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Method for enriching and separating listeria monocytogenes

A technology of Listeria monocytogenes, which is applied in the field of food-borne pathogenic bacteria separation, can solve the problems of separation failure, large concentration of miscellaneous bacteria, changes, etc., to increase the chance of contact, stabilize the reaction solution, and improve separation. The effect of efficiency

Inactive Publication Date: 2015-05-20
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. 4) The traditional immunomagnetic separation technology often directly couples antibody molecules to immunomagnetic beads. The steric hindrance effect between large 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, and micron magnetic beads are prone to non-specific adsorption, making it difficult to achieve the purpose of food sample liquid Specific separation of bacteria; 6) Excessive 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; (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

Method used

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  • Method for enriching and separating listeria monocytogenes
  • Method for enriching and separating listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. The dendrimer-antibody complex is prepared according to the following steps:

[0031] (1) Dissolve 1.0 mg dendrimer in 2 mL phosphate buffer (PBS, 0.02mol / L, pH 6.5), add 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3) -Dimethylamino) carbodiimide hydrochloride EDC, placed on a mixer at room temperature and stirred, activated for 15 min;

[0032] (2) Take 10.5 mg Lm The specific antibody is added to the above reaction solution and placed on the mixer at room temperature and stirred for 30 min;

[0033] (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.

[0034] 2. The long-chain biotin-dendrimer-antibody complex is prepared according to the following steps:

[0035] (1) Dissolve 15 mg long-chain biotin, 3.6 mg NHSS, and 2.4 mg EDC in 2 mL 0.02 M pH 6.5 PBS buffer;

[0036] (2) Add 0.53 mg of dendrimer-antibody compl...

Embodiment 2

[0040] Example 2 Enrichment effect experiment

[0041] (1) Take 1 mL concentration as 10 4 cfu / mL Lm Centrifuge at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

[0042] (2) Enrichment and capture: Set up the technical solution groups of the present invention ( Lm Dendrimer group modified by antibody and long-chain biotin), Lm Nano magnetic beads modified by specific antibodies, Lm The micron magnetic beads modified by specific antibodies enrich the target bacteria.

[0043] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated Lm The immunomagnetic beads were washed twice with PBST, mixed well, and resuspended the immunomagnetic bead complex with 1 mL sterile PBS solution.

[0044] (4) Calculation of capture rate: After gradient dilution of the resuspension of the target bacteria enriched in each group, count each gradient with a plate, ...

Embodiment 3

[0057] Example 3 Enrichment and capture experiment

[0058] The conventional magnetic stand separation time is 30 min, and the rest is the same as in Example 2.

[0059] The capture rate of each group is as follows:

[0060] Lm Capture rate of micron magnetic beads modified by specific antibody Lm Capture rate of specific antibody-modified nano magnetic beads Lm Capture rate of dendrimers co-modified by antibody and long-chain biotin 60.1% 40.6% 92.8%

[0061] 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 has been improved, especially Lm The capture efficiency of the nanomagnetic bead group modified by the specific antibody has the most obvious improvement, which shows that the capture efficiency of the nanomagnetic bead group can be greatly improved by prolonging the time, but it is still lower than the short time separation (3 min). Lm Captu...

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Abstract

The invention discloses a method for enriching and separating listeria monocytogenes (Listeria monocytogenes, Lm), 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 dendrimer and an antibody, coating a long-chain biotin molecule on the antibody-modified dendrimer, capturing the target bacteria in a sample solution through the dendrimer modified by the antibody and the long-chain biotin, identifying streptavidin-modified nano magnetic beads and coupling the long-chain biotin dendrimer 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. From Listeria monocytogenes ( Listeria monocytogenes , Lm 2) Poisoning incidents caused by it happen from time to time. It is extremely necessary to develop a fast and efficient technology for enriching and separating Listeria monocytogenes in samples. [0003] The immunomagnetic separation technology is one of the important components of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/01
Inventor 熊勇华许恒毅郭亮江湖
Owner NANCHANG UNIV
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