| Food safety has always been one of the people's livelihood issues that have been widely concerned by leaders and people around the world,and it has long threatened the health and safety of human beings.Among the many food safety incidents that have occurred,foodborne diseases caused by foodborne pathogenic microorganisms are one of the common causes.Therefore,preventing and controlling the occurrence of foodborne diseases caused by foodborne pathogenic microorganisms has become the top priority of governments all over the world.In order to solve the need for rapid detection of pathogenic microorganisms in the supervision of foodborne diseases,to overcome the disadvantages of traditional detection methods such as plate culture and enzyme-linked immunoassay,such as time-consuming,labor-intensive,long cycle,and insufficient sensitivity.It is very important to develop new detection methods that are easy to operate,sensitive and convenient to prevent the occurrence of foodborne diseases.Glycopeptides antibiotics(GPAs)have a strong affinity with the cell wall of Gram-positive bacteria,they can not only be used to treat Gram-positive bacteria infections,but also serve as recognition molecules for the detection of Gram-positive bacteria.Although antibodies and phages with high specificity are used in the detection of food borne pathogens,GPAs is also favored by researchers for its advantages of easy preparation and modification,high stability and low cost,and used in the isolation and detection of foodborne pathogens.In this paper,teicoplanin(Teic)was used as the recognition molecule and modified on carboxylated magnetic beads(MBs)by PEG and BSA“dual”mediated coupling,it was used to enrich and separate Staphylococcus aureus(S.aureus)and Listeria monocytogenes(L.monocytogenes)in phosphate buffer solution(PBS)or food spiked samples,and to construct“sandwich”complexes with immunofluorescence quantum dot microspheres,the specific detection of the two target bacteria was achieved by detecting the fluorescence intensity of the complex.The contents of each chapter are as follows:In Chapter 1:The application of GPAs in the detection of foodborne pathogens and the coupling methods of recognition molecules and magnetic nanomaterials were reviewed.In Chapter 2:Based on“dual”mediated teic functional magnetic beads(MBs-PEG-BSA-Teic),combined with biotinylated pig Ig G modified quantum dot fluorescent microspheres(QBs-SA-Bio-Ig G,emission wavelength:525 nm),a specific detection method for S.aureus was constructed.In the study,the coupling method of teic modification on the surface of MBs was optimized,and the material with the best separation efficiency was used for subsequent experiments,namely MBs-PEG-BSA-Teic.Combine MBs-PEG-BSA-Teic with S.aureus,and realize the enrichment and separation of S.aureus by means of external magnetic field.Under the optimal conditions,the separation efficiency of this material for S.aureus with a bacterial concentration of 3.3×10~1-3.3×10~6CFU/m L in PBS is higher than 89.34%,and the separation efficiency of S.aureus is also higher than 75.62%in he actual spiked samples of beef within this concentration range.In this method,teic was used as the recognition molecule of S.aureus,and adopts a“dual”mediated coupling method,which improves the separation efficiency of target bacteria,reduces the amount of materials,and save the costs.QBs-SA-Bio-Ig G is used as a signal output molecule,it realizes the sensitive detection of S.aureus.The sensitivity of S.aureus in PBS and actual spiked samples of beef was 3.3×10~1CFU/m L and 3×10~1CFU/g.This study shows that teic has the ability to recognize S.aureus and the feasibility of using teic as recognition molecule in magnetic separation technology.At the same time,it demonstrates the significant advantages of the“dual”mediated coupling method in improving reaction efficiency and saving detection costs.In Chapter 3:Developed a method for the specific detection of L.monocytogenes based on MBs-PEG-BSA-Teic combined with polyethylene glycol(PEG)mediated L.monocytogenes antibody modified quantum dot fluorescent microspheres(QBs-PEG-Ab,emission wavelength:565 nm).In this study,MBs-PEG-BSA-Teic was bond to L.monocytogenes to realize the enrichment and isolation of L.monocytogenes under the action of external magnetic field.Under the optimal conditions,the separation efficiency of L.monocytogenes from PBS with the concentration of 2.6×10~1-2.6×10~6CFU/m L was higher than 87.58%,the separation efficiency of L.monocytogenes was higher than 81.57%in the spiked samples of beef in this concentration range.After the enrichment and isolation of L.monocytogenes by MBs-PEG-BSA-Teic,QBs-PEG-Ab was used as the signal output molecule to realize the sensitive detection of L.monocytogenes.The limits of detection of L.monocytogenes in PBS and beef were 2.6×10~1CFU/m L and 2.6×10~1CFU/g.This study confirmed that teic has the ability to recognize L.monocytogenes and further demonstrated the feasibility of its application in magnetic separation technology.In Chapter 4:MBs-PEG-BSA-Teic combined with QBs-SA-Bio-Ig G and QBs-PEG-Ab were used to simultaneously detect S.aureus and L.monocytogenes.In this study,MBs-PEG-BSA-Teic was first bond to S.aureus and L.monocytogenes,the enrichment and isolation of S.aureus and L.monocytogenes were realized under the action of external magnetic field.Under the optimal conditions,the separation efficiency of this material for S.aureus from PBS with the concentration of 3.3×10~1-3.3×10~6CFU/m L was higher than 83.33%,the separation efficiency of L.monocytogenes from 2.6×10~1-2.6×10~6CFU/m L was higher than 77.96%.But,the separation efficiency of S.aureus and L.monocytogenes were higher than 62.54%and61.17%in the spiked samples,respectively.In this study,the method was used for simultaneous detection of various actual spiked samples.The results showed that the detection limit of the method was 10~1CFU/m L for the actual spiked samples of PBS and juice,and 10~2CFU/g for the actual spiked samples of spinach and beef.This study showed that the established method is suitable for the simultaneous detection of two target bacteria in a variety of actual spiked samples,and provides a certain guiding significance for the detection of other Gram-positive bacteria in actual spiked samples. |
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