High-efficiency rapid vibrio parahemolyticus enrichment and separation method

Abstract

The invention discloses a high-efficiency rapid enrichment and separation method, mainly relates to a method for enriching and separating vibrio parahemolyticus from a matrix, aims at providing basis for follow-up study on target bacteria, and relates to the field of biotechnology. The method comprises the following steps of: carrying out covalent coupling on an arborescence hyper-branched polymer and an antibody, enveloping the antibody-modified arborescence hyper-branched polymer with long-chain biotin molecules, capturing the target bacteria in a sample liquid by using the arborescence hyper-branched polymer which is co-modified by the antibody and the long-chain biotin, recognizing and coupling the long-chain biotin arborescence hyper-branched polymer in the sample liquid by using streptavidin-modified nano magnetic beads, separating and heavy-suspending the captured bacteria, and the like. The captured bacteria can be subjected to follow-up analysis directly; and compared with the conventional bacterium separation method, the method disclosed by the invention is more applicable to magnetic separation of bacteria in a complex matrix, and the target bacterium separation efficiency 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] Vibrio parahaemolyticus ( vibrio parahaemolyticus, VP), belonging to the genus Vibrio, is a Gram-negative, halophilic, polymorphic bacterium. It is widely distributed in seawater near the coast, seabed sediment, plankton and seafood such as fish, shrimp and shellfish. It can cause acute gastroenteritis and primary sepsis. According to the report of the Ministry of Health, in the second quarter of 2008, food poisoning caused by Vibrio parahaemolyticus ranked second in microbial food poisoning, second only to wax samples. Bacillus. It is particularly urgent to establish a new method for efficient and rapid detection of Vibrio parahaemolyticus. In view of the need to establish an efficient and rapid detection method, immunomagnetic separation technology has been rapidly d...

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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