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Method for detecting escherichia coli based on visual BPE-ECL technology

A technology of Escherichia coli, technical detection, applied in the field of biosensing, to achieve the effect of reducing detection cost, low cost and strong specificity

Active Publication Date: 2021-06-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report showing that E. coli in food can be detected directly by using the characteristics of the strain itself without the help of other chemical reactions.

Method used

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  • Method for detecting escherichia coli based on visual BPE-ECL technology
  • Method for detecting escherichia coli based on visual BPE-ECL technology
  • Method for detecting escherichia coli based on visual BPE-ECL technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Cultivation of Escherichia coli O157:H7

[0058] 1. Take out the strain Escherichia coli O157:H7 preserved in 50% (v / v) glycerol from the -80°C refrigerator. After thawing, inoculate 10 mL of LB (Luria-Bertani) liquid medium with 1% inoculum , cultivated in a constant temperature shaker at 37°C and 160rpm for 18-24h;

[0059] 2. Transfer the bacterial solution obtained above into 10mL LB liquid medium according to the same ratio, and culture at 37°C and 160rpm for 2.5h;

[0060] 3. Take 1mL of the bacterial liquid into a 1.5mL centrifuge tube, use a refrigerated centrifuge at 4°C, 8000rpm, centrifuge for 10min, and discard the supernatant to obtain bacterial sediment;

[0061] 4. Add 1 mL of sterile normal saline to resuspend the cells, centrifuge at 4°C and 8000 rpm for 10 minutes to wash away the residual culture medium, add 1 mL of sterile normal saline (0.85% NaCl) to resuspend the cells; repeat this operation for 2 Second-rate;

[0062] 5. Gradiently ...

Embodiment 2

[0063] The preparation of embodiment 2 screen printing bipolar electrodes

[0064]Firstly, polyethylene terephthalate (PET) cheap electrically inert material is selected as the substrate, and then two working electrode leads are printed with silver ink at both ends of the substrate; The carbon electrode is printed with carbon paste and dried as a bipolar electrode; then the electrode specification layer is printed with photo-curable insulating paste and cured with ultraviolet light; finally, the electrode insulating layer is printed with photo-solid insulating paste and cured with ultraviolet light. The screen-printed electrodes are about 3.4 cm long and 1 cm wide, and the bipolar electrode wires are about 12 mm long. Such as figure 2 shown.

Embodiment 3

[0065] Example 3: Electrochemiluminescent Stability Measurement of Screen-Printed Dual-Electrode Anode Luminescent Agent

[0066] 10 μL of electrochemiluminescence reagent and co-reactant (50 mM Ru(bpy) 3 Cl 2 ·6H 2 O, 250mM TPA, ready to use), drop 10 μL IMBs-E.coliO157:H7 on the cathode (wherein, the concentration of enriched E.coli O157:H7 is 10 4 ), connected to an external electrochemical workstation, applied a constant voltage of 5.5V, coupled with a fluorometer to scan at a constant wavelength of 620nm for 70s, and quantitatively detected the signal value of the anode luminescent agent, such as image 3 As shown, the luminescence value of IMBs-E.coliO157:H7 reached the peak at the beginning, gradually decreased within 25s and slowly reached a stable level, and then stabilized, so the ECL measurement was performed when the luminescence was selected for 30s.

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Abstract

The invention discloses a kit based on immunomagnetic bead separation, and application of the kit. The invention further discloses a method for detecting the concentration of escherichia coli based on a visual BPE-ECL technology. The invention further discloses application of the kit and the detection method in detection of the concentration of escherichia coli in food. According to the established method for detecting the concentration of the escherichia coli through the visual BPE-ECL technology, whether the escherichia coli exists in a sample to be detected or not can be observed through naked eyes, the method has the advantages of being simple in principle, rapid (the experimental period is 45 min), high in specificity and high in detection sensitivity (the detection limit is 101 CFU / mL), the method is easy and convenient to operate, actual liquid samples (milk and tap water) do not need to be preprocssed, and a solid actual sample (minced beef) is simply preprocessed according to the national standard; and meanwhile, biochemical modification does not need to be carried out on the surface of a bipolar electrode, and the electrode can be repeatedly used, so the detection cost can be effectively reduced.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and in particular relates to a method for detecting Escherichia coli based on the visualized BPE-ECL technology, in particular to a method for detecting the concentration of Escherichia coli O157:H7 based on the visualized BPE-ECL technology. Background technique [0002] Food-borne pathogens refer to pathogenic bacteria introduced during food processing and distribution. These pathogenic bacteria survive in food, grow and metabolize and cause food deterioration and destruction. At the same time, some pathogenic bacteria secrete toxic substances, which directly or indirectly cause human illness. , common foodborne pathogens include pathogenic Escherichia coli (especially hemorrhagic Escherichia coli O157: H7), Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter sakazakii , Salmonella, etc. The harm of food-borne pathogens does not decreas...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N21/64G01N27/12G01N33/569
CPCG01N21/76G01N21/6486G01N27/12G01N33/56916G01N2333/245
Inventor 刘元建王敏惠熊晓辉
Owner NANJING UNIV OF TECH
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