Development of novel hairpin-mediated isothermal amplification assay for rapid detection of Aeromonas hydrophila in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758)

Rapid and field-deployable nucleic acid amplification is essential in pathogen detection. Here, we developed a novel fluorescence-based isothermal amplification assay termed Hairpin-Mediated Isothermal Amplification (HAMP) to detect Aeromonas hydrophila, a zoonotic and motile pathogenic bacterium in Nile tilapia, Oreochromis niloticus. In the Philippines, infection caused by A. hydrophila is a major concern in the tilapia aquaculture industry, as it can cause Motile Aeromonas Septicemia. The newly developed HAMP assay demonstrated a sensitive and fast detection method for this highly virulent bacterium. The method used computationally designed primers with hairpin structures to maintain strong target specificity. The suitable incubation temperature is 63°C and it can detect bacteria within 45 minutes. The assay can detect A. hydrophila DNA with a concentration as low as 1.43x10^-5 ng/µL. When compared side-by-side with the standard polymerase chain reaction assay, the newly optimized fluorescence-based method reduced detection time by 45 minutes. Non-target aeromonads were not amplified in both methods, exemplifying the specificity of the assay while maintaining reliable pathogen detection. Both methods produced clear and specific bands for the positive control, and there were no bands for the negative control. The method requires no specialized equipment beyond a portable heating device, highlighting it as a promising solution for fast, sensitive and accurate nucleic acid detection, particularly useful in resource-limited settings such as fish farms.