Identification of conserved SARS-CoV-2 gene regions for diagnostic tool development through whole-genome sequencing

The high mutation frequency of the SARS-CoV-2 spike (S) gene undermines the reliability of S-based diagnostics. Identifying conserved protein epitopes that remain stable across SARS-CoV-2 variants is paramount in resource-limited settings. This study aims to identify conserved gene regions of SARS-CoV-2 using whole-genome sequencing and immunoinformatics to support the development of monoclonal antibody-based rapid diagnostics. SARS-CoV-2 positive samples (Ct ≤ 30) collected in Ethiopia (June–August 2022) from a retrospective study (n = 70) were sequenced using Illumina NextSeq-550. Conserved regions were identified via Multiple Alignment using Fast Fourier Transform, entropy plots, and mutation profiling. B-cell epitopes were predicted and assessed for antigenicity and cross-reactivity. Structural modeling was conducted using AlphaFold and visualization tools. High-quality whole-genome sequencing of 63 SARS-CoV-2 samples revealed conserved regions in the nucleocapsid gene across major variants. Three surface-exposed, antigenic, and non-allergenic B-cell epitopes were identified with low cross-reactivity to common human coronaviruses. Structural modeling and docking confirmed their accessibility and strong antibody binding, supporting their potential for robust diagnostic applications. In conclusion, conserved N protein epitopes identified in this study are promising targets for developing robust, variant-resistant rapid diagnostic tools.