Real-Time PCR Analysis of Vibrio vulnificus from Oysters

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10² to 10⁸ CFU ml⁻¹), with a lower limit of 72 fg of genomic DNA μl of PCR mixture⁻¹ or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r² = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.     

Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR

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). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.     

Real-time PCR detection of Vibrio vulnificus in oysters: comparison of oligonucleotide primers and probes targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus

The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.     

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.     

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.     

Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe.

An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters.

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

Distribution of Vibrio vulnificus in the Chesapeake Bay.

Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.     

Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C_T) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10³ V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 10³ CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China

Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.     

New approach for discrimination of Vibrio vulnificus by real-time PCR before and after gamma-irradiation

The effects of gamma-irradiation on the destruction of Vibrio vulnificus by real-time PCR were studied. gamma-irradiation was found to result in extensive reduction in the molecular size of DNA. Irradiation of viable cells (1 x 10(6) CFU/ml) at 1.08 KGy resulted in 100% destruction determined by plate counts, with most of the DNA from the irradiated cells having a bp-length of less than 1000. The use of a pair of primers to amplify a 1000-bp sequence of DNA from cells exposed to 1.08 KGy failed to yield amplification. In contrast, primers designed to amplify sequences of 700, 300, and 70-bp yielded amplification with C(t) values resulting in 13.4, 27.6, and 45.4% detection of genomic targets. When viable cells of V. vulnificus were exposed to 1.08, 3.0, and 5.0 kGy, the average molecular size of genomic DNA visualized in an agarose gel decreased with increasing dose, corresponding to an increased probability of amplification with primers targeting sequences of decreasing size.     

Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR

Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.     

A real-time PCR assay for the rapid determination of 16S rRNA genotype in Vibrio vulnificus

In a terminal restriction fragment polymorphism (T-RFLP) study, we recently reported a significant association between the type B 16S rRNA gene and clinical strains of Vibrio vulnificus associated with the consumption of raw oysters. In the present study we describe a real-time PCR assay for the rapid determination of the 16S rRNA type of V. vulnificus isolates. This assay was used to reexamine the 16S rRNA gene type in the strains studied previously by T-RFLP and additional isolates from selected sources. Analyses revealed that 15 of the strains (10 environmental and 5 clinical) previously found to be 16S rRNA type A actually appear to possess both the type A and B genes. The presence of both alleles was confirmed by cloning and sequencing both gene types from one strain. To our knowledge, this is the first report of 16S rRNA sequence heterogeneity within individual strains of V. vulnificus. The findings confirm the T-RFLP data that 16S rRNA type may be a useful marker for determining the clinical significance of V. vulnificus in disease in humans and cultured eels. The real-time PCR assay is much more rapid and less resource-intensive than T-RFLP, and should facilitate further study of the occurrence and distribution of the 16S rRNA genotypes of V. vulnificus. These studies should provide more definitive estimates of the risks associated with this organism and may lead to a better understanding of its virulence mechanism(s).     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

A. DALSGAARD, I. DALSGAARD, L. HØI AND J.L. LARSEN. 1996. Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus .     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.