Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 20;60(4):e0228321.
doi: 10.1128/jcm.02283-21. Epub 2022 Mar 24.

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

Affiliations

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

Jeremy Ratcliff et al. J Clin Microbiol. .

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

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Performance of ASP-PCR and NGS in REMAP-CAP trial. (A to C) Individual method performance on REMAP-CAP samples. Integers indicate total samples tested in 1-log data bins. (D) Probit regression of likelihood of lineage designation success for ASP-PCR, NGS-SNP, and NGS-Pangolin with 95% confidence intervals derived from REMAP-CAP samples.
FIG 2
FIG 2
Performance of ASP-PCR and NGS in COV003 trial. (A to C) Individual method performance on COV003 samples. Numbers above indicate total samples in 1-log data bins. (A) Probit regression of likelihood of lineage designation success for ASP-PCR, NGS-SNP, and NGS-Pangolin with 95% confidence intervals derived from COV003 samples.
FIG 3
FIG 3
Impact of degraded RNA in COV003 samples on method performance. (A) Genome coverage of COV003 samples plotted versus sample viral load. Samples with ≤20,000 bases with ≥2 reads and viral loads of >106 IU/mL were defined as degraded (red box). (B to D) Individual method performance on COV003 degraded and nondegraded samples. Numbers above indicate total samples in 1-log data bins.
FIG 4
FIG 4
Effect of molecular modification on S:D1118H/Alpha ASP-PCR performance. Percentage of total samples successfully typed using approach described in Materials and Methods. P values from Fisher’s exact test. Lineage designations (e.g., wild type [WT] or Alpha/B.1.1.7) were concordant for all samples for all methods.

References

    1. World Health Organization. 2021. Genomic sequencing of SARS-CoV-2: a guide to implementation for maximum impact on public health. World Health Organization, Geneva, Switzerland.
    1. Lythgoe KA, Hall M, Ferretti L, de Cesare M, MacIntyre-Cockett G, Trebes A, Andersson M, Otecko N, Wise EL, Moore N, Lynch J, Kidd S, Cortes N, Mori M, Williams R, Vernet G, Justice A, Green A, Nicholls SM, Ansari MA, Abeler-Dörner L, Moore CE, Peto TEA, Eyre DW, Shaw R, Simmonds P, Buck D, Todd JA, on behalf of the Oxford Virus Sequencing Analysis Group (OVSG), Connor TR, Ashraf S, da Silva Filipe A, Shepherd J, Thomson EC, The COVID-19 Genomics UK (COG-UK) Consortium , Bonsall D, Fraser C, Golubchik T. 2021. SARS-CoV-2 within-host diversity and transmission. Science 372:eabg0821. 10.1126/science.abg0821. - DOI - PMC - PubMed
    1. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Hengartner N, Giorgi EE, Bhattacharya T, Foley B, Hastie KM, Parker MD, Partridge DG, Evans CM, Freeman TM, de Silva TI, Sheffield COVID-19 Genomics Group, McDanal C, Perez LG, Tang H, Moon-Walker A, Whelan SP, LaBranche CC, Saphire EO, Montefiori DC. 2020. Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell 182:812–827.e19. 10.1016/j.cell.2020.06.043. - DOI - PMC - PubMed
    1. Tao K, Tzou PL, Nouhin J, Gupta RK, de Oliveira T, Kosakovsky Pond SL, Fera D, Shafer RW. 2021. The biological and clinical significance of emerging SARS-CoV-2 variants. Nat Rev Genet 22:757–773. 10.1038/s41576-021-00408-x. - DOI - PMC - PubMed
    1. Chiara M, D’Erchia AM, Gissi C, Manzari C, Parisi A, Resta N, Zambelli F, Picardi E, Pavesi G, Horner DS, Pesole G. 2021. Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities. Brief Bioinform 22:616–630. 10.1093/bib/bbaa297. - DOI - PMC - PubMed

Publication types

Supplementary concepts