Fast magnetic separation method of mycobacterium tuberculosis (MT)

Abstract

The invention discloses a method for enrichment separation of mycobacterium tuberculosis (MT), and better provides a basis to the follow-up study of target bacteria, relating to the field of biotechnology. The method comprises the following steps of: performing covalent coupling of a hyperbranched polymer and an antibody; coating a long-chain biotin molecule with the antibody-modified hyperbranched polymer; capturing target bacteria in sample liquid by use of the hyperbranched polymer co-modified by the antibody and the long-chain biotin; identifying streptavidin-modified nano magnetic beads, and coupling with the long-chain biotinylation hyperbranched polymer in the sample liquid; separating and re-suspending the captured bacteria, and the like, wherein the re-suspension liquid can be directly subjected to follow-up analysis. Compared with a traditional magnetic bacterium separation method, the method disclosed by the invention is more suitable for the magnetic separation of bacteria in a complicate matrix, and improves the separation efficiency of target bacteria in a sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating pathogenic bacteria based on nano magnetic beads. Background technique [0002] Tuberculosis is currently one of the major infectious diseases that cause death to humans, and about one-third of the world's population has been infected with Mycobacterium tuberculosis ( Mycobacterium tuberculosis , MT ), and is developing at a rate of 9 million new cases per year. In my country, about 550 million people have been infected with this bacteria, and about 1.5 million cases of active tuberculosis patients are newly added every year in the country. Especially in recent years, with the continuous emergence of drug-resistant and multidrug-resistant tuberculosis, tuberculosis has become a public problem that seriously endangers human health. Due to the low detection rate of MDR-TB patients, the treatment time is long, the cure rate is low, and the mortality rate is hi...

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), which usually takes longer to specifically capture bacterial cells in the matrix; 3) Micron magnetic beads have poor monodispersity and are prone to self-aggregation or Precipitation; 4) The traditional immunomagnetic separation technology often directly couples antibody molecules to immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and changes in the spatial direction of the antibody, increasing the inter-antibody interaction. steric hindrance effect, which reduces the capture efficiency of the antibody 5) The matrix is ​​complex in nature and the concentration of non-target pathogenic bacteria is large, and the micron magnetic beads are prone to non-specific adsorption, making it difficult to achieve specificity for the target bacteria in the sample liquid 6) If the concentration of micron magnetic beads is too high, the bacterial cells will be damaged (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 the failure of the separation; 7) When the magnetic beads are coupled to antibodies Generally, the active antibody is attached to the surface of the magnetic bead by hydrophobic adsorption or chemical coupling.

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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