Comparison Between Reverse Transcription Droplet Digital and Quantitative Polymerase Chain Reactions for the Determination of Viral RNA Copy Numbers in COVID-19 Patient Plasma.

INTRODUCTION

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Studies have shown the association of viral RNAemia with the severity of COVID-19 and poor patient outcomes. Here, we compared 2 reverse transcription PCR assays: droplet digital PCR (ddPCR) and quantitative PCR (qPCR), in determining the viral RNA copy numbers in patient plasma.

MATERIAL AND

METHODS

A total of 229 plasma samples collected on days 0, 1, 4, 7, and 28 (when available) from 33 COVID-19 ICU patients were analyzed. RNA was extracted from samples using the MagNA Pure 96 DNA and Viral NA small volume kit (Roche Diagnostic) on the MagNA Pure 96 platform with 100 mL starting and elution volumes. For ddPCR, the quantification of viral RNA copies was performed using a SARS-CoV-2 ddPCR kit (Bio-Rad Laboratories Inc.) following the manufacturer's instruction. For qPCR, the quantification of viral RNA copies was determined using the CDC 2019-Novel Coronavirus real-time RT-PCR diagnostic panel on an Applied Biosystems 7500 Fast Dx real-time PCR system with a set of known copy-positive controls as standard.

RESULTS

Our results showed that not all patients had detectable viral RNA in their plasma samples by both methods, with only 23 (70%) and 31 (94%) patients having detectable viral RNA by ddPCR and qPCR, respectively. The qPCR detected viral RNA amplicons of both N1 and N2 targets in 87 specimens, and when these N1 and N2 copy numbers were averaged, the median was 447.7 copies/mL of plasma with a range of 56.8 to 192,495.5 copies/mL of plasma. The ddPCR detected viral RNA amplicons in 64 specimens, the median was 173.4 copies/mL of plasma with a range of 39.7-108,326.6 copies/mL of plasma when the N1 and N2 copy numbers were averaged.

CONCLUSION

Both ddPCR and qPCR methods can detect viral RNA in plasma samples from severe COVID-19 patients. The qPCR was able to detect more positive specimens, suggesting higher sensitivity compared to the ddPCR method.