Development and Validation of a Colorimetric LAMP Assay Targeting the Plasmid pBI143 for Human Fecal Pollution Tracking in Urban Waterways

Abstract This study developed and validated a colorimetric loop-mediated isothermal amplification (cLAMP) assay targeting the mobA gene of the human fecal-associated plasmid pBI143 for rapid detection of sewage pollution. Analytical sensitivity was evaluated using synthetic controls, untreated sewage, and environmental waters seeded with sewage, with results benchmarked against a quantitative PCR (qPCR) assay. Host sensitivity and specificity were assessed using untreated sewage and nonhuman fecal samples collected in Australia and the United States. Environmental validation used urban waterway samples collected under wet- and dry-weather conditions. The cLAMP assay consistently detected pBI143 at ≥ 103 gene copies (GC)/μL in control material and all untreated sewage samples, yielding an overall host sensitivity of 1.00. Host specificity was 0.79 for cLAMP compared with 0.69 for qPCR/dPCR. Sewage dilution experiments demonstrated detection down to approximately 2.45–2.54 log10 GC, while environmental seeding experiments showed consistent detection at sewage inputs ≥2 μL/200 mL sample. Field application showed good qualitative agreement between cLAMP and qPCR. The pBI143 marker was strongly correlated with the human-associated fecal marker Bacteroides HF183. Overall, the pBI143 cLAMP assay provides a rapid and inexpensive qualitative approach for detecting sewage pollution and could complement existing MST tools for environmental water quality monitoring.