SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

  1. DR Ganguly
  2. S Rottet
  3. S Yee
  4. WY Hee
  5. AB Smith
  6. NC Khin
  7. AA Millar
  8. AM Fahrer

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Abstract

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×10 6 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30 ) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

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  1. Version published to 10.1101/2020.05.29.109702v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.05.29.109702: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    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:
    Aspects of this method could be combined with community-driven open source protocols (for example, BEARmix: https://gitlab.com/tjian-darzacq-lab/bearmix) to achieve even lower costs while addressing technical limitations, such as appropriate methods for virus inactivation as discussed below. Despite promising progress, we must highlight the critical need for validation against patient samples alongside clinical certification in order for results to be considered diagnostic. While we attempted to mimic patient samples by spiking synthetic RNA into non-infectious saliva, this cannot replace validation using patient saliva. We propose the combined use of Tween20 and heat treatment (95 °C for 5 mins) for viral inactivation (Roberts et al. 2009; Darnell et al. 2004), and for increased release of viral RNA from patient saliva. However, this needs to be validated with samples from patients with known SARS-CoV-2 infection. We therefore report a theoretical limit of detection based on reactions using synthetic virus RNA while controlling for non-specific amplification. Primer pairs CCDC-N and SG2 demonstrated the best specificity (Figure 4 E) and sensitivity (Figure 5 A, C) towards the synthetic SARS-CoV-2 RNA. Based on the amplification of synthetic SARS-CoV-2 RNA in serial dilutions (no saliva), we estimate the LoD for CCDC-N and SG2 primer pairs to be 64-256 and 16-64 molecules per reaction, respectively. Therefore, we propose these primers to be best suited for SARS-CoV-2 screenin...

    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.

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  3. Version published to 10.1101/2020.05.29.109702v1 on bioRxiv