A steam-mediated isothermal amplification and flocculation-based detection platform for electricity-free point of care diagnostics

Abstract

Approximately 9% of the global population lacks access to reliable electricity, and the absence of affordable, electricity-free diagnostic tools hinders early detection of infectious diseases, exacerbating public health burdens in resource-limited settings. We introduce SteamFloc-LAMP – an electricity-free molecular diagnostic platform engineered for instrument-free detection of pathogenic nucleic acid targets. The platform leverages steam-mediated heating from boiling water to sustain the isothermal conditions required for Loop-Mediated Isothermal Amplification (LAMP). Thermal characterization of the steam-mediated heating system identified parameters that enable the consistent maintenance of optimal temperatures for LAMP reactions with minimal fluctuations. Visual end-point detection was achieved through a bridging flocculation mechanism, which exploits the interaction between LAMP amplicons, spermine, and charcoal particles, leading to visible aggregation in positive samples, thus enabling naked-eye detection without the need for specialized equipment or expensive reagents like fluorophores or colorimetric dyes. The SteamFloc-LAMP assay targeted the lipL32 gene, recognized for its exclusivity to pathogenic Leptospira strains. The assay achieved a detection limit of 100 fg of genomic DNA per reaction (∼90 genome copies). Specificity tests using lipL32-specific primers demonstrated the assay's ability to distinguish pathogenic Leptospira accurately, with no cross-reactivity with ligB, ligA, or lipL41 genes found in nonpathogenic strains. A blind test involving DNA extracted from Leptospira reference cultures further validated the assay's diagnostic accuracy, aligning with PCR results. These findings demonstrate the SteamFloc-LAMP assay as a reliable, simple, and cost-effective field deployable diagnostic tool with significant implications for point-of-care detection.