A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities.

Automated summary

The emergence of SARS-CoV-2 variants of concern (VOCs) with mutations linked to increased transmissibility, vaccine escape, and virulence has led to the need for widespread genomic surveillance. We have developed multiplex high-resolution melting assays to identify Alpha, Beta, Delta, and Omicron VOCs. The assays were evaluated against whole genome sequencing and showed high sensitivity and specificity. These multiplex assays have the potential to be used for high throughput surveillance of SARS-CoV-2 VOCs, especially in areas with limited genomics facilities.