Rapid monitoring of SARS-CoV-2 variants through high resolution melt analysis

ABSTRACT

Since September 2020 the current global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus is characterized by a succession of waves of infection due to the emergence of new variants of the original virus, presenting various genomic mutations. Many mutations are present in the gene encoding the Spike protein, the main target of the nucleic acidbased vaccines. The Variants of Concern that have been reported since autumn 2020 include Alpha/ B.1.1.7 and sublineages, Beta/B.1.351, Gamma/P.1 and sublineages, Delta/B.1.617.2 and sublineages, Omicron/ B.1.1.529 and sublineages. The rapid and cheap variant monitoring in the population is pivotal for epidemiological studies and for the prompt detection of SARS-CoV-2 variants characterized by high transmissibility or reduced susceptibility to neutralizing antibodies induced by vaccination. Surveillance of genomic variants is currently based on viral whole genome sequencing (WGS) performed on a random fraction of samples positive to molecular tests. WGS involves high costs and extended analysis time compared to a PCR-based diagnostic test, as well as specialized staff and expensive instruments. To rapidly identify the variant in samples positive to SARS-CoV-2, different rapid tests based on real-time PCR and high-resolution melting (HRM) were designed and applied on 88 oropharyngeal swab samples collected from October 2020 to February 2022 (84 positive samples and 4 negative samples). The HRM results were confirmed by PCR product sequencing. Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR assay for COVID-19 testing. Moreover, 83 samples out of 84 (98.8%) were correctly identified as follows 8 Wuhan (wild type), 12 Alpha, 23 Delta, 37 Omicron BA.1, 1 Omicron BA.1.1, 2 Omicron BA.2. With our lab equipment, about 10 samples can be processed every 3 hours at the cost of 8.5 per sample, including RNA extraction. The identified variants overlapped with mutation and case prevalence over time in Italy (as reported in outbreak.info, which collects genomic data from the GISAID Initiative), accounting for the feasibility of this approach.