Development Of DHPLC Detecting For Pathogenic Vibrio And Rapid Typing Methods For Vibrio Cholerae

The vibrios are harmful bacteria commonly found in animal food products.There are many vibrios which have been proven to be the most important human pathogenic bacteria, such as Vibrio cholerae,Vibrio vulnificus,Vibrio parahaemolyticus,Vibrio alginolyticus, Vibrio mimicus,Vibrio metschnikovii et al.Among these vibrios,Vibrio vulnificus,Vibrio parahaemolyticus and Vibrio mimicus are occasionally pathogenic to humans,while Vibrio metschnikovii rarely infects humans,which only a few isolates are separated from human urine and blood.These vibrios are forbidden in the detection of export and import food. Currently,the biochemical identification is widely used in the detection of pathogenic vibrios. However,the method is tedious,needing long time and it can't be used in the big batch of sample test.Additionally,the reaction isn't special to some samples isolated.Above all,it is difficult to rapidly identify and detect the vibrios using the present method,which can not meet the practical needs of international import and export trade.The purpose of this study is to establish a rapid,accurate,efficient and low cost method,which can be used for rapid typing detection methods of Vibrio cholerae, meeting the needs of import and export food inspection detection.Six pathogenic vibrios were selected as model in the research,including Vibrio cholerae,Vibrio vulnificus,Vibrio parahaemolyticus,Vibrio alginolyticus, Vibrio mimicus and Vibrio metschnikovii.The genome DNAs of the six pathogenic vibrios were extracted by kit method and the extractions were measurement using spectrophotometer.According to their unique gene sequences of the pathogenic vibrios,specific primers were designed and prepared,and then the sensitivity, specificity and actual sample PCR-DHPLC of six pathogenic vibrios were investigated.Meanwhile,collagenase gene of Vibrio cholerae(vcc gene),O1 and O139 group sharing group virulence genes(ctxA and tcpA genes)and the O139 group-unique virulence genes(LPSgt gene)were designed and prepared,according to the toxin-related genes of Vibrio cholerae.After optimizing the PCR annealing temperature and reaction system for four pairs of primers,the Multiplex PCR-DHPLC rapid typing detection for Vibrio cholerae was revealed.First,the specific primers of six vibrios and specific primers related to the toxin of Vibrio cholerae were designed,synthesized,the reaction system and annealing temperature was optimized. Then,the PCR-DHPLC method for the detection of six vibrios and the MPCR-DHPLC rapid typing method of V.choleare(O1,O139,non-O1/non-O139strains)were developed in this paper,and rapid detection sensitivity of PCR-DHPLC for all of the six pathogenic vibrios was as low as 60 cfu/mL.Compared with the traditional approach of microbiological and PCR-gel electrophoresis, good accuracy and efficiency can be obtained using the method developed in this study.The method would be extensively applied in the food detection of pathogenic vibrio cholerae and rapid typing for V.choleare,with the merits of high detection accuracy,convenient and low cost.