Evaluation of DNA Colony Hybridization and Real-Time PCR for Detection of Vibrio parahaemolyticus and Vibrio vulnificus in Postharvest-Processed Oysters

2009 ◽  
Vol 72 (10) ◽  
pp. 2106-2109 ◽  
Author(s):  
JESSICA L. JONES ◽  
KATHY E. NOE ◽  
ROBIN BYARS ◽  
ANGELO DePAOLA
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Colony Hybridization ◽  
Alkaline Peptone Water ◽  
Peptone Water

The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.

Enumeration of Vibrio parahaemolyticus and Vibrio vulnificus in Various Seafoods with Two Enrichment Broths

1994 ◽  
Vol 57 (5) ◽  
pp. 403-409 ◽  
Author(s):  
CURTIS J. HAGEN' ◽  
EDNA M. SLOAN ◽  
GAYLE A. LANCETTE ◽  
JAMES T. PEELER ◽  
JOHN N. SOFOS
Keyword(s):  
Cold Stress ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
Most Probable Number ◽  
Geometric Means ◽  
Probable Number ◽  
Alkaline Peptone Water ◽  
Peptone Water ◽  
Stressed Cells

This study compares recoveries of Vibrio parahaemolyticus and Vibrio vulnificus with salt-polymyxin B broth (SPB) and alkaline peptone water (APW) from samples of crab legs, oysters, shrimp, lobster and shark, which were inoculated at three levels (approximately 101 to 102, 102 to 103 and 104 to 105/g) with each of the pathogens. Six samples of each product were analyzed [3-tube most probable number (MPN)] with each broth. Inoculated samples of oysters and slurries of crab and lobster were also tested after cold stress (refrigerated at 2 to 4°C, 3 or 7 days, or frozen at −15°C for 21 or 28 days). For each seafood, geometric means of cells recovered with APW were significantly (P < 0.05) higher than the corresponding means of recovery with SPB. In addition, 12 of 15 calculated estimates of 50% relative detectable levels (RDL50) were lower (P < 0.05) for APW than for SPB. In these samples, the level of detection by APW was found to be 40 to 32,000 and 6- to 42-fold lower for V parahaemolyticus and V. vulnificus, respectively, than the level of detection by SPB. In cold-stored samples, overall detection of the pathogens was greatly reduced, but APW was also more efficient than SPB in recovering stressed cells.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

Effect of Frozen Storage and Vacuum-Packaging on Survival of Vibrio Vulnificus in Gulf Coast Oysters (Crassostrea virginica)

1994 ◽  
Vol 57 (7) ◽  
pp. 604-606 ◽  
Author(s):  
ROGER W. PARKER ◽  
ELLEN M. MAURER ◽  
A. BILL CHILDERS ◽  
DONALD H. LEWISI
Keyword(s):  
Vibrio Vulnificus ◽  
Gulf Coast ◽  
Health Hazard ◽  
Frozen Storage ◽  
Polymyxin B ◽  
Aerobic Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
Alkaline Peptone Water ◽  
Peptone Water

Vibrio vulnificus contamination of raw oysters is a serious public health hazard, therefore, it is necessary to investigate the persistence of V. vulnificus in harvested and stored oysters. For this study, triplicate oyster samples were split into four treatment groups: control, normal-packaged; control, vacuum-packaged; inoculated, normal-packaged; and inoculated, vacuum-packaged. Oysters in the inoculated groups were individually injected with V. vulnificus to a level of approximately 1 × 106 CFU/g. Control oysters were already naturally contaminated to a level of approximately 1 × 104 CFU/g. Oysters were then packaged, frozen and stored at −20°C. On day 0 and days 7, 14, 30 and 70 post-freezing, concentrations of total aerobic bacteria and V. vulnificus were determined using a 3-tube most probable number (MPN) estimation from enrichment Alkaline Peptone Water tubes with subsequent presumptive V. vulnificus growth on modified Cellobiose-Polymyxin B-Colistin agar. Length of frozen storage had a significant effect on decreasing total aerobic bacteria (from approximately 106 CFU/g to approximately 102.5 CFU/g) and V. vulnificus (from approximately 105 CFU/g to approximately 101 CFU/g). Also, vacuum-packaged samples showed significantly lower concentrations of V. vulnificus over the length of the study than did the normal-sealed samples.

Development of a Quantitative Real-Time PCR Method for Estimation of the Total Number of Vibrio parahaemolyticus in Contaminated Shellfish and Seawater

2005 ◽  
Vol 68 (5) ◽  
pp. 1083-1088 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
YOSHITO IWADE ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
Vibrio Parahaemolyticus ◽  
Plate Count ◽  
Most Probable Number ◽  
Cycle Number ◽  
Probable Number ◽  
The Real ◽  
Pcr Method

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 101 to 107 CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.

Vibrio vulnificus and Vibrio parahaemolyticus in U.S. Retail Shell Oysters: A National Survey from June 1998 to July 1999

2002 ◽  
Vol 65 (1) ◽  
pp. 79-87 ◽  
Author(s):  
DAVID W. COOK ◽  
PAUL O'LEARY ◽  
JEFF C. HUNSUCKER ◽  
EDNA M. SLOAN ◽  
JOHN C. BOWERS ◽  
...  
Keyword(s):  
United States ◽  
North Atlantic ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Gulf Coast ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Hemolysin Gene ◽  
Thermostable Direct Hemolysin

