Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay

  1. Viera Kováčová
  2. Kristína Boršová
  3. Evan D Paul
  4. Monika Radvánszka
  5. Roman Hajdu
  6. Viktória Čabanová
  7. Monika Sláviková
  8. Martina Ličková
  9. Ľubomíra Lukáčiková
  10. Andrej Belák
  11. Lucia Roussier
  12. Michaela Kostičová
  13. Anna Líšková
  14. Lucia Maďarová
  15. Mária Štefkovičová
  16. Lenka Reizigová
  17. Elena Nováková
  18. Peter Sabaka
  19. Alena Koščálová
  20. Broňa Brejová
  21. Edita Staroňová
  22. Matej Mišík
  23. Tomáš Vinař
  24. Jozef Nosek
  25. Pavol Čekan
  26. Boris Klempa

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Abstract

Background

The emergence of a novel SARS-CoV-2 variant of concern called B.1.1.7 lineage sparked global alarm due to evidence of increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect lineage B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant.

Aim

Since many countries lack robust genomic surveillance programs and failed assays detect multiple unrelated variants containing similar mutations as B.1.1.7, we sought to develop an RT-qPCR test that can accurately and rapidly differentiate the B.1.1.7 variant from other SARS-CoV-2 variants.

Methods

We used bioinformatics, allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop a test that differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a surveillance study of B.1.1.7 lineage prevalence in Slovakia.

Results

Our multiplexed RT-qPCR test showed 97% clinical sensitivity at detecting lineage B.1.1.7. The assay was used in a country-wide surveillance of B.1.1.7 lineage spread in Slovakia. Retesting nearly 7,000 SARS-CoV-2 positive samples obtained during three campaigns performed within a one month period, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%.

Conclusion

Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.09.21251168: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study has been approved by the Ethics committee of Biomedical Research Center of the Slovak Academy of Sciences, Bratislava, Slovakia (Ethics committee statement No. EK/BmV-02/2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    We cut the locus encoding the spike protein and used the MAFFT alignment tool (with the parameter - auto)[12] to align all the sequences against the WUHAN reference (NCBI ID: NC_045512.2).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Using SeaView [13], we called 95% consensus sequences for the ΔH69/ΔV70 and ΔY144 group and the No deletions group that were subsequently used to design primer and probe sets specific to either B.1.1.7 or all other SARS-CoV-2 variants, respectively.
    SeaView
    suggested: (SeaView, RRID:SCR_015059)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    assay remains to be validated on clinical samples, the test still relies on target gene failures and therefore retains the limitations for SGTF assays and necessity to confirm lineage status by sequencing. To differentiate B.1.1.7, we took an alternative approach by targeting the ΔY144 deletion using allele-specific PCR methods combined with judicious placement of LNA oligonucleotides. This allowed us to stabilize the 5’-end and mismatched base, and to shorten the 3’-terminal of the reverse primer. Together, these modifications provided us with a primer/probe set that retained specificity for B.1.1.7 variants and reduced specificity to other variants containing the ΔH69/ΔV70 deletion. Our test, instead of relying on target failures to identify putative variants, provides a positive signal in the presence of B.1.1.7 and ΔH69/ΔV70 deletion variants that can easily be differentiated by comparing their relative Ct values to a common SARS-CoV-2 S gene primer/probe set that serves as a benchmark. We have provided interested users with the primer and probe sequences to implement this B.1.1.7 assay in their own laboratories with the hope this can rapidly scale the ability of countries to identify the B.1.1.7 variant and implement epidemiological measures to mitigate its spread. This test is also available as a research use only kit called rTest COVID-19 B.1.1.7 qPCR kit (https://www.multiplexdx.com/products/rtest-covid-19-b-1-1-7-qpcr-kit, MultiplexDX, Inc., Bratislava, Slovakia) tha...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  2. Version published to 10.1101/2021.02.09.21251168 on medRxiv