Synthesis Of Vancomycin Functionalized Magnetic Beads And Its Application In The Separation And Detection Of Listeria Monocytogenes And Staphylococcus Aureus

At present,food safety has become a common public health problem all over the world.Traditional culture methods,immunological methods and molecular biological methods can not meet the demand of rapid and sensitive detection of pathogenic bacteria in food.As a fast and effective enrichment method,traditional immunomagnetic separation has a good application value in the rapid detection of pathogenic bacteria.However,the cost of antibody is expensive,and the preparation and storage is difficult,which greatly limits the wide application of traditional immunomagnetic separation.Vancomycin(Van)is a glycopeptide antibiotic,its binding force with target bacteria is similar to antigen-antibody binding force.It has many advantages such as good stability,easy preparation and preservation.It has been widely applied in the isolation and detection of gram-positive bacteria in recent years.This paper used Van modified magnetic beads(MBs)as carriers for enriching and separating of the pathogenic bacteria,and then combined with polymerase chain reaction,flow cytometry,multiplex quantitative polymerase chain reaction for detecting L.monocytogenes and S.aureus in food.The contents of each chapter are described as follows:In the first chapter:the application of vancomycin for detection of foodborne pathogens was reviewed.In the second chapter:vancomycin(Van)functionalized PEGylated-magnetic nanoparticles(Van-PMs)combined with polymerase chain reaction(PCR)showed great potential for highly efficient detection of L.monocytogenes at low infectious dose.Herein,Van-PMs played as universal molecular probes for targeting bacterial cells,while PEG was used to help Van close to D-alanyl-D-alanine(D-Ala-D-Ala)moieties on surface of L.monocytogenes.Under optimum conditions,the developed Van-PMs complexes exhibited extremely high capture efficiency(CE)in a 10 mL bacterium suspension with the value was higher than 90%and 83%in the phosphate-buffered saline(PBS)solution and lettuce samples,respectively.Meanwhile,gram-negative bacteria slightly influenced CE even under concentrations as high as 10~6 CFU/mL.The assay of Van-PMs-PCR was applied to detect L.monocytogenes with detection limits of 30 CFU/mL and 30 CFU/g in PBS solution and lettuce samples,respectively.The whole experiment was completed within 4 h.This assay demonstrated the potential of this modified method in practical application involving the detection of L.monocytogenes or other gram-positive pathogenic bacteria.In the third chapter:a novel sandwich strategy was designed to detect S.aureus.The strategy is based an antibacterial agent that captures bacterial cells and a fluorescein-labeled antibody that acts as the signal-output probe.Van,which exerts a strong antibacterial effect on gram-positive bacteria,was utilized as a molecular recognition agent to detect pathogenic bacteria.To effectively concentrate S.aureus,bovine serum albumin(BSA)was used as the amplification carrier to modify magnetic beads(MBs),which were then functionalized with Van.To improve the specificity of the method for S.aureus detection,fluorescein isothiocyanate(FITC)-tagged pig immunoglobulin G(FITC-pig IgG)was adopted as the signal probe and the second recognition agent that bound between the Fc fragment of pig IgG and protein A in the surface of S.aureus.To quantify S.aureus,the fluorescence signal was measured by flow cytometry(FCM).The use of multivalent magnetic nanoprobes(Van–BSA–MBs)showed a high concentration efficiency(>98%)at bacterial concentrations of only 33 CFU/mL.The detection limit for S.aureus was 3.3×10~1 CFU/m L and the total detection process could be completed within 120 min.Other gram-positive bacteria and gram-negative bacteria,including L.monocytogenes,Bacillus cereus,Cronobacter sakazakii,Escherichia coli O157:H7,and Salmonella Enteritidis,negligibly interfered with S.aureus detection.The proposed detection strategy for S.aureus possesses attractive characteristics,such as high sensitivity,simple operation,short testing time,and low cost.In the fourth chapter:established a simple,rapid,and ultrasensitive Van and nanoparticles-based method to isolate and detect bacteria in milk based on a novel multivalent binding strategy.The new method involved Van functionalized poly(amidoamine)(G4 PAMAM)dendrimers to generate multivalent interactions between specific multiple-ligand–receptor and capture target bacteria from milk samples.Streptavidin(SA)modified MBs were used to isolate the Van-bacteria complex that was subjected to multiplex quantitative polymerase chain reaction(m-qPCR).The new Van-based simultaneous detection method was highly sensitive with a faster bacteria isolation time at 2 min.The results gave a linear range at 3.2×10~1–3.2×10~6 CFU/mL for L.monocytogenes and 4.1×10~1–4.1×10~6 CFU/mL for S.aureus,respectively.The actual LOD was 32 and 41 CFU/mL,respectively.Other gram-negative bacteria and gram-positive bacteria revealed insignificant interference in the simultaneous detection of L.monocytogenes and S.aureus.This novel Van-dendrimer-based magnetic separation nanoplatform with a multivalent interaction exhibited high sensitivity and specificity in a fast and simple pathogenic bacteria detection process.In addition,it is the first time that used Van as a recognition molecule for two gram-positive bacteria simultaneous detection.