Establishment Of Detection Methods For Vibrio Parahaemolyticus And Associated Virulence Factors

Vibrio species are naturally diverse bacteria that inhabit aquatic environments and marine animals as symbionts and commensals. Among more than 70 identified Vibrio species, Vibrio cholerae, V. parahaemolyticus and V. vulnificus are major concerns as they are pathogenic to animals, including humans. Infection by V. cholerae, V. parahaemolyticus and V. vulnificus occurs through ingestion of contaminated seafood or exposure to aquatic environments. V. parahaemolyticus is recognised as the leading cause of human gastroenteritis associated with seafood consumption, major virulence factor thermostable direct hemolysin (TDH) and heat-related hemolysin (TRH).According to groEL gene sequences of V. cholerae, V. parahaemolyticus and V. vulnificus, we designed and synthesized three pairs of primers groVc, groVp and groVv, and established a multiplex PCR detection for three vibrios species. Polymerase chain reaction conditions were optimized using 25μL reaction mixture for each tube containing 2μL of DNA template,2×PCR TaqMix 10μL and 0.5μL of each primer set. PCR conditions were optimized as follows:initial denaturation of 4 min at 94℃ followed by 30 cycles each having denaturation for 30s at 94℃, annealing for 30s at 66℃ and extension for 30s at 72℃, and final extension step for 5 min at 72℃ in a PCR thermal cycler.The test result showed that the detection has high specificity. The detection limit of mixed genomic DNA in multiplex PCR was 1ng/μL. This method can detect the three species of Vibrios, which provides technical means for detection of V. cholerae, V. parahaemolyticus and V. vulnificus.According to the sequences of toxR gene, heat direct hemolysin (TDH) gene and heat-related hemolysin (TRH) geness, three pairs of primers of V. parahaemolyticus were designed. After optimization, polymerase chain reaction conditions for a multiplex PCR were optimized using 25μL reaction mixture for each tube containing 2μL of DNA template, 2×PCR TaqMix 10μL and trh peimer 2μL, tdh primer 0.5μL, toxR primer 0.5μL. PCR conditions were optimized as follows:initial denaturation of 4 min at 94℃ followed by 30 cycles each having denaturation for 45s at 94℃,annealing for 45s at 60℃ and extension for lmin at 72℃, and final extension step for 10 min at 72℃ in a PCR thermal cycler. The detection limit of genomic DNA in multiplex PCR was 0.1ng/μL of trh and tdh, and 1×10-2 ng/μL of toxR. The specificity experiment showed that the designed primers are specific. This multiplex PCR can be used as a rapid detection method for pathogenic V. parahaemolyticus.Dot-ELISA was established to detect the direct heat hemolysin (TDH) of V. parahaemolyticus. Serum antibody was developed by our laboratory. The best reaction conditions were as follows:the PVDF membrane was blocked for lh with 20% skim milk, serum antibody of 1:100 for 30min,1:1000 HRP-goat anti-rabbit IgG for 1h. The specificity and sensitivity detection indicated that the method is simple, and intuitive, and does not require special equipment for the grassroots and field testing.