| Vibrio parahaemolyticus(Vp),a rod-shaped or multiple forms halophilic Gram-negative bacterium,belongs to Vibrio genus,is a common foodborne pathogen all over the world.It’s widely distributed in marine and estuarine,and virulent V parahaemolyticus strains are transmitted by consumption of raw or undercooked seafood causing acute gastroenteritis.V parahaemolyticus-caused food poisoning incidents occurred every year in China,and it’s took the first place in the food-borne pathogens.Rapid detection and epidemiological investigation of V.parahaemolyticus play key roles to control the foodborne infection.The disadvantage of classical detection method were complicated operation,long period and high costs,which encouraged the development of molecular detection technology targeting species-specific genes.Several PCR assays,based on virulence and housekeeping genes and housekeeping genes,have been developed for the identification of V.parahaemolyticus,which may leads to leak detection or false positives.Threrfore,it’s imminent to establish a sensitive and specific molecular detection methods.In this study,according to the phylogenetic and homologous relationships of toxR and toxS genes in Vibrio genus,three pairs of PCR primers(primers-S,primers-R and primers-R-S)were designed,targeting at the toxR,toxS and region spanning toxR and toxS genes,respectively.A total of 57 V.parahaemolyticus isolates and 19 non-V.parahaemolyticus strains were used to evaluate these 3 pairs of primers.The results showed that the primers-R-S was more stability and species-specificity than the other two pairs of primers.Therefore,the toxR-S PCR based on primers-R-S was refined as rapid V parahaemolyticus detection method in this study.The detected limitation of toxR-S PCR was 2pg genomic DNA,which showed an ideal sensitivity.Compare analysis between toxR-S PCR and classical method was performed in 359 seafood samples,and showed 100%sensitivity,92.39%specificity and 95.82%coincidence rate.The further study confirmed that toxRS PCR was specific and rapid,and the toxR-S PCR is suitable for.rapid identification of the V.parahaemolyticus isolates and seafood samples.V.parahaemolyticus epidemiological investigation of Jiangsu,Zhoushan,Shanghai,et al,had been reported.But little investigation and analysis of East China has been reported.It’s significance for the control of vibriosis to conduct the molecular epidemiological study of V.parahaemolyticus in this area.The detection of pandemic virulence genes and Multilocus Sequence Typing(MLST)for V.parahaemolyticus isolates was carried out in this section.4 pathogenic strains and 2 pandemic strains were detected in a total of 152 iaolates.Positive rate of MTase was 1.3%.No isolates carried the complete III secretion system and 78.3%of the isolates carried the complete set of VI secretion system.152 Vp strains was parted into 84 STs,which had 69 new STs.Cluster analysis showed that part of the environment isolates and clinical pathogenic strains had close relatives,and the STs that sources of the strains belongs the same species was more likely to generate clustering relations.It’s not necessary or sufficient for pathogenic isolates to be clustered in homologous complex CC3.On the basis of phylogenetic analysis,the distribution of virulence genes and the source hosts of environmental isolates showed molecular characteristics of polymorphism.The stydies of V.parahaemolyticus epidemiological in East China suggested,4 pathogenic strains and 2 pandemic strains isolated from seafood samples may lead to pandemic potential hazard,and molecular characteristics of polymorphism provides epidemic condition without regularity.V.parahaemolyticus environmental isolates made a potential threat to people’s healthy of East China. |
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