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是重要的 包括疾病诊断、基因表达谱分析。核酸扩增检测（NAAT）， 酶促聚合反应扩增特定核酸序列的优点，提供了高的 灵敏度和特异性，并已成为许多传染病诊断的金标准。然而，在这方面， 美国国家自动测试系统一直受到以下因素的严重阻碍： 床旁（POC）诊断，特别是用于资源有限的环境。建议的目标 研究是通过开发一种简单，负担得起的方法来量化核酸， 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， 微流控方法（被称为“nuclemeter”）用于定量靶核酸分子。的量 可以通过聚合位置定量读出原始临床样品中的目标分析物 反应扩散前沿在核子计的终点，几乎一样简单，阅读温度在一个 “玻璃水银”温度计。作为一个应用实例，HIV感染中的病毒载量检测将用于 评价和验证其临床应用。这项研究背后的假设是， 酶促聚合反应-扩散前沿的“λ”表示样品中的靶核酸浓度。 为了验证我们的假设，并证明其适用性作为一个新的，负担得起的，核酸为基础的， 诊断方法，我们组建了一个多学科的研究团队，并提出了以下具体目标： i）研究用于核酸终点定量的基于反应-扩散的微流体装置和方法，ii） 开发基于手机的无标签生物发光检测平台;以及iii）评估和验证 核酸检测仪用于病毒载量检测临床应用的可行性。所提出的工作既具有创新性 并且立即有用，因为它：i）引入了一种新颖的、简单的、负担得起的用于核酸终点的方法 量化，ii）开发一个最小仪器，基于手机的检测平台，以及iii）提出一个 新的两阶段等温扩增检测，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，我们的核酸仪系统作为核酸定量的技术平台， 也将具有广泛的适用性，许多其他生物医学研究，如高通量DNA 筛选

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.