An expanded high throughput RT-PCR assay to rapidly identify all known SARS-CoV-2 variants of concern using melting temperature coding

  1. Padmapriya P Banada
  2. Raquel Green
  3. Sukalyani Banik
  4. Deanna Streck
  5. Ibsen Montalvan
  6. Robert Reiss
  7. Robert Jones
  8. Salvatore A. E. Marras
  9. Soumitesh Chakravorty
  10. David Alland

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Abstract

Background

The rapid emergence of new vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires an equally rapid deployment of diagnostic tests to specifically identify each VOC as soon as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) Alpha/Beta/Gamma RT-PCR melting temperature (Tm) signature-based assay, which now includes modifications that allow specific detection of Delta (B.1.617.2) and Omicron (B.1.529) VOCs.

Methods

We developed a dual SMB assay (SMB-452), which targeted the T22917G (L452R) mutation in the SARS-CoV-2 spike protein to specifically detect the Delta VOC. We also identified a Tm profile in our existing SMB-501 and SMB-484 assays (which detect mutations in codons 501 and 484 of the SARS-CoV-2 spike protein, respectively) that differentiate the Omicron-specific N501Y (A23063T) and E484A (A23013C) mutations from both wild type (WT) and other VOCs. The entire six SMB three-codon assay was tested using reference SARS-CoV-2 RNAs. The assay was then validated using clinical samples from COVID-19 patients tested with a LightCycler 480 (LC480) (74 samples), Bio-Rad CFX96 (34 samples), Rotor-Gene Q (Qiagen) (34 samples) and an ABI-7500 (34 samples) RT-PCR instruments. Six SMB Tm results were then inputted into an Excel Analysis tool to generate specific VOC identifications.

Results

The limit of detection (LOD) for the new SMB-452 assay, which specifically identified the Delta variant was 1 genomic equivalent (GE) per reaction. The LODs of the SMB-501 and SMB-484 assays, which detect Omicron were 100 and 10 3 GE respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% of the samples as WT or VOCs in clinical samples and 6% of the tests producing indeterminate results. None of the samples were incorrectly identified as WT or as a different VOC. Thus, excluding samples with indeterminant results, the assay was 100% sensitive and 100% specific compared to sequencing. There was also 100% concordance between the LC480, BioRad, ABI and Qiagen results, excluding negative or indeterminate results; however, the Qiagen assay had significantly more indeterminates than the other assays.

Conclusion

This new assay can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.18.22269424: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical considerations: The usage of de-identified clinical samples from RT-PCR confirmed COVID-19 positive and negative patients in this study was approved by the Rutgers University institutional Review Board under protocol numbers 20170001218 and 2020001541.
    Field Sample Permit: The remaining 40 specimens collected in November and December 2021 were tested only with the LC480 instrument.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Briefly, a total of 412,389 high quality SARS-CoV-2 genome sequences deposited in GISAID (24) as of Feb 19, 2021, were analyzed using BLAST (25) and aligned with MAFFT (26).
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Primers and probes were designed on the basis of sequence conservation using Primer3 program (27) to amplify a 122 bp region flanking the position 22917 (452 codon) in the reference strain (GenBank accession number MN908947).
    Primer3
    suggested: (Primer3, RRID:SCR_003139)
    Tm values obtained from the instrument for each SMB-assay from both WT and MT probes, were exported and identified using the Excel analyze tool.
    Excel
    suggested: None
    Sequencing data were analyzed using Ugene (ver 37) or MegAlign Pro software (DNAStar, ver16).
    MegAlign Pro
    suggested: None
    Statistical analysis: Standard statistical analyses (average, standard deviation) and graphing were performed using Microsoft excel (ver 2102),
    Microsoft excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    , GraphPad Prism 8.4.3 for Windows, R version 4.1.1 and ggplot2 package.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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 10.1101/2022.01.18.22269424v1 on medRxiv