Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

  1. Orchid M. Allicock
  2. Devyn Yolda-Carr
  3. John A. Todd
  4. Anne L. Wyllie

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Abstract

The key to limiting SARS-CoV-2 spread is to identify virus-infected individuals (both symptomatic and asymptomatic) and isolate them from the general population. Hence, routine weekly testing for SARS-CoV-2 in all asymptomatic (capturing both infected and non-infected) individuals is considered critical in situations where a large number of individuals co-congregate such as schools, prisons, aged care facilities and industrial workplaces. Such testing is hampered by operational issues such as cost, test availability, access to healthcare workers and throughput. We developed the SalivaDirect RT-qPCR assay to increase access to SARS-CoV-2 testing via a low-cost, streamlined protocol using self-collected saliva. To expand the single sample testing protocol, we explored multiple extraction-free pooled saliva testing workflows prior to testing with the SalivaDirect RT-qPCR assay. A pool size of five, with or without heat inactivation at 65 °C for 15 min prior to testing resulted in a positive agreement of 98% and 89%, respectively, and an increased Ct value shift of 1.37 and 1.99 as compared to individual testing of the positive clinical saliva specimens. Applying this shift in Ct value to 316 individual, sequentially collected, SARS-CoV-2 positive saliva specimen results reported from six clinical laboratories using the original SalivaDirect assay, 100% of the samples would have been detected (Ct value < 45) had they been tested in the 1:5 pool strategy. The availability of multiple pooled testing workflows for laboratories can increase test turnaround time, permitting results in a more actionable time frame while minimizing testing costs and changes to laboratory operational flow.

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  1. Version published to 10.1038/s41598-023-34662-2
  2. ScreenIT

    SciScore for 10.1101/2022.01.16.22269390: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: The use of de-identified saliva specimens from healthy or asymptomatic individuals was approved by the Institutional Review Board of the Yale Human Research Protection Program (Protocol ID. 2000028394) (11).
    Consent: Study participants were informed in writing about the purpose and procedure of the study, and consented to study participation through the act of providing the saliva sample; the requirement for written informed consent was waived by the Institutional Review Board.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Additionally, the Institutional Review Board of the Yale Human Research Protection Program determined that the use of de-identified, remnant COVID-19-positive clinical samples obtained from laboratory partners for the RT-qPCR testing conducted in this study is not research involving human subjects (Protocol ID: 2000028599).
    Yale Human Research Protection Program
    suggested: None
    All statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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

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  3. Version published to 10.1101/2022.01.16.22269390v1 on medRxiv