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Method for enriching staphylococcus aureus by magnetic beads

A staphylococcus and golden yellow technology, applied in the biological field, can solve the problems of poor environmental tolerance and expensive antibody cost, and achieve the effect of high load capacity, low cost and controllable quality

Pending Publication Date: 2022-07-22
南昌大学第一附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the widely used technology is immunomagnetic separation technology, but the antibody used in immunomagnetic separation is relatively expensive and has poor environmental tolerance

Method used

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  • Method for enriching staphylococcus aureus by magnetic beads
  • Method for enriching staphylococcus aureus by magnetic beads
  • Method for enriching staphylococcus aureus by magnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. A polyoxyethylene diamine-mediated cefepime functionalized magnetic nanoparticle complex is prepared according to the following steps:

[0048] (1) Draw 1 mL of magnetic nanoparticles (10 mg / mL) with a particle size of 180 nm, add it to 9 mL of sterile PBS solution with pH 7.4, and then wash the magnetic nanoparticles under the action of an external magnetic field, repeating 3 times , the washed magnetic nanoparticles were resuspended in 9 mL of sterile PBS solution to obtain a magnetic nanoparticle solution.

[0049] (2) Weigh 2.9 mg of EDC and dissolve it in 290 μL of sterile PBS solution, weigh 3.25 mg of NHSS and dissolve it in 325 μL of sterile PBS solution, then add the dissolved EDC and NHSS to the magnetic nanoparticle solution, and then add 9 mL of sterile PBS solution was added, and activated at 25° C. for 1 h to obtain activated magnetic nanoparticles.

[0050] (3) The activated magnetic nanoparticles were washed three times with sterile PBS solution, and...

Embodiment 2

[0057] Enrichment effect experiment

[0058] (1) Take 1mL concentration as 10 4 CFU / mL of Staphylococcus aureus was transferred to a 1.5 mL sterile centrifuge tube, centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and resuspended with an equal volume of sterile PBS solution.

[0059] (2) Experimental grouping: magnetic nanoparticles, cefepime functionalized magnetic nanoparticles, and cefepime functionalized magnetic nanoparticle complexes mediated by polyoxyethylene diamine were used to enrich and capture the target bacteria.

[0060] The scheme of enrichment and capture of target bacteria in each group is as follows:

[0061] a. The polyoxyethylenediamine-mediated cefepime functionalized magnetic nanoparticle complex group (the preparation method is the same as that of Example 1) enrichment and capture of the target bacteria scheme is as follows:

[0062] Add 50 μg of polyethylene glycol diamine-mediated cefepime functionalized magnetic nanoparticle comp...

Embodiment 3

[0080] Cut the center piece of beef freshly purchased from the supermarket in a sterile manner, confirm its sterility through selective medium, and then prepare it according to the method in GB 4789.10-2016 National Food Safety Standard for Food Microbiological Inspection Staphylococcus aureus Inspection. into the sample solution to be tested, add Staphylococcus aureus to adjust the colony concentration to 10 4 CFU / mL for use.

[0081] The prepared polyoxyethylene diamine-mediated cefepime functionalized magnetic nanoparticle complex (50 μg) was added to the sample solution, placed on a mixer, and incubated at 37° C. for 30 min at a speed of 180 rpm. Placed on a 0.4T magnetic stand for magnetic separation for 6min. The supernatant was poured into a sterile centrifuge tube, and the separated captured Staphylococcus aureus-cefepime-polyoxyethylenediamine-magnetic nanoparticle complexes were washed twice with sterile PBS solution, and washed with 1 mL of Resuspend in PBS soluti...

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Abstract

The invention discloses a method for enriching staphylococcus aureus by magnetic beads, and belongs to the technical field of biology. Comprising the following steps: washing and resuspending magnetic nanoparticles; activating the magnetic nanoparticles to obtain activated magnetic nanoparticles; carrying out reaction on polyoxyethylene diamine and the activated magnetic nanoparticles to obtain a polyoxyethylene diamine modified magnetic nanoparticle compound; the preparation method comprises the following steps: reacting glutaraldehyde with a polyoxyethylene diamine modified magnetic nanoparticle compound to obtain a substance A; the method comprises the following steps: reacting cefepime with a substance A to obtain a polyoxyethylene diamine mediated cefepime functionalized magnetic nanoparticle compound; and enriching and capturing. According to the present invention, the polyoxyethylene diamine is adopted as the mediator so as to reduce the steric hindrance, the cefepime is adopted as the recognition element so as to improve the binding force with bacteria, and the nano-scale magnetic beads with the large specific surface area are adopted to improve the loading capacity of the recognition element so as to improve the bacteria capture efficiency. Compared with the traditional method, the method has the advantages of short incubation time, less material consumption, low cost and wide adaptability.

Description

technical field [0001] The invention relates to the field of biotechnology, and more particularly to a method for enriching Staphylococcus aureus with magnetic beads. Background technique [0002] Staphylococcus aureus is a recognized food-borne pathogen that contaminates a wide variety of foods and is prevalent in milk, raw meat, meat products, and cakes. Enterotoxins produced by Staphylococcus aureus can cause symptoms of food poisoning in humans, such as acute vomiting, severe diarrhea, and gastroenteritis. However, the composition of food matrix is ​​complex, which is easy to cause great interference to subsequent detection methods and reduce the detection limit. Therefore, a rapid, efficient and sensitive method for rapid screening of Staphylococcus aureus in contaminated food must be established. [0003] In view of the need to establish a fast, efficient and sensitive detection method, the separation technology based on functionalized magnetic nanoparticles has been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/24C12R1/445
CPCC12Q1/24G01N2333/31
Inventor 刘洋许恒毅黄瑾肖芳斌白雪锟孔蕴源魏丹丹孙珑彦苏康太张伟
Owner 南昌大学第一附属医院
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