Establishment Of A Rapid Detection Method For Salmonella And Development Of A Multipathogen Enrichment Medium

Food-borne disease is a worldwide public health problem,and food-borne pathogens are the main cause of food-borne disease.Salmonella,Staphylococcus aureus and Listeria monocytogenes are common food-borne pathogens,and food-borne disease caused by them have attracted widespread attention.Accurate and rapid detection of these pathogens is the key to prevent and control related foodborne diseases.Traditional methods for detecting food-borne pathogens have complicated procedures,and thus are time consuming and heavy workload.Therefore,it is urgent to establish a fast,easy-to-use modern detection method to detect food-borne pathogens.At present,the detection of food-borne pathogens requires a pre-enrichment process,but each pathogen has its own growth habits and nutritional requirements.Different pathogens require different media.To a large extent,the application in practical detection is limited,so it is also urgent to develop a universal medium that can enrich them simultaneously.In the first part of this study,Salmonella was used as the target bacteria,and a rapid detection method based on biolayer interferometry(BLI)was established to achieve rapid and accurate detection of Salmonella in food,providing a new technical means for the monitoring system of foodborne pathogens in China.In the second part,a universal medium for rapid and simultaneous enrichment of Salmonella,Staphylococcus aureus,and Listeria monocytogenes was developed.It laid the foundation for the subsequent detection of the above three pathogenic bacteria using multi-channel biomolecular interaction instrument high-throughput detection technology.The main research contents are as follows:(1)A real-time,label-free rapid detection method for Salmonella enteritidis based on BLI was established.Optimal pH of acetate buffer used for antibody immobilization is 5,and optimal antibody concentration for antibody immobilization is 50?g/mL.The limit of detection for Salmonella enteritidis in buffer is 1.90×10~6CFU/mL,1.29×10~6 CFU/mL,8.77×10~5 CFU/mL,5.64×10~5 CFU/mL respectively at binding time of 60 s,120 s,180 s,300 s.In addition,this method is highly specific.In the detection of Salmonella enteritidis in composite matrix(whole egg and beef)as a whole,most of the recovery rates at different times were above 95%,and the relative standard deviations were lower than 7.71%.We believe that this BLI-based method is expected to become a new diagnostic tool for the rapid,label-free,specific,real-time detection of Salmonella enteritidis in food safety and other related fields.The method developed in this research can be easily applied to the rapid detection of other food-borne pathogens and harmful substances.(2)A universal multipathogen enrichment medium for rapid and simultaneous enrichment of Salmonella,Staphylococcus aureus and Listeria monocytogenes was prepared.Single factor experiments were used to select the best accelerators and inhibitors,and optimized by orthogonal experiments.Finally,Salmonella,Staphylococcus aureus and Listeria monocytogenes co-enrichment medium(SSL)formula was obtained:tryptone 17.0 g/L,peptone 3.0 g/L,yeast extract 6.0 g/L,Dipotassium hydrogen phosphate 2.5 g/L,disodium hydrogen phosphate 9.6 g/L,potassium dihydrogen phosphate 1.35 g/L,peptone peptone 15.0 g/L,beef meal 20.0g/L,soybean peptone 10.0 g/L,put Neomycin 0.3 g/L,mannitol 3.5 g/L,sodium pyruvate 4.5 g/L,fosfomycin sodium 0.00115 g/L,and lithium chloride 1.0 g/L.(3)Through the analysis of the enrichment effect of the compound enrichment medium of Salmonella,Staphylococcus aureus and Listeria monocytogenes,the above three target bacteria were cultured in SSL for 12 h,the bacteria concentration basically reached 10~7 CFU/mL,and after 16 h of cultivation,the bacteria concentration reached 10~8 CFU/mL,which was higher than the concentration of bacteria after TSB culture for 24 h.The growth of non-target bacteria is inhibited.The enriched bacteria concentration can fully meet the requirements of subsequent high-throughput detection technologies such as multiplex PCR,multi-channel BLI biosensor,and multi-channel Surface plasmon resonance biosensor.