Droplet Digital PCR or Real-Time PCR as a Method for Quantifying SARS-CoV-2 RNA in Plasma-Is There a Difference?

Abstract

The aim of this study is to ascertain whether qRT-PCR (reverse transcriptase real-time PCR) or RT-ddPCR (reverse transcriptase digital droplet PCR) is more effective for detecting SARS-CoV-2 RNA (severe acute respiratory syndrome coronavirus 2 RNA) in blood plasma from COVID-19 (coronavirus infectious disease-19) patients. The E-gene of SARS-CoV-2 RNA was quantified using both methods in 128 plasma samples from 70 hospitalized patients, followed by a statistical analysis to compare the sensitivity and concordance between the methods. Out of the 128 samples, 89 yielded consistent results irrespective of the method used, whereas 39 samples showed discrepancies between the two different methods. RT-ddPCR frequently registered higher viral quantities compared to qRT-PCR; however, the results did not demonstrate a clear superiority in sensitivity for RT-ddPCR. Although RT-ddPCR registered higher viral quantities, this study concludes that both methods provide comparable results for detecting SARS-CoV-2 E-gene RNA in plasma.

Keywords: COVID-19; E-gene; RNAemia; RT-ddPCR; SARS-CoV-2; plasma; qRT-PCR.

Figure 1

Flow chart from top to bottom, describing sample selection criteria.

Figure 2

Standard curves for SARS-CoV-2 E-gene qRT-PCR and RT-ddPCR, generated through simple linear regression, where each dot represents one replicate. The standards are the same in both panels, as is their expected concentration in log10 copies/mL. The left panel displays how the standard’s expected concentration correlates with Cq values when analyzed using qRT-PCR. The right panel displays how the standard’s expected concentration correlates with observed concentration when analyzed with RT-ddPCR.

Figure 3

(a) XY scatterplot of all samples determined to be positive using both methods (n = 29), with the sample number on the x-axis and the concentration (log 10 copies/mL) on the y-axis. Each pair of results (RT-ddPCR result versus qRT-PCR result for one sample) is represented by the same color. (b) Box and whisker plot showing the mean and 5–95% percentile of positive results based on RT-ddPCR versus qRT-PCR.

Figure 4

Box and whisker plot showing mean and 5–95% percentile of disconcordant samples; qRT-PCR-positive (q pos) versus RT-ddPCR-negative (dd neg) and qRT-PCR-negative (q neg) versus RT-ddPCR-positive (dd pos), n = 39.