Postharvest processing (PHP) is used to reduce levels of Vibrio vulnificus in oysters, but process validation is labor-intensive and expensive. Therefore, quantitative PCR was evaluated as a rapid confirmation method for most-probable-number enumeration (QPCR-MPN) of V. vulnificus bacteria in PHP oysters. QPCR-MPN showed excellent correlation (R 2 ‫؍‬ 0.97) with standard MPN and increased assay sensitivity and efficiency.Vibrio vulnificus can cause life-threatening, systemic disease (2, 9, 14) that is associated with the consumption of raw oysters. The bacterium is distributed throughout temperate estuaries worldwide (6,7,17,24,25), and environmental conditions of warmer water temperature and lower salinity favor its growth in molluscan shellfish (5,13,16,21,23,25). Warning labels on oyster products and educational programs have not been effective in reducing disease mortality rates for at-risk individuals with underlying diseases, such as cirrhosis, hemochromatosis (iron overload), diabetes, or immune system dysfunction (8). Therefore, the FDA and the Interstate Shellfish Sanitation Conference (ISSC) have mandated postharvest processing (PHP) of oysters harvested from Gulf Coast states in order to reduce V. vulnificus infections (11). Application of PHP methodology requires validation and verification in order to ensure that the process will substantially reduce numbers of V. vulnificus bacteria to levels that are below the predicted threshold for disease. Validation trials for PHP are laborintensive and cost-prohibitive (10, 12), and improved protocols for industry compliance and for risk assessment of V. vulnificus in PHP oysters are urgently needed.The standard method for validating PHP requires that three independent lots of oysters meet the specification of Ͻ30 most probable numbers (MPN)/g of V. vulnificus by using the geometric mean of 10 samples/lot. Levels of V. vulnificus bacteria in oysters are enumerated by MPN endpoint titration of replicate samples in enrichment broth cultures (12), and speciesspecific growth is determined by isolating typical V. vulnificus colonies on selective medium, with subsequent confirmation by DNA probe (26). The present study evaluated a real-time quantitative PCR (QPCR) assay for detection of V. vulnificus growth in MPN enrichment cultures (QPCR-MPN). QPCR increases assay throughput by using automated species-specific confirmation, which is not available with standard PCR. Also, the limit of detection for direct QPCR enumeration of V. vulnificus bacteria in oysters without enrichment is generally 100 CFU/g, but QPCR-MPN assays generally increase the sensitivity of detection to 1 bacterium/g after enrichment (1,18,19,20) and permit detection of V. vulnificus at levels (30 CFU/g) required for validation protocols.Field trials were conducted to assess application of QPCR-MPN to oyster PHP validation, as application of QPCR-MPN to enumerate V. vulnificus bacteria in PHP oysters has not been examined previously and merits further scrutiny. For example, large numbers of dead b...