Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material

Q1 Biochemistry, Genetics and Molecular Biology

Biomolecular Detection and Quantification Pub Date : 2016-09-01 DOI:10.1016/j.bdq.2016.08.002

Liesbet Deprez, Philippe Corbisier, Anne-Marie Kortekaas, Stéphane Mazoua, Roxana Beaz Hidalgo, Stefanie Trapmann, Hendrik Emons

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引用次数: 53

Abstract

Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use.

Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

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通过使用认证的参考物质，验证用于DNA拷贝数浓度定量的数字PCR方法

数字PCR已成为一种新兴的核酸序列特异性检测和定量技术，应用于各种领域。在过去的几年里，已经发表了许多关于新型数字PCR方法发展的报告。这些发展成熟为适用于诊断或其他常规检测目的的可靠分析方法，需要对其预期用途进行验证。在这里，液滴数字PCR方法的内部验证的结果被提出。本方法用于定量纯化的线性化质粒在核酸背景溶液中的绝对拷贝数浓度。研究了测量过程中哪些因素对测量结果有显著影响，并估计了对总体测量不确定度的贡献。在执行数字PCR方法的内部方法验证时，应调查的所有方面提供了全面的概述。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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