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. 2021 Oct 8;13(10):2028.
doi: 10.3390/v13102028.

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

Richard Hale  1 , Peter Crowley  2 , Samir Dervisevic  3 , Lindsay Coupland  3 , Penelope R Cliff  4 , Saidat Ebie  4 , Luke B Snell  4 , Joel Paul  5 , Cheryl Williams  5 , Paul Randell  6 , Marcus Pond  6 , Keith Stanley  2
Affiliations

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

Richard Hale et al. Viruses. .

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.

Keywords: SARS-CoV-2; in vitro diagnostic test; variants.

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Conflict of interest statement

R.H., P.C. and K.S. are employees of AusDiagnostics. Other authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Screenshot of assay output using AusDiagnostics results software. The upper right panel shows the amplification curves for the individual assays, while the lower right panel shows their respective amplicon melt curve. The left panels show sample information and the output of the diagnostic algorithm.
Figure 2
Figure 2
ORF8 deletion (Delta 394) observed in the cluster of samples from Oldham, UK.
See this image and copyright information in PMC

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