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

AMPHEUS Project, REMAP-CAP Immunoglobulin Domain UK Investigators, and Oxford COVID-19 Vaccine Trial Group, Jeremy Ratcliff, Farah Al-Beidh, Sagida Bibi, David Bonsall, Sue Ann Costa Clemens, Lise Estcourt, Amy Evans, Matthew Fish, Pedro M Folegatti, Anthony C Gordon, Cecilia Jay, Aislinn Jennings, Emma Laing, Teresa Lambe, George MacIntyre-Cockett, David Menon, Paul R Mouncey, Dung Nguyen, Andrew J PollardMaheshi N Ramasamy, David J Roberts, Kathryn M Rowan, Jennifer Rynne, Manu Shankar-Hari, Sarah Williams, Heli Harvala, Tanya Golubchik, Peter Simmonds

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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 ( C T ) 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 SARS-CoV-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
Pages (from-to)e0228321
JournalJournal of Clinical Microbiology
Issue number4
Early online date24 Mar 2022
Publication statusPublished - 20 Apr 2022

Keywords / Materials (for Non-textual outputs)

  • Alleles
  • COVID-19/diagnosis
  • Humans
  • Polymerase Chain Reaction
  • SARS-CoV-2/genetics


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