Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters

Abstract A direct plating method and an FDA method were compared for enumeration of Vibrio vulnificus in Gulf Coast oysters. The direct plating method was based on hybridization of colony lifts and used an alkaline phosphatase-labeled oligonucleotide probe that targeted the cytolysin gene (Direct-VVAP). The FDA method employs most probable number analysis with confirmation of suspect isolates by enzyme immunoassay (MPN-EIA). Indigenous V . vulnificus levels in oysters harvested from Florida, Alabama and Louisiana throughout the year and in Gulf Coast market oysters ranged from 5 g −1 . Similar V. vulnificus levels ( r =0.66) were found with these two procedures in freshly harvested oysters collected between April and October. Measurement variance was much greater, however, with the MPN procedure (0.118) than with the DNA probe procedure (0.004). In market oysters, the MPN-EIA procedure gave 0.4 log 10 higher estimates than the Direct-VVAP method, but the methods were closely correlated ( r =0.83). Confirmation procedures were in agreement >90% of the time, as suspect isolates confirmed by one procedure were identified by the other procedure. Except when V . vulnificus densities are low ( −1 ), the Direct-VVAP method provides an alternative procedure that is more rapid and precise than the MPN-EIA analysis.