Non-targeted Separation Using Vancomycin Functionalized Magnetic Beads Combined With Rolling Circle Amplification For Specific Detection Of Staphylococcus Aureus

Food safety is a global hot issue,and food-borne pathogens are the main cause of frequent food safety problems.At present,the gold standards for the detection of foodborne pathogens were mostly based on traditional culture methods,but traditional culture methods took a long time and were complicated to operate,which was contrary to the concept of rapid identification and detection of food-borne pathogens.Therefore,the establishment of rapid and sensitive pathogen detection technology was particularly important.Antibiotic magnetic separation(AMS)was a simple and fast pre-treatment method for the detection of pathogenic bacteria.It used antibiotics as recognition molecules to modify the surface of magnetic particles directly or indirectly.It could capture and isolate the non-target bacteria and eliminate the time required for bacterial enrichment.In addition,antibiotic magnetic separation had lower cost and more stable reagents than immunomagnetic separation,so it had a better application prospect in the detection of foodborne pathogens.In this study,Vancomycin(Vancomycin,Van)modified nano magnetic beads(Magnetic nanobeads,MBs)were used as pretreatment methods for the isolation and enrichment of food-borne pathogenic bacteria,and DNA nanoflowers(DNFs)or RCA single-stranded products prepared by Rolling circle amplification(RCA)were used.Without the need to extract the DNA template of the target bacteria,load the enzyme or fluorescent dye to achieve colorimetric or fluorescent detection of Staphylococcus aureus(S.aureus).The content of each chapter is follows:The first chapter summarized the application of antibiotic magnetic separation technology as a pretreatment method in the detection of pathogenic bacteria.The second chapter established Van-MBs combined with DNFs colorimetry to detect S.aureus.In this study,bovine serum protein(BSA)-mediated vancomycin(Van)-functionalized magnetic beads combined with biotinylated immunoglobulin G(Biotin-IgG)recognized S.aureus to form a "sandwich structure",and the biotin modified by IgG serves as a "bridge":on the one hand,through the biotin-avidin system(Biotin-avidin system(BAS),directly combined with streptavidin modified horseradish peroxidase(Streptavidin-horseradish peroxidase,SA-HRP)to catalyze the color development of 3,3',5,5'-tetramethylbenzidine(TMB)to achieve the colorimetric detection of S.aureus;On the other hand,the single-stranded nucleic acid products obtained by the RCA reaction were paired with biotin-modified probe 2(Biotin-probe2)to prepare DNFs.The DNFs were connected to the "sandwich structure" through the BAS system.The surface of the DNFs could be loaded with a large amount of SA-HRP,which efficiently catalyzed the color development of the TMB color developing solution,and realizes the expanded colorimetric detection of S.aureus signals.In this experiment,a series of optimization and characterization of the magnetic separation and colorimetric detection sections were performed,and specificity verification was performed.Under the optimal experimental parameters,common colorimetric detection of phosphate buffered saline(Phosphate-buffered saline,PBS)and the minimum detection limit of S.aureus in juice sample was 3.3 ×105 CFU/mL,and the minimum detection limit of S.aureus in PBS and juice samples was 3.3 ×103 CFU./mL.The minimum detection limit of S.aureus in PBS and juice samples by Van-MBs-DNFs signal amplification colorimetric detection was 3.3×103 CFU/mL.The third chapter established the detection of S.aureus by Van-MBs combined with RCA fluorescence staining.In this study,Van-MBs and biotin-IgG were used to identify S.aureus,and then the BAS system was used to introduce the prepared RCA probe into the "sandwich structure",which was triggered by the action of phi29 DNA polymerase RCA reaction,the nucleic acid single strand produced by the reaction was stained with the nucleic acid dye SYBR GREEN ? to achieve fluorescence detection of S.aureus.In this study,a series of parameters were optimized for the Van-MBs combined with RCA fluorescence staining method,and the specificity of the method was verified.Under the optimal experimental parameters,the minimum detection limit of this method for S.aureus in PBS was 3.3 × 102 CFU/mL,the lowest detection limit in spiked juice sample was 3.3 × 102 CFU/mL.