Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

  1. Jennifer R. Hamilton
  2. Elizabeth C. Stahl
  3. Connor A. Tsuchida
  4. Enrique Lin-Shiao
  5. C. Kimberly Tsui
  6. Kathleen Pestal
  7. Holly K. Gildea
  8. Lea B. Witkowsky
  9. Erica A. Moehle
  10. Shana L. McDevitt
  11. Matthew McElroy
  12. Amanda Keller
  13. Iman Sylvain
  14. Ariana Hirsh
  15. Alison Ciling
  16. Alexander J. Ehrenberg
  17. Bradley R. Ringeisen
  18. Garth Huberty
  19. Fyodor D. Urnov
  20. Petros Giannikopoulos
  21. Jennifer A. Doudna
  22. IGI SARS-CoV-2 Consortium

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Abstract

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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  1. Version published to 10.1371/journal.pone.0255690
  2. ScreenIT

    SciScore for 10.1101/2021.01.10.21249151: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The WHHS institutional review board (IRB) waiver and consent form for NP/saliva comparison are presented in Supplemental Figure 6.
    Consent: The WHHS institutional review board (IRB) waiver and consent form for NP/saliva comparison are presented in Supplemental Figure 6.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Following incubation, the 1mL virus/OMNIgene mix was divided to inoculate eight T175 flasks containing Vero-E6 cells at 80% confluency (125ul/flask)
    Vero-E6
    suggested: None

    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: 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.

    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.

  3. Version published to 10.1101/2021.01.10.21249151 on medRxiv