Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.
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Authors
Al-Beidh, Farah
Bibi, Sagida
Costa Clemens, Sue Ann
Estcourt, Lise
Evans, Amy
Fish, Matthew
Folegatti, Pedro M
Gordon, Anthony C
Jennings, Aislinn
Laing, Emma
Lambe, Teresa
MacIntyre-Cockett, George
Menon, David
Mouncey, Paul R
Nguyen, Dung
Pollard, Andrew J
Ramasamy, Maheshi N
Roberts, David J
Rowan, Kathryn M
Rynne, Jennifer
Williams, Sarah
Harvala, Heli
AMPHEUS Project, REMAP-CAP Immunoglobulin Domain UK Investigators, and Oxford COVID-19 Vaccine Trial Group
Publication Date
2022-04-20Journal Title
J Clin Microbiol
ISSN
0095-1137
Publisher
American Society for Microbiology
Volume
60
Issue
4
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Ratcliff, J., Al-Beidh, F., Bibi, S., Bonsall, D., Costa Clemens, S. A., Estcourt, L., Evans, A., et al. (2022). Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.. J Clin Microbiol, 60 (4) https://doi.org/10.1128/jcm.02283-21
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 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.
Keywords
Diagnostics, Next-generation Sequencing, Variant Identification, Sars-cov-2, Variants Of Concern, Allele-specific Probe Pcr
Sponsorship
Wellcome Trust (215522)
Identifiers
35321556, PMC9020347
External DOI: https://doi.org/10.1128/jcm.02283-21
This record's URL: https://www.repository.cam.ac.uk/handle/1810/336409
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