Detection of enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus: performance of real-time PCR kits in an interlaboratory study

2017 ◽  
Vol 243 (8) ◽  
pp. 1335-1342 ◽  
Author(s):  
Erik Eschbach ◽  
Annett Martin ◽  
Jennifer Huhn ◽  
Constanze Seidel ◽  
Ralf Heuer ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Interlaboratory Study

scholarly journals Light-scattering sensor for real-time identification of Vibrio parahaemolyticus , Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate

2012 ◽  
Vol 5 (5) ◽  
pp. 607-620 ◽  
Author(s):  
Karleigh Huff ◽  
Amornrat Aroonnual ◽  
Amy E. Fleishman Littlejohn ◽  
Bartek Rajwa ◽  
Euiwon Bae ◽  
...  
Keyword(s):  
Light Scattering ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Agar Plate ◽  
Solid Agar ◽  
Real Time Identification

scholarly journals Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

2007 ◽  
Vol 56 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Dobryan M. Tracz ◽  
Paul G. Backhouse ◽  
Adam B. Olson ◽  
Joanne K. McCrea ◽  
Julie A. Walsh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Vibrio Vulnificus ◽  
Comparative Sequence Analysis ◽  
Molecular Techniques ◽  
Array Technology ◽  
Species Specific ◽  
Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

scholarly journals Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhenpeng Li ◽  
Hongxia Guan ◽  
Wei Wang ◽  
He Gao ◽  
Weihong Feng ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Health Concern ◽  
Specific Gene ◽  
Amplification Efficiency ◽  
Pcr Assay ◽  
Percent Agreement ◽  
Stool Samples

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

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.

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.

scholarly journals Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

2006 ◽  
Vol 72 (3) ◽  
pp. 2031-2042 ◽  
Author(s):  
Linda N. Ward ◽  
Asim K. Bej
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Detection Methods ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Reading Frame ◽  
Initial Inoculum ◽  
Hemolysin Gene ◽  
Pure Cultures

ABSTRACT We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

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