Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods

Karunasagar, Iddya; Sugumar, G.; Reilly, Peter J.

doi:10.1016/0168-1605(96)00974-9

Cited by 36 publications

(22 citation statements)

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“…In recent years, a number of researchers have drawn attention to the need for more sensitive and rapid techniques using molecular approaches for detection of V. parahaemolyticus in clinical samples, as well as in seafood. PCR-based methods that amplify regulatory toxR sequences (22), conserved sequences such as gyrB (34), conserved chromosomal sequences (16,17,23), and hemolysin sequences such as tdh (7,20,28,32), trh (32), or tlh (thermolabile hemolysin) (2) have been used by various workers. Enumeration of V. parahaemolyticus from seafood are important in the context of current FDA guidelines which indicate that shellfish should contain less than 10,000 V. parahaemolyticus cells per g (25).…”

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confidence: 99%

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

Deepanjali¹,

Kumar²,

Karunasagar³

2005

Appl Environ Microbiol

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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 10 4 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.Vibrio parahaemolyticus is a halophilic bacterium that occurs in estuarine environments worldwide (5,13,14,16,18,19). This organism was first discovered in Japan in 1950 in association with a food poisoning case (9). V. parahaemolyticus infection can cause gastroenteritis in humans, and the illness is most frequently associated with the consumption of raw or undercooked seafood and seafood recontaminated with the bacterium after cooking (31). V. parahaemolyticus accounts for about 70% of the gastroenteritis associated with seafood in Japan (14), and in India about 10% of the cases of gastroenteritis in patients admitted to the Infectious Diseases Hospital in Kolkata are due to V. parahaemolyticus (3). However, not all strains of V. parahaemolyticus are pathogenic. It has been demonstrated that the Kanagawa phenomenon, a beta-hemolysis in high-salt blood agar (Wagatsuma agar), is associated with most clinical strains but with very few environmental strains (32, 38). The hemolysis is due to the production of a thermostable direct hemolysin (TDH) (30). A TDH-related hemolysin (TRH) produced by a Kanagawa phenomenon-negative strain was discovered during investigation of an outbreak of gastroenteritis in the Maldives Islands in 1985 (10, 11). TDH and TRH, encoded by the tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus (31). It has been reported that more than 90% of clinical isolates but less than 1% of environmental isolates produce TDH (7,22,33). In recent years, the incidence of V. parahaemolyticus infection has been increasing in many parts of the world, and this has been attributed to the emergence of a new clone of the O3:K6 serotype carrying only the tdh gene (24). Although various serovars of the...

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

Deepanjali¹,

Kumar²,

Karunasagar³

2005

Appl Environ Microbiol

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175

122

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“…Identification of Vibrios species on the basis of biochemical reactions are pose difficult due to their atypical type of reactions pattern [27]. Ben Kahla-Nakbi et al in 2007 reported that most of the Vibrios species are indole (IND) negative.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Differentiation and Molecular Characterization of Biofield Treated <i>Vibrio parahaemolyticus</i>

Trivedi¹,

Branton²,

Trivedi³

et al. 2015

AJCEM

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Abstract:The recent emergence of the Vibrio parahaemolyticus (V. parahaemolyticus) is a pandemic. For the safety concern of seafood, consumer monitoring of this organism in seafood is very much essential. The current study was undertaken to evaluate the impact of Mr. Trivedi's biofield energy treatment on [ATCC-17802] strain of V. parahaemolyticus for its biochemical characteristics, biotype and 16S rDNA analysis. The lyophilized strain of V. parahaemolyticus was divided into two parts, Group (Gr.) I: control and Gr. II: treated. Gr. II was further subdivided into two parts, Gr. IIA and Gr. IIB. Gr. IIA was analyzed on day 10, whereas, Gr. IIB was stored and analyzed on day 142 (Study I). After retreatment of Gr. IIB on day 142 (Study II), the sample was divided into three separate tubes. The tubes first, second and third were analyzed on day 5, 10, and 15, respectively. The biochemical reaction and biotyping were performed using automated MicroScan Walk-Away ® system. The 16S rDNA sequencing was carried out to correlate the phylogenetic relationship of V. parahaemolyticus with other bacterial species after the treatment. The results of biochemical reactions were altered 24.24%, out of thirty-three in the treated groups with respect to the control. Moreover, negative (-) reaction of urea was changed to positive (+) in the revived treated Gr. IIB, Study II on day 15 as compared to the control. Besides, biotype number was substantially changed in all the treated groups as compared to the control. However, change in organisms were reported in Gr. IIA on day 10 and in Gr. IIB; Study II on day 5 as Shewanella putrefaciens and Moraxella/Psychrobacter spp., respectively with respect to the control i.e. Vibrio sp. SF. 16S rDNA analysis showed that the identified sample in this experiment was V. parahaemolyticus after biofield treatment, and the nearest homolog genus-species was observed as Vibrio natriegens with 98% gene identity. The results envisaged that the biofield energy treatment showed an alteration in biochemical reaction pattern and biotype number on the strain of V. parahaemolyticus.

