Droplet digital PCR and real-time PCR for the sensitive and specific detection of Vibrio vulnificus based on the novel target genes

Vibrio vulnificus poses a significant risk to public health due to its high pathogenicity and mortality rates as a common seafood-borne pathogen. Therefore, rapid, accurate, and specific detection methods for V. vulnificus are crucial for ensuring human safety and minimizing economic losses. This study identified vvhA and vv08030 as promising targets for V. vulnificus detection using bioinformatics screening and PCR analysis. Based on these specific target genes, a duplex real-time PCR (qPCR) assay and a duplex droplet digital PCR (ddPCR) assay were developed and evaluated for the rapid quantitative detection of V. vulnificus. These methods showed 100% specificity to V. vulnificus and no cross-reaction with other strains. It was proved in this research that the qPCR method can detect genomic DNA as low as 31.4 fg/μL, and the quantification range of the bacterial suspension was between 8.25 × 10² and 8.25 × 10⁷ CFU/mL. On the other hand, ddPCR exhibited greater sensitivity with genomic sensitivity reaching 3.14 fg/μL and could accurately quantify bacterial suspensions between 8.25 × 10¹–8.25 × 10⁵ CFU/mL. The feasibility of the ddPCR method in detecting V. vulnificus was assessed in spiked food samples. The lowest detectable V. vulnificus in salmon was 5.74 × 10¹ CFU/g without any pre-enrichment. These methods demonstrated high sensitivity, accuracy and rapidity, with potential applications in V. vulnificus detection strategies.