Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

Vibrio vulnificus is a gram-negative halophilic bacterium commonly found in warm coastal waters throughout the world (37, 44). In the United States, this microorganism is indigenous to Gulf of Mexico water (gulf water) and thrives during the warmer months. V. vulnificus is known to cause gastroenteritis and, in some cases, septicemia when it is ingested in raw or poorly cooked oysters (25, 26). Infection by V. vulnificus often results in fatal consequences, and the mortality rate is up to 60%, primarily in individuals who are immunocompromised (10, 11, 17, 18, 28, 31). Since infection by V. vulnificus is one of the leading causes of seafood-related illnesses in the United States, the Interstate Shellfish Sanitation Conference (ISSC) has proposed that illnesses caused by V. vulnificus due to consumption of shellfish must be reduced by at least 60% by the year 2007 (21). In order to achieve this goal, the ISSC suggests that consumable oysters should not contain more than 3 CFU of V. vulnificus per g of oyster meat (21). Therefore, to meet this objective, a rapid, reliable, and sensitive method for detection of this pathogen is as important as the methods of treatment for reducing the numbers of V. vulnificus in oysters to an acceptable level. Detection of V. vulnificus by conventional biochemical and microbiological culture methods, such as the most-probable-number method or the use of selective agar media, are time-consuming and require several days to obtain confirmatory results (42). A genetically based colorimetric colony hybridization method targeting the V. vulnificus-specific hemolysin gene, vvh, has been described previously and has been recommended as an alternative method for detection of this microorganism in shellfish (24, 33, 52). Although the DNA-DNA colony hybridization method in which the vvh gene segment is used as a probe is reliable and specific for detection of this pathogen, it takes at least 3 days to complete. A conventional PCR method for detection of V. vulnificus and other microbial pathogens in shellfish has also been shown to be effective (8, 22). However, this PCR approach requires analysis of the amplified DNA in an agarose gel or by DNA-DNA hybridization to confirm the results, which again is time-consuming and laborious. Recently, introduction of a real-time PCR amplification method in which SYBR Green I fluorescent dye is used has made detection of microbial pathogens such as Legionella (46), Escherichia coli (23), Vibrio parahaemolyticus (7), and Campylobacter (29) rapid and cost-effective and the analysis of results simple. The SYBR Green I fluorescent dye binds to the minor grooves of the amplified DNA during the primer annealing and extension steps of each PCR cycle. Accumulation of amplified DNA is measured by determining the increase in fluorescence over time, and this is followed by confirmation of results by melting curve analysis (48). In this paper, we describe development of a method for rapid and sensitive detection of V. vulnificus in shellfish and gulf water by real-time PCR with SYBR Green I fluorescent dye and oligonucleotide primers targeting a segment of the hemolysin (vvh) gene.