Enrichment-Based Duplex PCR for Early Detection of Potentially Virulent Vibrio harveyi in Aquaculture

Vibrio harveyi is an important bacterial pathogen in marine aquaculture, responsible for outbreaks affecting a wide range of fish species. Early detection of potentially virulent isolates in fish and environmental samples is essential for effective disease prevention and management, particularly under field conditions where the pathogen may be present at low abundance. In this study, we developed and evaluated the analytical and diagnostic performance of a two-step detection protocol that combines a short alkaline peptone water (APW-1) enrichment step with a duplex PCR assay targeting the species-specific toxR gene and the plasmid-associated fpcrp gene, a putative marker linked to increased virulence in some fish-pathogenic vibrios. This marker was detected in a diverse set of publicly available genomes from virulent V. harveyi isolates as well as in virulent isolates used in this study. This integrated approach enables reliable detection of V. harveyi in complex biological and environmental matrices while simultaneously providing virulence-associated screening. The duplex PCR showed high analytical specificity for V. harveyi based on toxR amplification when tested against a panel of closely related Vibrio species and allowed the identification of V. harveyi isolates carrying the putative virulence-associated marker. Without enrichment, the limit of detection was approximately 10⁵ CFU/g or CFU/mL in fish tissues and water samples, whereas incorporation of an 8 h APW-1 enrichment step improved the analytical sensitivity to around 10² CFU/g or CFU/mL. The diagnostic performance of the complete two-step protocol was evaluated using experimentally spiked samples and field samples from Mediterranean aquaculture settings, including fish tissues and aquatic environments. Overall, this enrichment-duplex PCR assay provides a rapid, sensitive and practical tool for the early detection and risk-oriented monitoring of potentially virulent V. harveyi isolates in aquaculture environments, supporting surveillance programmes and control strategies.