OBJECTIVETo investigate the occurrence of important foodborne pathogens in shellstock Pacific oysters in the food markets in South China.

METHODSFrom July 2007 to June 2008, retail oysters were collected in different seasons from South China and analyzed for the prevalence and levels of Listeria monocytogenes, Vibrio vulnificus and Vibrio parahaemolyticus.

RESULTSNone of L. monocytogenes could be detected in any of the 202 oyster samples tested, while E vulnificus and V. parahaemolyticus could be detected in 67 (54.9%) and 109 (89.3%) of the 122 oyster samples analyzed, respectively, with an MPN (most probable number) value greater than or equal to 3. V. vulnificus and V. parahaemolyticus with a more than 102 MPN/g were found in 36 (29.5%) and 59 (48.4%) of the 122 oyster samples, respectively. The tdh and trh genes were detected in 4 (0.3%) and 8 (0.6%) of the 1 349 V parahaemolyticus isolates, respectively. Of the 122 samples, 4 (3.3%) was positive for either tdh or trh. The levels of V. vulnificus and total V. parahaemolyticus in oysters in South China varied in different seasons.

CONCLUSIONV. vulnificus and pathogenic V. parahaemolyticus are frequently found in oysters in south China, which may pose a potential threat to public health. Data presented here will be useful for the microbiological risk assessment in oysters in China.

Animals ; China ; Commerce ; Food Microbiology ; Listeria monocytogenes ; isolation & purification ; Ostreidae ; microbiology ; Vibrio parahaemolyticus ; isolation & purification ; Vibrio vulnificus ; isolation & purification

OBJECTIVEThis study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of Vibrio parahaemolyticus (V. parahaemolyticus).

METHODSThe specificity of this assay was evaluated by using a panel of 33 strains of V. parahaemolyticus and 22 strains of other species bacteria. The sensitivity was determined by using serial dilutions of V. parahaemolyticus (ATCC 17802) chromosomal DNA (5×10(0) - 5×10(5) copies/µl). The samples were also tested by using qualification PCR assay and Taqman real-time PCR assay in parallel for comparison with LAMP.

RESULTSBoth sensitivity and specificity of LAMP assay, PCR assay and Taqman real-time PCR assay were 100% (22/22, 33/33, respectively). The detection limits of above three methods assay were 5×10(1) copies/µl, 5×10(3) copies/µl and 5×10(2) copies/µl, respectively. The reaction period of time needed of the above three assays was 22 min, 3 h, 50 min, respectively.

CONCLUSIONCompared to qualification PCR assay and Taqman real-time PCR assay, the established LAMP assay was better in low detection limit and less reaction time, which made it an ideal method for quick detection of V. parahaemolyticus.

Nucleic Acid Amplification Techniques ; methods ; Sensitivity and Specificity ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVEA novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.

METHODSThe Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.

RESULTSThe capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.

CONCLUSIONSThe Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Food Microbiology ; methods ; Immunomagnetic Separation ; methods ; Nanotechnology ; Real-Time Polymerase Chain Reaction ; methods ; Seafood ; analysis ; microbiology ; Vibrio cholerae ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVETo apply pulse-field gel electrophoresis analysis(PFGE) in analysing a case of food poisoning caused by Vibrio parahaemolyticus.

METHODSPFGE using restriction enzyme Not I was employed in molecular subtyping of thirty strains of V. parahaemolyticus isolated from a case of food poisoning in Guangzhou city and PFGE patterns were analyzed by using BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA).

RESULTSThirty strains were of the same type of pulsotype.

CONCLUSIONSMolecular subtyping by PFGE might disclose the epidemiological relationships of the strains from humans, food and the environment, giving a strong molecular epidemiological evidence and a support for the source-tracking of outbreak events.

Bacterial Typing Techniques ; methods ; China ; Electrophoresis, Gel, Pulsed-Field ; methods ; Foodborne Diseases ; microbiology ; Humans ; Vibrio parahaemolyticus ; classification ; genetics ; isolation & purification

OBJECTIVETo improve the key technology of immunesensors in immobilizing bio-sensitive element and keeping its bioactivity, an enzyme immunosensor based on chitosan-SiO(2) (CS-Sio(2)) hybrid membrane was fabricated. To estimate the new immunosensor Vibrio parahaemolyticus which was the main pathogens of aquatic products.

