Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR.
Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR.
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DOI：
10.1007/s00216-017-0625-y
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发表时间：
2017-11
影响因子：
4.3
通讯作者：
Žel J
中科院分区：
文献类型：
--
作者：
Košir AB;Divieto C;Pavšič J;Pavarelli S;Dobnik D;Dreo T;Bellotti R;Sassi MP;Žel J
准确和精确的核酸定量对临床和诊断决策至关重要，因为高估或低估可能导致疾病的错误治疗或产品的错误标签。数字聚合酶链式反应是核酸绝对拷贝数测定的最佳工具之一。然而，数字聚合酶链式反应需要在准确性和不确定性来源方面有很好的特点。利用液滴数字聚合酶链式反应，先前的研究已经表明，制造商分配的液滴体积与独立实验室测量的液滴体积之间存在差异。在本研究中，我们报告了基于光学显微镜成像并可追溯到国际单位制的不同液滴体积测定方法的实验室间比较结果。这种比较是在相同的DNA材料上进行的，考察了液滴发生器、混合液、操作器、盒间和盒内的可变性以及液滴测量协议等参数的影响。用QX200型™AutoDG™液滴数字™聚合酶链式反应系统和两套QX100型™液滴数字™聚合酶链式反应系统测量平均液滴体积。数据显示了这两个系统之间的显著音量差异，以及当使用不同的混音时音量的显著差异。我们还表明，这两种液滴发生器系统产生的液滴体积(分别为13.1%和15.9%)明显低于制造商声明和之前由其他实验室测量的液滴体积。这表明，为了确保准确的量化，应评估每个系统的液滴体积。本文的在线版本(10.1007/s002160170625-y)包含补充材料，授权用户可以使用。
Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system. The online version of this article (10.1007/s00216-017-0625-y) contains supplementary material, which is available to authorized users.
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影响因子：
2.5
作者：
Schindelin J;Rueden CT;Hiner MC;Eliceiri KW
通讯作者：
Eliceiri KW
影响因子：
4.3
作者：
Corbisier, Philippe;Pinheiro, Leonardo;Mazoua, Stephane;Kortekaas, Anne-Marie;Chung, Pui Yan Jenny;Gerganova, Tsvetelina;Roebben, Gert;Emons, Hendrik;Emslie, Kerry
通讯作者：
Emslie, Kerry
影响因子：
4.6
作者：
Dobnik D;Štebih D;Blejec A;Morisset D;Žel J
通讯作者：
Žel J
影响因子：
7.4
作者：
Dobnik, David;Spilsberg, Bjorn;Zel, Jana
通讯作者：
Zel, Jana
评估数字PCR的人类基因组DNA的定量：可访问的放大靶标。
影响因子：
7.4
作者：
Kline, Margaret C.;Romsos, Erica L.;Duewer, David L.
通讯作者：
Duewer, David L.