A Reaction- Diffusion-Based Approach for Nucleic Acid Quantification

摘要 核酸序列(DNA或RNA)的定量检测在许多生物医学应用中都很重要 包括疾病诊断、基因表达谱分析。核酸扩增检测(NAAT)，它需要 酶促聚合反应扩增特定核酸序列的优点，提供了很高的 敏感性和特异性，并已成为许多传染病诊断的黄金标准。然而， NAAT严重受制于应用程序的仪器成本和复杂性 护理点(POC)诊断，尤其适用于资源有限的环境。建议的目标是 研究是通过开发一种简单、负担得起的方法来量化正在经历的核酸来弥合这一差距。 酶促聚合反应。为此，我们建议研究一种新的、基于反应扩散的、 用于定量目标核酸分子的微流控方法(被称为“核子计”)。金额的多少 可以通过聚合位置定量读出原始临床样本中的目标分析物 核子计终点的反应-扩散前沿，几乎和读取温度一样简单 “玻璃水银”温度计。作为一个示例应用，HIV感染中的病毒负载测试将用于 评价和验证其临床应用。这项拟议研究背后的假设是， 酶聚合反应扩散前沿的大小表明样品中的目标核酸浓度。 来检验我们的假设，并证明它作为一种新的、负担得起的、基于核酸的分子 通过诊断学方法，我们组建了一个多学科研究团队，并提出了以下具体目标： I)研究一种基于反应扩散的微流控装置和核酸终点定量方法，ii) 开发基于手机的、无标签的生物发光检测平台；以及iii)评估和验证 核子仪临床应用于病毒载量检测的可行性。建议的工作既有创新性，又有创新性 并立即有用，因为它：i)引入了一种新的、简单的、负担得起的核酸终点方法 量化，ii)开发最少的仪器，基于手机的检测平台，以及iii)提出 新的两阶段等温扩增分析，用于高灵敏度、特异性、多重核酸检测。如果 如果成功，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，我们的核子仪系统作为核酸量化的技术平台， 也将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 放映。

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.