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

Control of SARS-CoV-2 (SCV-2) transmission is a major priority that requires understanding SCV-2 replication dynamics. We developed and validated novel droplet digital PCR (ddPCR) assays to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from full-length genomic RNAs (gRNAs) in a multiplexed format. We applied this multiplex ddPCR assay to 144 cross-sectional nasopharyngeal samples. sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was remarkably stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Interestingly, adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Longitudinal daily testing of 6 persons for up to 14 days revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. These data indicate stable viral transcription during infection. More work is needed to understand why cultures are negative despite persistence of viral RNAs.