Development of a dye-based enzymatic recombinase amplification method for rapid and visual detection of acute...

Acute hepatopancreatic necrosis disease (AHPND) has become one of the most devastating threats to global shrimp farming, causing widespread economic losses. As there is currently no effective treatment, early detection plays a crucial role in preventing disease outbreaks. However, current diagnostic methods are often complex, expensive, and impractical for on-site use in resource-limited environments. In this study, we developed a visual detection method based on enzymatic recombinase amplification (ERA) combined with the Diamond Nucleic Acid Dye, offering a rapid, cost-effective, and high-sensitivity solution for AHPND detection. By optimizing the key parameters such as primer concentration, dye-to-ERA ratio, amplification temperature, and reaction time, we established optimal conditions that resulted in strong fluorescence signals and minimal background noise. The method demonstrated remarkable specificity for AHPND, with no cross-reactivity observed with other known pathogens. Additionally, the detection limitation was as low as 5.27 × 10⁰ copies/μL, and the technique maintained stable amplification even in the presence of 800 ng of interfering healthy shrimp DNA. The comparison with the gold standard nested PCR showed a high concordance rate of 84.6%, validating the reliability of this method for rapid field diagnosis. This method provides a promising alternative for early, on-site detection of AHPND in shrimp farming, offering a significant tool for controlling disease outbreaks and minimizing economic impact in the shrimp culture industry.