Rapid Detection ofVibrio vulnificusin Shellfish and Gulf of Mexico Water by Real-Time PCR

Abstract

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water 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-probablenumber method or the use of selective agar media, are timeconsuming 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 cyc...

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

“…such as V. parahaemolyticus, V. vulnificus, V. cholerae and V. penaeicida from different resources (Blackstone et al 2003;Panicker et al 2004;Gubala 2006;Goarant and Merien 2006). However, so far, no real-time PCR assay was reported for quantification of V. alginolyticus.…”

“…Number of V. alginolyticus (log CFU ml -1 of samples) more sensitive than detection of V. vulnificus (Takahashi et al 2005), V. cholerae (Gubala 2006) and as sensitive as detection of V. parahaemolyticus (Blackstone et al 2003;Cai et al 2006) and V. vulnificus (Panicker et al 2004). Vibrio alginolyticus may enter into a viable but nonculturable (VBNC) state, and enrichment can help the recovery and multiplication of the cells, avoiding false-negative detection.…”

“…Fast, sensitive and quantitative detection of aquatic and seafood microbial pathogens such as V. parahaemolyticus (Blackstone et al 2003), Vibrio vulnificus (Panicker et al 2004), V. cholerae (Gubala 2006), Vibrio penaeicida (Goarant and Merien 2006) was achieved by SYBR Green I real-time PCR. The fluorescent dye SYBR Green I emits strong fluorescence upon binding to the minor groove of the DNA double helix.…”

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

“…Although sample enrichment does increase sensitivity, it essentially renders RT-PCR non-quantitative. RT-PCR has also been used to quantify ammonia-oxidizing bacteria in soil (Hermansson and Lindgren 2001), enterococci and human adenovirus in water (He and Jiang 2005), Vibrio vulnificus in shellfish and water (Panicker et al 2004), Lactobacillus salivarius in broiler chickens (Harrow et al 2007) and Clostridium tyrobutyricum in milk (López-Enríquez et al 2007).…”

Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX)

“…Real-time PCR assays for detection of other marine microorganisms have been reported, including assays for toxic dinoflagellates (Bowers et al 2000, Gray et al 2003, Galluzzi et al 2004, human pathogens (Guy et al 2003, Myers et al 2003, Panicker et al 2004, and pathogens of other bivalve molluscs (Audemard et al 2004). In order to enhance the QPX qPCR assay, unique aspects were incorporated into its development, including a pathogen-spiked marine aggregate dilution series to demonstrate applicability with environmental samples and the use of an electronic particle counter to obtain absolute quantitative results.…”