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

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

doi:10.1016/j.bdq.2016.08.002

Liesbet Deprez, Philippe Corbisier + Show 5 more

Open Access

https://doi.org/10.1016/j.bdq.2016.08.002

Copy DOI

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.

Highlights

Accurate quantification of the copy number concentration of specific nucleic acid sequences is important for several applications both within the fields of red biotechnology, and green biotechnology (e.g. GMO testing)
The primers and probes of the breakpoint cluster region gene (BCR)-abl oncogene 1 (ABL) droplet digitalTM PCR (ddPCR) method are used in a standardised quantitative real-time PCR (qPCR) method developed during a large inter-laboratory study and the absence of nonspecific amplification artefacts in qPCR has been shown [20,21]
The selectivity of the BCRABL ddPCR method was experimentally assessed by performing 4 replicate measurements of a matrix blank consisting of 1 × T1E0.01 buffer with the nucleic acid background of the ERM-AD623 samples and 4 replicates of a positive control consisting of an undiluted sample of ERM-AD623a at a PCR copy number concentration of 54000 cp/␮L

Summary

Introduction

Accurate quantification of the copy number concentration of specific nucleic acid sequences is important for several applications both within the fields of red biotechnology, (e.g. oncology and infectious diseases) and green biotechnology (e.g. GMO testing). The measurement principle of dPCR relies on partitioning the PCR mix across a large number of small individual reaction volumes, such that the distribution of the target sequence follows a binominal distribution function and that a part of the reaction volumes does not contain a copy of the target sequence [3]. Following an end-point PCR, partitions containing one or more copies of the target sequence are labelled positive and counted. The proportion of positive partitions is used to estimate the copy number concentration of the target sequence, taking into account the statistics of the binominal distribution [4]. Available dPCR systems are based on two different approaches to partition the PCR mix: some use microfluidic chips on which the PCR mix is distributed over premanufactured chambers [5,6] while others are based on oil-water emulsions to separate the solution into droplets [7,8]

Methods

Results

Discussion

Conclusion