Real-TimePCR Analysis of Vibrio vulnificus fromOysters

Abstract

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 (10 2 to 10 8 CFU ml ؊1 ), with a lower limit of 72 fg of genomic DNA l of PCR mixture Vibrio vulnificus produces a rapidly fatal septicemia, which is primarily associated with the ingestion of raw oysters (3, 16). Contact of wounds with seawater or shellfish can also lead to serious infections that can progress to septicemia or require limb amputation. Methods for examination of seafood safety currently rely on fecal coliform analysis; however, V. vulnificus is not associated with fecal contamination (30). Although current standards do not regulate the number of V. vulnificus organisms in shellfish, the Food and Drug Administration supports postharvest treatments that will greatly reduce or eliminate the numbers of this organism. Therefore, quantitative methods are needed to accurately and efficiently validate application of these treatments to the seafood industry.V. vulnificus is indigenous to estuaries worldwide and can be readily isolated from the environment (24, 30, 37); however, standard detection methods require enrichment and selective plating media to reduce the growth of background organisms (10). Species identification requires additional time-consuming assays that are frequently inaccurate and laborious. Standard plate count based on DNA probe hybridization for colony identification can also be used for enumeration in oyster tissues (19,36,37), but this assay still requires overnight growth of bacteria and usually involves several days of processing time for large numbers of samples. Numerous studies have indicated that, under conditions of reduced temperatures (4 to 5°C) and nutrient availability, vibrios become nonculturable on standard media while retaining viability (8,13,26,32,33). Although the contribution of a viable but nonculturable (VBNC) population to V. vulnificus disease is unknown, human infections have been reported with VBNC V. cholerae (8). Therefore, enumeration assays that do not require cultivation may be useful for risk assessment. Several PCR assays (5, 6, 23), as well as reverse transcription-PCR (RT-PCR) (9) for detection of vibrios without ...

Rapid Detection of Vibrio harveyi in Seawater by Real-Time PCR

“…However, after 14 days, the determination coefficient (R 2 ) was low at 0.747, indicating that the efficiency of extraction had declined. This result supported that such starved cells in the environment are unlikely to be lysed 3) , causing underestimation of the real-time quantification.…”

“…The detection limits (10 2 cells/ml) of direct counts were not changed during the 14 day experiment, showing determination coefficients (R 2 ) of 0.971, 0,976, and 0.747, at 0 day, 7 days, and 14 days, respectively. It was reported that nonculturable cells of V. vulnificus after incubation in ASW at 4°C were detected by real-time PCR, unless complete degradation of membranes or loss of nucleic acid occurred 3) . However, after 14 days, the determination coefficient (R 2 ) was low at 0.747, indicating that the efficiency of extraction had declined.…”

Genotype Is Correlated with but Does Not Predict Virulence of Vibrio vulnificus Biotype 1 in Subcutaneously Inoculated, Iron Dextran-Treated Mice

“…V. vulnificus biotype 1 strains added to the collection included nine environmental strains from the Tampa Bay region of the Gulf of Mexico and one clinical strain from the Florida Department of Public Health, kindly provided by Valerie Harwood; previously described clinical strains MO6-24/0 (25,51,60) and ATCC 23907 (25); MLT365, MLT367, MLT403, VV1009, 2400112, and LL728, previously examined by our group for virulence (51,52); and genomically sequenced strains CMCP6 (29) and YJ016 (13). All strains were confirmed as V. vulnificus by species-specific vvhA PCR and DNA probe as previously described (10,59).…”

Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

“…Cultures that did not amplify by PCR at this locus were also analyzed for the presence of individual genes using primers derived from either wza (wzaF1 and wzaR [5Ј-TCGCGT TATCTGATCAACCA-3Ј]), wzb (wzbF [5Ј-GGTTGATCAGATAACGCGAA-3Ј] and wzbR [5Ј-AAGGAATACAAGCGTCTAGG-3Ј]), wzc (wzcF2 [5Ј-CCC GGAAATGAACGAGACAATG-3Ј] and wzcR2), or wbfV (wbfVF [5Ј-CTA TCGTAGATGTGGATATTGAG-3Ј] and wbfVR). Additionally, vvhA primers were used to test the stability of chromosomal DNA, using PCR conditions as previously described (6).…”