Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX^® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+ V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+ V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX^® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX^® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+ V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+ V. parahaemolyticus.     

Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.     

Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India

The levels of total and tdh⁺ Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh⁺ V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh⁺ V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.     

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.     

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.     

Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log₁₀ (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter (P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.     

DETECTION OF VIBRIO ALGINOLYTICUS AND VIBRIO PARAHAEMOLYTICUS IN SHELLFISH SAMPLES USING COLLAGENASE-TARGETED MULTIPLEX-PCR

The increase of Vibrio infections especially associated with the consumption of contaminated seafood and fish underlines the necessity of an efficient monitoring system for Vibrio spp. supported by appropriate detection tools both in food products and in clinical samples. The aim of this study was the evaluation of the reliability of the collagenase‐targeted multiplex‐polymerase chain reaction (m‐PCR) for the detection of Vibrio alginolyticus, Vibrio cholerae and Vibrio parahaemolyticus in shellfish samples enriched in alkaline peptone water (APW). The coupling of a pre‐enrichment phase of the samples with the specificity of the PCR‐based assay was applied successfully to the detection of V. alginolyticus and V. parahaemolyticus, demonstrating that collagenase‐target m‐PCR may be used as a valid molecular target to discriminate the three Vibrio species.     

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D = 0.942), while that of sequence analysis of the gyrB gene was minimal (D = 0.702). The discriminatory ability was greatly enhanced (D = 0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.     

Rapid and sensitive detection of Vibrio parahaemolyticus in sea foods by electrochemiluminescence polymerase chain reaction method

Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. Because of the safety concerns, detection and characterization of V. parahaemolyticus have attracted much attention. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) method combined with universal probes hybridization technique was applied to rapid detection of V. parahaemolyticus, infected and uninfected sea foods for the first time. Whether the sea food samples were infected was discriminated by detecting the gyrase B (gyrB) gene. We detect V. parahaemolyticus both in artificially contaminated sea foods and natural samples. The experiment results show that the infected and uninfected sea food samples can be clearly identified and the detection limit for V. parahaemolyticus is 1.6 pg purified genomic DNA in the presence of 1 μg non-specific background DNA. The technique may provide a new means in V. parahaemolyticus detection due to its simplicity and high efficiency.     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Susceptibility of Vibrio parahaemolyticus to disinfectants after prior exposure to sublethal stress

In the present study, Vibrio parahaemolyticus 690 in phosphate buffered-saline containing 3% NaCl was subjected to sublethal stresses: heat shock at 42 °C for 15 min, acid adaptation at pH 5.0 for 30 min, or cold shock at 20 °C for 4 h. The effect of sublethal stress on the susceptibility of V. parahaemolyticus to a chlorine-containing disinfectant (Clidox-S) and a quaternary ammonium compound (Quatricide) at 25 and 40 °C was investigated. It was found that the sublethal stresses examined enhanced the resistance of V. parahaemolyticus 690 to both disinfectants. Depending on the kinds of sublethal stress, V. parahaemolyticus 690 showed various degrees of enhanced resistance to disinfectants. Furthermore, the phenomenon of enhanced resistance to the disinfectants was more marked at 40 than at 25 °C.     

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.     

Biological characterization of two marine Bdellovibrio-and-like organisms isolated from Daya bay of Shenzhen, China and their application in the elimination of Vibrio parahaemolyticus in oyster

Bdellovibrio-and-like organisms (BALOs) are a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. However, nothing is known of the application potential of marine BALOs in safeguarding seafood safety. Here, biological characterization of two marine BALOs strains and their application in the elimination of Vibrio parahaemolyticus in oyster (Crassostrea ariakensis) at the laboratory scale were investigated.BALOs strains BDH12 and BDHSH06 were isolated from sediment of Daya bay in Shenzhen of China, with Shewanella putrefaciens strain 12 and V. parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16S rDNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bdellovibrio, Bacteriolyticum, Bacteriovorax, or Peredibacter.Biological characterizations revealed that both strains had the optimal pH, salinity and temperature at 7.2, 3% and 30 °C, correspondingly. They could not utilize autoclaved, dead cells as hosts. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 82.5% (47 strains) and 84.2% (48 strains) of the total 57 preys tested respectively. In combination, they lysed 98.2% (56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains.A 7-day laboratory-scale V. parahaemolyticus elimination experiment in oyster showed that in the control, the cell counts of total vibrios and V. parahaemolyticus strain Vp plus in water and in oyster intestines were on the rise, whereas in the BALOs treated groups, their numbers were down from 8.09 ± 0.05 log CFU/ml and 8.02 ± 0.04 log CFU/ml to 2.39 ± 0.01 log CFU/ml and 2.33 ± 0.01 log CFU/ml, respectively. The same patterns could also be observed in oyster intestines. Results of this study indicate the feasibility of using BALOs to biologically control or even eliminate V. parahaemolyticus in seafood oyster.     

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1–10³ cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10²–10³ cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.     

Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China

A total of 1293 seafood samples from fishing farm, retail markets, restaurants and cooking rooms of hotels in Jiangsu province and Shanghai city of China were collected and analyzed for the prevalence of Vibrio parahaemolyticus during July to October in 2007. Two hundred and fifty one isolates of V. parahaemolyticus were identified, of which 8 isolates were positive for tdh and 2 were positive for trh gene. Three tdh positive isolates were identified from low-temperature preserved seafood samples and 5 isolates from fresh seafood samples, of these tdh positive isolates, 3 were positive in ORF8-PCR test. The genetic diversity among V. parahaemolyticus isolates was assessed using random amplified polymorphic DNA (RAPD)-PCR and the results showed that there were 33 different genetic patterns that were clustered into nine groups (groups A to I) at 82% similarity level. About 31.9% of the isolates belong to type III9d that were widely distributed in fresh, iced, frozen, dried and salted seafood samples. Seven tdh positive isolates belonged to group A and one belonged to group C, 2 trh positive isolates were type I10d belonging to group F, which was identical to that of reference strains isolated from patients. This study demonstrated genetic variability within V. parahaemolyticus isolates from seafood in Chinese markets and confirmed the presence of toxigenic V. parahaemolyticus not only in fresh but also in iced and frozen seafood products indicating that low-temperature preserved seafood might be also a vehicle for transmitting pathogenic V. parahaemolyticus.     

A review of the current status of cultural and rapid detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus is ubiquitous in estuarine environments and can be commonly found in seafood products. This bacterial pathogen continues to emerge as an important cause of foodborne illness, and several foodborne disease outbreaks caused by V. parahaemolyticus have been linked to the consumption of contaminated seafood, in particular those consumed raw such as oysters. In response to these outbreaks, especially during the 1990s, several cultural, immunological‐based and molecular detection methods have been developed, which allow for rapid detection and quantification of total and pathogenic V. parahaemolyticus. The development of molecular methodology has allowed for clinical and environmental isolates of V. parahaemolyticus to be subtyped, thus providing the framework for risk‐based strategies aimed at controlling foodborne outbreaks cause by this pathogen. It is important that the detection and typing methods strive to accomplish detection and differentiation of the pathogenic strains from environmental (non‐pathogenic) ones, as well as to detect the presence of the organism and not just the presence of V. parahaemolyticus produced toxins, which can also be produced by closely related species. This review covers the current status of detection and typing methodology for identification and characterisation of V. parahaemolyticus from seafood.     

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR^® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.     

Genome Sequencing of Seafood-borne Vibrio Parahaemolyticus VP49 Reveals the Presence of Novel Virulence Attributes

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium autochthonous to the marine environments and responsible for seafood-borne gastroenteritis. Though the hemolysins TDH and/or TRH are classical virulence factors, several other potential virulence factors may contribute to their pathogenicity. In this study, V. parahaemolyticus was isolated from the seafood harvested along southwest coast of India and confirmed by standard biochemical and molecular methods. Genome sequencing of V. parahaemolyticus revealed the presence of T3SS2 operon in an approximately 44 kb region in close proximity to the hemolysin gene trh. The annotation of T3SS2 operon revealed the presence of genes encoding apparatus proteins VscC2/R2/S2/T2/U2/N2, VcrD2, an ATPase VscN2, transloconsVopB2/D2, and effectors VopA/C/L. To the best of knowledge, this is the first report on sequencing and characterization of a T3SS gene cluster in seafood isolate of V. parahaemolyticus and this information will be of assistance in future studies to determine the different virulence attributes as well as mechanisms that enhance environmental or host fitness of V. parahaemolyticus. The presence of such virulence attributes in V. parahaemolyticus isolated from seafood suggests the potential of these isolates to cause infection in humans upon ingestion of contaminated seafood and questions the safety of seafood to consumers.     

Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water

The safety and accessibility of drinking water are major concerns throughout the world. Consumption of water contaminated with infectious agents, toxic chemicals or radiological hazards represents a significant health risk and is strongly associated with mortality. Therefore, we have developed an oligonucleotide-based microarray using the sequences of 16S-23S rDNA internal transcribed spacer regions (ITS) and the gyrase subunit B gene (gyrB) found in the most prevalent and devastating waterborne pathogenic agents. This new diagnostic contains 26 specific probes and can simultaneously detect Aeromonas hydrophila, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio choleraeo, Vibrio parahaemolyticus, Yersinia enterocolitica and Leptospira interrogans. Testing was carried out against a total of 218 bacterial strains, including 53 representative strains, 103 clinical isolates and 62 strains of other bacterial species belonging to 10 genera and 48 species. The results were specific and reproducible, with a detection sensitivity of 0.1 ng DNA or 10⁴ CFU/ml achieved for pure cultures of each target organism. The diluted cultures and real drinking water samples were tested by the microarray with 100% accuracy. This novel diagnostic method is superior in time- and labor-efficiency to conventional bacterial culture and antiserum agglutination, and can be readily applied to epidemiological surveillance and other food safety applications.     

Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.