Comparative performance of TCBS and TSA for the enumeration of trh+ Vibrio parahaemolyticus by direct colony hybridization

2019 ◽  
Vol 157 ◽  
pp. 37-42 ◽  
Author(s):  
Karanth Padyana Anupama ◽  
Kundar Deeksha ◽  
Ariga Deeksha ◽  
Iddya Karunasagar ◽  
Indrani Karunasagar ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Comparative Performance ◽  
Colony Hybridization

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 < 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 < 0.05; and oyster: r = 0.99, P < 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 < 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.

Effect of Ploidy on Vibrio parahaemolyticus and Vibrio vulnificus Levels in Cultured Oysters

2020 ◽  
Vol 83 (11) ◽  
pp. 2014-2017
Author(s):  
JESSICA L. JONES ◽  
KERI A. LYDON ◽  
WILLIAM C. WALTON
Keyword(s):  
Crassostrea Virginica ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Vibrio Vulnificus ◽  
High Quality ◽  
Colony Hybridization ◽  
Naturally Occurring ◽  
Rapid Growth Rate ◽  
Vibrio Spp ◽  
Half Sibling

ABSTRACT Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring human pathogenic bacteria commonly found in estuarine environments where oysters are cultured. The use of triploid oysters has increased due to their rapid growth rate and because they maintain a high quality throughout the year. Previous work suggested levels of Vibrio spp. may be lower in triploid oysters than diploid oysters. Therefore, this study aimed to determine whether there is a difference in the abundances of V. parahaemolyticus and V. vulnificus between half-sibling diploid and triploid American oysters (Crassostrea virginica). In four trials, 100 individual oysters (either iced or temperature abused) were analyzed for V. parahaemolyticus and V. vulnificus by using direct plating followed by colony hybridization. Mean levels of V. parahaemolyticus in iced and abused diploid oysters were 3.55 and 4.21 log CFU/g, respectively. Mean levels in iced and abused triploid oysters were 3.49 and 4.27 log CFU/g, respectively. Mean levels of V. vulnificus in iced and abused diploid oysters were 3.53 and 4.56 log CFU/g, respectively. Mean levels in iced and abused triploid oysters were 3.54 and 4.55 log CFU/g, respectively. The differences in Vibrio spp. abundances between diploid and triploid oysters was not significant (P > 0.05). However, the differences across treatments were significant (P < 0.05), with the exception of V. parahaemolyticus levels in trial 3 (P = 0.83). Variation between individual oysters was also observed, with 12 of 808 measurements being outside of the 95th percentile. This phenomenon of occasional statistical outliers (“hot” or “cold” oysters) has been previously described and supports the appropriateness of composite sampling to account for inherent animal variability. In summary, the data indicate that abundances of V. parahaemolyticus and V. vulnificus are not dependent on the ploidy of cultured oysters but vary with the type of handling. HIGHLIGHTS

Evaluation of an Alkaline Phosphatase–Labeled Oligonucleotide Probe for the Detection and Enumeration of the Thermostable-Related Hemolysin (trh) Gene of Vibrio parahaemolyticus

2006 ◽  
Vol 69 (11) ◽  
pp. 2770-2772 ◽  
Author(s):  
JESSICA L. NORDSTROM ◽  
RACHEL RANGDALE ◽  
MICHAEL C. L. VICKERY ◽  
ANDREA M. B. PHILLIPS ◽  
SHELLEY L. MURRAY ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Vibrio Parahaemolyticus ◽  
Oligonucleotide Probe ◽  
The United States ◽  
Bacterial Strains ◽  
Specific Detection ◽  
Colony Hybridization ◽  
The United Kingdom ◽  
Pathogenic Vibrio ◽  
Reliable Methods

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase–labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.

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 and Differentiation of Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA Colony Hybridization Using the Hydrophobic Grid Membrane Filtration Technique for Isolation

1994 ◽  
Vol 57 (2) ◽  
pp. 163-165 ◽  
Author(s):  
CHARLES A. KAYSNER ◽  
CARLOS ABEYTA ◽  
KAREN C. JINNEMAN ◽  
WALTER E. HILL
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Membrane Filtration ◽  
Gene Probe ◽  
Colony Hybridization ◽  
Rapid Technique ◽  
Gene Probes ◽  
Radiolabeled Probe ◽  
Filtration Technique

We have developed a means of differentiating and enumerating Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA colony hybridization directly on HGMF filters. V. parahaemolyticus can be detected by a tdh-3-radiolabeled gene probe and V. vuinificus detected by a specific cytotoxin-hemolysin-radiolabeled probe with enumeration directly from autoradiograms. This procedure is more rapid than current techniques allowing enumeration and identification of these two species in samples as diverse as seawater, oyster (Crassostrea gigas), and shrimp (Pandalidae family) within 4 d. Our method is based on a rapid technique (18 h) for isolation and enumeration of V. parahaemolyticus from food using a membrane filtration technique with hydrophobic grid filters (HGMF). With the HGMF method, however, it is not possible to differentiate V. parahaemolyticus from V. vuinificus since on the HGMF-sucrose-based agar used, the two species are indistinguishable as both species are unable to ferment sucrose. Using a combination of the HGMF and selective gene probes, these two species can be differentiated.

scholarly journals Identification of Vibrio parahaemolyticus Strains at the Species Level by PCR Targeted to the toxR Gene

1999 ◽  
Vol 37 (4) ◽  
pp. 1173-1177 ◽  
Author(s):  
Yung Bu Kim ◽  
Jun Okuda ◽  
Chiho Matsumoto ◽  
Naoki Takahashi ◽  
Satoru Hashimoto ◽  
...  
Keyword(s):  
Phylogenetic Relationships ◽  
Vibrio Parahaemolyticus ◽  
Sequence Variation ◽  
Vibrio Alginolyticus ◽  
Species Level ◽  
Specific Detection ◽  
Colony Hybridization ◽  
Hybridization Test ◽  
Toxr Gene ◽  
Positive Results

The DNA colony hybridization test with the polynucleotide probe forVibrio parahaemolyticus toxR gene was performed. All 373 strains of V. parahaemolyticus gave positive results, and the strains belonging to four other Vibrio species including Vibrio alginolyticus gave weakly positive results, suggesting that toxR sequence variation may reflect the phylogenetic relationships of Vibrio species. We then established a toxR-targeted PCR protocol for the specific detection of V. parahaemolyticus.

scholarly journals Seasonal Variation in Abundance of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters along the Southwest Coast of India

2005 ◽  
Vol 71 (7) ◽  
pp. 3575-3580 ◽  
Author(s):  
A. Deepanjali ◽  
H. Sanath Kumar ◽  
I. Karunasagar ◽  
I. Karunasagar
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Seasonal Abundance ◽  
Southwest Coast Of India ◽  
Colony Hybridization ◽  
Southwest Coast ◽  
Alkaline Peptone Water ◽  
Pathogenic Vibrio ◽  
Hemolysin Gene ◽  
Peptone Water

ABSTRACT The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 104 organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.

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