Development of a Multiplex Tandem PCR (MT-PCR) Assay for the Detection of Emerging SARS-CoV-2 Variants

Abstract

The emergence of variants of SARS-CoV-2 has created challenges for the testing infrastructure. Although large-scale genome sequencing of SARS-CoV-2 has facilitated hospital and public health responses, access to sequencing facilities globally is variable and turnaround times can be significant, so there is a requirement for rapid and cost-effective alternatives. Applying a polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) approach enables rapid (<4 h) identification of SARS-CoV-2 lineages from nucleic acid extracts, through the presence or absence of a panel of defined of genomic polymorphisms. For example, the B.1.1.7 lineage (“UK”, “Alpha”, or “Kent” variant) is characterised by 23 mutations compared to the reference strain, and the most biologically significant of these are found in the S gene. We have developed a SARS-CoV-2 typing assay focused on five positions in the S gene (HV69/70, N501, K417, E484 and P681). This configuration can identify a range of variants, including all the “Variants of Concern” currently designated by national and international public health bodies. The panel has been evaluated using a range of clinical isolates and standardised control materials at four UK hospitals and shows excellent concordance with the known lineage information derived from full sequence analysis. The assay has a turnaround time of about three hours for a set of up to 24 samples and has been utilised to identify emerging variants in a clinical setting.

Broad Neutralization of SARS-CoV-2 Variants, Including Omicron, following Breakthrough Infection with Delta in COVID-19-Vaccinated Individuals

“…Delta variant infection was confirmed using whole-genome sequencing as previously described ( 18 ) or using multiplex tandem-PCR (MT-PCR) ( 31 ).…”

Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification

“…Overall, these reactions showed high concordance with WGS, demonstrating PPA 96-100% and NPA 95-100% for all targeted mutations. Several groups have previously described similar approaches to SARS-CoV-2 variant determination by RT-qPCR and digital droplet RT-PCR, particularly for the spike del69-70, E484K, and N501Y positions (32)(33)(34)(35)(36)(37)(38)(39). Some of these assays included additional mutation sites that were not in our study, such as spike del144 or ORF1a Δ3675-3677 (32,38).…”

“…Several groups have previously described similar approaches to SARS-CoV-2 variant determination by RT-qPCR and digital droplet RT-PCR, particularly for the spike del69-70, E484K, and N501Y positions (30-37). Some of these assays included additional mutation sites that were not in our study, such as spike del144 or ORF1a Δ3675–3677 (30, 36).…”

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification

“…Several groups previously described similar approaches to SARS-CoV-2 variant determination by RT-qPCR and digital droplet RT-PCR, particularly for the spike del69–70, E484K, and N501Y mutations ( 32 – 39 ). Some of those assays included additional mutation sites that were not in our study, such as spike del144 or open reading frame 1a (ORF1a) Δ3675–3677 ( 32 , 38 ).…”