912154
• 关键词: Base Sequence; Biological Assay; Bioluminescence; Biomedical Research; Blinded; Blood; Blood specimen; Body Fluids; Clinical; Communicable Diseases; DNA; Data Analyses; Detection; Devices; Diagnostic; Diffusion; Disease; Eye; Feces; Forensic Medicine; Funding; Gel; Gene Expression Profiling; Glass; Gold; HIV; HIV Infections; Health; Healthcare; Human Resources; Human immunodeficiency virus test; Immunoassay; Infrastructure; Interdisciplinary Study; Label; Lateral; Measures; Mediating; Mercury; Methods; Microfluidic Microchips; Microfluidics; Molecular; Molecular Diagnosis; Monitor; Nucleic Acid Amplification Tests; Nucleic Acids; Positioning Attribute; Reaction; Reading; Reproducibility; Research; Resources; Saliva; Sampling; Sensitivity and Specificity; Signal Transduction; Spottings; System; Technology; Temperature; Testing; Thermometers; Time; Training; Urine; Viral Load result; Virus; Work; Zika Virus; base; clinical application; complex data; cost; cost effective; cost shifting; design; digital; disease diagnosis; drug discovery; global health; innovation; instrument; instrumentation; laboratory facility; molecular diagnostics; novel; nucleic acid detection; operation; pathogen; point-of-care diagnostics; polymerization; programs; rapid diagnosis; screening

Abstract Quantitative detection of nucleic acid sequences (DNA or RNA) is important in many biomedical applications including disease diagnosis, gene expression profiling. Nucleic acid amplification testing (NAAT), which takes advantage of enzymatic polymerization reaction to amplify specific nucleic acid sequences, provides high sensitivity and specificity and has become the gold standard for many infectious disease diagnostics. However, the NAAT has been severely hindered by the cost and complexity of the instrumentation for applications to point-of-care (POC) diagnostics, especially for use in resource-limited settings. The objective of the proposed research is to bridge this gap by developing a simple, affordable approach to quantify nucleic acids undergoing enzymatic polymerization reaction. To this end, we propose to study a new, reaction-diffusion-based, microfluidic method (dubbed the “nuclemeter”) for quantifying target nucleic acid molecules. The amount of target analytes in raw clinical samples can be quantitatively read out through the position of polymerization reaction-diffusion front in the nuclemeter at the endpoint, nearly as simply as reading temperature in a “mercury in glass” thermometer. As an example application, viral load testing in HIV infection will be used to evaluate and validate its clinical application. The hypothesis behind the proposed research is that the position of the enzymatic polymerization reaction-diffusion front indicates target nucleic acid concentration in samples. To test our hypothesis and demonstrate its suitability as a new, affordable, nucleic acid-based, molecular diagnostics approach, we assemble a multidisciplinary research team and propose the following specific aims: i) study a reaction-diffusion-based microfluidic device and method for endpoint quantification of nucleic acids, ii) develop a cellphone-based, label-free, bioluminescent detection platform; and iii) evaluate and validate the feasibility of clinical application of the nuclemeter for viral load testing. The proposed work is both innovative and immediately useful because it: i) introduces a novel, simple, affordable approach for nucleic acid endpoint quantification, ii) develops a minimally-instrumented, cellphone-based detection platform, and iii) proposes a new two-stage isothermal amplification assay for highly sensitive, specific, multiplex nucleic acid testing. If successful, it would open the door to affordable, mobile, personalized, molecular diagnosis and treatment. Beyond disease diagnostics, our nuclemeter system, as a technology platform of nucleic acid quantification, would also have broad applicability for many other biomedical research, such as high throughput DNA screening.

抽象的 核酸序列（DNA 或 RNA）的定量检测在许多生物医学应用中非常重要 包括疾病诊断、基因表达谱检测（NAAT）。 酶促聚合反应的优点是扩增特定的核酸序列，提供了高 敏感性和特异性已成为许多传染病诊断的金标准。 NAAT 的应用受到仪器成本和复杂性的严重阻碍。 护理点 (POC) 诊断，特别适用于资源有限的环境。 研究的目的是通过开发一种简单、负担得起的方法来定量核酸，从而弥补这一差距。 为此，我们建议研究一种新的、基于反应扩散的、 微流控方法（称为“核计”）用于定量目标核酸分子的量。 通过聚合位置可以定量读出临床原始样品中的目标分析物 核子计终点处的反应扩散前沿，几乎与读取温度一样简单 “玻璃水银”温度计作为示例应用，将用于 HIV 感染的病毒载量检测。 评估和验证其临床应用所提出的研究背后的假设是该立场。 酶促聚合反应-扩散前沿的大小指示样品中的目标核酸浓度。 为了检验我们的假设并证明其作为一种新的、负担得起的、基于核酸的分子的适用性 诊断方法，我们组建了一个多学科研究团队，并提出以下具体目标： i) 研究基于反应扩散的微流体装置和核酸终点定量方法，ii) 开发基于手机的无标记生物发光检测平台；以及 iii) 评估和验证 核子计用于病毒载量检测的临床应用的可行性拟议的工作都是创新的。 并且立即有用，因为它：i) 引入了一种新颖、简单、经济实惠的核酸终点方法 量化，ii) 开发一个最少仪器化的、基于手机的检测平台，以及 iii) 提出一个 用于高灵敏度、特异性、多重核酸检测的新型两阶段等温扩增测定。 如果成功的话，它将为负担得起的、移动的、个性化的分子诊断和治疗打开大门。 除了疾病诊断之外，我们的核测量仪系统作为核酸定量的技术平台， 也对许多其他生物医学研究具有广泛的适用性，例如高通量 DNA 筛选。