Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis

Abstract

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned a droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNAs from anonymized COVID-19 positive subjects and we found a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Further, we observed that expression profiles of sgmRNAs analyzed in a subset of 110 samples subjected to meta-transcriptomics sequencing were highly correlated with those obtained by ddPCR. Our results, providing a comprehensive and dynamic snapshot of SARS-CoV-2 sgmRNAs expression and replication, may contribute to provide a better understanding of SARS-CoV-2 transcription and expression mechanisms, and support the development of more accurate molecular diagnostic tools and for the stratification of COVID-19 patients.

Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed ddPCR

“…Due to limited yield of viral RNA from swab samples and the presence of potential inhibitors in the collection medium, droplet digital PCR is ideal for sensitive, reliable, and simultaneous quantitation of sgRNA and gRNA. Few studies have used ddPCR to examine viral kinetics on limited number of subjects or time points per participant (12, 16, 18, 19); therefore, more extensive cross-sectional and longitudinal sample analyses are desired for full characterization of viral kinetics. To address these knowledge gaps and to link active SCV-2 transcription with infectiousness, we developed a multiplex ddPCR assay to quantify SCV-2 sgRNA and gRNA simultaneously and applied this assay to cross-sectional and longitudinal samples obtained from participants with recent COVID-19 who also had viral culture performed.…”