Emulsion digital PCR

项目摘要 该项目旨在开发一种新型的数字化聚合酶链式反应平台，消除 需要均匀的体积，从而降低了数字聚合酶链式反应的复杂性、成本和运行时间 同时增加了它的动态范围。其结果是产生了更高性能的数字PCR系统 与实时聚合酶链式反应的简单性、速度和低的每次检测成本相匹配。 实时聚合酶链式反应在临床诊断中仍然很受欢迎，但数字聚合酶链式反应优于 实时聚合酶链式反应，因为它提供绝对定量，在低浓度下更准确， 以及更大的重现性。然而，因为假设均匀体积是 目前的数字聚合酶链式反应平台需要精确的微流控 芯片和控制，这导致低产量和高每次检测成本。在此，我们建议 通过开发一种使用可变体积的数字PCR系统来解决这些限制 只需摇动样品、聚合酶链式反应试剂和油/乳化剂混合物即可产生液滴 来创造一种乳剂。进行聚合酶链式反应，并对乳状液中的液滴进行成像和大小调整。 识别具有一个或多个核酸拷贝的液滴，以及核酸浓度 是有决心的。我们称之为edPCR(乳胶数字聚合酶链式反应)。 我们通过计算机模拟来表征这种edCPR方法，并对其进行验证 试验性的。使用模拟来研究液滴占有率与 分析物浓度和液滴大小分布，并估计 在液滴体积测量存在误差的情况下测量的浓度。 模拟还提供了给定液滴尺寸分布的动态范围的估计和 统计学上的力量。实验结果表明，该方法具有较高的准确度和精密度 动态范围，并排除有偏差的潜在来源，如液滴收缩和 聚合酶链式反应过程中的液滴融合。 在拟议的工作中，我们将开发一个商业edPCR平台，该平台将为 在提供高通量(例如384井板操作)的同时实现数字聚合酶链式反应 以及较低的每次检测成本，我们相信这将消除 数字聚合酶链式反应广泛应用于临床分析、医疗机构和大规模检测。

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.