Detection of<i>Vibrio parahaemolyticus</i>in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

Ward, Linda N.; Bej, Asim K.

doi:10.1128/aem.72.3.2031-2042.2006

Cited by 121 publications

(90 citation statements)

References 50 publications

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“…However, this assay may not be useful for the detection of pandemic strains that are devoid of orf8. Pyrolysis metastable atom bombardment mass spectrometry was shown to identify various phenotypic characteristics of V. parahaemolyticus (96). By targeting specific phenotypic markers, this technique may differentiate pandemic and nonpandemic V. parahaemolyticus strains.…”

Section: Methods For Detectionmentioning

confidence: 99%

Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

et al. 2007

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SUMMARY Vibrio parahaemolyticus is recognized as a cause of food-borne gastroenteritis, particularly in the Far East, where raw seafood consumption is high. An unusual increase in admissions of V. parahaemolyticus cases was observed at the Infectious Diseases Hospital in Calcutta, a city in the northeastern part of India, beginning February 1996. Analysis of the strains revealed that a unique serotype, O3:K6, not previously isolated during the surveillance in Calcutta accounted for 50 to 80% of the infections in the following months. After this report, O3:K6 isolates identical to those isolated in Calcutta were reported from food-borne outbreaks and from sporadic cases in Bangladesh, Chile, France, Japan, Korea, Laos, Mozambique, Peru, Russia, Spain, Taiwan, Thailand, and the United States. Other serotypes, such as O4:K68, O1:K25, and O1:KUT (untypeable), that had molecular characteristics identical to that of the O3:K6 serotype were subsequently documented. These serotypes appeared to have diverged from the O3:K6 serotype by alteration of the O:K antigens and were defined as “serovariants” of the O3:K6 isolate. O3:K6 and its serovariants have now spread into Asia, America, Africa, and Europe. This review traces the genesis, virulence features, molecular characteristics, serotype variants, environmental occurrence, and global spread of this unique clone of V. parahaemolyticus.

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Section: Methods For Detectionmentioning

confidence: 99%

Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

et al. 2007

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“…This could also be the reason why the detection rate of pathogenic V. parahaemolyticus with the trh gene in the environment is low. Ward and Bej used a multiplexed real-time PCR to detect V. parahaemolyticus in oysters, with results that were 51% positive for the tlh gene and 12.1% for the tdh gene but negative for the trh gene (45). In the study conducted by Rizvi and Bej, their results showed that 58% of the 24 oysters tested were positive for the tlh gene, 21% for the tdh gene, and 0.7% for the trh gene (46).…”

Section: Discussionmentioning

confidence: 99%

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

Liao

Yong

Wang

2017

Appl Environ Microbiol

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This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus. Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log 10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log 10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (Ϫ0.245, Ϫ0.152, and Ϫ0.121 log 10 CFU/day, respectively) were higher than those estimated with the MMs (Ϫ0.134, Ϫ0.0887, and Ϫ0.0732 log 10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during lowtemperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

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“…However, in the reaction with amounts of V. rotiferianus DNA 10 times higher than that of V. harveyi DNA, V. harveyi were out of range from less than 1% of the initial amounts. Ward and Bej 16) reported that co-existence of >2,500-fold nonspecific DNA caused a coprecipitation effect and inhibited the PCR in the real-time PCR system for V. parahaemolyticus targeting tlh, ORF8, tdh, and trh genes. However, in this experiment, we did not use such a huge amount of DNA as the competitor.…”

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confidence: 99%

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

Fukui

Sawabe

2008

Microb. Environ.

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Vibrio harveyi cause mass mortalities of cultured marine fish. To address the ecology of V. harveyi in aquaculture, intensive monitoring is needed. We first optimized a quantitative real-time PCR method to determine V. harveyi abundance. The designed TaqMan probe and primers based on the 16S rRNA gene were specific at 68°C of annealing and extension. Furthermore, the method using a chelating resin method was able to enumerate 1.7×10 2 CFU/ml in breeding seawater at an abalone farm. This method represents a good tool for monitoring the ecology of V. harveyi in marine environments within 5 h.

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Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

Cited by 121 publications

References 50 publications

Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

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

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