The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19

  1. Noah B. Toppings
  2. Lisa K. Oberding
  3. Yi-Chan Lin
  4. David Evans
  5. Dylan R. Pillai

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Abstract

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.

Author Summary

This paper provides insight into a drawback of the standard method of testing for COVID-19 (RT-PCR). The results presented here propose an explanation for why inconclusive results sometimes occur with this method. These results can aid microbiologists in the interpretation of inconclusive test results. These results can also aid in decisions about which COVID-19 test a laboratory should use, as there are a plethora of options available. This is important because this standard testing method will remain a critical tool – globally – for managing the COVID-19 pandemic and any future viral pandemics and epidemics. Thus, it is important to investigate every facet of the testing method. The findings presented here are applicable to any virus which makes sub-genomic transcripts as part of its life cycle. Trends observed in viral cultures are presented alongside the same trends observed in clinical samples. Unlike similar papers in the field, this paper did not strive to develop a new methodology or tool.

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  1. ScreenIT

    SciScore for 10.1101/2021.12.14.21267750: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: Clinical Sample Collection and Ethics: This research involved human participants and was performed in accordance with the relevant guidelines and regulations.
    Consent: Informed consent was obtained from all participants as required and was approved by Conjoint Health Research Ethics Board (CHREB) at the University of Calgary (REB18-0107, REB20-0402 and REB20-0444).
    IRB: Informed consent was obtained from all participants as required and was approved by Conjoint Health Research Ethics Board (CHREB) at the University of Calgary (REB18-0107, REB20-0402 and REB20-0444).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    For virus culture, Vero cells were infected at MOI = 0.01 in 500 µL inoculant/well.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis was performed in R Studio (R 4.1.2) and MATLAB R2020b.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A limitation of this study is its small sample size of eleven clinical nasopharyngeal swabs. A larger number of clinical samples – and different types – should be tested in the future to confirm the conclusions made here. Furthermore, this study did not show that samples with less ORF1 copies than N gene copies would in fact be more likely to be ORF1 negative, N gene positive, in an RT-PCR test. Future studies could test for a relationship between copy number ratios and rates of inconclusive test results. A strength of this study is that it used two different ddPCR targets for each of the three ORFs of interest. In conclusion, this study proposes a biological explanation for certain inconclusive RT-PCR results. Namely, sub-genomic RNA in clinical samples may increase the likelihood of an N gene target from being positive by RT-PCR. Further studies are required to validate this finding in a larger and more diverse sample set.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.12.14.21267750 on medRxiv