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Method for enriching and separating helicobacter pylori

A Helicobacter pylori enrichment technology, applied in the biological field, can solve the problems of separation failure, poor monodispersity of micron magnetic beads, and large concentration of miscellaneous bacteria, so as to increase the chance of contact, shorten the separation time, and improve the capture efficiency.

Inactive Publication Date: 2013-09-04
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 isolation of target bacteria; 6) Too high 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

Method used

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  • Method for enriching and separating helicobacter pylori
  • Method for enriching and separating helicobacter pylori

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. The dendritic hyperbranched polymer-antibody complex is prepared according to the following steps:

[0029] (1) Weigh 4.5 mg of dendritic polyamide-amine amined with dendritic hyperbranched polymer, and dissolve it in 4 mL of phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 25% pentane. 545 μL of dialdehyde aqueous solution to make the final concentration of glutaraldehyde 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;

[0030] (2) Add Helicobacter pylori dropwise to the above solution HP The specific antibody is 12 mg, and its final concentration is about 3 mg / mL. React at room temperature for 24 hours at a speed of 150 r / min on a shaker;

[0031] (3) The above solution was spin-dried solvent under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 d; after the dialysis, the obtained solution was freeze-dried.

[0032] 2. The long-chain biotin-dendritic hyperbranched polymer...

Embodiment 2

[0038] Example 2 Enrichment effect experiment

[0039] (1) Take 1 mL concentration as 10 4 The cfu / mL Helicobacter pylori was centrifuged at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, the supernatant was discarded, and resuspended in an equal volume of sterile PBS solution.

[0040] (2) Enrichment and capture: The technical solution group of the present invention (the dendritic hyperbranched polymer group co-modified by Helicobacter pylori antibody and long-chain biotin), the Helicobacter pylori specific antibody-modified nanomagnetic bead group, and Helicobacter pylori Micron magnetic beads modified by bacteria-specific antibodies enrich the target bacteria.

[0041] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated immunomagnetic beads that trap Helicobacter pylori are washed twice with PBST, mixed well, and resuspended with 1 mL sterile PBS solution Immunomagnetic bead complex.

[0042] (4) Calculation of capture r...

Embodiment 3

[0055] Example 3 Enrichment and capture experiment

[0056] The conventional magnetic stand separation time is 30min, and the rest is the same as in Example 2.

[0057] The capture rate of each group is as follows:

[0058] The capture rate of Helicobacter pylori specific antibody modified micron magnetic beads The capture rate of Helicobacter pylori specific antibody modified nano magnetic beads Capture rate of dendritic hyperbranched polymer co-modified with Helicobacter pylori antibody and long-chain biotin 53.1% 34.8% 91.0%

[0059] The experimental results show that the separation of 3 minutes in Comparative Example 2 and when the separation time reaches 30 minutes, the capture efficiency of the three groups is improved, especially the Helicobacter pylori-specific antibody-modified nanomagnetic bead group has the most obvious improvement. This indicates that the capture efficiency of the nanomagnetic bead group can be greatly improved by prolonging the time, but it is s...

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Abstract

The invention discloses a method for enriching and separating helicobacter pylori (Helicobacter pylori, Hp), 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 hyperbranched polymer and an antibody, coating a long-chain biotin molecule on the antibody-modified hyperbranched polymer, capturing the target bacteria in a sample solution through the dendriform hyperbranched polymer modified by the antibody and the long-chain biotin, identifying streptavidin-modified nano magnetic beads and coupling the long-chain biotin dendriform hyperbranched polymer 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 separating food-borne pathogenic bacteria based on nano magnetic beads. Background technique [0002] Food-borne 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 foodborne diseases each year, and 2.2 million people worldwide die each year due to foodborne diseases. In my country, there are 200,000 to 400,000 food poisoning cases each year. Except for accidents, most of them are caused by food-borne pathogens. Helicobacter pylori (Helicobacter Pylor, HP) It is a microaerobe, which requires 5-8% of environmental oxygen. It cannot survive in the atmosphere or absolutely anaerobic environment, but it can be transmitted through hands, unclean food, unclean tableware, feces, etc. It is chronic active gastritis, digestion The main pathogenic bacteria of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20G01N33/569C12R1/01
Inventor 魏华
Owner NANCHANG UNIV
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