Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR

  1. Shuo Shen
  2. Andrew Y. Fu
  3. Maidar Jamba
  4. Jonathan Li
  5. Zhen Cui
  6. Larry Pastor
  7. Daniel Cataldi
  8. Qing Sun
  9. Joseph A. Pathakamuri
  10. Daniel Kuebler
  11. Michael Rohall
  12. Madison Krohn
  13. Daniel Kissinger
  14. Jocelyn Neves
  15. Isaac Archibeque
  16. Aiguo Zhang
  17. Chuanyi M. Lu
  18. Michael Y. Sha

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Abstract

Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants.

IMPORTANCE

We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

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  1. Version published to 10.1128/spectrum.04248-23
  2. Version published to 10.21203/rs.3.rs-1879627/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2021.04.01.21254484: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was approved by the institutional review board (IRB) at UCSF (UCSF IRB #11-05207) as a no-subject contact study with waiver of consent and as exempt under category 4.
    Consent: This study was approved by the institutional review board (IRB) at UCSF (UCSF IRB #11-05207) as a no-subject contact study with waiver of consent and as exempt under category 4.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    and conserved regions in each target gene were identified using BioEditor 7.2.5.
    BioEditor
    suggested: None
    Primers and probes were designed to target the most conserved regions of each of the target genes of the viral genome, using Primer3plus software and following general rules of real-time PCR design.
    Primer3plus
    suggested: (Primer3Plus, RRID:SCR_003081)

    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.

  4. Version published to 10.1101/2021.04.01.21254484v1 on medRxiv