Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains

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.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.00460-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5840-5847 ◽

Cited By ~ 227

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.

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Development of a multiplex real-time PCR method for pathogenic Vibrio parahaemolyticus detection (tdh+ and trh+)

Food Control ◽

10.1016/j.foodcont.2011.09.015 ◽

2012 ◽

Vol 24 (1-2) ◽

pp. 128-135 ◽

Cited By ~ 26

Author(s):

Alejandro Garrido ◽

María-José Chapela ◽

Martiña Ferreira ◽

Miroslava Atanassova ◽

Paula Fajardo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Pathogenic Vibrio ◽

Pcr Method

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

Journal of Microbiological Methods ◽

10.1016/s0167-7012(03)00020-4 ◽

2003 ◽

Vol 53 (2) ◽

pp. 149-155 ◽

Cited By ~ 126

Author(s):

George M Blackstone ◽

Jessica L Nordstrom ◽

Michael C.L Vickery ◽

Michael D Bowen ◽

Richard F Meyer ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Pathogenic Vibrio

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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

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.639473 ◽

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.

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Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR

Canadian Journal of Microbiology ◽

10.1139/cjm-2018-0083 ◽

2018 ◽

Vol 64 (11) ◽

pp. 809-815 ◽

Cited By ~ 7

Author(s):

Deshun Xu ◽

Lei Ji ◽

Xiaofang Wu ◽

Wei Yan ◽

Liping Chen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Virulence Genes ◽

Annealing Temperature ◽

Cross Reactivity ◽

Bacterial Strains ◽

Pcr Assay ◽

Pure Cultures ◽

Sporadic Cases

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 104 and 105 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.

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