Purpose: Currently, approved regulatory methods for Vp either lack quantitative capability, or rely on biochemical identification. As Vp is a ubiquitous organism, quantitation is increasingly important and biochemical identification has been found unreliable for these bacteria isolated from food and environmental samples. The intent of this study was to validate two MPN-real-time PCR methods: one for enumeration of total Vp (tlh+) and one for pathogenic Vp (tdh+/trh+).
Methods: Oysters processed to reduce vibrio levels were homogenized by standard procedures. Aliquots of the homogenates were spiked with known concentrations of tdh+/trh+ Vp strains. Appropriate dilutions of strains were spread plated in triplicate on TSA to determine spike levels. Spiked homogenates were used to inoculate a standard 3-tube MPN in APW. After overnight incubation, aliquots of turbid MPN tubes were tested for the presence of total or tdh+/trh+ Vpby real-time PCR on the Cepheid SmartCycler II and AB 7500 Fast platforms.
Results: The PCR portions of the methods were determined to have 100% inclusivity and exclusivity. Differences between the spike level and MPN-PCR values of the test methods were not statistically significant (P>0.35). Recovery of the test methods was determined to be between 100-102%. A significant correlation (P<0.001) was observed between spike levels and MPN-PCR values, across a range of concentrations (0.15-6.74 log Vp/g), with correlation coefficients between 0.97-0.98.
Significance: The results of this study demonstrate the validity of data obtained using these MPN-real-time PCR methods for enumeration of total and pathogenic Vp in oysters. Based on this study, these methods have been accepted for regulatory use by the National Shellfish Sanitation Program.