Method for separating streptococcus pneumoniae in complicated substrate

Abstract

The invention discloses a method for enriching and separating streptococcus pneumoniae in order to provide a basis for the follow-up study of target bacteria, relating to the field of biotechnology. The method comprises the steps of: covalent coupling between a dendritic polymer and an antibody, exposed amino of long-chain biotin blocking antibody-dendritic polymer, capturing of target bacteria in sample liquid by the antibody coupled with the dendritic polymer, identification of streptavidin coupled with nano magnetic beads and coupling with the long-chain biotinylated dendritic polymer in the sample liquid, magnetic separation and re-suspension of bacteria and the like. The re-suspension liquid can be directly subjected to follow-up analysis; and compared with the traditional magnetic bacterial separation method, the method disclosed by the invention is more suitable for the magnetic separation of bacteria in a complicated substrate, and improves the separation efficiency of the 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] Streptococcus pneumoniae ( Streptococcus spneumoniae , SP ), as a clinically important pathogen, is widely distributed in nature, and its carrier rate in healthy people is between 5% and 50%, and it is closely related to colonization and infection. SP In addition to causing sinusitis and otitis media, it is also the number one pathogen that causes serious diseases such as meningitis, lobar pneumonia, and sepsis. In addition, it can cause severe neurotoxicity, such as focal neurological deficits, cognitive impairment, and hearing loss. In recent years, 700,000 to 1,000,000 deaths have been caused by Streptococcus pneumoniae worldwide, and its main incidence is concentrated in the elderly and children. In addition, China is one of the countries with the largest number of infectio...

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Problems solved by technology

However, this separation technology has many limitations: 1) The specific surface area of ​​micron magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; (Multiphase reaction) combination, it usually takes longer to specifically capture bacterial cells in sputum and other substrates; 3) Micron magnetic beads have poor monodispersity, and are prone to self-aggregation or precipitation in sputum and other substrate fluids; 4) The antibody molecules are directly coupled to the immunomagnetic beads. This process often leads to a great reduction in the activity of the antibody and a change in the spatial direction of the antibody, which increases the steric hindrance effect between the antibodies, thereby reducing the capture of the antibody. Efficiency 5) Due to the complex nature of the matrix and the high concentration of non-target pathogenic bacteria, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specific separation of target bacteria in the sample liquid; 6) The concentration of micron magnetic beads is too high High levels 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

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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