The Enterotoxin Genes Detection And Genotyping Study Of Foodborne Staphylococcus Aureus

Objective:1To establish a multiplex PCR method for the detection of14kinds offoodborne Staphylococcus aureus enterotoxin genes, and to investigate thedistribution of Staphylococcus aureus enterotoxin genes in Shijiazhuang city.2Design a MLVA typing method for foodborne Staphylococcus aureus,compare the advantages and disadvantages of MLST, MLVA, PFGEgenotyping method in foodborne Staphylococcus aureus gene diversity study.Methods:1Subculture93foodborne Staphylococcus aureus strains, and extractgenomic DNA with extraction kit;2Design14pairs of PCR primers for type A, B, C, J, E, G, H, J, K, L, M,N, Q, and U enterotoxin genes, and establish a multiplex PCR method byoptimizing the reaction conditions, then detect enterotoxin genes of93foodborne Staphylococcus aureus isolated from Shijiazhuang city;3Design a MLVA typing method for Staphylococcus aureus genotypingby referencing the relevant literatures and the website data base, genotype26foodborne Staphylococcus aureus strains by MLST, MLVA, PFGE methods,and compare the typing results.Results:193foodborne Staphylococcus aureus strains were successfullyrecoveried; the concentration and purity of extracted genomic DNA wereaccorded with the experiment request.2In addition to SEE,14pairs of PCR primers for enterotoxin genes weresuccessfully amplified with target gene fragments; and the bidirectionalsequencing results were proved to be the enterotoxin gene fragments byBLAST in GeneBank. 3The designed Multiplex PCR reaction system were successfullyamplified to the detection of foodborne Staphylococcus aureus enterotoxingenes; in93foodborne Staphylococcus aureus strains,79strains were provedcontaining enterotoxin genes, and the enterotoxin gene detection rate was84.95%;55(59.14%) strains contained SEA, then SEG32strains(34.41%),SEM32strains(34.41%), SEK25strains(26.88%), SEB22strains(23.66%),SEU21strains(22.58%), SEN20strains(21.51%), SEH16strains(17.20%),SEJ15strains(16.13%), SEC14strains(15.05%), SED13strains(13.98%),SEQ13strains(13.98%), SEL7strains(7.53%);57(61.29%) strains weredetected containing two or more kinds of enterotoxin genes simultaneously,and a strains were detected containing10kinds of the enterotoxin genessimultaneously,3strains contained9simultaneously,3strains contained8simultaneously.426Staphylococcus aureus strains were divided into8ST types byMLST typing, of which type ST59contained11strains, all of which wereisolated from the same food poisoning incident; type ST5contained5strains,type ST464contained3strains, type ST7and ST15contained2strains; anewly discovered ST type was also detected, which has been submitted to theMLST international database for confirming and recording.5The designed MLVA method for foodborne Staphylococcus aureustyping was confirmed with good typing ability and repeatability;26strainswere successfully divided into10genotypes, type VA05contained11strains,all of which were isolated from the same food poisoning incident; type VA07contained4strains, type VA01, VA09and VA10contained2strains, each oneof other types contained1strain.626Staphylococcus aureus strains were divided into13genotypes of9groups by PFGE typing, group A contained11strains, all of which wereisolated from the same food poisoning incident; group B contained4strains,which were divided into4subtypes; group C contained3strains, which weredivided into2subtypes; group D and group E contained2strains; each one ofother groups contained1strain. Conclusion:114pairs of designed PCR primers for the detection of enterotoxin genesand multiplex PCR reaction system have good stability, which can be used forthe rapid detection of enterotoxin genes in foodborne Staphylococcus aureus.2The designed MLVA typing method has better typing ability thanMLST, but worse than PFGE.3Compaired with MLST and PFGE, the design MLVA typing methodwas quick and easy, and had good stability and genotyping ability, which canbe used for rapid genotyping of foodborne Staphylococcus aureus.