Evaluation of DNA colony hybridization and real-time PCR for detection of Vibrio parahaemolyticus and Vibrio vulnificus in postharvest-processed oysters.

Abstract

The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.