Development Of Rapid Isolation Detection Methods And Detection Kits For Listeria Monocytogenes In Food

Listeria monocytogenes（L.monocytogenes）,a Gram-positive pathogenic bacterium that may be present in a variety of foods.Because of its strong resistance to acid and alkali,low temperature growth and reproduction,it is difficult to be killed and cause continuous harm.In recent years,there have been numerous foodborne illnesses caused by L.monocytogenes infections,so there is an urgent need to develop sensitive detection methods for L.monocytogenes to screen foods and prevent the risks they cause.Compared with the"gold standard"culture method,which is time-consuming and laborious,immunology,which is costly,and molecular biology,which requires professionals,it is necessary to establish a rapid,sensitive,and easy-to-use instant detection method to complete the early detection of L.monocytogenes and prevent the occurrence of foodborne diseases.However,the complex food matrix causes extremely serious interference to the assay,so efficient pre-treatment methods need to be established to mitigate matrix effects.Magnetic separation technology can be used as a rapid and simple means of isolation and enrichment of bacteria.It can be used to prepare antibiotic-functionalized magnetic beads for the identification,enrichment,and isolation of L.monocytogenes in combination with the specific recognition ability of antibiotics to bacteria to exclude the interference of food substrates.Ampicillin（Amp）,aβ-lactam antibiotic,has a lactam ring structure that binds to penicillin binding proteins（PBPs）on bacterial cell membranes specifically.So it can be used as a recognition molecule for the preparation of antibiotic-functionalized magnetic beads and subsequent synthesis of immune glucose oxidase composites（e.g.,metal-organic frameworks and graphene oxide）were synthesized separately and combined with Amp functionalized magnetic beads to form a"sandwich"sandwich complex for glucose catabolism,and glucose concentration was measured using a blood glucose meter for sensitive and immediate detection of L.monocytogenes.The chapters are as follows:The first chapter reviews the mechanism for the action of Amp on bacteria and the study on the detection of foodborne pathogenic bacteria by magnetic separation technique combined with multiple detection assays.In Chapter 2,to achieve efficient enrichment of L.monocytogenes in food,a method for the preparation of functionalized magnetic beads（Amp-MBs）with Amp coupled directly on MBs was developed,and it was applied to the enrichment of L.monocytogenes in food samples.Under optimal conditions,Amp-MBs exhibited a capture efficiency higher than 90%for L.monocytogenes in juice.For the highly sensitive detection of L.monocytogenes in food,a zeolitic imidazolate framework-8（ZIF-8）with Zn²⁺as the metal backbone encapsulating glucose oxidase（GOD）was synthesized.The complex（GOD@ZIF-8@Ab）exhibited excellent performance for L.monocytogenes recognition and glucose catalysis.In combination with Amp-MBs,it forms a"sandwich"structure with L.monocytogenes and can be used to catalyze glucose catabolism.The sensitive detection of L.monocytogenes was completed by glucose concentration measurement using a blood glucose meter.The limit of detection（LOD）of L.monocytogenes in juice was 10¹ CFU/m L under optimal conditions.In this study,we successfully completed the sensitive detection of L.monocytogenes in juice.Chapter 3:To achieve efficient enrichment of L.monocytogenes in food,a method for the preparation of functionalized magnetic beads（Amp-PEG-MBs）with polyethylene glycol（PEG）as a mediator to"indirectly"couple Amp was developed,and it was applied to the enrichment of L.monocytogenes in food samples.Under the optimal conditions,Amp-PEG-MBs were able to capture L.monocytogenes in juice efficiently（﹥90%）at a lower dosage（40μg）.In order to achieve highly sensitive detection of L.monocytogenes in food,this study immobilized Ab and GOD on the surface of graphene oxide（GO）,so that the functionalized composite（GOD@GO@Ab）has both L.monocytogenes recognition ability and glucose catalytic performance.In combination with magnetic separation technology,the GOD@GO@Ab@L.monocytogenes@Amp-PEG-MBs"sandwich"complex was successfully formed,which can catalyze glucose catabolism.Under the optimal conditions,the LOD of L.monocytogenes in juice was 10¹ CFU/m L.The magnetic separation method established in this study can effectively reduce the amount of magnetic beads compared with the"direct"coupling method,which can effectively save the experimental cost while ensuring the detection results and laying the foundation for the development of the kit.In Chapter 4,for the on-site application of rapid isolation and detection of L.monocytogenes,based on the studies in Chapters 2 and 3,this chapter assembles Amp-PEG-MBs with GOD@GO@Ab and other required reagents into a stand-alone assay kit to accomplish the standardized and sensitive on-site detection of L.monocytogenes in food.This study included reproducibility tests,stability tests and shelf-life tests,and the kit was applied to the detection of L.monocytogenes in a variety of samples.The results demonstrated that the kit has good reproducibility and stability,maintains excellent detection performance after 240 days of storage,and has potential for practical application.In summary,this study combined Amp-based magnetic separation technology with immunoglucose oxidase composite materials to establish two sensitive detection methods and rapid detection kits.The performance of them were evaluated in food samples.The results demonstrate that the established methods and kits have good accuracy and sensitivity,which provide theoretical reference and technical means for the detection of food safety in our country.