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.
ABSTRACT
The current global pandemic (COVID-19) caused by the new Betacoronavirus SARS-CoV-2 is characterized by successive waves of infection due to new variants that include mutations in the gene encoding the Spike protein, the main target of the nucleic acidbased vaccines. In fact, as of autumn 2020, several countries have reported the detection of SARS-CoV-2 variants that have spread more efficiently (referred to as variants of concern by WHO). Such variants include the Alpha variant (English variant, B.1.1.7), the Beta variant (South African variant, B.1.351), the Gamma variant (Brazilian variant, P.1), and the more recent Delta variant (Indian variant, B.1.617. 2). Therefore, it is pivotal to monitor the virus and the onset of SARSCoV-2 variants characterized by high transmissibility or reduced susceptibility to neutralizing antibodies induced by vaccination.Surveillance of genomic variants is currently based on sequencing of viral genomes performed on a random fraction of samples positive by molecular test. The sequencing of 228 SARS-CoV-2 positive samples by ASUR Marche Area Vasta 1 (Fano-Pesaro-Urbino) from February to June 2021 highlighted the progressive increase of variants (mainly B.1.1.7 and to a lesser extent P.1) from early February until March 18th. From March 18th onwards, only variants B.1.1.7 and P.1 were detected. DNA sequencing involves high costs and extended analysis time compared to a PCR-based diagnostic test. To rapidly identify the samples containing virus variants to be sequenced for complete characterization, in synergy with the University of Urbino, five rapid tests based on real-time PCR and high-resolution melting (HRM) were designed on the gene encoding the Spike protein. Preliminary results indicated that the sensitivity of the assays was not significantly different from that of commercial molecular tests. Furthermore, through HRM analysis, it was possible to discriminate amplicons with mutation 1709 C > A causing the amino acid substitution A570D, specific for the alpha variant.