Fast and Low-Cost Detection of SARS-CoV-2 Variant-Associated Mutations by One-Step RT-qPCR Assay with High Resolution Melting

The emergence of more virulent and transmissible variants of the type 2 coronavirus causing severe acute respiratory syndrome (SARS-CoV-2) has triggered expensive genomic surveillance that proves difficult to sustain over time.Development of a simple and low-cost one-step RT-qPCR assay with High Resolution Melting (HRM-RTqPCR) analysis for the detection of mutations associated with SARS-CoV-2 Gamma (P.1) and Lambda (C.37) variants. In silico melting simulation was performed to select amplicons in spike-gene to identify mutations associated with Gamma and Lambda variants. Twenty four SARS-CoV-2 positive samples were sequenced and analyzed by HRM-RTqPCR. Two candidate amplicons were selected, amplicon 1 for identification of Gamma and amplicon 2 for Lambda. Clinical samples were associated as Alpha, Gamma or Lambda variants by sequencing. Melting temperature of amplicon 1 for samples associated with Gamma was 77.50 °C ± 0.1, while for the remaining samples (Alpha and Lambda) was 78.33 °C ± 0.09. Amplicon 2 showed a Tm of 76.53 °C ± 0.07 for samples associated with Lambd, while the rest of samples (Alpha and Gamma) showed a Tm of 77.28 °C ± 0.09. An assay was developed to identify mutations associated with SARS-CoV-2 variants by a simple one-step HRM-RTqPCR. Bioinformatic tools helped in the selection of targets. This work contributes to optimizing time and resources in the active surveillance of variants.