Detection Of Vibrio Parahaemolyticus By Qpcr And Screening And Functional Study Of T6SS-1 Effectors

Vibrio parahaemolyticus(Vp)and Vibrio cholerae(Vc)belong to gram-negative bacteria and are important pathogens that causes food-borne gastroenteritis or sepsis.They are widely distributed in estuaries,coastal waters,and sediments.With the increasing consumption of seafood,it is important to establish a rapid and accurate detection method that can simultaneously detect Vibrio parahaemolyticus and Vibrio cholera.Pathogenic V.parahaemolyticus recognized virulence factors are thermostable direct hemolysin(TDH)and TDH-related hemolysin(TRH).The type VI secretion systems(T6SS)discovered in recent years are widely found in gram-negative bacteria and play an important role in the survival and pathogenicity of bacteria.The various proteins delivered by T6SS,called"effectors," have now been identified and exhibit various toxic activities in target cells.This study was the first to establish a dual-fluorescence quantitative PCR method for the simultaneous and quantitative detection of Vibrio parahaemolyticus(toxR)and Vibrio cholerae(ompW)based on the TaqMan probe method,which is an effective method for rapid detection of Vibrio parahaemolyticus and Vibrio cholerae simultaneously.Using the ultrafiltration tube concentration method,Label-free proteomics analysis and Western-blot technique,we successfully screened and identified the secreted proteins VP0837 and VP0285 from wild strain SH112 and double deletion strain ?vipAl-hcpl belonging to Vibrio parahaemolyticus T6SS-1 effectors.In addition,by analyzing the biological characteristics and pathogenicity of the effector protein VP0837 gene-deleted strain,it was shown that T6SS-1 plays an important role in pathogenicity of Vibrio parahaemolyticus.Experiment ? Establishment and application of the duplex real-time PCR of Vibrio parahaemolyticus and Vibrio choleraIn this study,a TaqMan-based duplex real-time PCR assay was established for the detection of Vibrio parahaemolyticus(Vp)and Vibrio cholera(Vc).The specific primers and probes were designed based on toxR gene of V.Parahaemolyticus and ompW gene of V.cholera.The results showed that this PCR assay could unambiguously detect V.parahaemolyticus and V.cholera with a minimum detection limit of 10 CFU/mL and was with high sensitivity.The specificity of the assay showed that the two bacteria had no cross reaction with other pathogens(Such as Escherichia coli 0157,Salmonella,Vibrio mimicus and Vibrio vulnificus).In addition,it had good repeatability between the batch and within the group and the coefficient of variation was less than 1.5%.When the developed method applied for assaying food samples artificially contaminated by VP or VC and detected immediately,the detection limit was found to be 100 CFU/mL(Vp)and 50 CFU/mL(Vc)in shrimp samples or 50 CFU/mL(Vp)and 50 CFU/mL(Vc)in shellfish samples per reaction without an enrichment step.The minimum detection limit of method was 1 CFU/mL in shrimp contaminated by VP or VC enriching for 2 hours.It was drawn a conclusion that this newly established duplex real-time PCR assay has the characteristics of high sensitivity,strong specificity,and good repeatability and the entire detection procedure including DNA extracted of this method could be completed within 1 to 3 hours.It's an effective method for rapid detection of V parahaemolyticus and V.cholera.Experiment ? Screening and identification of type VI secretion system 1 effectors of Vibrio parahaemolyticusFor the initial screening and identification of type VI secretion system 1 effectors Vibrio parahaemolyticus.Based on the previous research results of this laboratory,the secretory proteins of the wild-type SH112 and delta vipA1-hcpl double-deleted strains of V parahaemolyticus were extracted by ultrafiltration tube concentration method.After proteolysis for LC-MS/MS detection,the screened proteins were subjected to Gene Ontology(GO)analysis and LFQ(Label Free Quantitation)analysis.In addition,the two secreted proteins VP0837 and VP0285 were selected for prokaryotic expression,polyclonal antibodies were prepared,and the secreted proteins extracted were identified by Western-blot.The results showed that 52 differential proteins were successfully identified by LC MS/MS method according to the secreted proteins extracted from the wild strains SH112 and ?vipAl-hcpl double deletion strains,of which 36 were down-regulated in?vipAl-hcpl and 16 were up-regulated;GO results proved that the identified proteins were mainly involved in the process of metabolism,biological location,signaling molecules,redox,biological binding and so on.Western-blot showed that the mouse anti-VP0837 and VP0285 sera can specifically recognize the supernatant protein of the wild strain of V.parahaemolyticus SH112,but failed to identify the supernatant protein of the double deletion strain?vipA1-hcp1;indicating the secretion protein VP0837 and VP0285 belong to the effector protein of Vibrio parahaemolyticus T6SS-1 and play an important role in the secretory function.The results laid the foundation for the further study of the function of VP0837 and VP0285 proteins and the rapid detection method of Vibrio parahaemolyticus.Experiment ? Construction and analysis of pathogenicity of T6SS-1 effector VP0837 of Vibrio parahaemolyticusThe aim of this study was to study the role of T6SS-1 in the biological characteristics and pathogenicity of V parahaemolyticus.The T6SS-1 effector VP0837 was used as the study object to construct the vp0837 gene-deleted strain and the complement strain C?vp0837.By analyzing the differences in growth characteristics,motility,and biofilm formation ability of each strain,the effects of deletion strains on cell adhesion invasion and toxicity,differences in transcript levels of cytokines,and lethality in mice were compared.The test results showed that compared with the wild strain SH112 and the complement strain C?vp0837,the motility and biofilm formation ability of the deletion strain vp0837 were not significantly different;and the toxicity,adhesion and invasion of Caco-2 cells by?vp0837 were significantly reduced.However,the expression of IL-1? and IL-6 in Caco-2 cells was significantly up-regulated by ?vp0837;the lethality of ICR mice was significantly decreased.The above results suggest that the secretory protein vp0837 gene of V.parahaemolyticus T6SS-1 not only participates in the host cell cytotoxicity and inflammatory process,but also has a significant effect on the lethality of mice.It is fully demonstrated that the vp0837 gene plays an important role in the infection pathogenicity of V parahaemolyticus.