Emulsion digital PCR

乳化数字PCR

• 批准号: 10699081
• 负责人: Peter B Allen
• 金额: $ 49.43万
• 依托单位: DPLEXBIO INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-17 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10699081
• 关键词: 3-Dimensional; Address; Archives; Biological Assay; COVID detection; COVID-19; Clinical; Clinical Research; Complex; Computer Simulation; Computer software; Concentration measurement; Custom; DNA; Data Collection; Dedications; Dependence; Diagnostic; Emulsions; Exhibits; Head; Image; Infection; Laboratories; Light; Measurement; Measures; Methods; Microfluidic Microchips; Microscopy; Noise; Nucleic Acids; Oils; Optics; Outcome; Performance; Reagent; Reproducibility; Reproducibility of Results; Resolution; Running; Sampling; Side; Source; Speed; System; Techniques; Testing; Three-Dimensional Imaging; Time; Uncertainty; Viral Genome; Work; clinical diagnostics; coronavirus disease; cost; data acquisition; detection sensitivity; digital; fluorescence imaging; fluorescence microscope; genome integrity; improved; innovation; instrument; microscopic imaging; molecular diagnostics; next generation; operation; point of care; simulation

Project Summary This proposed project aims to develop a new type of digital PCR platform which eliminates the need for uniform volumes and thereby reduces the complexity, cost, and run time of digital PCR while increasing its dynamic range. The result is a higher performance digital PCR system that matches the simplicity, speed, and low per-assay cost of real-time PCR. Real-time PCR maintains great popularity in clinical diagnostics, but digital PCR is superior to real-time PCR because it provides absolute quantitation, greater accuracy at low concentrations, and greater reproducibility. However, because it was hypothesized that uniform volumes are required for digital PCR quantitation, current digital PCR platforms require precise microfluidic chips and control, which result in low throughput and high per-assay costs. Here we propose to address these limitations by developing a digital PCR system that employs variable volume droplets created simply by shaking a sample along with PCR reagents and an oil/emulsifier mix to create an emulsion. PCR is performed, and droplets in the emulsion are imaged and sized. Droplets with one or more copies of a nucleic acid are identified, and nucleic acid concentration is determined. We call this edPCR (emulsion digital PCR). We characterized this edCPR method via computational simulations and validated it experimentally. Simulations were used to investigate the dependence of droplet occupancy on analyte concentration and droplet size distribution, and to estimate the accuracy of the measured concentration in the presence of errors in measurement of droplet volume. Simulations also provided an estimate of dynamic range for a given droplet size distribution and statistical power. The method was validated experimentally in terms of accuracy, precision, and dynamic range, and to rule out potential sources of biased error, such as droplet shrinking and droplet fusion during PCR. In the proposed work, we will develop a commercial edPCR platform that will offer the superior performance of digital PCR while providing the high-throughput (e.g. 384 well plate operation) and low per-assay cost of real-time PCR, which we believe will remove the barriers to the widespread use of digital PCR in clinical assays, point-of-care settings, and large-scale testing.

项目摘要 该提出的项目旨在开发一种新型的数字PCR平台，以消除 需要统一的卷，从而减少数字PCR的复杂性，成本和运行时间 同时增加其动态范围。结果是更高的性能数字PCR系统 匹配实时PCR的简单性，速度和低每次测定成本。 实时PCR在临床诊断方面保持广泛欢迎，但数字PCR优于 实时PCR，因为它提供了绝对定量，在低浓度下的精度更高， 和更大的可重复性。但是，因为假设均匀的体积是 数字PCR量化所需，当前的数字PCR平台需要精确的微流体 芯片和控制，导致吞吐量低和每测的较高成本。在这里我们建议 通过开发使用可变体积的数字PCR系统来解决这些限制 仅通过与PCR试剂和油/乳化剂混合物一起摇动样品而创建的液滴 创建乳液。进行PCR，并成像乳液中的液滴。 鉴定出具有一个或多个副本副本的液滴，核酸浓度 确定。我们称此EDPCR（乳液数字PCR）。 我们通过计算模拟表征了此EDCPR方法并验证了它 实验。模拟用于研究液滴占用率对 分析物浓度和液滴尺寸分布，并估计 在液滴体积测量中存在误差的情况下测量的浓度。 模拟还为给定的液滴尺寸分布和 统计能力。该方法是根据准确性，精度和 动态范围，并排除偏见错误的潜在来源，例如液滴收缩和 PCR期间的液滴融合。 在拟议的工作中，我们将开发一个商业EDPCR平台，该平台将为上级提供 在提供高通量的同时，数字PCR的性能（例如384井板操作） 和每次测定的实时PCR成本低，我们认为这将消除障碍 在临床测定，护理点环境和大规模测试中广泛使用数字PCR。