Materials Measurement Laboratory , National Institute of Standards and Technology , Gaithersburg, Maryland 20899, United States.
Anal Chem. 2017 Apr 18;89(8):4648-4654. doi: 10.1021/acs.analchem.7b00240. Epub 2017 Apr 6.
Digital polymerase chain reaction (dPCR) end point platforms directly estimate the number of DNA target copies per reaction partition, λ, where the partitions are fixed-location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both λ and the partition volume, V, must be metrologically traceable to some accessible reference system, ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the critical dPCR assumptions that are essential for establishing the SI traceability of λ. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable partition volume specifications. Our colleagues at the National Institute of Standards and Technology (NIST) have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus, neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split samples with both cdPCR and ddPCR platforms can transfer the λ traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR estimates of CRM target concentration.
数字聚合酶链反应 (dPCR) 终点平台直接估计每个反应分区（λ）中的 DNA 靶标拷贝数，其中分区是固定位置的腔室（cdPCR）或漂浮在油中的液滴（ddPCR）。为了在初级校准标准物质（CRM）中目标浓度的认证中使用，λ 和分区体积（V）都必须与一些可访问的参考系统具有计量可追溯性，理想情况下，是国际单位制（SI）。cdPCR 腔室的固定空间分布可实现 PCR 扩增的实时监测。对所得反应曲线的分析可验证对于建立 λ 的 SI 可追溯性至关重要的关键 dPCR 假设。我们不知道用于验证 ddPCR 平台这些假设的直接方法。我们可获得的 cdPCR 和 ddPCR 系统的制造商未提供可溯源的分区体积规格。我们在国家标准与技术研究院（NIST）的同事已经开发出一种可靠的方法来确定 ddPCR 液滴体积，并证明不同的 ddPCR 试剂产生的液滴大小略有不同。因此，没有任何一个 dPCR 平台本身可以提供可计量溯源的目标浓度估计。我们在这里表明，使用 cdPCR 和 ddPCR 平台评估拆分样本可以将 cdPCR 测定的 λ 可追溯性特征转移到其 ddPCR 类似物上，从而建立完全可溯源的 CRM 目标浓度 ddPCR 估计值。
