Study on the applicability of novel molecular targets of Vibrio species by pan-genome mining in the field of nucleic acid detection

Vibrio spp. are ubiquitous in marine areas worldwide and can cause diseases in humans. Aqueous environments favor the transfer of genes between strains, resulting in the loss of strain-specific target genes. This study aimed to explore new molecular targets for the detection of Vibrio strains at the species level using pan-genome analysis for nucleic acid detection and differentiation of major pathogenic Vibrio in aquatic products. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: three for Vibrio parahaemolyticus, one for Vibrio alginolyticus, three for Vibrio vulnificus, three for Vibrio fluvialis. We constructed primers for the selected genes, and we verified their specificity using various Vibrio species and non-Vibrio strains. After systematic optimization, a novel molecular target-based 4-multiplex PCR (4-mPCR) method was developed, exhibiting a detection limit of 103 CFU/mL for pure cultures and 104 CFU/g for fish samples. The initial inoculum was approximately 1 Log10 CFU/g, and all samples were positive after 4 h of enrichment. Not only that, the feasibility of the mined new targets in the field of nucleic acid detection was further validated by the 4-mqPCR and LAMP techniques. These results suggest that novel targets mined by pan-genomic have good generalizability for use in a variety of nucleic acid detection techniques and can help improve the accuracy of nucleic acid-based detection techniques in the detection of pathogenic Vibrio species in aquatic samples, with potential applications for food safety investigations and disease diagnosis.