Innovative biomarker design for cholera diagnosis through bioinformatics analysis

Cholera, caused by Vibrio cholerae , remains a significant public health challenge, especially in resource-limited settings. Existing diagnostic methods like stool culture are accurate but impractical due to time constraints and infrastructure requirements which limits their practicality in resource-limited settings. Rapid diagnostic tests (RDTs) offer quicker results but have shown variability in sensitivity and specificity. This study aimed to improve cholera diagnosis by identifying and evaluating four key V. cholerae genes— ctxA, ompW, toxR, and tcpA —as potential targets for a rapid immunochromatographic assay (ICA)-based diagnostic kit. More than 160 Protein sequences were retrieved globally and screened using computational tools for antigenicity, allergenicity, membrane topology, and conserved epitope prediction. Two peptide biomarkers were designed and validated through in silico analyses, including physicochemical property evaluation, molecular docking, protein-protein interaction studies, and molecular cloning simulations. Biomarker 1 has an antigenic score of 0.9412 while Biomarker 2 has an antigenic score of 0.7126, both are non.allergen and solubility scores of 0.605 and 0.482 respectively. Based on the physicochemical properties, biomarker 1 contains 977 total amino acids, and the molecular weight is 97169.32 and biomarker 2 contains 289 total amino acids, and the molecular weight is 27738.70. Biomarkers 1 and 2 showed favorable Z-scores of -2.74 and -1.82, respectively, indicating structural reliability. The identified biomarkers demonstrate strong potential for incorporation into a robust, rapid, and accessible cholera diagnostic kit. Such a tool could facilitate early detection, improve patient outcomes, and support public health surveillance efforts in endemic regions. Field validation is recommended to confirm diagnostic performance under real-world conditions.