Establishment And Application Of A Salmonella Detection Method Based On Subtractive Inhibition Theory Of Surface Plasmon Resonance Technique

Salmonella is a common foodborne zoonotic pathogen that poses a great threat to human health and the breeding industry.Salmonellosis is an animal epidemic disease that is required to be reported by the World Organization for Animal Health and is also a Class Ⅱ animal epidemic disease controlled by the Chinese government.After being infected with Salmonella,it can quickly colonize host cells and cause persistent infections.Therefore,rapid detection of Salmonella is of great significance for the prevention and control of diseases.The traditional detection method is microbiological culture,which has complicated steps and a long cycle.Surface Plasmon Resonance(SPR)technology can make up for this shortcoming.Since SPR is very sensitive to the refractive index of the dielectric on the metal surface,when the mass of the substance attached to the metal surface changes,the SPR response value will also change accordingly.Therefore,biological recognition molecules can be coupled to the chip surface and the binding and dissociation process of the analyte with biological recognition molecules in solution can be monitored by SPR technology.SPR technology based on the subtractive inhibition assay can achieve rapid,sensitive,high-throughput and semiquantitative detection.Based on this,this study established a subtractive inhibition SPR method for detecting Salmonella through condition optimization and preliminary application,providing technical support for rapid semi-quantitative detection of Salmonella.1 Establishment of a subtractive inhibition assay for the detection of Salmonella using surface plasmon resonanceThe mouse-specific monoclonal antibodies against Salmonella membrane protein PagN developed in our laboratory were used as the target.Salmonella was first incubated with the antibody,and then the unbound free antibody(analyte)was flowed through the chip surface coupled with anti-mouse IgG(ligand),and the SPR signals generated were inversely proportional to the concentration of Salmonella in the sample.Through optimization of the technique,in terms of ligand coupling effect,the results showed that pH 5.0 sodium acetate solution is the best coupling buffer for anti-mouse IgG coupling on CM5 chip surface,and 7000 RU is the best coupling amount.In order to reuse the chip,regeneration with pH 1.7 GlyHCl solution at a flow rate of 20 μL/min for 180 s is the best regeneration condition.In order to fully obtain unbound free antibodies,by comparing different separation methods,it was found that centrifugation at 200,400,800,1200 and 1600 ×g for 2 min each is the best separation method.In terms of selection of the monoclonal antibody against PagN,it was found that 3B3 is more suitable for detection by this method,and the optimal concentration of monoclonal antibody is 150 μg/ml,indicating that 3B3 is a monoclonal antibody against PagN membrane surface epitope.The specificity analysis showed that this method is sensitive to different serotypes of Salmonella but not sensitive to non-Salmonella,indicating it has good specificity.Using this method to detect Salmonella with different concentrations and fitting the curve equation with bacterial concentration as X-axis and R/R0 as Y-axis,it was found that the limit of detection(LOD)of this method is 300 CFU/ml.Through the above analysis,a semi-quantitative detection technology for rapid detection of Salmonella was initially established.2 Salmonella-contaminated samples and phage lysis effect on Salmonella analyzed by the SPR subtractive inhibition assay based on monoclonal antibody(3B3)against PagNIn order to evaluate the application prospects of the above method in clinical detection,this study carried out preliminary application of the method by simulating contamination of Salmonella Enteritis in water and milk powder.The results showed that the LOD of SPR subtractive inhibition method was 102 and 103 CFU/ml in water and milk powder solution contaminated with Salmonella Enteritis,respectively,which was consistent with the bacterial plate count results.Under the condition of co-contamination of Salmonella Enteritis and 3 non-Salmonella bacteria,when the bacterial concentration was higher than 103 CFU/ml,the SPR subtractive inhibition method could detect Salmonella contamination in the sample.In addition,the lysis effect of phages showed that the results using this method could well reflect the lysis effect of phages on Salmonella and were consistent with the bacterial plate count results.The above analysis further verified that this method can be used for clinical detection of Salmonella contamination.In summary,this study established a SPR subtractive inhibition method for rapid detection of Salmonella based on Salmonella PagN monoclonal antibody(3B3)and carried out preliminary application,thus providing technical support for rapid semi-quantitative detection of Salmonella.