Droplet Volume Variability and Impact on Digital PCR Copy Number Concentration Measurements
Droplet Volume Variability and Impact on Digital PCR Copy Number Concentration Measurements
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DOI：
10.1021/acs.analchem.8b05828
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发表时间：
2019-03-19
影响因子：
7.4
通讯作者：
Burke, Daniel G.
中科院分区：
文献类型：
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作者：
Emslie, Kerry R.;McLaughlin, Jacob L. H.;Burke, Daniel G.
数字聚合酶链反应（dPCR）越来越多地被标准物质生产商和计量机构用于核酸标准物质的赋值以及均一性和稳定性研究。参考方法程序应满足若干要求，并且应完全了解不确定度和偏倚。先前已经记录了当在液滴dPCR测量方程中使用不准确的液滴体积时靶浓度的偏差。在本研究中，我们使用光学显微镜表征了孔内和孔间液滴体积变异性，并确定了这两种变异性来源对目标浓度估计值的影响。测量了图像上的小的光学失真，在没有校正的情况下，其使液滴体积测量值有偏差。使用几批Mastermix在39周内对孔间液滴体积进行纵向监测，结果表明平均液滴体积为0.786 nL，中间精密度为1.7%。孔内液滴体积的频率分布不同。一些威尔斯孔显示出偏态分布，导致模拟dPCR的估计靶浓度存在小偏倚，靶浓度在62和8000拷贝mu L-1之间。该偏差的大小和方向受孔内液滴体积的分布模式的影响。主混合物在dPCR混合物中的比例影响液滴体积。在预混物和主混合物混合期间，10%的移液误差导致液滴体积变化2.6%，因此，浓度测量偏差突出显示了dPCR混合物重量法制备用于高准确度测量的优势。
Digital polymerase chain reaction (dPCR) is increasingly being adopted by reference material producers and metrology institutes for value assignment, and for homogeneity and stability studies of nucleic acid reference materials. A reference method procedure should fulfill several requirements, and the uncertainty and biases should be completely understood. A bias in target concentration when inaccurate droplet volume is used in the droplet dPCR measurement equation has previously been documented. In this study, we characterize both intrawell and interwell droplet volume variability using optical microscopy and determine the impact of these two sources of variability on target concentration estimates. A small optical distortion across the image was measured which, without correction, biased droplet volume measurements. Longitudinal monitoring of interwell droplet volume over 39 weeks using several lots of Mastermix demonstrated a mean droplet volume of 0.786 nL and intermediate precision of 1.7%. The frequency distribution of intrawell droplet volumes varied. Some wells displayed a skewed distribution which resulted in a small bias in estimated target concentration for a simulated dPCR with target concentrations of between 62 and 8000 copies mu L-1. The size and direction of this bias was influenced by the distribution pattern of the droplet volumes within the well. The proportion of Mastermix in dPCR mix affected droplet volume. A pipetting error of 10% during mixing of the premix and Mastermix resulted in a 2.6% change in droplet volume and, consequently, a bias in concentration measurements highlighting the advantages of gravimetric preparation of dPCR mixes for high accuracy measurements.