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。