METHODSA CS-SiO(2) hybrid membrane was prepared using sol-gel method. The enzyme immunosensor was fabricated by coating the membrane and horseradish peroxidase labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) on the surface of four-channel screen-printed carbon electrode. The immunosensor was characterized by cyclic voltammetry. Vibrio parahaemolyticus could be detected according to the decrease percentage (DP) of peak current before and after immune response, while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction. Seven kinds of bacteria, like Vibrio alginolyticus, were selected for specific experiments.

RESULTSBy studying the infrared spectrum of three kinds of films, the CS-SiO(2) hybrid membrane was prepared and HRP-anti-VP was fixed in the hybrid membrane. Under the optimum conditions of immunoreaction and electrochemical detection, the DP of peak current before and after immune response showed a linear relation with lgC in the range of 10(4) - 10(9) cfu/ml, while the linear regression equation was: DP = 6.5 lgC-3.319, the correlation coefficient was 0.9958 and the detection limit was 6.9 x 10(3) cfu/ml (S/N = 3). The immunosensor possessed acceptable specificity, reproducibility (RSD < 6%), stability (the amperometric response was 95% of the initial response after a week) and accuracy (96.7% of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003).

CONCLUSIONThe enzyme immunosensor based on CS-SiO(2) hybrid membrane gave a good performance in rapid detection of Vibrio parahaemolyticus.

Biosensing Techniques ; instrumentation ; methods ; Electrochemical Techniques ; instrumentation ; methods ; Equipment Design ; Immunoenzyme Techniques ; instrumentation ; methods ; Silicon Dioxide ; Vibrio parahaemolyticus ; isolation & purification

We developed single base extension-tags (SBE-tags) microarray to detect eight common food-borne pathogens, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki, Shigella, Escherichia coli O157:H7 and Campylobacter jejuni. With specific PCR primers identified and integrated for eight food-borne pathogens, target sequences were amplified and purified as template DNA of single base extension-tags reaction. The products were hybridized to microarrays and scanned for fluorescence intensity. The experiment showed a specific and simultaneous detection of eight food-borne pathogens. The system limits is 0.1 pg for a genomic DNA and 5x10(2) CFU/mL for Salmonella typhimurium cultures. The single base extension-tags assay can be used to detect food-borne pathogens rapidly and accurately with a high sensitivity, and provide an efficient way for diagnosis and control of disease caused by food-borne pathogens.
Food Contamination ; analysis ; Food Microbiology ; Humans ; Listeria monocytogenes ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; methods ; Salmonella typhimurium ; isolation & purification ; Staphylococcus aureus ; isolation & purification ; Vibrio parahaemolyticus ; isolation & purification

OBJECTIVETo explore the phenetic and genetic features of clinical Vibrio parahaemolyticus strains from 2007-2009 in China.

METHODSA total of 135 clinical Vibrio parahaemolyticus strains, isolated from Zhejiang, Jiangsu, Sichuan, Guangxi, Liaoning Provinces during 2007 to 2009, were selected for the research. The occurrence of virulence genes thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh), species-specific genes thermolabile hemolysin (tlh), toxR, VPM and gyrB, the pandemic clone gene markers(GS-PCR, PGS-PCR, orf8 and HU-α) in 135 Vibrio parahaemolyticus strains was detected by PCR. The antimicrobial susceptibilities to eight antimicrobial agents of the experimental strains were determined by the broth microdilution method. All strains were serotyped and underwent the cluster analysis with pulsed-field gel electrophoreses.

RESULTSThe results of PCR methods claim that all experiment strains carry species-specific genes such as tlh, toxR, gyrB, VPM. Among clinical strains, 85.9% (116/135) carry tdh and/or trh. 85.2% (115/135) were positive for tdh, and 3.0% (4/135) were positive for trh; while 3 strains carried both.66.7% (90/135) , 80.7% (109/135) , 65.2% (88/135) , 66.7% (90/135) clinical strains carried the genes of GS-PCR, PGS-PCR, orf8, HU-α, respectively. The results of antibiotics susceptibility test showed that 8.1% (11/135) strains were resistant to at least one agent, including 9 strains were resistant to ampicillin, 2 strains were resistant to trimethoprim and sulphamethoxazole, and 1 strain were resistant to tetracycline. All clinical strains were sensitive to cefotaxime, ceftazidime, gentamicin, ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 29 serotypes were identified for clinical strains, predominantly O3, O4 and O1 groups, accounting for 89.6% (121/135). O3: K6 was dominant serotype, accounting for 56.3% (76/135). The pandemic flora in China included O3: K6, O4: K68, O1: K36, O1: K25, O1: K5 and O3: K29 serotypes.Genomic DNAs of 135 clinical strains were digested with SfiI and NotI, the molecular size of PFGE restriction fragments used for analysis mainly ranged from 30-700 kb.When subjected to UPGMA clustering, 6 and 9 clusters were grouped by SfiI and NotI, and the minimal similarity was 52.6% and 58.7%, and pandemic flora were located in C groups and D group, respectively.

