Use of amplicon melt temperature to detect SARS-CoV-2 Lineage B.1.1.7 variant through the G28048T mutation in the ORF 8 gene

Abstract

A new variant of SARS-CoV-2 (Lineage B.1.1.7) was identified in the UK in December 2020 which was associated with higher transmissibility of COVID-19. The AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay is used at sixteen UK hospitals and detects part of the ORF8 gene (together with a segment from the ORF1a gene). The objective of this study was to determine if the recently identified mutation in ORF8 (G28048T) in the B.1.1.7. lineage could be used to identify the new variant quickly in clinical cases with PCR positive results. The melt data from SARS-CoV-2 positives from two hospitals (October through December 2020) were reviewed, and distribution over time and location was evaluated. A low melt variant of the ORF8 amplicon started to appear in samples from Guy's and St. Thomas’ NHS Trust, London, at the start of November, and grew as a proportion of the total positives during the subsequent two months. These low melt variants were very rare during the same period at the Northern Care Alliance, Greater Manchester, North West of UK. It was confirmed that these carried the G28048T mutation. The geographic and temporal distribution of the low melt amplicons makes it very likely that these are lineage B.1.1.7 strains. The melt temperature of this amplicon could be used to discriminate between the original and new variants in advance of the full sequencing of the isolate. However, the appearance of other mutations in the same amplicon means that this approach would be of diminishing value over time.

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

“…The format of the assay, where both the "wildtype" and variant SNP are measured simultaneously, has the potential to allow early identification of other SNPs, such as E484A (a potential "escape" mutation [12]), as it will exhibit delayed amplification (typically 6-10 cycles) in both assays. Automated melt curve analysis is also provided for all the amplicons and this has the potential to give further information about a variant [13]. The selection of the SNPs for this panel was strongly predicated on the guidance of the UK Government, but the same approach could, in principle, be made for any set of SNPs/deletions.…”