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

Area of Science:

• Microbiology
• Aquatic Animal Health
• Molecular Diagnostics

Background:

• Aeromonas veronii is a significant fish pathogen impacting global aquaculture.
• Rapid and accurate detection methods are vital to mitigate economic losses.
• Existing detection methods can be time-consuming or require specialized equipment.

Purpose of the Study:

• To develop a rapid, colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting Aeromonas veronii.
• To evaluate the specificity, sensitivity, and accuracy of the developed LAMP assay.
• To compare the performance of the LAMP assay with conventional PCR.

Main Methods:

• A colorimetric LAMP assay was designed targeting a GGDEF domain-containing diguanylate cyclase gene in A. veronii.
• The assay was optimized for reaction time (30 min) and temperature (65°C).
• Specificity was tested against common fish pathogens, and sensitivity was determined using purified plasmid and genomic DNA.

Main Results:

• The LAMP assay provided a clear color change (pink to yellow) within 30 minutes at 65°C.
• 100% specificity was observed against other fish pathogens.
• The limit of detection (LOD) was 500 pg/μL for plasmid DNA and 1.72 × 10^5 cfu/mL for genomic DNA.
• The assay showed high agreement with conventional PCR (94.7% accuracy, 0.888 Cohen's kappa).
• LOD in spiked water samples was 1.72 × 10^5 cfu/mL.

Conclusions:

• The developed colorimetric LAMP assay is a rapid, specific, and sensitive tool for detecting Aeromonas veronii.
• This assay offers a valuable alternative to conventional PCR for routine diagnostics in aquaculture.
• It facilitates timely interventions to control A. veronii outbreaks and minimize economic impact.