Rapid Quantitative Detection of Vibrio parahaemolyticus in Seafood by MPN-PCR

Luan, Xiaodong; Chen, Jixiang; Yu, Lie; Li, Yun; Jia, Jingdun; Liu, Rui; Zhang, Xiaohua

doi:10.1007/s00284-008-9177-x

Cited by 40 publications

(21 citation statements)

References 12 publications

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“…Enumeration of the three Vibrio spp. in water samples was accomplished by enrichment in alkaline peptone water (APW) and using a combined most-probable-number (MPN)-real-time PCR method (15,37). Water samples (1 liter, 100 ml, and 10 ml) were filtered in triplicate through 0.45-m-pore-size nitrocellulose membranes (Whatman, GE Healthcare, Versailles, France), and the filters were incubated in APW at 41.5°C for 18 h. Smaller volumes (i.e., 1 ml of undiluted sample and 1 ml of 10-fold and 100-fold dilutions) were inoculated, in triplicate, directly into APW broth and incubated at 41.5°C for 18 h.…”

Section: Methodsmentioning

confidence: 99%

Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Esteves

Hervio-Heath

Mosser

et al. 2015

Appl Environ Microbiol

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Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae are Gram-negative halophilic bacteria autochthonous to marine and estuarine environments and components of those ecosystems (1). These vibrios are recognized throughout the world as agents of gastroenteritis resulting from consumption of raw or undercooked seafood and serious infections caused by exposure of skin wounds to seawater (2).Subpopulations of V. parahaemolyticus and V. vulnificus are potential agents of disease outbreaks. For example, enteropathogenic strains of V. parahaemolyticus produce a thermostable direct hemolysin (TDH) and/or a TDH-related hemolysin (TRH), and the genes tdh and trh code for TDH and TRH, respectively (3). Other factors are associated with virulence of V. vulnificus, including the vvhA gene encoding hemolytic cytolysin (4, 5). It is important to note that V. parahaemolyticus is the leading cause of bacterial human gastroenteritis associated with seafood consumption, especially in the United States and Japan (6,7,8). In the United States, V. vulnificus is responsible for 95% of all seafoodrelated deaths, with a mortality rate of around 50% (5). V. cholerae, the etiologic agent of cholera, has been detected in natural freshwater and brackish water worldwide, even in areas where no clinical cases of cholera have been reported (1). Most environmental isolates of V. cholerae are of the non-O1/non-O139 serotype but are capable of causing diarrheal outbreaks (7, 9). The presence and isolation of these three Vibrio spp. have also been documented to occur in coastal waters and shellfish-rearing areas in Europe, i.e., Spain (10), Italy (11, 12), Denmark (13), and Norway (14). These vibrios have been isolated in French coastal waters and shellfish at different locations along the English Channel, the Atlantic Ocean, and the Mediterranean Sea (15,16,17,18).Vibrios are less frequently associated with outbreaks of disease in Europe than in the United States and Asia, and specifically, the risk of V. parahaemolyticus infection is considered to be low (8,19). In France, 100 cases of V. parahaemolyticus infection were reported in 2001, following consumption of mussels imported from Ireland (20). Since then, only sporadic cases of Vibrio infections have been described (21,22).From the perspective of vibrio ecology, the spatiotemporal distribution of vibrios has been linked to environmental factors, with temperature being one of the most important, because it is related to seasonal distribution in temperate coastal areas, with maximal abundance during summer through early fall (23,24,25,26,27,28). Many studies have shown that these bacteria enter a viablebut-nonculturable state when water temperatures average less than 15°C and temperatures above 20°C favor their growth (23,24,25,26,28). Salinity is also an important parameter influencing the dynamics of human-pathogenic vibrios in aquatic systems.

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Section: Methodsmentioning

confidence: 99%

Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Esteves

Hervio-Heath

Mosser

et al. 2015

Appl Environ Microbiol

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“…3 It is therefore a leading cause of seafood-borne gastroenteritis, especially in countries where seafood consumption is high 4 as well as being responsible for foodborne disease outbreaks, with fatalities in Japan in 1950, 5,6 India, 2,7 China, 6,8,9 Taiwan, 10 Korea 11 and Malaysia. 12 The mechanism by which V. parahaemolyticus infects humans has yet to be entirely determined.…”

Section: Asian Journal Of Medical Sciences 5(2014) 33-39mentioning

confidence: 99%

Investigating Brunei’s Seafood Markets for Vibrio parahaemolyticus using the Most Probable Number-Polymerase Chain Reaction

