Innovative multiplex diagnostic strategies for Vibrio harveyi and Vibrio campbellii: a comparative study of droplet digital PCR, real-time PCR, and point-of-care PCR

Rapid and accurate detection of Vibrio campbellii and Vibrio harveyi, two major pathogens responsible for disease outbreaks in aquaculture systems, is critical for mitigating economic and ecological losses. However, existing diagnostic methods often fail to differentiate between these closely related species, increasing the risk of misdiagnosis and ineffective control. In this study, novel species-specific pathogenic markers were identified through bioinformatics analysis, enabling the development of multiplex PCR-based diagnostic assays, including real-time PCR (qPCR), point-of-care PCR (POC PCR), and droplet digital PCR (ddPCR). All three PCR methods demonstrated high species specificity. ddPCR achieved the highest sensitivity with 95 % LODs below 3 CFU/ml, while qPCR and POC PCR detected down to 15–20 CFU/ml. In spiked samples, ddPCR detected 10⁰ CFU/ml, outperforming qPCR and POC PCR, which reliably detected 10¹ CFU/ml. Comparative performance analysis highlighted POC PCR as the most suitable for on-site aquaculture diagnostics due to its speed (20 min) and low cost ($0.73/sample), while ddPCR proved effective for detecting low pathogen loads. These diagnostic tools provide a rapid and robust means for species-level identification of Vibrio infections in aquaculture, supporting timely management and disease control strategies.