Method for enriching and separating listeria monocytogenes

Abstract

The invention discloses a method for enriching and separating listeria monocytogenes (Listeria monocytogenes, Lm), provides a basis for subsequent research on target bacteria and relates to the technical field of biology. The method comprises the following steps: performing covalent coupling on a dendrimer and an antibody, coating a long-chain biotin molecule on the antibody-modified dendrimer, capturing the target bacteria in a sample solution through the dendrimer modified by the antibody and the long-chain biotin, identifying streptavidin-modified nano magnetic beads and coupling the long-chain biotin dendrimer in the sample solution, separating and suspending the captured bacteria, wherein suspension can be directly analyzed later. Compared with the traditional bacteria magnetic separation method, the method is suitable for performing magnetic separation on the bacteria in a complex substrate, and the separation efficiency of 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. Listeria monocytogenes ( Listeria monocytogenes , L m ) poisoning incidents occur from time to time, and it is extremely necessary to develop a rapid and efficient technology for enriching and isolating Listeria monocytogenes in samples. [0003] Immunomagnetic separation technology is one of the important compo...

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. 4) The traditional immunomagnetic separation technology often directly couples antibody molecules to immunomagnetic beads. The steric hindrance effect between large 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, and micron magnetic beads are prone to non-specific adsorption, making it difficult to achieve the purpose of food sample liquid Specific separation of bacteria; 6) Excessive concentration of micron magnetic beads will cause damage to bacterial cells (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in failure of separation; (7) Magnetic beads When coupling antibodies, hydrophobic adsorption or chemical coupling is generally used to couple 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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