Magnetic bead separation method of escherichia coli O157

Abstract

The invention discloses an enriched enrichment and separation method of escherichia coli O157 (E.colio157), provides a basis for the follow-up study of target bacteria, and relates to the field of biotechnology. The method comprises the following steps of: carrying out covalent coupling on a broom molecule and an E.coli O157 antibody, enveloping a long chain biotin molecule by using the antibody modified broom molecule, capturing the target bacteria in a sample solution by using the broom molecule co-modified by the antibody and the long chain biotin, recognizing and coupling the long chain biotin modified broom molecule in the sample solution by a streptavidin modified nanometer magnetic bead, and subjecting the captured bacteria to separation and heavy suspension and the like, wherein a heavy suspension solution can be directly subjected to subsequent analysis. Compared with a traditional bacteria magnetic separation method, the method disclosed by the invention is more applicable to magnetically separating the bacteria in a complex matrix, and the separation efficiency of the target bacteria in the sample is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating food-borne pathogenic bacteria based on nano magnetic beads. Background technique [0002] Foodborne pathogen contamination is one of the major problems of food safety in my country. According to WHO statistics, about one-third of people in developed countries are infected with food-borne diseases every year, and 2.2 million people in the world die every year due to food-borne diseases. In my country, the number of cases of food poisoning is between 200,000 and 400,000 per year, most of which are caused by food-borne pathogens except for accidents. Among them, Escherichia coli O157 ( Escherichia coli O157 , E. coli O157 ) as one of the main pathogenic bacteria, the poisoning incidents caused by it occur from time to time, and the harm is serious, mainly manifested in diarrhea, hemorrhagic enteritis, and secondary hemolytic uremic syndrome and thrombotic thr...

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; Bacterial cells are combined through a multiphase reaction (multiphase reaction), and it usually takes longer to specifically capture bacterial cells in the food matrix; 3) Micron magnetic beads have poor monodispersity and are prone to self-disruption in the food matrix solution. Aggregation or formation of precipitates; 4) Traditional immunomagnetic separation techniques often directly couple antibody molecules to immunomagnetic beads, which often leads to greatly reduced antibody activity and changes in the spatial direction of antibodies. The steric hindrance effect between antibodies reduces the capture efficiency of antibodies. 5) The nature of the food matrix is ​​complex and the concentration of non-target pathogenic bacteria is large. Micron magnetic beads are prone to non-specific adsorption, and it is difficult to realize the detection of food samples. Specific separation of target bacteria; 6) Too high concentration of micron magnetic beads will cause damage to bacterial cells (magnetic field causes magnetic beads on the cell surface to attract each other, causing cells to be squeezed or even ruptured), resulting in failure of separation 7) Magnetic beads even When linking antibodies, hydrophobic adsorption or chemical coupling is generally used to link active antibodies on the surface of magnetic beads

The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

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