Study On Three Kinds Of Foodborne Bacteria Immunomagnetic Beads Combined With ATP Chemiluminescence Detection

As foodborne pathogens,salmonella,Listeria monocytogenes and Staphylococcus aureus have the possibility of epidemic outbreaks and the severity of infection in china.However,the traditional plate culture method is time-consuming,laborious and tedious,so it is urgent to develop a method for rapid detection.This study investigated the current field of international biological sensors of many new technologies,will eventually ATP biological method and immunomagnetic separation technology as the main research method and content,and has carried on the deep discussion and research.Research 1:The optimal conditions for the coupling of antibodies with magnetic beads were investigated by different temperature and time.Through a combination of orthogonal test comparison of amino modified magnetic beads and Salmonella antibody is the best reaction conditions are pH 9.6,temperature 37 C,reaction time 2h;combined with the mono antibody Lester bacteria optimum reaction conditions for pH 9.6,temperature 37 C,reaction time is 1.5h;combined with Staphylococcus aureus antibody the optimum reaction conditions for pH 9.6,temperature 37 C,reaction time is 1h.The best use of Salmonella,List Rand,and Staphylococcus aureus beads was 80 L,100 L and 60 mu L,respectively.Through the optimization of the coupling conditions,stable Salmonella,List Rand,and Staphylococcus aureus immunomagnetic beads were obtained.Research 2:Taking Salmonella as the object of study,we optimized the best buffer for ATP bioluminescence and screened the best luminescent chemicals.Through the influence of Tris-HCI,Na2HPO4-C6H8O7,KH2PO4-NaOH,PBS and four buffer solutions on the reaction of pH=7.4,it was concluded that Tris-HCI buffer had the best dilution effect on the broth.By comparing the concentration of 10 g/L five,NaCl,NaH2PO4 chemical NaOH,KCI,MgC12 on the chemiluminescence reaction,the MgCl2 has an enhanced effect on the other four kinds of light emitting light,there is a certain inhibitory effect,inhibition of activity:NaOH>NaCl>NaH2PO4>KCl.Research 3:Detection by immunomagnetic enrichment of Salmonella,Listeria monocytogenes and Staphylococcus aureus List Rand condition optimization and ATP lighting technology,the establishment of Salmonella,Listeria monocytogenes and Staphylococcus aureus List Rand immunomagnetic beads combined with ATP lighting technology,and to detect the simulated polluted samples.Results:(1)comparison of different bacteria-immunomagnetic concentration ratio of 1:6.34,1:12.69,1:25.38,1:30.07 immune magnetic separation technology capture rate,results show that when-IMB concentration ratio was 1:30,nearly 100%capture rate of Salmonella,Listeria monocytogenes bacteria Lester.When the concentration of-IMB was 1:25,the capture rate of Staphylococcus aureus was nearly 100%.(2)the same,in a total volume of 0.4 mL the amount of IMB at the same time,compared with 3 kinds of tested bacteria and Bacillus Subtilis in different volume ratio of the capture rate was decreased first and then increased the capture.(3)technology combined with ATP bioluminescence technique to detect Salmonella,Listeria monocytogenes Lester bacteria and Staphylococcus aureus immune magnetic separation,this technology combined with traditional bacterial culture method has good linear correlation,the correlation coefficients were R2=0.99762,P<0.0001;R2=0.9866,P<0.0001;R2=0.9911,P<0.0001.The detection limit is as low as 102CFU/mL.(4)using the immunomagnetic capture combined with ATP luminescence detection method in simulated samples to capture bacteria,the results showed that the concentration of 3 kinds of bacteria,106,105 simulation samples 107,104,103 and 102CFU/mL,ATP luminescence detection was 108,107,respectively,105,104 and 106 103 CFU/mL.