Development Of An Immunossay For Listeria Monocytogenes

Listeria monicytogenes is a foodborn pathogen that can infect human and animals with high virulence.This paghogen widely exists in environment,and can tolerate high osmotic pressure.The bacterium exists in soil,sewage and rotten vegetables.Animal foods are main sourses contaminated by this pathogen,the fatality rate caused by L.monocytogenes is higher.Therefore,it has important practical significance for the detection of the pathogen.In this study,a ELISA detection method specific for L.monocytogenes was developed using a surface protein as target.Based on the 16 immune proteins identified previously in L.monocytogenes ATCC19111 in our laboratory,one surface protein with high specificity was selected.Through analying the sequence and the three-dimensional structure of the protein,a specific part of this protein was selected and expressed in E.coli BL21(DE3)with a molecular weight of about 26.3 kDa.The protein was puried by immunoaffinity chromatography and injected into New Zealand white rabbits and SPF hens as immunogen to prepare polyclonal antibodies(pAb).A ELISA method for L.monocytogenes was established using the purified rabbit-pAb(IgG)and chicken-pAb(IgY).The optimal conditions of the ELISA method were determined.The rabbit-pAb was used as capture antibody with coating amount of 0.5 ?g/well,while the chiken-pAb was used as the detection antibody with 400 folds dilution.Blocking solution was determined as 0.5%skim milk powder in PBS and the incubation time was determined as 1h,HRP-IgY was diluted for 10,000 folds and the incubation time was determined as 30min.Using the optimal conditions,the detection limit of the method was 6�106 CFU/mL and the limit of quantification was 1.2�107 CFU/mL.Five different strains of L.monocytogenes.five different species of Listeria and five stains of non-Listeria were used to evaluate the specificity of the method and the result showed that the detection method had good specificity.Raw beef and skim milk were selected as the actual samples,and different amounts of L.monocytogenes were added to the samples to evaluate the practicality of the method.The results showed that 101 CFU/mL and 102 CFU/mL L.monocytogenes contaminated in the two food samples be detected after enrichment for 24 h.The detection method in this study has high specificity and sensitivity,which can achieve a rapid and effective detection purpose in samples.Therefore,it is necessary to develop rapid and effective detection methods for preventing and controlling L.monocytogenes.