From June 1998 to July 1999, 370 lots of oysters in the shell were sampled at 275 different establishments (71%, restaurants or oyster bars; 27%, retail seafood markets; and 2%, wholesale seafood markets) in coastal and inland markets throughout the United States. The oysters were harvested from the Gulf (49%), Pacific (14%), Mid-Atlantic (18%), and North Atlantic (11%) Coasts of the United States and from Canada (8%). Densities of Vibrio vulnificus and Vibrio parahaemolyticus were determined using a modification of the most probable number (MPN) techniques described in the Food and Drug Administration's Bacteriological Analytical Manual. DNA probes and enzyme immunoassay were used to identify suspect isolates and to determine the presence of the thermostable direct hemolysin gene associated with pathogenicity of V. parahaemolyticus. Densities of both V. vulnificus and V. parahaemolyticus in market oysters from all harvest regions followed a seasonal distribution, with highest densities in the summer. Highest densities of both organisms were observed in oysters harvested from the Gulf Coast, where densities often exceeded 10,000 MPN/g. The majority (78%) of lots harvested in the North Atlantic, Pacific, and Canadian Coasts had V. vulnificus densities below the detectable level of 0.2 MPN/g; none exceeded 100 MPN/g. V. parahaemolyticus densities were greater than those of V. vulnificus in lots from these same areas, with some lots exceeding 1,000 MPN/g for V. parahaemolyticus. Some lots from the Mid-Atlantic states exceeded 10,000 MPN/g for both V. vulnificus and V. parahaemolyticus. Overall, there was a significant correlation between V. vulnificus and V. parahaemolyticus densities (r = 0.72, n = 202, P &lt; 0.0001), but neither density correlated with salinity. Storage time significantly affected the V. vulnificus (10% decrease per day) and V. parahaemolyticus (7% decrease per day) densities in market oysters. The thermostable direct hemolysin gene associated with V. parahaemolyticus virulence was detected in 9 of 3,429 (0.3%) V. parahaemolyticus cultures and in 8 of 198 (4.0%) lots of oysters. These data can be used to estimate the exposure of raw oyster consumers to V. vulnificus and V. parahaemolyticus.

scholarly journals Effects of Intertidal Harvest Practices on Levels of Vibrio parahaemolyticus and Vibrio vulnificus Bacteria in Oysters

2016 ◽  
Vol 82 (15) ◽  
pp. 4517-4522 ◽  
Author(s):  
J. L. Jones ◽  
T. P. Kinsey ◽  
L. W. Johnson ◽  
R. Porso ◽  
B. Friedman ◽  
...  
Keyword(s):  
New Jersey ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Ambient Air ◽  
The United States ◽  
Washington State ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Handling Practices

ABSTRACTVibrio parahaemolyticusandVibrio vulnificuscan grow rapidly in shellfish subjected to ambient air conditions, such as during intertidal exposure. In this study, levels of total and pathogenic (tdh+and/ortrh+)V. parahaemolyticusand totalV. vulnificuswere determined in oysters collected from two study locations where intertidal harvest practices are common. Samples were collected directly off intertidal flats, after exposure (ambient air [Washington State] or refrigerated [New Jersey]), and after reimmersion by natural tidal cycles. Samples were processed using a most-probable-number (MPN) real-time PCR method for total and pathogenicV. parahaemolyticusorV. vulnificus. In Washington State, the mean levels ofV. parahaemolyticusincreased 1.38 log MPN/g following intertidal exposure and dropped 1.41 log MPN/g after reimmersion for 1 day, but the levels were dependent upon the container type utilized. PathogenicV. parahaemolyticuslevels followed a similar trend. However,V. vulnificuslevels increased 0.10 log MPN/g during intertidal exposure in Washington but decreased by >1 log MPN/g after reimmersion. In New Jersey, initial levels of all vibrios studied were not significantly altered during the refrigerated sorting and containerizing process. However, there was an increase in levels after the first day of reimmersion by 0.79, 0.72, 0.92, and 0.71 log MPN/g for total,tdh+andtrh+V. parahaemolyticus, andV. vulnificus, respectively. The levels of all targets decreased to those similar to background after a second day of reimmersion. These data indicate that the intertidal harvest and handling practices for oysters that were studied in Washington and New Jersey do not increase the risk of illness fromV. parahaemolyticusorV. vulnificus.IMPORTANCEVibrio parahaemolyticusandVibrio vulnificusare the leading causes of seafood-associated infectious morbidity and mortality in the United States.Vibriospp. can grow rapidly in shellfish subjected to ambient air conditions, such as during periods of intertidal exposure. When oysters are submersed with the incoming tide, the vibrios can be purged. However, data on the rates of increase and purging during intertidal harvest are scarce, which limits the accuracy of risk assessments. The objective of this study was to help fill these data gaps by determining the levels of total and pathogenic (tdh+and/ortrh+)V. parahaemolyticusandV. vulnificusin oysters from two locations where intertidal harvest practices are common, using the current industry practices. The data generated provide insight into the responses ofVibriospp. to relevant practices of the industry and public health, which can be incorporated into risk management decisions.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 &gt; 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

Selectivity and Specificity of a Chromogenic Medium for Detecting Vibrio parahaemolyticus†

2005 ◽  
Vol 68 (7) ◽  
pp. 1454-1456 ◽  
Author(s):  
YI-CHENG SU ◽  
JINGYUN DUAN ◽  
WEN-HSIN WU
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Bile Salts ◽  
Vibrio Vulnificus ◽  
Enterobacter Cloacae ◽  
Most Probable Number ◽  
Chromogenic Medium ◽  
Probable Number ◽  
Vibrio Fluvialis ◽  
Vibrio Mimicus ◽  
Vibrio Spp

The thiosulfate–citrate–bile salts–sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Oyster Tissue ◽  
Pcr Assay ◽  
Colony Hybridization ◽  
The Real ◽  
Mantle Fluid ◽  
June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P &lt; 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P &lt; 0.05; and oyster: r = 0.99, P &lt; 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P &lt; 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

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