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

It happens that people get food-borne diseases caused by eating foods contaminated with food-borne pathogens such as Listeria monocytogenes,Escherichia coli O157:H7,and Salmonella in recent years.The control and detection of foodborne pathogens have been highly valued all over the world.It’s crucial to master the fast and accurate detection and control technology for the prevention of diseases and deaths caused by foodborne pathogens.The traditional detection methods for pathogenic bacteria are often time-consuming and labor-intensive.Therefore,a sensitive,convenient,accurate,and easy-to-operate detection method needs to be developed urgently.The number of food-borne pathogens that exist in food may be relatively small under normal circumstances,in order to ensure the accuracy of the detection results,pre enrichment process is needed no matter what kind of detection technology.Since different types of pathogenic bacteria have different nutritional requirements,the pre enrichment samples can only be used for the detection of single foodborne pathogens,which greatly limits the application of high-throughput detection technologies such as multi-channel BLI biosensor in the detection of foodborne pathogens.Therefore,it’s very necessary to develop a new universal enrichment medium which can rapidly multiply multiple pathogenic bacteria at tone time,so as to improve the detection efficiency of pathogenic bacteria.In the first part of this study,Listeria monocytogenes was used as the target bacteria,a rapid detection method based on biolayer interferometry(BLI)was established to achieve rapid and accurate detection of Listeria monocytogenes in food by using phage and antibody as specific biological recognition elements,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 Listeria monocytogenes,Escherichia coli O157:H7,and Salmonella was developed.The main research contents are as follows:(1)A real-time,label-free rapid detection method for Listeria monocytogenes based on BLI was established.(1)Using antibody as recognition elements.The conditions of antibody immobilization on the surface of AR2G sensor,detection limit and specificity of this method were explored.Optimal p H of acetate buffer used for antibody immobilization is 4,and optimal antibody concentration for antibody immobilization is 50μg/m L.The limit of detection for Listeria monocytogenes in buffer is 4.60×10~4CFU/m L,1.20×10~4CFU/m L,1.00×10~4CFU/m L and 7.83×10~3CFU/m L respectively at binding time of 3min,6min,9min,12min.In addition,this method is highly specific.(2)Using phage as recognition elements.The conditions of phage immobilization on the surface of SA sensor and the detection limit of this method were explored.The optimal concentration of biotinylation reagent for modified phage is 2%,The best phage titer is 10~9PFU/m L.The limit of detection for Listeria monocytogenes in buffer is 3.89×10~5CFU/m L,2.25×10~5CFU/m L and 1.35×10~5CFU/m L respectively at binding time of 3 min,6 min,9 min.(2)A universal multipathogen enrichment medium for rapid and simultaneous enrichment of Listeria monocytogenes,Escherichia coli O157:H7,and Salmonella was prepared.Single factor experiments were used to select the best accelerators and inhibitors,and optimized by orthogonal experiments.Finally,Listeria monocytogenes,Escherichia coli O157:H7,and Salmonella co-enrichment medium(LES)formula was obtained:Tryptone 17 g/L,yeast extract 6.0 g/L,potassium dihydrogen phosphate 2.5 g/L,potassium dihydrogen phosphate 1.35 g/L,disodium hydrogen phosphate 9.6 g/L,sodium chloride 5.0 g/L,peptone 3.0 g/L,peptone peptone 25 g/L,beef powder 15 g/L,soybean peptone 14 g/L,glucose 3 g/L,actinomycin 0.3 g/L,mannitol 3 g/L,sodium pyruvate 6 g/L,acridine yellow 3 mg/L,fosfomycin sodium 3.125 mg/L.Through the analysis of the enrichment effect of the compound enrichment medium of Listeria monocytogenes,Escherichia coli O157:H7,and Salmonella,the above three target bacteria were cultured in LES for 12 h,the bacteria concentration basically reached 10~7CFU/m L.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 multi-channel BLI biosensor,multiplex PCR and multi-channel Surface plasmon resonance biosensor.The new rapid detection method based on BLI technology established in this study can realize the real-time rapid unlabeled detection of Listeria monocytogenes,and the exploration also has important guiding significance for the rapid detection of other foodborne pathogens other than Listeria monocytogenes.The developed general culture medium can realize the rapid proliferation of Listeria monocytogenes,Escherichia coli O157:H7 and Salmonella,which lays a foundation for the simultaneous detection of the above three pathogens by using multi-channel biomolecular interaction instrument high-throughput detection technology.