Rapid Detection of Vibrio vulnificus with Recombinase Polymerase Amplification Assays Based on Specific Sequence Tags of Core Genome

<i>Vibrio vulnificus</i> (<i>V. vulnificus</i>) is a motile, Gram-negative, opportunistic human pathogen capable of causing severe to life-threatening infections in individuals with predisposing conditions. It has the highest mortality rate among foodborne pathogens. Rapid and accurate detection of <i>V. vulnificus</i> is crucial for preventing and controlling acute deaths caused by infection with this bacterium. However, identifying <i>V. vulnificus</i> is challenging due to its high genomic plasticity. We analyzed 518 <i>V. vulnificus</i> genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish <i>V. vulnificus</i> from its closely related species. Specifically, one specific sequence tag with the minimal mutations was selected for <i>V. vulnificus</i> detection, combined with a recombinase polymerase amplification (RPA) method. The results showed that the developed RPA detection method displayed high specificity and enabled the identification of a specific 462 bp band from <i>V. vulnificus</i>. The reaction involved isothermal incubation at 39 °C for 20 min with a compact portable instrument. The sensitivity of the method was determined to be as low as 0.5 aM (1.65 fg/μL) of genomic DNA, 0.96 copies/μL of pUC57-Vv plasmid and 1 CFU/mL of <i>V. vulnificus</i> cells. The RPA method accurately detected target DNA within 5-10 min. Additionally, specificity testing was performed using 33 different strains of <i>V. vulnificus</i>. In conclusion, the established RPA method exhibits excellent high sensitivity and rapid discriminative capability, making it suitable for clinical applications and rapid detection in the field.