SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

  1. Steffen Klein
  2. Thorsten G. Müller
  3. Dina Khalid
  4. Vera Sonntag-Buck
  5. Anke-Mareil Heuser
  6. Bärbel Glass
  7. Matthias Meurer
  8. Ivonne Morales
  9. Angelika Schillak
  10. Andrew Freistaedter
  11. Ina Ambiel
  12. Sophie L. Winter
  13. Liv Zimmermann
  14. Tamara Naumoska
  15. Felix Bubeck
  16. Daniel Kirrmaier
  17. Stephanie Ullrich
  18. Isabel Barreto Miranda
  19. Simon Anders
  20. Dirk Grimm
  21. Paul Schnitzler
  22. Michael Knop
  23. Hans-Georg Kräusslich
  24. Viet Loan Dao Thi
  25. Kathleen Börner
  26. Petr Chlanda

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Abstract

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

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  1. Version published to 10.3390/v12080863
  2. ScreenIT

    SciScore for 10.1101/2020.07.08.20147561: (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

    Software and Algorithms
    SentencesResources
    2.3 RNA extraction using QIAcube: To compare the performance of the magnetic bead RNA extraction, the QIAamp Viral RNA body fluid kit was carried out with manual lysis according to the manufacturer’s protocol (Qiagen, Germany).
    QIAcube
    suggested: (Qiagen QIAcube, RRID:SCR_020414)
    Data analysis was performed in Excel and GraphPad Prism (GraphPad Software, USA), and 95 % “exact” Clopper-Pearson confidence intervals were calculated using MedCalc (MedCalc Software, Belgium). 2.5 Estimation of detection limit of magnetic bead RNA extraction using MS2 RNA spike-in: Five µl diluted MS2 RNA (Roche, Switzerland) containing 6.1 × 101 to 6.1 × 106 molecules was spiked into 140 µl lysis buffer before a 140 µl patient sample was added.
    Excel
    suggested: None
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    MedCalc
    suggested: (MedCalc, RRID:SCR_015044)

    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

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  3. Version published to 10.1101/2020.07.08.20147561v1 on medRxiv