2013

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Background: Vibrio parahaemolyticus (V. parahaemolyticus) is the commonest source of seafood poisoning and has a very high incidence in the countries of Asian. Aims and Objectives:This study aims to investigate the presence of V. parahaemolyticus in seafood from Brunei seafood market using the Most Probable Number-Polymerase Chain Reaction (MPN-PCR).Results: None of the twnety-three seafood samples that were purchased at random from unselectively chosen wet markets and hypermarkets in Brunei Darussalam yielded V. parahaemolyticus. Conclusion:This could be due to any or a combination of: the absence or low level of V. parahaemolytius from where the samples were harvested, inactivation of V. parahaemolyticus during the processing and preservation of the samples, possible sampling factors as well as good hygienic practices in Brunei's seafood market The mechanism by which V. parahaemolyticus infects humans has yet to be entirely determined. However, pathogenic V. parahaemolyticus is known to produce either thermostable direct haemolysin (TDH), TDHrelated haemolysin (TRH) or both.13, 14 TDH and TRH are encoded by tdh and trh genes, respectively. Thus, they are recognized as major virulent factors of V. parahaemolyticus and either or both of these virulent factors can cause illness. The toxR gene is well conserved among V. parahaemolyticus which are nonpathogenic in the absence of tdh or/and trh gene, and consequently used as target for its specific detection. 15According to the Food and Agriculture Organization report, 16 seafood constitutes the main source of animal protein for a significant percentage of world population. Therefore, as V. parahaemolyticus is known to cause gastroenteritis which could be fatal if complications occur, it is important to determine its degree of contamination of seafood to prevent food poisoning in consumers. In addition as a very high level of contamination with V. parahaemolyticus has been reported in bivalves and shrimp culture environment in Malaysia, 12 it is highly likely that seafood in Brunei is also contaminated with V. parahaemolyticus due to the proximity of the two countries and importation of seafood into Brunei.Thus, this study investigated the presence of V. parahaemolyticus in seafood from both wetmarkets and hypermarkets in Brunei Darussalam using MPN-PCR method so as to provide insight into the biosafety assessment of V. parahaemolyticus in the Sultanate.

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“…Sample 1, water used as a negative control; and samples 2-8 represent periodic enrichment cultures, i.e., 8-, 7-, 6-, 5-, 4-, 3-and 2-h enrichment culture of V. parahaemolyticus ATCC 17802, respectively. Utilization of the enrichment-PCR method 27,28) also requires more time than the newly developed assay of this study, as additional gel-run and detection procedures are required after PCR step which is avoided by the use of real-time turbidimeter in the LAMP assay. A recent study has reported successful utilization of real-time PCR method after a 6 h enrichment period in detecting less than 5 V. parahaemolyticus cells/g of seafood, 29) however, this method is more expensive and complex, and requires more time than the LAMP assay of the present study.…”

Section: Specificity and Sensitivity Of Lamp Detectionmentioning

confidence: 99%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

Neogi

et al. 2015

Biol. Pharm. Bull.

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The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n 61) but none of the non-target strains (n 34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n 70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains.Key words Vibrio parahaemolyticus; loop-mediated isothermal amplification; rapid detection; enrichment culture; seafood Vibrio parahaemolyticus is one of the most important seafood-borne pathogen causing gastrointestinal disorders to humans. This halophilic Gram-negative bacterium was first identified as a causative agent of human gastroenteritis in Japan in 1950.1) However, V. parahaemolyticus can occur ubiquitously in the brackish coastal environment of most continents and infections associated with seafood contaminated by this pathogen have occurred throughout the world.2-4) Particularly, the worldwide spread of the V. parahaemolyticus O3 : K6 serotype after its emergence in Asia in 1995, it has become very important to identify this pathogenic species in a rapid and sensitive manner.5) At present, the outbreak of V. parahaemolyticus infections is a significant public health concern in many countries including China, Japan and U.S.A. 6,7)Seafood is very popular in many countries, and simple and specific identification of a pathogen from seafood samples is essential for taking preventive and curative measures. Conventional methods for the diagnosis of V. parahaemolyticus include cultivation of bacteria on selective media followed by biochemical tests. However, the traditional phenotypic identification is problematic, it is very time-consuming requiring 3-7 d and not highly specific because several Vibrio species display similar biochemical characteristics, which make it difficult for rapid identification of V. parahaemolyticus. 8) To circumvent this problem, the identification of V. parahaemolyticus by rapid and specific molecular techniques targeting the genes encoding thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) were developed.9-11) However, these genes are only found in pathogenic strains and most V. parahaemolyticus isolates from the environment sources do not produce TDH or TRH. 3,12) Besides, there are reports of toxigenic factors other than TDH or TRH, e.g., type III secretion system 2, associated with clinical V. parahaemolyticus stra...

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Rapid Quantitative Detection of Vibrio parahaemolyticus in Seafood by MPN-PCR

Cited by 40 publications

References 12 publications

Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Rapid Proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Investigating Brunei’s Seafood Markets for Vibrio parahaemolyticus using the Most Probable Number-Polymerase Chain Reaction

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

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