Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR

Abstract

A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples (n = 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results. The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another. When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two. Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads. Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values.

FIG 1

Regression analysis of measured values of ddPCR against nominal values.

FIG 2

Pairwise linear regressions for clinical samples.

FIG 3

Quantitative differences between assays. Values are log₁₀ copies/ml with differences between assays on the y axis and the average on the x axis. The mean difference between assays is represented by the solid line, and ±2 SDs is represented by the dotted lines.