Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction
Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction
复制标题
统计体积校正的多分散液滴乳液聚合酶链反应与微流控液滴数字聚合酶链反应的比较
DOI：
10.1021/acs.analchem.8b01988
复制
发表时间：
2018-08-07
影响因子：
7.4
通讯作者：
Nichols, Kevin P.
中科院分区：
文献类型：
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作者：
Byrnes, Samantha A.;Chang, Tim C.;Nichols, Kevin P.
聚合酶链式反应 (PCR) 等核酸扩增技术实现了高灵敏度和特异性的疾病检测和定量，从而实现更准确的诊断和治疗方案。芯片实验室应用已经开发出将单个生物分子（例如 DNA 和 RNA）分割成皮升大小的液滴的方法。这些单独的反应容器实现了 PCR 的数字化，无需标准曲线即可缩短核酸检测和直接定量的时间，从而简化测定分析。尽管影响很大，但这些改进通常仅限于拥有训练有素的人员和昂贵设备的集中实验室。为了解决这些限制并使该技术更适用于各种设置，我们开发了一个统计框架，适用于在无需任何专门设备的情况下制备的多分散液滴中进行的液滴 PCR。多分散液滴系统可实现液滴数字 PCR (ddPCR) 和逆转录酶液滴数字 PCR (RT-ddPCR) 的精确定量，与 BioRad 的 ddPCR 等市售系统相当。此外，该方法与一系列输入样本量兼容，将检测动态范围扩展到商业 ddPCR 系统之外。在这项工作中，我们证明这些 ddPCR 检测可以减少总体检测时间，同时仍然提供定量结果。我们还报告了多重 ddPCR 测定，并演示了同时在多个样品中快速制备液滴的概念验证方法。我们简单的多分散液滴制备和统计框架可以扩展到复杂样品中核酸定量的各种设置。
Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.