Hot-Nucleic Acid Lateral Flow Assay (Hot-NALFA): A Novel Method for Point Mutation Detection

This study presents "hot-nucleic acid lateral flow assay" (Hot-NALFA), an innovative method for detecting point mutations in nucleic acid sequences. Hot-NALFA integrates the temperature-sensitive allelic discrimination properties of molecular beacons into a lateral flow assay format operating at elevated temperatures (~45–55°C). This approach enables end-point, visual detection of mutations, eliminating the requirement for real-time fluorescence instrumentation traditionally associated with molecular beacon-based allelic discrimination. The method exhibits high specificity, generating a visible test line exclusively in the presence of mutant targets. The technique was validated with synthetic sequences representing two clinically significant mutations: P681H in the S gene of SARS-CoV-2 and H526Y in the rpob gene of Mycobacterium tuberculosis. Optimal binary discrimination between wild-type and mutant sequences was achieved within the temperature range of 45–55°C for both mutations. By enabling direct allelic discrimination in a simple and cost-effective lateral flow assay format, Hot-NALFA addresses key limitations of current mutation detection methods and holds promise for point-of-care diagnostics.