Emulsion digital PCR

乳化数字PCR

基本信息

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
项目类别：
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

项目摘要

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.

项目概要该项目旨在开发一种新型数字 PCR 平台，消除需要均匀的体积，从而降低数字 PCR 的复杂性、成本和运行时间同时增加其动态范围。结果是更高性能的数字 PCR 系统与实时 PCR 的简单性、速度和每次检测成本低相匹配。实时 PCR 在临床诊断中保持着广泛的流行，但数字 PCR 优于实时 PCR，因为它提供绝对定量，在低浓度下具有更高的准确性，和更高的再现性。然而，由于假设体积均匀数字 PCR 定量所需的当前数字 PCR 平台需要精确的微流控芯片和控制，导致通量低和每次检测成本高。在此我们建议通过开发采用可变体积的数字 PCR 系统来解决这些限制只需将样品与 PCR 试剂和油/乳化剂混合物一起摇动即可产生液滴形成乳液。进行 PCR，并对乳液中的液滴进行成像并确定尺寸。鉴定具有一个或多个核酸拷贝的液滴，并确定核酸浓度已确定。我们称之为 edPCR（乳液数字 PCR）。我们通过计算模拟描述了这种 edCPR 方法并对其进行了验证实验性地。模拟用于研究液滴占据率的依赖性分析物浓度和液滴尺寸分布，并估计分析的准确性在液滴体积测量存在误差的情况下测量浓度。模拟还提供了给定液滴尺寸分布的动态范围的估计，以及统计功效。该方法在准确度、精密度等方面均经过实验验证动态范围，并排除潜在的偏差误差来源，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，该平台将提供卓越的数字 PCR 的性能，同时提供高通量（例如 384 孔板操作）实时 PCR 的每次检测成本较低，我们相信这将消除检测的障碍数字 PCR 在临床检测、现场护理环境和大规模检测中的广泛使用。