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

Vibrio vulnificus (V. vulnificus) 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 V. vulnificus is crucial for preventing and controlling acute deaths caused by infection with this bacterium. However, identifying V. vulnificus is challenging due to its high genomic plasticity. We analyzed 518 V. vulnificus genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish V. vulnificus from its closely related species. Specifically, one specific sequence tag with the minimal mutations was selected for V. vulnificus 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 V. vulnificus. 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 V. vulnificus cells. The RPA method accurately detected target DNA within 5-10 min. Additionally, specificity testing was performed using 33 different strains of V. vulnificus. 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.