Sun Chen, Liu Leqian, Vasudevan Harish N, Chang Kai-Chun, Abate Adam R
Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA 94158, USA.
Department of Radiation Oncology, University of California San Francisco, San Francisco, CA 94158, USA.
bioRxiv. 2021 Jan 15:2021.01.13.424628. doi: 10.1101/2021.01.13.424628.
Droplet digital PCR provides superior accuracy in nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource or clinical settings. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.
液滴数字PCR在核酸定量方面具有更高的准确性。然而，微流体技术对乳液的生成和分析要求是其应用的一个障碍，特别是在资源匮乏或临床环境中。在此，我们报告了一种通过涡旋制备ddPCR液滴并通过对回收的扩增子进行批量分析来读取结果的新方法。我们通过完全批量处理且不使用微流体技术准确地定量SARS-CoV-2序列来证明该方法。我们用于定量反应的方法应适用于所有产生扩增子的数字检测，包括在液滴、微腔或纳升孔中进行的数字PCR和环介导等温扩增（LAMP）。更广泛地说，我们的方法将ddPCR的重要特性（包括更高的准确性和对抑制的稳健性）与定量PCR的大容量样本处理能力结合在一起。
