Study On Rapid Detection System Of Vibrio Parahaemolytious And Vibrio Alginolyticus

The thesis has established the rapid detection system of Vibrio parahaemolytious and Vibrio alginolyticus firstly,including Multiplex Polymerase Chain Reaction, Nanogold-assisted PCR,dot immunogold filtration assay.A series of parameters were optimized,and they formed ripe reagent kits.The reagent kits have been applied to investigate on Vibrio parahaemolytious and Vibrio alginolyticus contamination of the products of lovestock.They shall short the detection period from4d-5d to2d.The research abstract was summarized as follows:1、Establishment and primary application of Multiplex Polymerase Chain Reaction for Detection of Vibrio parahaemolytious and Vibrio alginolyticus In this study,A multiplex polymerase chain reaction(M-PCR) was established and optimized to detect Vibrio parahae-molytious (VP)and Vibrio alginolyticus(VA) simultaneously. Two sets of specific primers were designed according to the toxR sequence of VPand VA in the GenBank. It was showed that all sampies which contained VP or VA could be amplified by the multiplex PCR using these two sets primers. Different methods were used for the preparation of DNA,and the column reagent kit method was the best methods than adding bacteria、hot cleavage、 freezing-thaw again and again、phenol-CHCl3extractio.Two specific bands of VP208bp and VA159bp were detected using2.0%agarose gel electrophoresis in this multiplex PCR.The method had a better specificity and no cross-reaction with other enterobacteria such as Citro Bacter、salmonella. Vibrio damsela、V.cholera、Vibrio metschnikovii. The detection limit of the M-PCR assay for pure culture was50cfu for VP and60cfu for VA and for clinic samples was2cfu for VP and3cfu for VA. It can be stored at-20℃for9months and maintain good detecting result.The method provided a simple、fast、strongly specific characteristics、high sensitive and inexpensive way of identifying VP or VA. The method could prove very useful for identification and inspection of VP or VA from fishery product in import and export.2、Establishment and primary application of nanoparticle-based assisted PCR for Detection of Vibrio parahaemolytious Nanogold-assisted PCR was established and optimized to detect Vibrio parahaemolytious(VP). One set of specific primers were designed according to the toxR sequence of VP in the GenBank by Primer Premier6.0. The reaction conditions%cycle numbles、sensitivity and specialty of the method was optimized and assessed. It was showed that all sampies which contained VPcould be amplified by the nanogold-assisted PCR using this set primers. One specific band of VP208bp was detected using2.0%agarose gel electrophoresis in this nanogold-assisted PCR and accorded with designed result,other bacterial(such as Vibrio alginolyticus、V.cholera. Vibrio metschnikovii. salmonella、Citro Bacter) controls gave negative results. The nanogold-assisted PCR was able to detect as little as3cfu/reaction system for VP from pure culture samples and more sensitive than polymerase chain reaction. The experiment proved that the method is better than ordinary PCR. The method was more sensitive than ordinary PCR about10times under the same contions. The method provided a fast and reliable way of identifying VP. The method could prove very useful for identification and inspection of VP from fishery product in import and export.3、Establishment and primary application of nanoparticle-based assisted PCR for Detection of Vibrio alginolyticus Nanogold-assisted PCR was established and optimized to detect Vibrio alginolyticus (VA). One set of specific primers were designed according to the toxR sequence of VA in the GenBank by Primer Premier6.0. The reaction conditions、sensitivity and specialty of the method was optimized and assessed. It was showed that all sampies which contained VA could be amplified by the nanogold-assisted PCR using this set primers. One specific band of VA159bp was detected using2.0%agarose gel electrophoresis in this nanogold-assisted PCR and accorded with designed result,other bacterial(such as Vibrio parahaemolyticus、 V.cholera、Vibrio metschnikovii、salmonella、Citro Bacter) controls gave negativ、 results. The nanogold-assisted PCR was able to detect as little as6cfu/reaction system for VA from pure culture samples and more sensitive than polymerase chain reaction. The method was more sensitive than ordinary PCR about10times under the same contions. The method provided a fast and reliable way of identifying VA. The method could prove very useful for identification and inspection of VA from fishery product in import and export.4、Gold immunochromatography assay for detection of Vibrio-parahaemolytious To establish a more rapid and simple method of detecting Vibrio-parahaemolytious(VP),the gold immunochromatography assay(GICA) was developed by coating rabbit anti-VP IgG-2on nitrocellulose(NC)membrane,spraying sheep anti-rabbit IgG on control line,labeling rabbit anti-VP.IgG-1with colloidal gold.If detected sample contains VP.,the detection line appeared red line.The whole test procedure could be finished only15-30minutes.The method had a better specificity and no cross-reaction with other enterobacteria such as Vibrio alginolyticus、V.cholera.、Vibrio damsela.、Ciro Bacter and Salmonella. The minimum amount of VP. giving a positive reaction was3.592×104cfu/ml. It could be stored at4℃for4months,at room temperature for2month,at37℃for3weeks and maintain good detecting result. So the trip had a simple、rapid、high sensitive、strongly specific characteristics,didn’t need specific instruments and facilities,and its result was easily observed and determined.It was good worthy to use it widely in small diagnostic laboratories.