The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19.
Reverse transcription-PCR (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when one RT-PCR target is positive for SARS-CoV-2 and one RT-PCR target is negative within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. It was hypothesized that concentration differences across targets may be due to the transcription of sub-genomic RNA, as this would result in an increase in the concentration of gene targets near the 3’ end of the SARS-CoV-2 genome. A panel of six digital droplet (dd)PCR assays was designed to quantitate the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity in such a way as to maximize gene-to-gene copy ratios. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. Both the highest specific infectivity (copies/pfu) and the highest differences between gene targets were observed at 6 hours post-infection (eclipse phase) in infected cells. The same trends in the relative abundance of copies across different targets observed in infected cells was observed in clinical samples, though trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by this data.