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.

Heterogeneity and Coexistence of T790M and T790 Wild-Type Resistant Subclones Drive Mixed Response to Third-Generation Epidermal Growth Factor Receptor Inhibitors in Lung Cancer

“…Positive and negative droplet counts were used to calculate the concentration of target and reference DNA sequences as previously described. 10 Normal control DNA (human genomic DNA [Promega, Madison, WI]), and no DNA template controls were included. Samples with insufficient positive events were independently repeated at least twice to validate results.…”

Evaluation of T Cell Immunity against Human Cytomegalovirus: Impact on Patient Management and Risk Assessment of Vertical Transmission

“…Modern quantitative molecular methods mostly rely on real-time PCR for clinical viral load testing. Recently, digital droplet PCR (ddPCR) was evaluated as a method allowing precise direct quantification without requiring a calibration curve [47, 48]. It relies on limiting partition of the PCR volume in a large number of droplets, each of which may be envisioned as a PCR reaction giving a positive or negative result according to whether a single target molecule was present in the droplet or not.…”

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Applications of Digital PCR for Clinical Microbiology

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