PCR-basad Vibrio Parahaemolyticus Serotyping And Pathogenicity Analysis Model Construction

Vibrio parahaemolyticus belongs to Gram-negative halophilic Vibrio,which can be adhered,aggregated and propagated in the intestinal tract of aquatic animals.Therefore,it is widely distributed in marine products and environments.It can also be detected in freshwater products and freshwater aquaculture environments.Poisoning can occur after people ingest food contaminated with the bacteria which not only causes huge losses to the aquaculture industry,but also seriously endangers human health.In recent years,the disease caused by Vibrio parahaemolyticus has leapt to the top of the food-borne pathogens in China.Not all Vibrio parahaemolyticus can cause disease in humans.The Vibrio parahaemolyticus causing outbreaks of foodborne diseases is mainly concentrated in a few serotypes,so the identification of Vibrio parahaemolyticus serotypes is particularly important for the screening of highly pathogenic strains.At present,the serotype identification method of Vibrio parahaemolyticus is traditional serum agglutination method which is complicated,time-consuming,high cost,and has high technical requirements for the inspectors.This study established a new serotype method of Vibrio parahaemolyticus based on polymerase chain reaction(Polymerase Chain Reaction,PCR)technology.And the new method will be used in the test of all Vibrio parahaemolyticus collected by China’s 2017 Food Safety Risk Monitoring Network.In order to comprehensively grasp the drug resistance distribution and toxigenic characteristics of Vibrio parahaemolyticus in aquatic products in China,this study also used micro-broth dilution method to determine the antibiotic sensitivity of the bacteria to 8 types of antibiotics commonly used in clinical treatment of bacterial infections.The multiplex real-time PCR method was used to detect six virulence genes such as tlh,tox R,tdh,trh1,trh2 and orf8 which were often carried by the strain.The main findings are as follows:(1)The strain identification and virulence gene detection of 1194 food-borne Vibrio parahaemolyticus in 2017 were carried out by real-time PCR method.The results showed that all the tested strains in this study were confirmed to be Vibrio parahaemolyticus.The positive rates of tlh gene and tox R gene were 100% and 98.2%,respectively.The positive rates of virulence genes tdh gene,orf8 gene,trh1 gene,and trh2 gene were 4.02%,1.59%,0.34%,and 3.35%,respectively.The virulence gene positive rate in Fujian Province was the highest(34%),followed by Zhejiang province(22%)and Henan province(22%).The virulence gene positive rates of the strains from Liaoning,Tianjin and Heilongjiang provinces were respectively 20%,15% and 13%.(2)The antibiotic susceptibility test results of 1194 strains of hemolysed strains showed that all strains were sensitive to cefotaxime and ceftazidime,and the resistance rate to cefazolin is the highest(93.2%),followed by Ampicillin(46.0%),cotrimoxazole(3.1%),tetracycline(1%).The resistance rates to gentamicin,imipenem,ciprofloxacin,amoxicillin/clavulanic acid,levofloxacin,chloramphenicol,etc.are less than 1%.The multi-drug resistance of all tested strains to the tested antibiotics was not serious.The resistance rates of Vibrio parahaemolyticus from different provinces were slightly different.The resistance rates of strains in Hainan,Tianjin and Jiangsu were 100%,and therates of strains in Fujian,Henan,Zhejiang,Hubei,Guangxi,Shandong and other provinces were above 90%,and the rate in Shanghai is 79%.(3)In order to establish a new method for the identification of Vibrio parahaemolyticus based on PCR technology,this study used PCR technology to test the 12 O-type antigens of Vibrio parahaemolyticus and used the fluorescent PCR technology to test 7 K-type antigens.The results showed that there were at least 12 O groups and 5 K groups in 14 provinces and states.Among them,O1,O2,O3,O4 totally account for a large proportion(56.1%).The distribution of several pathogenic serotypes was: 19 strains of O1:K25 serotype,17 strains of O3: K6 serotype,1 strain of O4: K8 serotype,total proportion 3.09%.The new method has a high review rate with traditional serotyping method.The above results indicate that the new serotype analysis method of Vibrio parahaemolyticus based on PCR technology established in this study is efficient,time-saving and accurate to meet the needs of serological typing experiments of Vibrio parahaemolyticus.