Detection Of Foodborne Eschenichia Coli O157:H7Using Protein Luminex Technology

Escherichia coli O157: H7（E.coli. O157: H7）is one of the most importantfoodborne pathogenic bacteria, the main route of transmission is food, especially is rawbeef. There have been large outbreaks that result in people’s lives and property sufferedheavy losses occurred in many developed countries. E.coli O157also is detected in manyprovinces of China, its potential risk of large-scale outbreaks in the country. To avoidmajor pathogenic bacteria outbreaks, it is very important method to detect beef productsaccurately and rapidly. Liquid chip become a hot spot of detection technology because ofits advantage of rapid, sensitivity and specificity. This study established E. coli O157: H7liquid protein chip detection method, preliminarily explored the function of N, N-dimethyl acetamide （DMAC） as solvent of1-Ethyl-3-（3-dimethylaminopropyl）carbodiimide hydrochloride （EDC） and N-Hydroxysuccinimide （NHS） to improve theconnection efficiency of the microspheres and monoclonal antibody and the sensitivity ofdetection, applied the established method to detect154actual beef samples.Specific studies in this paper are as follows:1. Isolation and culture of E.coli O157:H7standard strain, was inactivated byformaldehyde, ultrasonication to obtain inactivated vaccine antigen, use Freund’s adjuvantemulsion method prepare vaccine, immune Balb/C mice，method of ascite to obtainspecific monoclonal antibody and evaluation of monoclonal antibody characteristics.（1） Preparation of two hybridoma cell strain E.coli O157: H7of monoclonal antibodywhich stable secretion, its chromosome number is90, and that determined fusion issuccessful, and it have a stable genetic.（2） According to identify the characteristics of monoclonal antibodiescomprehensive，the results prove that obtained monoclonal antibody is IgG; titer are1:512000and1:1024000; protein content of antibody are18.459mg/mL and23.014mg/mL; specific of monoclonal antibody to18kinds of intestinal bacteria isfavorable; affinity constant is1.01×10⁸, it means high-affinity antibody; SDS-PAGE has clear objective strips and few miscellaneous that prove the monoclonal antibody is highpurity; monoclonal antibody titer did not change after one month, three months, six months,demonstrate it stability.2. Preparation of vaccine to immunize New Zealand rabbits, took sterile blood fromheart, isolated and purified serum to polyclonal antibody of E.coli O157:H7pathogenicbacteria, applied biotin labeling kit to do biotinylate and calculate levels of biotin label.（1） Antibody titer is1:64000; it showed negative reaction with18kinds of intestinalbacteria, means good specificity; after purifying protein content is44.18mg/mL.（2） The best purification conditions is35%and uses100%elution buffer two-stepelution.（3） Preparation of biotin labeled polyclonal antibody,3molecules biotin can belabeled in per molecule protein, it’s suitable for continue experiment.3. To explore DMAC that is applied as lytic agent of EDC, NHS to couplemicrospheres with monoclonal antibody, improve coupling efficiency.（1） The microspheres and monoclonal antibody coupled successful, it is the basis formaking a success of E. coli O157: H7detection.（2） Separately applied DMAC and H₂O to dissolve EDC and NHS for activatingmicrospheres, coupling monoclonal antibody. By the measured we know that Medianfluorescent instensity （MFI） were3012and2253, that is the DMAC can significantlyimprove the couple efficiency of microspheres and the protein.（3） For each text （2μL microspheres） the best amount of protein coupling is95μg.4. Established the liquid protein chip method to detecte E.coli O157: H7, respectivelyexplore the monoclonal antibody amount of coupling, the addition of biotin-labeledmonoclonal antibody, Streptavidin-R-Phycoerythrin （SA-PE） additon and the reaction timeof SA-PE have influence on the detection result. Drawing the detection curve andevaluated the specificity, sensitivity, and reproducibility of this detection method. Applicatethis method and H₂O as solvent to detect actual beef samples, and compared the positivedetectable rate of this method and bacterial isolation and culture method. Explore DMACinfluence on positive rate.（1） Optimize the conditionsof detection experimental, get the best amount of biotin-labeled monoclonal antibody is76μg; the optimum SA-PE content is0.5μg; SA-PE optimum reaction time is30min.（2） To establish the detection methods in application of the optimum reactionconditions. The minimum bacteria concentration can be detect positive is10³cfu/mL （afterculture）, the LOD is100cfu/mL （after culture）; no cross-reaction with18kinds of intestinalbacteria means good specificity; after three repeated experiment the inter-assay variationcoefficient is7.61that indicates good reproducibility.（3） Before culture the minimum bacteria concentration can be detected positive is0.5cfu/mL which applicated establish method to detect artificially added bacteria in beefsampls.（4） Applicated establish method to detect154actual beef samples, get19positivesamples,16positive samples with the bacterial culture method.（5） Compare the minimum bacteria concentration can be detected positive and LODof DMAC and H₂O as solvent of EDC and NHS, the DMAC is10³cfu/mL and100cfu/mL;the H₂O is104cfu/mL and10³cfu/mL.（6） Detecting154actual beef samples which used separately DMAC and H₂O assolvent of EDC and NHS to activate microspheres and couple protein.It can be detected19positive samples when used DMAC as solvent and17positive samples when used H₂O.