A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant.

High-resolution melting (HRM) analysis is a PCR-based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A-T to T-A transversion. As the N501Y substitution results from A-T to T-A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 103-106 copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 105-1010 copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.

Rapid Identification of SARS-CoV-2 Omicron BA.5 Spike Mutation F486V in Clinical Specimens Using a High-Resolution Melting-Based Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.5 emerged as of February 2022 and replaced the earlier Omicron subvariants BA.1 and BA.2. COVID-19 genomic surveillance should be continued as new variants seem to subsequently appear, including post-BA.5 subvariants. A rapid assay is needed to differentiate between the currently dominant BA.5 variant and other variants. This study successfully developed a high-resolution melting (HRM)-based assay for BA.4/5-characteristic spike mutation F486V detection and demonstrated that our assay could discriminate between BA.1, BA.2, and BA.5 subvariants in clinical specimens. The mutational spectra at two regions (G446/L452 and F486) for the variant-selective HRM analysis was the focus of our assay. The mutational spectra used as the basis to identify each Omicron subvariant were as follows: BA.1 (G446S/L452/F486), BA.2 (G446/L452/F486), and BA.4/5 (G446/L452R/F486V). Upon mutation-coding RNA fragment analysis, the wild-type fragments melting curves were distinct from those of the mutant fragments. Based on the analysis of 120 clinical samples (40 each of subvariants BA.1, BA.2, and BA.5), this method's sensitivity and specificity were determined to be more than 95% and 100%, respectively. These results clearly demonstrate that this HRM-based assay is a simple screening method for monitoring Omicron subvariant evolution.

Discrimination of SARS-CoV-2 Omicron Sublineages BA.1 and BA.2 Using a High-Resolution Melting-Based Assay: a Pilot Study.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting curves of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variant BA.1/BA.1.1, and 8 Omicron variant BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2. IMPORTANCE This study seeks to apply a novel high-resolution melting (HRM) assay to identify and discriminate BA.1 and BA.2 sublineages of the SARS-CoV-2 Omicron variant. Variant BA.2 may cause more severe disease than variant BA.1, meaning that identifying this variant is an important step toward improving the care of patients suffering from COVID-19. However, screening for these variants remains difficult, as current methods mostly rely on next-generation sequencing, which is significantly costlier and more time-consuming than other methods. We believe that our study makes a significant contribution to the literature because we show that this method was 100% sensitive and over 97.5% specific in our confirmation of 128 clinical samples.

Simultaneous Screening of SARS-CoV-2 Omicron and Delta Variants Using High-Resolution Melting Analysis.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, the Omicron variant (Pango lineage B.1.1.529), was identified in South Africa in late September 2021. This variant has multiple spike protein deletions and mutations, with 15 amino acid substitutions detected in the receptor-binding domain (RBD). These RBD substitutions are hypothesized to increase infectivity and reduce antibody affinity, which is supported by recent data showing that the Omicron variant spreads faster than the Delta variant (Pango lineage B.1.617.2). Thus, this increase in infectivity should lead to Omicron being the dominant variant and developing screening tests that discriminate between Omicron and Delta variants is urgently needed. In this study, we successfully developed a novel screening assay using high-resolution melting analysis, in which two genotypes at G446/L452 and S477/T478 RBD were determined (G446S/L452 and S477N/T478K for Omicron; G446/L452R and S477/T478K for Delta). Using synthetic DNA fragments, we confirmed both melting point and melting peak shape of the RBD Omicron variant was distinguishable from those of wild-type and the Delta variant. Although this study was conducted without clinical samples, these results suggest that our high-resolution melting (HRM)-based genotyping method can readily identify the Omicron and Delta variants. This simple method should contribute to the rapid identification of SARS-CoV-2 variants and thus prevent potential widespread infection and inflow of the Omicron variant.

Development of a genotyping platform for SARS-CoV-2 variants using high-resolution melting analysis.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus causing coronavirus disease 2019 (COVID-19), has been expanding globally since late 2019. SARS-CoV-2, an RNA virus, has a genome sequence that can easily undergo mutation. Several mutated SARS-CoV-2 strains, including those with higher infectivity than others, have been reported. To reduce SARS-CoV-2 transmission, it is crucial to trace its infection sources. Here, we developed a simple, easy-to-use genotyping method to identify SARS-CoV-2 variants using a high-resolution melting (HRM) analysis. METHODS: We investigated five mutation sites, A23403G, G25563T, G26144T, T28144C, and G28882A, which are known strain determinants according to GISAID clades (L, S, V, G, GH, and GR). RESULTS: We first employed synthetic DNA fragments containing the five characteristic sites for HRM analysis. All sequences clearly differentiated wild-type from mutant viruses. We then confirmed that RNA fragments were suitable for HRM analysis following reverse transcription. Human saliva did not negatively affect the HRM analysis, which supports the absence of a matrix effect. CONCLUSIONS: Our results indicate that this HRM-based genotyping method can identify SARS-CoV-2 variants. This novel assay platform potentially paves the way for accurate and rapid identification of SARS-CoV-2 infection sources.