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

Richard Hale,Samir Dervisevic,Marcus Pond,Paul Randell,Keith Stanley,Luke B Snell,Penelope R Cliff,Lindsay Coupland,Cheryl Williams,Joel Paul,Saidat Ebie,Peter Crowley

doi:10.3390/v13102028

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.

Highlights

IntroductionWhole-genome sequencing provides high-resolution data that enable investigation of pathogen evolution and population structure
In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), was first isolated from Wuhan city, China and within three months of this discovery, the global community was challenged with a devastating pandemic [1].Whole-genome sequencing provides high-resolution data that enable investigation of pathogen evolution and population structure
Whilst tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission, sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations [2]

Summary

Introduction

Whole-genome sequencing provides high-resolution data that enable investigation of pathogen evolution and population structure. When combined with robust epidemiological data, it is possible to gain insights into SARS-CoV-2’s origins, transmission and responses to control measures. Since the start of the pandemic, sequencing efforts and data sharing have facilitated tracking of the pandemic, identifying multiple independent virus introductions into different countries and novel mutations in the SARS-CoV-2 genome. Whilst tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission, sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations [2]. PCR-based genotyping is a rapid, high-throughput and cost-effective alternative for screening positive SARS-CoV-2 samples in many settings

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