Magnetic bead separation method of escherichia coli O157

A technology for separation of Escherichia coli and magnetic beads, applied in the biological field, can solve the problems of separation failure, poor monodispersity of micro-magnetic beads, and large concentration of bacteria, and achieve the effect of increasing the chance of contact, shortening the separation time, and improving the capture efficiency.

Inactive Publication Date: 2013-09-18
NANCHANG UNIV
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  • 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

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  • Magnetic bead separation method of escherichia coli O157
  • Magnetic bead separation method of escherichia coli O157

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. The broom molecule-antibody complex is prepared according to the following steps:

[0037] (1) Dissolve 1 mg of aminated broom molecule in 2 mL of 0.02 M, pH 6.5 phosphate buffer PBS, add 0.6 mg of N-hydroxysuccinimide NHSS, 0.4 mg of ethyl 3-(3-dimethylamino ) carbodiimide hydrochloride EDC, placed on a mixer at room temperature and stirred, activated for 15 min;

[0038] (2) Take 11.5 mg E. coli O157 The specific antibody was added to the above reaction solution, placed on a mixer at room temperature and stirred for 30 min;

[0039] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

[0040] 2. The long-chain biotin-broom molecule-antibody complex is prepared according to the following steps:

[0041] (1) Dissolve 10 mg long-chain biotin, 3.6 mg NHSS, and 2.4 mg EDC in 2 mL 0.02 M pH 6.5 PBS buffer;

[0042] (2...

Embodiment 2

[0046] Example 2 Enrichment effect experiment

[0047] (1) Take 1 mL of concentration as 10 4 cfu / mL E. coli O157 Centrifuge at 12,000 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.

[0048] (2) Enrichment and capture: respectively set the technical solution group of the present invention ( E. coli O157 Antibody and long-chain biotin co-modified broom group), E. coli O157 Specific antibody-modified nano-magnetic bead set, E. coli O157 Specific antibody-modified micron magnetic bead group enriches target bacteria.

[0049] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and capture the E. coli O157 The immunomagnetic beads were washed twice with PBST, mixed well, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.

[0050] (4) Capture rate calculation: After gradient dilution of the enriched target bacteria resu...

Embodiment 3

[0063] Example 3 Enrichment capture experiment

[0064] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0065] The catch rate of each group is as follows:

[0066] E. coli O157 Specific antibody-modified micron magnetic beads Obtaining rate E. coli O157 Capture efficiency of specific antibody-modified nanomagnetic bead sets E. coli O157 Capture efficiency of broom groups co-modified with antibodies and long-chain biotin 58.7% 39.1% 92.1%

[0067] Experimental result shows, separates 3min among the comparative example 2, when separation time reaches 30min, the capture efficiency of three groups has all been improved, especially E. coli O157 The capture efficiency of the specific antibody-modified nano-magnetic bead group is the most obvious, which shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by extending the time, but it is still lower than the short-...

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Abstract

The invention discloses an enriched enrichment and separation method of escherichia coli O157 (E.colio157), provides a basis for the follow-up study of target bacteria, and relates to the field of biotechnology. The method comprises the following steps of: carrying out covalent coupling on a broom molecule and an E.coli O157 antibody, enveloping a long chain biotin molecule by using the antibody modified broom molecule, capturing the target bacteria in a sample solution by using the broom molecule co-modified by the antibody and the long chain biotin, recognizing and coupling the long chain biotin modified broom molecule in the sample solution by a streptavidin modified nanometer magnetic bead, and subjecting the captured bacteria to separation and heavy suspension and the like, wherein a heavy suspension solution can be directly subjected to subsequent analysis. Compared with a traditional bacteria magnetic separation method, the method disclosed by the invention is more applicable to magnetically separating the bacteria in a complex matrix, and the separation efficiency of the 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 isolating food-borne pathogenic bacteria based on nano magnetic beads. Background technique [0002] Foodborne 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 food-borne diseases every year, and 2.2 million people in the world die every year due to food-borne diseases. In my country, the number of cases of food poisoning is between 200,000 and 400,000 per year, most of which are caused by food-borne pathogens except for accidents. Among them, Escherichia coli O157 ( Escherichia coli O157 , E. coli O157 ) as one of the main pathogenic bacteria, the poisoning incidents caused by it occur from time to time, and the harm is serious, mainly manifested in diarrhea, hemorrhagic enteritis, and secondary hemolytic uremic syndrome and thrombotic thr...

Claims

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Application Information

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IPC IPC(8): G01N1/34G01N1/40
Inventor 许恒毅赖卫华熊勇华魏华
Owner NANCHANG UNIV
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