Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR

We used a rapid DNA extraction and purification method to obtain the DNA from Vibrio vulnificus seeded into clam tissue homogenates for real-time PCR quantification of the organism. Without enrichment, the limit of detection was 1 x 10(2) cfu/g of tissue with a linear detection range of 1 x 10(2) to 1 x 10(8) cfu/g. With a 5 h non-selective enrichment, the limit of detection was 1 cfu/g of tissue with a linear detection range of 1 to 1 x 10(6) cfu/g of tissue. We found a 10-fold higher detection limit with seeded clam tissue homogenates compared to pure culture in TSB(+). The detection limits with pure broth culture and seeded tissue homogenates were identical, 1 cfu/ml and 1 cfu/ml, respectively, following 5 h non-selective enrichment. However, the Ct value with tissue homogenates was about 3 threshold cycles higher than with pure culture.