Reverse transcription-polymerase chain reaction (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 for SARS-CoV-2 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.


To understand the role of sub-genomic RNA transcripts in discordant results from RT-PCR tests for COVID-19.


A panel of six droplet digital polymerase chain reaction (ddPCR) assays was designed to quantify 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. Eleven clinical nasopharyngeal swabs were also tested with this panel.


In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. 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.

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