CONCLUSIONMost of Vibrio parahaemolyticus strains isolated from clinical sources in China were pathogenic. The pandemic clone, especially O3: K6 was prevalent. The GS-PCR and HU-α genes were reliable markers to identify the pandemic flora. The serotype by PFGE was reliable to distinguish the pandemic flora and the sporadic strains.

China ; epidemiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Molecular Epidemiology ; Vibrio Infections ; epidemiology ; microbiology ; Vibrio parahaemolyticus ; genetics ; isolation & purification ; pathogenicity ; Virulence ; genetics

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10(7) CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.
ATP-Binding Cassette Transporters ; genetics ; DNA Primers ; chemistry ; metabolism ; Food Microbiology ; methods ; Genome, Bacterial ; Real-Time Polymerase Chain Reaction ; Seafood ; microbiology ; Vibrio ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVETo study the molecular epidemiology of Vibrio parahaemolyticus isolates from clinical and environmental samples collected in Hangzhou area during 2000 and 2002.

METHODSV. parahaemolyticus isolates from food-poisoning, sporadic diarrhea patients and seafood in market in Hangzhou during 2000 and 2002 were serotyped. From clinical isolates of serotype O3:K6 and environmental isolates of serotype O3:KUT, virulence genes, tdh and trh, were examined by PCR. Molecular typings [including ribotyping, random amplified polymorphic DNA analysis (RAPD), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR)], were also performed.

RESULTSAmong 13 food-poisoning outbreaks caused by V. parahaemolyticus, O3:K6 strains with tdh positive and trh negative were detected in 11 episodes (84.6%), and O4:K8 strains with tdh positive and trh negative were responsible for other 2 outbreaks (15.4%). In 34 V. parahaemolyticus isolates from sporadic diarrhea patients, O3:K6, O4:K8, and O1:KUT strains with tdh positive and trh negative were detected with proportions of 26.5% (9/34), 17.6% (6/34), and 38.2% (13/34), respectively, and other 6 strains were not able to be serotyped. Of 64 isolates from seafood in which both tdh and trh were negative except trh was positive in one O1:KUT strain, 37 belonged to 7 serotypes except O3:K6 or O4:K8, and 9 were not able to be serotyped. The fingprintings of ribotyping, ERIC-PCR, and RAPD showed that almost all of O3:K6 isolates from food-poisoning and sporadic diarrhea patients were genetically close to each other and there were obviously genetical difference between O3:K6 isolates from clinic and O3:KUT isolates from environment.

CONCLUSIONThere was a distinct serotype distribution between V. parahaemolyticus isolates from clinic patients and seafood. One group of close related V. parahaemolyticus O3:K6 strains with tdh positive and trh negative seemed to be prevailing in Hangzhou in those years.

China ; epidemiology ; DNA, Bacterial ; analysis ; Disease Outbreaks ; Food Contamination ; Foodborne Diseases ; Humans ; Molecular Epidemiology ; Polymerase Chain Reaction ; Seafood ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVESTo investigate the characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China.

METHODSThermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in a total of 174 strains of V. parahaemolyticus isolated from patients and environment (seafood) in Hangzhou area by PCR.

RESULTSThe tdh was found in 92 out of 94 V. parahaemolyticus strains from food poisoning patients and in 33 out of 34 strains from sporadic diarrhea patients, and trh was not detected in all above clinical strains. Meanwhile the tdh was negative in all V. parahaemolyticus strains from environment, and the trh was also negative except one strain with urease activity. All strains with trh negative had no the activity of urease.

CONCLUSIONSThe V. parahaemolyticus strains from food poisoning patients and sporadic diarrhea patients are tdh positive and trh negative. The V. parahaemolyticus strains with tdh negative and almost trh positive in environment might be a potential pathogen in Hangzhou.

Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; China ; Environmental Microbiology ; Foodborne Diseases ; microbiology ; Hemolysin Proteins ; genetics ; Humans ; Shellfish ; microbiology ; Urease ; genetics ; Vibrio Infections ; microbiology ; Vibrio parahaemolyticus ; classification ; genetics ; isolation & purification