Novel method for separation of Gram-negative pathogenic bacteria in sepsis

A gram-negative, separation method technology, applied in the field of gram-negative pathogenic bacteria separation based on magnetic nanoparticles, can solve the problems of long cycle time, manpower and material resources, etc., and achieve the effect of low cost, long shelf life and controllable quality

Inactive Publication Date: 2017-07-04
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method not only has a long period, but also consumes a lot of manpower and material resources.

Method used

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  • Novel method for separation of Gram-negative pathogenic bacteria in sepsis
  • Novel method for separation of Gram-negative pathogenic bacteria in sepsis
  • Novel method for separation of Gram-negative pathogenic bacteria in sepsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Concanavalin A-magnetic nanoparticle complex is synthesized, prepared according to the following steps:

[0028](1) Draw 2mL of magnetic nanoparticles (10mg / mL), add them to 8mL of sterile PBS solution with pH=7.4, and then wash the magnetic nanoparticles under the action of an external magnetic field, repeat 3 times, and wash The final magnetic nanoparticles were resuspended in 10 mL sterile PBS solution; (2) 5.8 mg EDC was dissolved in 290 μL sterile PBS, 6.5 mg NHSS was dissolved in 325 μL sterile PBS, and then the dissolved EDC and NHSS were added to the washed magnetic nanoparticles solution and activated for 1 h; (3) the activated magnetic nanoparticles were washed 3 times with sterile PBS and resuspended in 8 mL of sterile PBS solution; (4) weighed Take streptavidin 0.5 mg, dissolve it in 100 μL sterilized PBS solution, then add it into the activated magnetic bead solution, react for 2 hours, then wash 3 times with sterilized PBS, resuspend in 8 mL sterilized ...

Embodiment 2

[0031] Embodiment 2 enrichment effect experiment

[0032] (1) Take 1mL concentration as 10 5 CFU / mL Gram-negative pathogenic bacteria were placed in a 1.5mL sterile centrifuge tube, centrifuged at 12000rpm for 5min, discarded the supernatant, and resuspended with an equal volume of sterile PBS solution.

[0033] (2) Enrichment and capture: Take 10 mL of the sample solution to be tested, add 1 mg of concanavalin A-magnetic nanoparticle complex, place it on a mixer, and incubate at room temperature for 45 minutes at a speed of 200 rpm to form a Gram-negative pathogen-Concanavalin Protein A-magnetic nanoparticle complex; insert the centrifuge tube into a conventional magnetic stand for separation for 6 minutes;

[0034] (3) After magnetic separation, the supernatant was transferred to a sterile centrifuge tube, and the isolated Gram-negative pathogen-concanavalin A-magnetic nanoparticle complex was washed twice with PBS, mixed evenly, and Resuspend the Gram-negative pathogen-co...

Embodiment 3

[0040] The sample to be tested is sterile lysed blood, and Gram-negative pathogenic bacteria are added to adjust the colony concentration to 10 5 CFU / mL for use.

[0041] The prepared concanavalin A-magnetic nanoparticle complex (1 mg) was added to the sample solution (10 mL), placed on a mixer, and incubated at room temperature for 45 min at a speed of 200 rpm. Then the conventional magnetic stand was separated for 6 minutes. After magnetic separation, the supernatant was transferred into a sterile centrifuge tube, and the isolated Gram-negative pathogen-concanavalin A-magnetic nanoparticle complex was washed twice with sterile PBS, mixed evenly, and washed with 1 mL Resuspend the Gram-negative pathogen-concanavalin A-magnetic nanoparticle complex in sterile PBS solution. Capture efficiency is obtained as in Example 2, and the rest are the same as in Example 2. The results are shown in Table 1, indicating that this protocol can efficiently enrich the Gram-negative pathogen...

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Abstract

The invention discloses separation of Gram-negative pathogenic bacteria. The method provides basis for subsequent research on the Gram-negative pathogenic bacteria and relates to the technical field of biology. The method includes the steps of: 1) conjugating magnetic nano-particles to streptavidin; 2) conjugating concanavalin A to long-chain biotin; 3) conjugating the biotinylated concanavalin A to the magnetic nano-particles coated with the streptavidin; 4) capturing the Gram-negative pathogenic bacteria in a sample solution by means of the streptavidin and long-chain biotin induced concanavalin A combined magnetic nano-particle composition; and 5) under effect of an external magnetic field, separating the captured Gram-negative pathogenic bacteria from the sample solution and performing re-suspension and the like. The Gram-negative pathogenic bacteria, captured through the magnetic separation, can be directly subjected to subsequent analysis. Compared with a conventional magnetic separation method for bacteria, the method can be used for magnetically separating the Gram-negative pathogenic bacteria from food substrates and blood. The method not only improves separation efficiency of the Gram-negative pathogenic bacteria in the sample solution but also reduces the cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating Gram-negative pathogenic bacteria based on magnetic nanoparticles. Background technique [0002] Sepsis is a serious systemic infectious disease with complex and changeable conditions and high mortality. Pathogens in the blood of patients with sepsis mainly include Gram-negative pathogens and Gram-negative pathogens. In the course of treatment, Gram-negative pathogenic bacteria and Gram-negative pathogenic bacteria have great differences in cell wall structure, and there are certain differences in the selection of drugs. The traditional method to identify pathogenic bacteria in the blood of patients with sepsis is mainly the blood plate culture method, but this method takes a long period of time and takes about 5-7 days, which may further aggravate the patient's condition during this process. In addition, if the pathogenic bacteria in the blood are not classi...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N1/02C12N1/20
CPCC12N1/02C12N1/20C12N13/00
Inventor 许恒毅杨国泰刘洋孔蕴源孟祥玉徐天雄黄楠
Owner NANCHANG UNIV
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