Isolation, Identification, Lyses Properties And Preliminary Application Of Bacteriophages In Rapid Detection Of Vibrio Parahaemolyticus

Vibrio Parahaemolyticus, also called halophilic bacteria, widely exsite inocean environment. They can caused many aquatic animal diseases such asulcers, haemorrhage and so on. They are one of the most important pathogenicbacteria in aquaculture industry. People who eat aquatic product infected byVibrio Parahaemolyticus can cause acute intestinal tract disease, septicemia oreven death when the concentration of Vibrio Parahaemolyticus was more than106cfu/mL. As revealed in Foodborne disease monitoring network, food poisoningcaused by Vibrio Parahaemolyticus has already become the most important facter ofmicrobe infection.Presently, the detection method of Vibrio Parahaemolyticus is mainlyabout traditional method and modern method. The long period of traditionalmethod cannot meet rapid detection of aquatic product. Morden method hasmany advantages such as high sensitivity but also has many disadvantages suchas complicated pre-dispose. Consequently, it is fairly important to develop anew method with advantages such as high specificity, simple operation and shortperiod of time to realize the purpose of rapid and quantitative detection of Vibrioparahaemolyticus. So we start our work by sifting efficient bacteriophages in order torealize the purpose of high specificity. Meanwhile, the luciferase-FMN:NADHoxidoreductase bioluminescent system in vitro (the coupled enzymes system) wasintroduced to realize the purpose of rapid detection of Vibrio Parahaemolyticus.Themain contents as follow:1Three Vibrio Parahaemolyticus phages VPp1, VPp2and VPp3wereisolated from aquatic sewage water. Their titer can reach1010pfu/mL after proliferation. Plaques of VPp1have transparent center and large halo after12hours culture in double-layer agar plate. VPp2and VPp3’s plaques haven’tlarge halo.2Result of restriction analyses shows that VPp1, VPp2and VPp3are threedifferent phages with double stranded DNA. Electron micrographs indicated thatVPp1, VPp2and VPp3has an icosahedral head of44nm,64nm and89nm indiameter respectively. VPp1hasn’t tail and belongs to the family Tectivirus. VPp2and VPp3has tail of114nm and106nm respectively, belongs to the familyMyoviridae.3The optimum MOI of VPp1, VPp2and VPp3is0.0001,0.00001and0.001respectively. The latent period of VPp1, VPp2and VPp3is10min,5minand25min;lyese period of VPp1, VPp2and VPp3is20min、25min、75min;burst size of VPp1, VPp2and VPp3is90.3,11.1and45.3respectively.Comprehensive analysis all the factors, VPp1is selected as the candidate ofnext experiments because of its short latent period and large burst size. VPp1isvery sensitive to UV irradiation. After20s of UV irradiation, survival rate ofVPp1decline more than60%. Genome of VPp1includes64genes, homologysearch of every gene of VPp1shows that VPp1is a new phage.4To maximize NADH release, lyses condition was optimized by three maininfluencing factors including bacteriophage titer, the lysis time and volume ratio ofbacteriophage to its host bacterium. Results of this three experiments was1010pfu/mL,15min and1:1respectively.5VPp1was combined with the luciferase-FMN:NADH oxidoreductasebioluminescent system in vitro to realize the purpose of rapid and quantitativedetection of Vibrio Parahaemolyticus. A standard curve between the number ofbacteria and the luminescence intensity of the coupled enzymes system were studied,which revealed a good linear relationship(strandard curve is y=1938.3x+2906.1) whenthe Vibrio Parahaemolyticus concentration was between0.31×10~8~6.18×10~8 cfu/mL. The detection limit is2.64×10~7cfu/mL.The average labeled recovery forstandard Vibrio Parahaemolyticus solution and oyster sample is96.28%and91.92%respectively, which revealed that this method has high accuracy andfeasibity.The repeatability RSD for standard Vibrio Parahaemolyticus solution andoyster sample is0.72%and0.68%respectively, which revealed that this method hasgood repeatability and high precision.Results of pre-enrichment shows that100cfu·ml-1can be obviously detected with a4h pre-enrichment.All the detection processcan be finished within4.5h.In a word, three Vibrio Parahaemolyticus phages VPp1、VPp2、VPp3wereisolated, VPp1was selected and combined with the luciferase-FMN:NADHoxidoreductase bioluminescent system in vitro to realize the purpose of rapid andquantitative detection of Vibrio Parahaemolyticus.