Quantitative detection of Vibrio vulnificus in raw oysters by real-time loop-mediated isothermal amplification

Vibrio vulnificus is a significant cause of seafood-related morbidity and mortality in the United States. Rapid and sensitive detection assays are needed to facilitate better control of potential V. vulnificus infections from seafood consumption. Recently, loop-mediated isothermal amplification (LAMP) has been applied to detect V. vulnificus. In this study, the LAMP assay was coupled with either fluorescence- or turbidity-based real-time platforms for the quantitative detection of V. vulnificus in raw oysters. Both real-time platform assays possessed good specificity, with no false positive or false negative results among 38 V. vulnificus and 42 other bacteria tested. Comparable sensitivity was obtained using the two real-time platforms, with the assay limits of detection to be approximately 1-10 CFU/reaction of V. vulnificus in pure culture, up to 100-fold more sensitive than PCR. When applied to spiked oyster samples, the real-time LAMP assays were able to detect 6.4x10(4) CFU/g of oyster without enrichment, 1000 fold more sensitive than PCR. Standard curves generated for detecting V. vulnificus in both pure culture and spiked oyster samples showed good linear regression between cell counts and the fluorescence Ct or turbidity Tt values. In summary, the real-time LAMP assays developed in the present study could be adopted for the quantitative detection of V. vulnificus in raw oysters with high sensitivity and specificity.