Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification

Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent V. vulnificus strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent V. vulnificus strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 10 3 CFU/g of V. vulnificus ATCC 33815, while showing negative results for a nonvirulent V. vulnificus strain (515-4c2) spiked at 10 7 CFU/g. Afte r6ho fenrichment, the LAMP assay could detect 1 CFU/g of the virulent V. vulnificus strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent V. vulnificus strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent V. vulnificus in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of V. vulnificus risks associated with raw oyster consumption. Vibrio vulnificus is a Gram-negative, halophilic bacterium that inhabits warm coastal and estuarine waters worldwide and occurs in high numbers in filter-feeding bivalve mollusks such as oysters and clams (29). This bacterium is an uncommon but serious cause of human illness due to the consumption of raw or undercooked seafood, especially oysters (5). Following ingestion of raw oysters or exposure of open wounds to seawater, V. vulnificus infection may rapidly develop (24 h) into primary septicemia and wound infection, two fatal diseases with mortality rates of over 50% and 25%, respectively (4, 29). As a matter of fact, V. vulnificus has been regarded as the predominant cause (95%) of seafood-related deaths in the United States, responsible for approximately 30 deaths annually (5, 29). Additionally, the CDC’s recent FoodNet report suggested a continued increase in Vibrio incidences since 2001, pointing to a need for improved prevention measures (5). In this regard, rapid and reliable detection methods are particularly needed to facilitate better control of potential V. vulnificus risks in raw oysters. As an opportunistic human pathogen, V. vulnificus causes fatal infections predominantly among at-risk consumers, such as persons with immunocompromising conditions, diabetes, and an elevated serum iron concentration due to chronic liver disease or alcohol abuse (39). Besides host susceptibility, epidemiological data also support that only a small percentage of V. vulnificus strains in oysters are virulent (6, 21, 22, 41). Therefore, it is desirable that detection methods selectively target virulent V. vulnificus strains for accurate risk assessment and control. However, numerous V. vulnificus virulence factors examined to date appear to be present in both clinical and environmental strains (12, 22), and currently, there is a lack of unique virulence biomarkers that can be used to screen for