Enumerix, Inc., 4030 Fabian Way, Palo Alto, California 94303, United States.
Anal Chem. 2022 Dec 27;94(51):17868-17876. doi: 10.1021/acs.analchem.2c03649. Epub 2022 Dec 12.
Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.
数字 PCR（dPCR）最初是为单分子定量而设想的。然而，由于分区容量有限，目前的 dPCR 系统通常要求 DNA 模板共享分区。在这里，我们介绍了 UltraPCR，这是一种新一代的 dPCR 系统，通过使用快速和并行化的工作流程，在单个分子方案中以 6 个对数动态范围进行 DNA 计数。每个 UltraPCR 反应被分成>3000 万个分区，无需微流控技术即可实现单个模板占有率。结合独特的乳液化学，分区具有光学透明性，可使用三维成像技术快速检测 DNA 阳性分区。由于不存在分区特异性竞争，单分子占有率也使得更直接的多重分析开发成为可能。作为概念验证，我们开发了一个 222 重的 UltraPCR 分析，并证明了它作为一种快速、低成本的非侵入性产前测试筛选分析的潜力，对于低至 4%的三体分数样本，具有高精度、准确性和可重复性。
