An integrated filtration-preconcentration and LAMP workflow for rapid detection of faecal contamination in seawater.

Coastal contamination from untreated wastewater poses public health risks in recreational, and shellfish production, areas. Current monitoring relies on culture-based Escherichia coli quantification, which requires 24-48 h for results and features low sampling frequencies, thereby precluding early detection. Optimizing surveillance requires rapid, cost-effective, and in situ deployable methods like Loop-mediated isothermal amplification (LAMP). Here, a controlled proof concept coupling a multi-stage preconcentration framework with a robust colorimetric LAMP assay was developed for rapid and specific detection of E. coli in seawater. Initially, five candidate primer sets targeting uidA and ybbW genes were evaluated, selecting two highly efficient sets (uidA-4 and ybbW) with superior speed (Time to threshold (Tt) < 5 min). After optimisation, validation demonstrated 100% inclusivity across 50 E. coli isolates and complete exclusivity against 31 non-target bacteria. Purified DNA confirmed analytical sensitivity down to 10⁻³ pg/µL, though amplification efficiency dropped with whole cells. This limitation was overcome via sequential filtration through three decreasing pore sizes (20 µm, 3 µm, and 0.45 µm), followed by direct thermal lysis (100 °C, 15 min, under constant agitation) on the final membrane, avoiding biomass loss, or sample dilution. By tailoring processed water volumes to monitoring scenarios (250-500 mL for recreational waters; 1.5-2.0 L for shellfish harvesting zones), the system effectively compensated for low E. coli loads, detecting levels down to 1 CFU/mL in under 1 h at a running cost of approximately 5 € per sample. Thus, this methodology establishes a resilient, cost-effective foundation for the future development of autonomous, long-term early-warning systems for in situ marine faecal contamination monitoring.