Abstract

Acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus harboring specific virulence plasmids, results in high mortality in Litopenaeus vannamei and leads to substantial economic losses in shrimp farming. No effective treatments currently exist for AHPND, so the development of rapid and accurate pathogen detection methods is essential for early detection of infected individuals, allowing timely intervention to prevent large-scale outbreaks and ultimately reduce economic losses. Recombinase polymerase amplification (RPA) is an ideal on-site detection method, offering rapid results, high sensitivity, and the ability to operate at low temperatures. The RPA visualization method primarily depends on gel electrophoresis, which increases complexity and requires advanced equipment, thus limiting its field applicability. Therefore, improving the RPA visualization technique is crucial for enabling on-site detection. This study systematically assessed 14 nucleic acid dyes for visualizing RPA results. Diamond Nucleic Acid Dye, at a 1000× concentration, exhibited excellent color contrast and fluorescence intensity, making it the optimal choice for RPA-based visualization. Using this dye, a highly sensitive, specific, and stable AHPND visualization detection method was developed. Specificity testing demonstrated the method's ability to effectively distinguish AHPND from other major shrimp pathogens (EHP, IHHNV, and WSSV). Sensitivity analysis revealed a detection limit of 1 copies/μL, underscoring its exceptional sensitivity. Stability testing confirmed that the detection system is resistant to interference as high as 800 ng of host DNA. The method was further validated using 80 real samples, achieving an accuracy rate of 91.25%, confirming the high reliability of this visualization approach for field applications. These results provide valuable guidance for selecting suitable nucleic acid dyes and strongly support the development of more efficient