RT-RPA-Cas12a-based discrimination of SARS-CoV-2 variants of concern

  1. Guiyue Tang
  2. Zilong Zhang
  3. Wei Tan
  4. Fei Long
  5. Jingxian Sun
  6. Yingying Li
  7. Siwei Zou
  8. Yujiao Yang
  9. Kezhu Cai
  10. Shenwei Li
  11. Zhiyi Wang
  12. Jiakun Liu
  13. Guobing Mao
  14. Yingxin Ma
  15. Guo-Ping Zhao
  16. Zhen-Gan Tian
  17. Wei Zhao

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Abstract

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. However, current methods are limited by the low sensitivity, long turn-around time or high cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 VOCs by combining R T- R PA and C RISPR-Cas12a d etecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 204 clinical samples, showing 99% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% accuracy in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which may facilitate point-of-care testing of SARS-CoV-2 and its variants.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.11.22274884: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical approval of the study was given by the Bioethics Committee of Bio-X Institute of Shanghai Jiao Tong University (COA: M202007).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    DNA fragments containing wild-type S gene 501 with point mutations were synthesized in pUC19 backbone plasmid by Sangon Biotech.
    pUC19
    suggested: RRID:Addgene_50005)
    Software and Algorithms
    SentencesResources
    The RT-RPA products were loaded into 8% PAGE gel and the relative greyness of sample bands was quantified using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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.

    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 10.1101/2022.05.11.22274884v1 on medRxiv