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Updated: Oct 15, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics
Annarita Oranger1, Caterina Manzari1, Matteo Chiara2,3
1Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari Aldo Moro, Via Orabona 4, 70126, Bari, Italy.
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 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 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.
