Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Abstract

BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

Strategic Research on the Molecular Diagnostics Industry in China

“…A large amount of diluted DNA is dispersed into micro-reactors via microfluidic techniques. The number of DNA templates per reactor is less than or equal to 1 [6]. After the PCR is complete, the fluorescence signal in each micro-reaction well is measured.…”

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

“…However, digital PCR still suffers from several disadvantages. [102][103][104] The copy numbers of different targets in a sample are often not known a priori and can vary by several orders of magnitude. Digital PCR quantification is most accurate at very low copy numbers, when estimates by Poisson statistics are not necessary.…”

An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases

“…It was anticipated that ddPCR would be superior to TaqMan based allelic discrimination, as previous studies already show accurate detection of rare mutations at variant allele frequencies of 0.01% and below [35][36][37][38] . Sanger sequencing was adopted to confirm mutations present by MuPol-Seq at a frequency close to, or within the capabilities of Sanger sequencing detection and visualisation (≥20%).…”

“…As a key aim in this study was to produce a deep sequencing strategy that could be introduced into routine diagnostics, a low cost, highly multiplexed workflow was implemented. We applied this approach to distinguish low abundance TP53 variations that may predict an individual response to DNA damaging therapy from background errors in presentation CLL cases, and evaluated the use of digital droplet PCR (ddPCR), a highly sensitive technique capable of detecting rare mutant-alleles, [35][36][37][38] to validate low abundance mutant base calls. In addition, in one case where a relapse sample was available we were able to show that TP53 mutations present at CLL diagnosis were also present at relapse by ddPCR following treatment with fludarabine.…”