Two-stage Nested PCR Effectiveness for Direct Detection of Vibrio vulnificus in Natural Samples

ABSTRCT The nested primers designed to amplify a 222-base pair portion of the hemolysin gene, vvhA, were specific for all V. vulnificus strains tested. The nested PCR amplification, coupled with direct extraction of template DNA, revealed improved sensitivity sufficient for detection of 1 to 10 CFU V. vulnificus in 1 mL of seafood homogenates, and eliminated the need for enrichment culturing. Thereby, the nested PCR method achieved a broader applicability, making it effective for extensive use in identification of the pathogen in natural samples such as raw seafoods, seawater and sediments.