Study On High Throughput And Rapid Detecting And Typing Methods For Staphylococcal Enterotoxin In Foods

Objective:Staphylococcus aureus is an important pathogenic bacteria which can cause human infection and food poisoning.The Staphylococcal enterotoxins(SEs)which produced by Staphylococcus aureus can contaminate food and result in food poisoning.People can appear the symptom of acute gastroenteritis,characterized by nausea,vomiting and diarrhea,after eating food contaminated by SEs in one to five hours.The detection of SEs had been included in international food inspected regulations.SEs has a variety of genotypes,and different types of enterotoxins have different toxicity.Therefore,it is necessary to establish a rapid,strong specificity,high sensitivity and high throughput enterotoxin detection and classification method,which has the extremely vital significance to prevent and diagnose food poisoning caused by enterotoxins,and protect the safety of food hygiene.Methods:The appropriate DNA extraction method was improved and established by detecting the purity and concentration DNA of Staphylococcus aureus standard strains.The DNAs of eighty staphylococcus aureus strains preserved by glycerin were extracted after bacteria activating.Five pairs of primers for five classical enterotoxins which included SEA,SEB,SEC,SED and SEE were designed.The PCR-DHPLC detecting and typing methods for SEs was established after optimizing of the annealing temperature and DNA template amount.The MPCR-DHPLC method was also established by optimizing of the MPCR reaction system.The SEs were detected and typed respectively by PCR-DHPLC and MPCR-DHPLC methods in 80 Staphylococcus aureus strains isolated from foods.The detection result of the SEs by existing mini-VIDAS method was compared with those of PCR-DHPLC and MPCR-DHPLC methods.Results:The concentration of the DNA extracted was near to 100 ng/?L,and A260/A280 was between 1.7 to 1.9.The concentration and purity of the DNA were conformed to the requirement of experiment.Five pairs of specific primers were designed and synthesized according to the gene sequence of the 5 types of classical enterotoxin SEA,SEB,SEC,SED and SEE.Both PCR-DHPLC and MPCR-DHPLC detecting and typing methods for SEs were established after optimizing.Both methods could detect specifically and stably of SEs with detection limit of 100 cfu/mL.The detecting and typing results of PCR-DHPLC and MPGR-DHPLC methods were entirely consistent in 80 Staphylococcus aureus strains isolated from foods.47 strains were detected with enterotoxin genes and the detection rate was 58.75%.Of all the Staphylococcus aureus,only 33 strains had one enterotoxin gene with positive rate of 31.25%.20 strains had SEA gene.3 strains had SEB gene.9 strains had SEC gene and 1 strain had SEE gene.There were 13 strains carrying with two enterotoxin genes with positive rate of 16.25%.4 strains had SEA and SEC genes simultaneously.1 strain had SEA and SEE genes,and 7 strains had SEB and SEC genes,while 1 strain had SEC and SED genes simultaneously.Furthermore,1 strain had SEA,SEB and SEC genes with positive rate of 1.25%.The detection result of SEs by mini-VIDAS method showed that 39 stains were positive in 80 staphylococcus aureus strains with positive rate of 48.75%.The positive rate of mini-VIDAS method was lower than those of PCR-DHPLC and MPCR-DHPLC methods with positive rate of 58.75%.It indicated that the sensitivity of PCR-DHPLC and MPCR-DHPLC methods established was higher than mini-VIDAS method.Moreover,the established methods can be used to type of SEs.Conclusion:Both PCR-DHPLC and MPCR-DHPLC detecting and typing methods established in this paper had much advantage,such as high sensitivity,strong specificity,high throughput and good stability.The PCR-DHPLC and MPCR-DHPLC methods could be effectively applied to rapid and high-throughput detecting and typing for SEs in foods and had fine application value.