Emulsion digital PCR

项目摘要 本项目旨在开发一种新型的数字PCR平台， 需要统一的体积，从而降低了数字PCR的复杂性，成本和运行时间 同时增加其动态范围。其结果是更高性能的数字PCR系统， 与实时PCR的简单性、速度和低的每次测定成本相匹配。 实时PCR在临床诊断中保持了很大的普及，但数字PCR优于上级 实时PCR，因为它提供绝对定量，在低浓度下更高的准确性， 和更高的再现性。然而，由于假设均匀体积是 数字PCR定量所需的，目前的数字PCR平台需要精确的微流体 芯片和对照，这导致低通量和高的每次测定成本。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 通过将样品与PCR试剂和油/乳化剂混合物一起沿着摇动而产生的液滴 来制造乳液进行PCR，并对乳液中的液滴进行成像和大小测定。 鉴定具有核酸的一个或多个拷贝的液滴，并确定核酸浓度。 是有决心的我们称之为edPCR（乳液数字PCR）。 我们通过计算机模拟来表征这种edCPR方法，并对其进行了验证 实验性的模拟用于研究液滴占有率对 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浓度。 模拟还提供了对于给定液滴尺寸分布的动态范围的估计， 统计力量该方法在准确度、精密度和精密度方面进行了实验验证。 动态范围，并排除潜在的偏差误差源，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化的edPCR平台，该平台将提供上级的 数字PCR的性能，同时提供高通量（例如384孔板操作） 实时PCR的每次检测成本低，我们相信这将消除 数字PCR在临床测定、护理点设置和大规模测试中的广泛使用。

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.