Establishment And Evaluation Of Visual Detection Method For Staphylococcus Aureus

Objective:Staphylococcus aureus is a common foodborne pathogen that is not lethal in itself,but can produce staphylococcus aureus enterotoxin after it multiplies in food.Staphylococcus aureus enterotoxin is destructive to the intestinal tract and often causes staphylococcus aureus enteritis.The onset of the disease is urgent and the poisoning symptoms are serious.The clinical symptoms are mainly vomiting,fever,abdominal pain,diarrhea and shock,which can lead to circulatory failure.At present,the laboratory detection methods for staphylococcus aureus mainly include traditional biochemical identification methods,immunological methods and molecular biological methods.However,these methods have the disadvantages of complicated operations,time-consuming,low sensitivity,low specificity and high requirements on experimental conditions and operators.Therefore,it is necessary to establish a simple,rapid,sensitive and accurate detection method for staphylococcus aureus to prevent and control food safety problems caused by staphylococcus aureus.The purpose of this study is to establish a rapid visual detection method for staphylococcus aureus by yolk antibody technique,immune magnetic separation technology,and TMB?3,3',5,5'-tetramethyl benzidine?-HRP?horseradish peroxidase?-H₂O₂?hydrogen peroxide?color system,and to evaluate the established test method,which provide certain technical support in order to ensure the food safety in our country.Methods:1.Preparation and identification of chicken yolk antibody against staphylococcus aureus.The inactivated staphylococcus aureus solution was used as antigen,and the vaccine was prepared by mixing it with freund's complete adjuvant/freund's incomplete adjuvant in a ratio of 1:1.The immunized SPF layer chickens were immunized by multi-point injection of chicken breast,and collected the immunized eggs.PEG-6000precipitation method was used to separate and extract chicken yolk antibody from egg yolk;antibody titer and specificity were determined by indirect ELISA;the purity of antibody was determined by SDS-PAGE electrophoresis;the protein content of antibody was determined by BCA protein concentration quantification.2.Preparation and characterization of immune magnetic beadsFe₃O₄ nanoparticles with surface modification of carboxyl group were synthesized by solvent heat method.The prepared carboxyl magnetic beads were conjugated with chicken egg yolk antibody using EDC and NHS as coupling agents to prepare immune magnetic beads that could specifically isolate and enrich staphylococcus aureus that to be examined.The composition,size,morphology and performance of the functionalized carboxyl and immunomagnetic beads were characterized by biological transmission electron microscopy?TEM?,fourier transform infrared spectroscopy?FTIR?,and magnetic measurement system?SQUID-VSM?.3.Establishment and evaluation of detection methods for staphylococcus aureusAfter rapid capture and enrichment separation of staphylococcus aureus in the test sample by using the immunomagnetic beads prepared above,H₂O₂ solution was added to the immunomagnetic complex.The small molecule H₂O₂ will quickly pass through the cell membrane of staphylococcus aureus and be catalyzed by catalase.After magnetic separation,the remaining solution was taken for color reaction with HRP and TMB,and the change of absorbance of the system was detected by uv-vis,and the color depth of the formed product was observed by naked eye,so as to realize quantitative and rapid visual detection of staphylococcus aureus in the sample to be tested.In order to achieve the best detection effect,the dosage of immune magnetic beads,enrichment time,dosage of H₂O₂,decomposition reaction time of H₂O₂,pH value of chromogenic solution,HRP concentration and TMB concentration were optimized,and the sensitivity,accuracy,specificity and repeatability of the detection system were evaluated.Finally,the detection method was used to detect artificially simulated milk samples.Results:1.Preparation and identification of chicken yolk antibody against staphylococcus aureus.The results showed that the antibody titer was 1:64000.It has good specificity and can specifically identify staphylococcus aureus,but it cannot combine with Escherichia coli O157:H7,Vibrio parahaemolyticus,Shigella bogdii,Salmonella,Listeria monocytogenes,Klebsiella pneumoniae,Enterobacter sakazakii,and Bacillus mirabilis;the results of electrophoresis showed that there were few stray bands and the target bands were clear;the antibody protein content was 21.78 mg·mL^-1.2.Preparation and characterization of immune magnetic beadsThe results of transmission electron microscopy showed that the synthesized nanoparticles are spherical,uniform and dispersed,with slightly rough surface.The average particle size of carboxyl beads was 240nm,and that of immunomagnetic beads was 281.7nm.Fourier transform infrared spectroscopy showed that the characteristic absorption peak of C=O in the peptide bond appeared at 1612 cm-1,indicating that IgY successfully conjugated on the surface of carboxyl beads.The magnetic measurement system showed that both functional carboxyl beads and immune magnetic beads had good superparamagnetism,and the saturation susceptibility of immunomagnetic beads decreased slightly after IgY was coupled to the surface of carboxyl beads,from 59.286emu/g to 52.039 emu/g,but still had the ability of rapid response of external magnetic field.3.Establishment and evaluation of detection methods for staphylococcus aureusCondition optimization results showed that the enrichment efficiency was the highest when the concentration of immune beads was 0.6 mg·mL^-1 and the enrichment time was 60 min,reaching 91.4%;the optimal amount of H₂O₂ was 50 liters;the optimal decomposition reaction time of H₂O₂ was 2 min.the optimal pH value of the chromogenic solution was 4.5;the optimal HRP concentration was 2.5 g·ml^-1.the optimal concentration of TMB was 2.0 mg·mL^-1.Under the optimal detection conditions,the concentration of staphylococcus aureus ranged from 10³ to 10⁷CFU·mL^-1,and the absorbance value of the reaction product at 654nm was linearly related to the logarithm of the concentration of staphylococcus aureus.The linear equation was y=-0.256x+1.864?R²=0.992?,and the detection limit was 10³ CFU·mL^-1.Using this method with good accuracy and specificity to detect milk samples,the detection limit was 10³ CFU·mL^-1,the standard recovery rate is 96.57%¹04.96%,and the relative standard deviation is 7.28%⁷.78%.This method can complete the detection within 75 min,and the detection result can be determined with the open eye,which meets the requirements of rapid spot detection of staphylococcus aureus.Conclusion:1.This study established a rapid visual detection method for staphylococcus aureus based on the yolk antibody technology,the immunomagnetic separation technology,and the color rendering system of TMB-HRP-H₂O₂.2.The detection limit of this method is 10³ CFU·mL^-1,with good specificity and stability,which lays an experimental foundation for the rapid detection of staphylococcus aureus in food.