The Study Of Molecular Detection And Genotyping Of Vibtio Parahaemolytcus

Vibrio parahaemolytcus is a Gram-negative halophilic bacterium distributed worldwide in the marine and estuarine environment,it infects the seafood products mostly and when people eat the seafood which is contaminated by Vibrio parahaemolytcus,it will cause acute gastroenteritis,such as diarrhea,vomiting,abdominal cramps and so on. It is recognized as the leading cause of human gastroenteritis throughout the world,in many Asian countries such as China,Japan,and in Europe as well as United States,this pathogen is a common cause of foodborne illnesses.The Thermostable direct hemolysin(TDH) and TDH-related hemolysin(TRH) have been recognized as the major virulence factors of this bacterium,and they are encoded by tdh and trh genes respectively, served as markers for pathogenic strains.The most strains of V. parahaemolyticus isolated from environment or seafood are not pathogenic,however over 90%of V.parahaemolyticus isolated from clinical patients produce TDH and they have pathogenicity.Recently,the study of V.parahaemolyticus is involved with the biochemical separation and identification,the rapid detection by molecule methods,the molecule polymorphism analysis with different source and phenotype of V. parahaemolyticus,the expression and structural analysis of virulence proteins and so on.Our country has the long coastline about 32 thousand kilometers,and the seafood is prevail in the southeast coastland, especially,in the area of Zhejiang Province,but the reports about food poisoning caused by V.parahaemolyticus are common occurance.So the detection of V.parahaemolyticus with rapid and correct method is helpful to prevent the contamination of V.parahaemolyticus.The objective of this assay is to analysis the characteristic of virulence genes and the genotyping of V.parahaemolyticus obtained from different sources in Zhejiang Province to help to establish the genomic fingerprinting,also new methods were developed to detect V.parahaemolyticus rapidly,and it will provide a platform to monitor and prevent the infection of V. parahaemolyticus.1.Using the PCR method to detect the tdh and trh genes of the V. parahaemolyticus isolated from clinical patients and seafood products in Zhejiang Province,2006.The results indicated that 92.31%and 1.54%of V.parahaemolyticus isolated from clinical patients possessed the tdh and trh gene respectively,the percent of the tdh-positive and trh-negative strains was 91.54%;In contrast,1.11%of V.parahaemolyticus isolated from seafood products harbored the tdh gene and 2.22%has the trh gene, 96.67%of the strains did not possess both the tdh and trh genes.It showed that the tdh-positive strains is the major pathogeny which was caused the food poisoning in Zhejiang,and the trh-positive strains is the potential pathogeny.2.RAPD technique was used to study the genotyping of the strains isolated form clinical patients and seafood products,four random primers were chosen by optimize the conditions of RAPD.A total of 53 loci, including 45 polymorphic ones were detected from 175 strains.The cluster analysis showed that the strains from three different hospitals and seafood products had a high similarity coefficient,the strains isolated from Hangzhou Hospital of Traditional Chinese Medicine and Sir Run Run Shaw Hospital were clustered into one group,then combined with the isolates from Hospital of Shaoxing,and last combined with the isolates from seafood products.The results of micro-analysis showed that most of the clinical strains had a higher genetic similarity than isolates from seafood products,and they were clustered into one group.43 clusters were obtained from 175 V.parahaemolyticus,the cluster A contained 74.6%of the clinical isolates and the clusterâ… included 52.1% of the isolates from seafood products,but several clusters just contained one strain which was isolated from seafood products.Also,91.1%of the clinical isolates which the serotype was O3 were belonged to the cluster A,and all of the isolates in this cluster were tdh-positive and trh-negative strains.It also indicated that the tdh-positve strains were the leading cause of food poisoning,and the strains which the serotype was O3 could infect people more easily.3.In order to develop the molecular method to detect V. parahaemolyticus rapidly and correctly.New PCR and real-time PCR primers were designed based on the sequence of RAPD regions.These two methods were specific to the V.parahaemolyticus,the detect limit was 5 pg by PCR method and 50 fg of purified DNA or 10~2 CFU per ml for pure cultures,it had the lower detect limit than the PCR method,and provided a technical platform to detect V.parahaemolyticus in the seafood products.