Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR

on behalf of the AMPHEUS Project REMAP-CAP Immunoglobulin Domain UK Investigators, Oxford COVID-19 Vaccine Trial Group

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4 Citations (Scopus)

Abstract

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARSCoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

Original languageEnglish
JournalJournal of Clinical Microbiology
Volume60
Issue number4
DOIs
Publication statusPublished - Apr 2022
Externally publishedYes

Bibliographical note

Funding Information:
The views expressed in this publication are those of the authors and not necessarily those of any funding bodies. We thank all volunteers who supported both studies presented in this article. We thank James Szymanski and Chak Foon Tso and the team at Dascena for the generous provision of raw viral load data from their respective studies. The COV003 trial is supported by the National Institute of Health Research (NIHR), the Lemann Foundation, Rede D’Or, the Brava and Telles Foundation, and AstraZeneca. The REMAP-CAP trial is supported by The Platform for European Preparedness Against (Re-) emerging Epidemics (PREPARE) consortium by the European Union, FP7-HEALTH-2013-INNOVATION-1 (no. 602525), the Australian National Health and Medical Research Council (no. APP1101719), the Australian Medical Research Future Fund (no. APP2002132), the New Zealand Health Research Council (no. 16/631), the Canadian Institutes of Health Research COVID-19 Rapid Research Funding Grant (no. 447335), the Canadian Institute of Health Research Strategy for Patient-Oriented Research Innovative Clinical Trials Program Grant (no. 158584), the National Institute for Health Research (UKRIDHSC COVID-19 Rapid Response Rolling Call, “The use of convalescent plasma to treat hospitalised and critically ill patients with COVID-19 disease” [COV19-RECPLAS]), the UK National Institute for Health Research (NIHR) and the NIHR Imperial Biomedical Research Centre, the Health Research Board of Ireland (CTN 2014-012), the UPMC Learning While Doing Program, the Translational Breast Cancer Research Consortium, the Pittsburgh Foundation, the French Ministry of Health (PHRC-20-0147), the Minderoo Foundation, and the Wellcome Trust Innovations Project (215522). Australian governments fund Australian Red Cross Lifeblood for the provision of blood products and services to Australia. Collection of UK plasma was funded by the DHSC through core funding under COVID-19 and EU SoHo Grants. J. Ratcliff is supported by Marshall and Clarendon Scholarships. M. Shankar-Hari is supported by the National Institute for Health Research Clinician Scientist Award (NIHR-CS-2016-16-011). Conceptualization, J. Ratcliff, P. Simmonds; methodology, D. Bonsall, T. Golubchik, H. Harvala, J. Ratcliff, P. Simmonds; software, T. Golubchik, J. Ratcliff; formal analysis, T. Golubchik, J. Ratcliff, P. Simmonds; investigation, C. Jay, G. MacIntyre-Cockett, D. Nguyen, J. Ratcliff, S. Williams; resources, F. Al-Beidh, S. Bibi, S. A. Costa Clemens, L. Estcourt, A. Evans, M. Fish, P. M. Folegatti, A. C. Gordon, A. Jennings, E. Laing, T. Lambe, D. Menon, P. R. Mouncey, A. J. Pollard, M. N. Ramasamy, D. J. Roberts, K. M. Rowan, J. Rynne, M. Shankar-Hari; data curation, T. Golubchik, J. Ratcliff, P. Simmonds; writing—original draft, J. Ratcliff; writing—review & editing, S. Bibi, H. Harvala, T. Golubchik, C. Jay, J. Ratcliff, A. J. Pollard, M. N. Ramasamy, M. Shankar-Hari, P. Simmonds; supervision, T. Golubchik, H. Harvala, P. Simmonds. The funders of REMAP-CAP had no role in the design, analysis, or interpretation of the data presented in this study. Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19. AstraZeneca did not have a role in reviewing the data for this study. A. J. Pollard is an NIHR senior investigator.

Funding Information:
The COV003 trial is supported by the National Institute of Health Research (NIHR), the Lemann Foundation, Rede D’Or, the Brava and Telles Foundation, and AstraZeneca. The REMAP-CAP trial is supported by The Platform for European Preparedness Against (Re-) emerging Epidemics (PREPARE) consortium by the European Union, FP7-HEALTH-2013-INNOVATION-1 (no. 602525), the Australian National Health and Medical Research Council (no. APP1101719), the Australian Medical Research Future Fund (no. APP2002132), the New Zealand Health Research Council (no. 16/631), the Canadian Institutes of Health Research COVID-19 Rapid Research Funding Grant (no. 447335), the Canadian Institute of Health Research Strategy for Patient-Oriented Research Innovative Clinical Trials Program Grant (no. 158584), the National Institute for Health Research (UKRIDHSC COVID-19 Rapid Response Rolling Call, “The use of convalescent plasma to treat hospitalised and critically ill patients with COVID-19 disease” [COV19-RECPLAS]), the UK National Institute for Health Research (NIHR) and the NIHR Imperial Biomedical Research Centre, the Health Research Board of Ireland (CTN 2014-012), the UPMC Learning While Doing Program, the Translational Breast Cancer Research Consortium, the Pittsburgh Foundation, the French Ministry of Health (PHRC-20-0147), the Minderoo Foundation, and the Wellcome Trust Innovations Project (215522). Australian governments fund Australian Red Cross Lifeblood for the provision of blood products and services to Australia. Collection of UK plasma was funded by the DHSC through core funding under COVID-19 and EU SoHo Grants. J. Ratcliff is supported by Marshall and Clarendon Scholarships. M. Shankar-Hari is supported by the National Institute for Health Research Clinician Scientist Award (NIHR-CS-2016-16-011).

Publisher Copyright:
© 2022 American Society for Microbiology. All Rights Reserved.

Keywords

  • SARS-CoV-2
  • allele-specific probe PCR
  • diagnostics
  • next-generation sequencing
  • variant identification
  • variants of concern

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