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“…In this context, many PCR-based techniques (1,8,18) targeting tdh and trh have been developed so far to improve the detection level of the pathogen in various types of seafood. One challenge presented by PCR-based methods is the interference of tdh or trh derived from dead cells present in seafood.…”

Section: Discussionmentioning

confidence: 99%

Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing Vibrio parahaemolyticus in Seafood

Hayashi

Okura

Osawa

2006

Appl Environ Microbiol

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A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producingVibrio parahaemolyticus is a motile gram-negative rodshaped, halophilic, facultative anaerobe that naturally inhabits estuaries and their fauna worldwide. The bacterium can cause one of the major food-borne gastroenteric infections, often associated with the consumption of raw or under-cooked seafood (2). Past epidemiological studies (6, 7, 16) revealed a strong association between gastroenteritis and both thermostable direct hemolysin (TDH) and another hemolysin, termed TDH-related hemolysin (TRH), produced by V. parahaemolyticus. Both hemolysins are thus considered to be major virulence factors of V. parahaemolyticus. Structural genes for TDH and TRH, tdh and trh, are encoded chromosomally, and PCRbased methods to detect the genes have been successfully developed (17, 19).TDH-or TRH-producing V. parahaemolyticus is usually found together with much larger populations of avirulent strains in the environment, and it has therefore been technically difficult to detect these virulent strains in seafood by conventional culture methods (18). In this context, the Food and Drug Administration (4) recommended a limit of 100 cells of V. parahaemolyticus per gram of seafood, expressed as the most probable number (MPN), based at least in part on the assumption that seafood below such a contamination level might not contain the virulent strain. However, the recommended MPN technique requires 4 to 7 days to complete, thereby posing a practical problem for the food safety program to be performed quickly enough to be of use. Furthermore, a recent study (5) has revealed that the total number of V. parahaemolyticus cells does not appear to correlate directly with the number (or the presence of) TDH-producing V. parahaemolyticus. As an alternative approach, several PCR-based methods targeting tdh and trh were developed for the more specific detection of the TDH-and TRH-producing V. parahaemolyticus in seafood (1, 18). Nevertheless these PCR assays do not distinguish between DNA derived from viable or dead cells. This potentially leads to false-positive results for food samples where intact DNA sequences of tdh or trh are present despite the absence of TDH-and TRH-producing V. parahaemolyticus cells due to chemical or heat treatment. Similar technical difficulties in the differentiation between viable and dead cells in DNA-targeted diagnostics have long been discussed for other bacterial species (10,12,14).In order to circumvent these technical obstacles, we describe here a novel combined culture and PCR method for the specific detection of viable TDH-or TRH-producing V. parahaemolyticus in seafood. The method employs two principals: (i) that only viable V. parahaemolyticus cells can penetrate through a soft-agar-coated filter due to their motility, and (ii) that DNAs released from ruptured virulent V. parahaemolyticus cells into enrichment medium can be eliminated by DNase pretreatment so that they do not i...

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Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods

Cited by 36 publications

References 5 publications

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

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

Biochemical Differentiation and Molecular Characterization of Biofield Treated <i>Vibrio parahaemolyticus</i>

Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing Vibrio parahaemolyticus in Seafood

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Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods

Cited by 36 publications

References 5 publications

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

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

Biochemical Differentiation and Molecular Characterization of Biofield Treated &lt;i&gt;Vibrio parahaemolyticus&lt;/i&gt;

Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing Vibrio parahaemolyticus in Seafood

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Biochemical Differentiation and Molecular Characterization of Biofield Treated <i>Vibrio parahaemolyticus</i>