Magnetic enrichment and separation method of Shigellaspp in complex matrix

Abstract

The invention discloses a magnetic enrichment and separation method of Shigellaspp in a complex matrix. The method provides the basis for the follow-up study on target bacteria and relates to the technical field of biology. The method comprises the following steps of: covalently coupling hyperbranched polymers with antibodies, enveloping long-chain biotin molecules by the hyperbranched polymers modified with the antibodies, capturing target bacteria in a sample solution by the hyperbranched polymers modified with the antibodies and long-chain biotin, identifying and coupling long-chain biotinylated hyperbranched polymers in the sample solution by nano magnetic beads modified with streptavidin, and carrying out separation and heavy suspension on the captured bacteria. The captured target bacteria can be directly used for the follow-up analysis. Compared with the traditional magnetic separation method of bacteria, the method is suitable for magnetic separation of the bacteria in the complex matrix, and the separation efficiency of the target bacteria in a sample is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Shigella separation method based on nano-magnetic beads. Background technique [0002] Shigella, commonly known as Shigella, is a Gram-negative intracellular pathogen and ranks first among infectious diarrhea pathogens in my country. Shigella mainly spreads through the digestive tract, invades intestinal epithelial cells, and causes typical dysentery symptoms characterized by fever, abdominal pain, and diarrhea. Every year, 160 million people are sick worldwide, and the high incidence of Shigella infection is mainly children aged 1-4. In adult patients, 10-100 cfu of Shigella can cause disease by infecting the intestinal tract, and it is particularly urgent to establish a new method for efficient and rapid detection of Salmonella typhimurium. In view of the need to establish an efficient and rapid detection method, Rapid Development of Immunomagnetic Separation Technology in Food...

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 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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