Distribution Of Foodborne Bacteria In Weihai Region And Establishment Of Multiplex Touchdown PCR For Detection Of Common Pathogenic Marine Vibrio

Background and Objective: Marine vibrio is widely distributed in the bay,coastal,foreign waters,marine organisms and sediments,and can infect fish or mammals and cause foodborne diseases in the population.Many vibrio species can cause diseases in humans or other animals.Among them,Vibrio cholerae,Vibrio vulnificus and Vibrio parahaemolyticus are most virulent.The range and frequency of bacterial diseases caused by marine vibrio gradually increased with the increasing consumption of seafood.Conventional culture method for detection of vibrio could not meet the needs of rapid diagnosis.Furthermore,some marine vibrio were difficult to culture.Therefore,the establishment of rapid detection technology for pathogenic vibrio is very important for the diagnosis and treatment of pathogenic marine vibrio infection as well as for the protection of public health.This study aims to understand the distribution of foodborne pathogenic bacteria in Weihai region and to establish a multiple touchdown PCR assay for rapid diagnosis of Vibrio vulnificus and Vibrio parahaemolyticus infection.Methods: A total of 357 feces specimens from patients with foodborne diseases were collected in Weihai Municipal Hospital from March 2017 to Oct 2017.Culture method and mass spectrometry were used to identify bacteria in feces specimens.Specific primers for vvh A gene of Vibrio vulnificus and col gene of Vibrio parahaemolyticus were designed and used to establish single touchdown PCR and multiplex touchdown PCR.The specificity and sensitivity of the multiple touchdown PCR method were determined by detecting the target species.The multiplex touchdown PCR was used to detect the Vibrio vulnificus and Vibrio parahaemolyticus in 103 feces specimens and compared with the culture method to evaluate its feasibility in detection of the specimens.Results:(1)A total 98 strains of pathogens were identified.The constituent ratio of the five-require-monitored foodborne pathogens,diarrheogenic Escherichia coli,Vibrio parahaemolyticus,Salmonella,Norovirus and Shigella,was 26.53%,21.43%,20.41%,10.20% and 7.14%,respectively.Fourteen(14.28%)other pathogenic marine vibrio were also identified,including 6(6.12%)strains of Vibrio vulnificus,3(3.06%)strains of Vibrio harnyi,3(3.06%)strains of Vibrio alginolyticus and 2(2.04%)strains of Vibrio vulnificus.The constituent ratio of all the pathogenic marine vibrio species was 35.71%.(2)The single touchdown PCR for Vibrio vulnificus or Vibrio parahaemolytics specifically amplified target species but did not cross-react with other Vibrio and non-Vibrio species.The detection limit was 104 CFU/ml for Vibrio vulnificus and 103 CFU/ml for Vibrio parahaemolytics.(3)The multiplex touchdown PCR specifically amplified target species of Vibrio vulnificus and/or Vibrio parahaemolytics but did not cross-react with other Vibrio and non-Vibrio species.The detection limit of was 104 CFU/ml for Vibrio vulnificus and 103 CFU/ml for Vibrio parahaemolytics.(4)Compared with bacterial culture method,the specificity and sensitivity of the multiplex touchdown PCR for detection of Vibrio vulnificus were 100% and 97.94% respectively,and those for detection of Vibrio parahaemolyticus was 100% and 95.12% respectively.Conclusion:(1)The detection rate of pathogenic marine vibrio in foodborne diseases in Weihai region is high,suggesting that the pathogenic marine vibrio may need to be monitored.(2)A multiplex touchdown PCR assay with high sensitivity and specificity is successfully established and it would be used for rapid diagnosis of infection caused by Vibrio vulnificus and Vibrio parahaemolytics.