The objectives of this proposal are: (1) to develop a rapid, easy-to-use and reliable V. vulnificus detection/quantification kit as an alternative to procedures currently accepted by ISSC, (2) to validate the test kit for the detection of V. vulnificus in raw oysters (live and PHP) using procedures currently accepted by ISSC as the reference method, (3) to introduce the innovative key technologies and their detection/quantification concepts to educate undergraduate and graduate students, and (3) transfer technology to oyster industry and institutions that conduct PHP validation and verification testing.
The V. vulnificus detection kit development is based on a novel two-phase culture technique, gradient concentration of selective antibiotics, biological indicator (pH indicator), cell motility/diffusion, oligosaccharide (cellobiose) utilization of V. vulnificus, and incubation temperature. These features promote the facultative anaerobic growth, migration and differentiation of V. vulnificus, while inhibiting the growth of other microorganisms. Utilizing the combination of gradient technology of antibiotics, two-phase culture technique and biological selectivity, the V. vulnificus detection kit can optimize expression of house-keeping genes; high sensitivity and specificity can then be evidenced through the single tube detection kit by altering the characteristics of a signal indicating medium which, in turn, will provide visible evidence of the presence of V. vulnificus. The kit can be applied qualitatively by using a single tube for presence/absence or quantitatively, using multiple tubes in an MPN format. The approximate price to produce the kit is less than $0.20/test, which is less than 10% the cost of currently accepted technologies.
Given the failure to meet the ISSC 60% V. vulnificus illness reduction goal and the FDA proposal for mandatory PHP of Gulf oysters, there will likely be a need for more efficient and cost effective vibrio enumeration methods for PHP validation and verification. The high cost of current methods deters progress in this area for all but the largest processors. We propose a simple, rapid and low cost alternative to currently accepted methods that will expand industry capacity to develop new PHP approaches such as high salinity relaying or depuration.