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Enterobacter sakazakii phage coupled nano-magnetic beads, preparation method thereof and application

A technology of Enterobacter sakazakii and nano-magnetic beads, which is applied in the direction of material analysis, coating, and instruments through electromagnetic means, can solve the problems of unsuitable for routine inspection work of grassroots enterprises, unable to meet rapid quantitative detection, and high requirements for operators , to achieve the effect of easy large-scale cultivation, simple structure and good selectivity

Inactive Publication Date: 2019-01-18
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection method of Enterobacter sakazakii used at home and abroad is still based on the conventional culture method, and the detection cycle takes 5-7 days. The operation procedure is complicated, the required reagents are various, and it cannot meet the needs of rapid quantitative detection.
The rapid detection technology based on PCR technology is widely used in the detection of food-borne pathogenic bacteria. It has high detection sensitivity, but the required equipment is expensive, and the requirements for operators are high. It is not suitable for basic-level enterprises to carry out routine inspection work.
Zhu Yinglian et al. applied Enterobacter sakazakii polyclonal antibody-coupled magnetic beads as a reagent for the enrichment and rapid detection of Enterobacter sakazakii, and used it in combination with immunofluorescence probes to achieve rapid qualitative detection of Enterobacter sakazakii in milk, but polyclonal Antibody preparation involves animal feeding, which is costly, long-term, and not suitable for large-scale preparation

Method used

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  • Enterobacter sakazakii phage coupled nano-magnetic beads, preparation method thereof and application
  • Enterobacter sakazakii phage coupled nano-magnetic beads, preparation method thereof and application
  • Enterobacter sakazakii phage coupled nano-magnetic beads, preparation method thereof and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Nano-magnetic beads coupled with Enterobacter sakazakii phage and its preparation

[0031] 1. Phage amplification, purification and concentration

[0032] Take 500 μL of Enterobacter sakazakii phage EspYZU05 stock solution and mix it with 500 μL of Enterobacter sakazakii CICC 21569 bacterial solution in the logarithmic growth phase, leave it at room temperature for 10 minutes, then add it to 100 mL of LB liquid medium, and cultivate overnight at 25°C and 150rpm constant temperature shaking Transfer the culture in the test tube to a sterilized centrifuge tube, centrifuge at 5000×g for 10 min, collect the supernatant, filter and sterilize it through a 0.22 μm microporous membrane to obtain a phage EspYZU05 proliferation solution. Add DNase and RNase to the proliferation medium to a final concentration of 1 μg / mL, and place at room temperature for 30 minutes; add solid NaCl to a final concentration of 1 mol / L (5.84 g of solid NaCl per 100 mL of culture medium), s...

Embodiment 2

[0036] Example 2 Capture of Enterobacter sakazakii by phage-coupled magnetic beads and detection by electrochemical sensor

[0037] Enterobacter sakazakii CICC 21569 was inoculated into LB medium, cultured at 37°C for 12h, centrifuged at 5000rpm for 10min to collect the bacteria, washed 3 times with sterile PBS buffer, resuspended in PBS, and the concentration of bacteria was adjusted to OD 600 = 1.0 (10 8 CFU / mL), prepare live cell suspension and store at 4°C for later use. Take 100 μL of the suspension of Enterobacter sakazakii CICC 21569 and dilute it 10 times with PBS to prepare 10 times gradient samples, and do 3 parallels for each concentration; PBS without bacteria is used as a blank control. Take 500 μL of serially diluted Enterobacter sakazakii CICC 21569 suspension and blank control sample, mix with 10 μL of phage-coupled magnetic beads prepared according to step (2), incubate at 37°C for 10 minutes, and remove the supernatant after magnetic separation for 2 minutes ...

Embodiment 3

[0040] Example 3 Detection of Enterobacter sakazakii Phage Coupled Magnetic Beads Electrochemical Sensor in Simulated Samples

[0041] In order to verify the practical application of the phage-coupled magnetic bead sensor, the milk simulated to be contaminated with Enterobacter sakazakii CICC21569 was selected as a detection sample, 5% skim milk was prepared, and the concentration of Enterobacter sakazakii in the milk was adjusted to 1.08×10 3 CFU / mL-1.08×10 7 CFU / mL. Preparation of simulated sample steps: take 10 μL concentration of 10 7 CFU / mL Enterobacter sakazakii suspension and 10 μL concentration of 10 7 CFU / mL of Escherichia coli was mixed, and 990 μL of sterilized skim milk was added and mixed to make a simulated sample, and Enterobacter sakazakii or Escherichia coli were added alone or not added as positive and negative controls; the bacterial content was determined by traditional gradient dilution plate method In addition, according to the above steps, the phage-c...

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Abstract

The invention discloses enterobacter sakazakii phage coupled nano-magnetic beads, a preparation method thereof and application to enterobacter sakazakii content detection. Compared with the prior art,the enterobacter sakazakii phage coupled nano-magnetic beads, the preparation method and the application have the advantages that through adoption of enterobacter sakazakii specific phage coupled nano-magnetic beads, selective absorption and separation can be carried out on enterobacter sakazakii cells in samples, so low content enterobacter sakazakii polluted samples are enriched; current detection is carried out on complexes of the phage coupled nano-magnetic beads and bacterial cells through utilization of an electrochemical sensor, so strain content is computed; the method is rapid and simple; conventional enrichment culture is avoided in a whole process; detection time is reduced to 2-3 hours; and a basic food supervision department and a foot enterprise can determine enterobacter sakazakii pollution sources and ways timely.

Description

technical field [0001] The invention relates to a nanometer magnetic bead coupled with Enterobacter sakazakii phage, a preparation method and an application thereof, and belongs to the technical field of detection of Enterobacter sakazakii. Background technique [0002] Enterobacter sakazakii (Enterobacter sakazakii) is an important food-borne pathogenic bacteria that parasitizes in the intestinal tract of humans and animals. This bacteria mainly causes neonatal meningitis, enterocolitis and bacteremia, etc. The population is also at serious risk. The source and transmission route of Enterobacter sakazakii in nature are very complicated. Studies have shown that infant formula milk powder and dairy products are the main source of infection for newborns and susceptible people. Therefore, it is an important means to prevent and control Enterobacter sakazakii infection by establishing a rapid and reliable detection and traceability technology to monitor the dairy production env...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/569G01N27/30G01N27/327
CPCG01N27/308G01N27/3278G01N33/54326G01N33/54346G01N33/56916G01N2333/265G01N2446/90
Inventor 杨振泉杨晓珺高璐胡钦饶胜其
Owner YANGZHOU UNIV
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