Rapid Detection of Aeromonas veronii Using GGDEF Gene-Based Colorimetric Loop-Mediated Isothermal Amplification.

Abstract

Aeromonas veronii has emerged as an important fish pathogen that poses threats to the aquaculture industry worldwide, and rapid detection is essential to minimise negative economic impact. In this study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect A. veronii, targeting a gene encoding GGDEF domain-containing diguanylate cyclase. The entire LAMP reaction could be completed in 30 min at 65°C based on colour change from pink to yellow. In addition, the assay demonstrated 100% specificity with no cross reaction with other common fish pathogens. The detection limit (LOD) was 500 pg/μL using purified plasmid and 1.72 × 10⁵ cfu/mL (equivalent to 1.72 × 10² cfu/reaction) using crude genomic DNA extracted from the pure culture of A. veronii ATCC 9071. LAMP demonstrated comparable performance to conventional PCR using 57 bacterial isolates, with 100% (20/20) specificity, 91.9% (34/37) sensitivity, 94.7% (54/57) accuracy and a 0.888 Cohen's kappa value. Lastly, the LOD of LAMP in a spiked water sample was 1.72 × 10⁵ cfu/mL (equivalent to 3.44 × 10³ cfu/reaction). Overall, our LAMP assay has a high level of diagnostic agreement with conventional PCR and can be used as a valuable tool for the rapid detection of A. veronii from environmental samples.