Development of a colorimetric loop-mediated isothermal amplification (cLAMP) assay for the rapid and visual detection of Aeromonas dhakensis infection in aquaculture

Abstract

Aeromonas dhakensis, an emerging zoonotic pathogen prevalent in aquatic environments, poses a significant threat, particularly in causing motile Aeromonas septicemia in aquaculture and food safety concerns. Timely detection of the pathogen is crucial for effective management. Herein, we develop a colorimetric loop-mediated isothermal amplification (cLAMP) assay for rapid, accurate, and convenient detection of A. dhakensis targeting ricin B lectin domain. The assay enabled naked-eye detection of the A. dhakensis nucleic acid within 20 min, based on a colour change from pink to yellow. The detection limit of the cLAMP assay was 50 pg of defined plasmid control DNA (equivalent to 1.43 × 10⁷ copies/μL) or approximately 1.0 × 10⁶ CFU/mL of whole genome DNA extracted from pure bacterial culture, in a 10 μL reaction. The assay demonstrated high specificity, yielding positive results in all 93 A. dhakensis samples and showing no cross-reaction with 24 non-target samples. Performance was further evaluated by comparing cLAMP results with qPCR in 117 bacterial samples, achieving 100 % concordance between the two methods. In summary, our findings demonstrate the simplicity, high sensitivity and accuracy of the cLAMP for A. dhakensis detection, making it as a valuable tool for rapid identification of this species within the Aeromonas genus, which is crucial for effective disease management.