Detection Of Vibrio Parahaemolyticus And Establishment Of Two Foodborne Bacterial Typing Methods

Foodborne diseases caused by foodborne pathogens are one of the main problems threatening human health and safety,among which Vibrio parahaemolyticus and Salmonella are common pathogens causing intestinal infection.Therefore,developing a rapid,simple and sensitive detection method and understanding the genetic distribution of pathogenic bacteria are of great significance to control and monitor foodborne pathogenic bacteria.Based on the sensitivity,specificity and short reaction time of isothermal amplification,this subject carries out specific visual detection of V.parahaemolyticus;At the same time,the whole genome sequencing was used to genotype V.parahaemolyticus and Salmonella,and a more suitable genotyping method for strains with similar genetic relationship was sought to finally understand the genetic relationship between strains.(1)Establishment of visual detection method for V.parahaemolyticus.Based on the specific recognition of V.parahaemolyticus by aptamer,a visual detection method of V.parahaemolyticus was established by using an efficient isothermal amplification method and the characteristics of alloy nanoparticles.The aptamer is used as the recognition unit(Apt-T).After specifically recognizing V.parahaemolyticus,the complementary strand T is released.T is used as the target sequence to further induce SDA amplification reaction and produce a large amount of DNA single strand(ss DNA).The gold nanoparticles electrostatically adsorb ss DNA and protect it from aggregation under the action of Na Cl.The solution is red,on the contrary,the solution is blue.Based on this,V.parahaemolyticus can be visually detected according to the change of solution color,and it can be quantitatively detected by using the change of UV characteristic absorption peak of gold nanoparticles.The results showed that the detection limit was 20 CFU/m L;It has good specificity and has no cross reaction with common foodborne pathogenic bacteria such as Listeria monocytogenes,Staphylococcus aureus,Escherichia coli O157: H7,Cronobacteria sakazakii and Salmonella typhimurium.This study shows that the system can be used for rapid identification of V.parahaemolyticus in shrimp.(2)Establishment of typing method for V.parahaemolyticus and Salmonella.Multilocus sequence typing(MLST),core genome multilocus sequence typing(cg MLST),drug resistance gene typing and virulence factor typing of V.parahaemolyticus showed that 106 strains of V.parahaemolyticus belonged to 45 ST types,and 55 strains of unknown ST,accounting for more than 50%,with genetic diversity.It was analyzed that the main reason was regional differences.cg MLST typing method shows high resolution and high allele difference.The common assumption inferred by the drug resistance gene and virulence factor typing method used in this subject is that the ancestors are the same as cg MLST method,and the typing results of most strains are the same as cg MLST method,and the clustering results in some bacterial groups are the same,showing high resolution,and the typing effect is better than MLST method.Serotype identification,Pulsed-field gel electrophoresis(PFGE),cg MLST typing and functional gene typing were carried out for 106 Salmonella strains.According to the whole genome data,8 serotypes and 8 ST were predicted,and the largest number of serotypes was S.enteritidis serotype,all of which belonged to ST11;Due to the close separation location and time,PFGE results showed that the strains were divided into 11 to 18 DNA fragments and clustered into 7 clusters.The similarity of strains exceeded89%,showing a high degree of genetic similarity;cg MLST typing method still showed high resolution for strains with similar genetic relationship.Salmonella can be divided into 7 clusters with allele difference between 0～2257;Functional genotyping divides 91 strains of S.enteritidis with the closest genetic relationship into 9 clusters.Compared with cg MLST method,the clustering accuracy rate is more than 82%.Although the accuracy of evolutionary relationship analysis based on 59 genes is lower than cg MLST method based on 2466 core genes,it can distinguish each strain more quickly by using fewer genes than cg MLST method,which is suitable for clustering and typing of S.enteritidis with closer genetic relationship.