Establishment And Evaluation Of Rapid Detection Method Of Brucella In Food Samples

Brucella spp.is facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide.Its spread and prevalent can cause huge threat for animal husbandry and human health.Thus it is important for disease prevention and control through enhancing the detection of the pathogenic bacteria and taking effective preventive measures.The current methods for detection of Brucella spp.have many disadvantages such as complicated operation,time-consuming,low sensitivity and low specificity.Herein,a new rapid,sensitive and specific detection method of Brucella needs to be developed.In this assay,a new rapid detection method based on polyclonal antibodies-conjugating quantum dots and antibody-modified magnetic beads was established and evaluated and it can provide a new way for rapid detection of Brucella.There are two parts of the assay:1.Preparation and identification of anti-Brucella polyclonal antibodyThe polyclonal antibody was produced through immune New Zealand white rabbits by Brucella 16 M and purified polyclonal antibody with saturated ammonium sulfate.The i ELISA was established and optimized with the concentration of antigen,the block solution,the block time,the concentration of serum,the reaction time of antigen-antibody,the concentration of HRP-goat anti-rabbit polyclonal antibody secondary antibody,the reaction time of HRP-goat anti-rabbit polyclonal antibody secondary antibody,the time of coloration,determined the titer and evaluated the specificity of polyclonal antibody with optimized i ELISA.The purity of the polyclonal antibody was evaluated with SDS-PAGE,determined the content of polyclonal antibody with Bradford Protein Assay Kit.The results of optimized i ELISA showed that the optimum coated condition was 37℃ incubated for 1 h,the optimum concentration of antigen was 1:40,the optimum block solution was 5% skim milk powder,the optimum block time was 1 h,the optimum concentration of serum was 1:16000,the optimum reaction time of antigen-antibody was 1.5 h,the optimum concentration of HRP-goat anti-rabbit polyclonal antibody secondary antibody was 1:10000,the optimum reaction time of HRP-goat anti-rabbit polyclonal antibody secondary antibody was 0.5 h,the optimum time of coloration was 10 min.The titers of the polyclonal antibody were determined by the optimized i ELISA.They were 1:256000 and 1:2048000.There was not so much cross reaction of polyclonal antibody with Melitensis M5,E.Coli O157,Listeria Monocytogenes,Proteus vulgaris,Enterobacter Cloacae,Enterobacter sakazakii,Klebsiella pneumonia,Shigella Bogdii,Staphylococcus aureus,Serratia marcescens,Salmonella Typhimurium.And the result of SDS-PAGE showed that there were not so many mixed stripes.The contents of the polyclonal antibody were 14.091 mg/m L and 17.454 mg/m L.2.The establishment and evaluation of the detection methodThe rabbit anti-Brucella polyclonal antibody was conjugated with IMB to prepare IMB-probe and conjugated Ig Y with QDs to prepare QDs-probe which can recognize the Brucella.The Brucella was separated by IMB-probe,and then the QDs-probe was used as a fluorescent label probe then we can get the IMB-probe-Brucella-QDs-probe “Sandwich” complex.To measure the fluorescence of the complex can determine the linear regression equation of the fluorescence intensity and bacteria concentration and made the rapid and quantify detection of Brucella come true.To establish the detection method,the steps of the method such as the amount of the IMB-probe,time of IMS,the amount of QDs-probe and label time was optimized.The sensitivity,specificity and repeatability of the method was evaluated and the milk and lamb-steeped liquor was detected with the new method.The 100 μL IMB was conjugated with 122.178 μg rabbit anti-Brucella polyclonal antibody incubated under 37℃ with 2 h.While the 10 μL QDs was conjugated with 262 μg Ig Y incubated under 37℃ with 2 h.And the new detection method took 100μL IMB-probe and 500 μL QDs-probe,the enrich time was 45 min and the label time was 60 min.The detection method takes 105 min totally with a limit of detection of 103 CFU/m L and range from 10 to 105 CFU/m L(R2=0.9983).The method can recognize the B.melitensis and B.suis while it cannot recognize the E.Coli O157 and Listeria Monocytogenes.The LOD of the method with milk and lamb-steeped liquor was 102 CFU/m L so it can be well used in real samples.In conclusion,I have successfully produced and characterized the rabbit anti-Brucella polyclonal antibody,IMB-probe and QDs-probe and established and optimized the rapid detection method.The stability and specificity of the new detection method is good and the LOD is low enough,it can meet the requirements of the application of food samples.And it provides a new idea of rapid detection.