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Rapid detection of Listeria monocytogenes through combined nanometer immunomagnetic bead technology-colloidal gold chromatography

A technology of Listeria monocytogenes and immune magnetic beads, which is applied in measuring devices, analytical materials, instruments, etc., can solve the problems of complex operation process, low sensitivity, time-consuming and labor-intensive biological detection methods, and achieve high sensitivity and less time-consuming , The effect of low detection cost

Active Publication Date: 2015-12-02
BEIJING UNIV OF AGRI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention mainly aims at the defects of time-consuming and labor-intensive, complicated operation process and low sensitivity of existing biological detection methods, and provides a test strip for rapid detection of Listeria monocytogenes by combining nano-immunomagnetic bead separation technology with colloidal gold immunochromatography method of preparation

Method used

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  • Rapid detection of Listeria monocytogenes through combined nanometer immunomagnetic bead technology-colloidal gold chromatography
  • Rapid detection of Listeria monocytogenes through combined nanometer immunomagnetic bead technology-colloidal gold chromatography
  • Rapid detection of Listeria monocytogenes through combined nanometer immunomagnetic bead technology-colloidal gold chromatography

Examples

Experimental program
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Embodiment example 1

[0029] Implementation Case 1 Preparation of Immunomagnetic Beads and Capture of Listeria Monocytogenes Detection

[0030] 1. Preparation of nano-immunomagnetic beads

[0031] (1) Antibody preparation and purification: 8-week-old healthy BALB / c female mice were immunized with autoclaved Listeria monocytogenes as an antigen, and monoclonal antibodies were prepared and screened by conventional hybridoma technology and limiting dilution method Cell lines, and then the specific antibody cell lines against Listeria monocytogenes were propagated and cultured, and injected into mice to prepare ascites, and the obtained ascites was purified by octanoic acid-ammonium sulfate precipitation; Listeria monocytogenes was treated with high pressure sterilization The bacterium was used as an antigen to immunize New Zealand white rabbits, and the obtained anti-Listeria monocytogenes serum was purified by ammonium sulfate precipitation, and the obtained polyclonal antibody was stored at -80 de...

Embodiment example 2

[0041] Implementation Case 2 Preparation of Colloidal Gold Immunochromatography Test Strip and Sample Detection

[0042] 1. Preparation of colloidal gold test strips

[0043] (1) Preparation of colloidal gold: Take 100mL of 0.01% HAuCl4 aqueous solution in a beaker, add 2mL of 1% trisodium citrate when heating and boiling, continue heating and boiling for 5min, add ultrapure water to 100mL after cooling, and prepare 40nm colloidal gold solution;

[0044] (2) Treatment of the conjugation pad: Label another anti-Listeria monoclonal antibody 3B10 prepared by pairing with the magnetic bead-conjugated monoclonal antibody 1B12 on the colloidal gold particles, so that the marking liquid is sprayed on the Combined with pads for later use;

[0045] (3) Coating of chromatographic membrane: use an automatic spraying machine to spray specific concentrations of polyantiserum and goat anti-mouse IgG on the T line and C line of the chromatographic membrane at a speed of 0.9 μL / cm When s...

Embodiment example 3

[0049] Implementation Case 3 Detection of Cultured Samples

[0050] Dilute the LM bacteria solution cultured for 12 hours to 0, 10, 50, 10 2 ,10 3 ,10 4 ,10 5 CFU / ml, 1 mL each was added to a centrifuge tube, and 0.1 mg of self-made immunomagnetic beads were added to each tube. At room temperature, incubate on a rotary mixer for 30 minutes, resuspend in 1 mL of LPB after magnetic separation, bathe in 85°C water for 15 minutes, suck out the supernatant after magnetic separation, and add 100 μL of the supernatant and the control bacteria solution to the test strips for sampling In the hole, observe the color development of the detection line and quality control line of the test strip for 5-15 minutes.

[0051] Such as Figure 4 As shown, after immunomagnetic bead separation and enrichment reaction, the concentration was 10 3 CFU / mL and 10 2 The test result of the CFU / mL bacterial solution was strongly positive, and the test result was weakly positive at a concentration ...

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Abstract

The invention provides a method for rapid detection of Listeria monocytogenes through combined nanometer immunomagnetic bead technology-colloidal gold chromatography. The method comprises the following steps: labeling a monoclonal antibody 1B12 by using a streptavidin-biotin mediated and coupled nanometer magnetic bead so as to prepare an immunomagnetic bead wherein the monoclonal antibody 1B12 has an accession number of CGMCC No. 10312 and is prepared and screened by using hybridoma technique; labeling another matching monoclonal antibody 3B10 with an accession number of CGMCC No. 10311 by using colloidal gold so as to obtain a labeled antibody, respectively using polyvalent antiserum and goat anti-mouse IgG as a detection line T and a quality control line C and constructing a colloidal gold immunochromatographic test strip from a conjugation pad of the colloidal gold-labeled antibody, a chromatographic membrane of the detection line T and the quality control line C and a water absorption pad; and in detection of a sample, subjecting the immunomagnetic bead and bacteria in the sample to specific capture and enrichment, then pouring an enrichment liquid onto a sample pad of the test strip and judging and reading chromogenic strips visible to naked eyes formed at the detection line T and the quality control line C according to the color of colloidal gold so as to realize rapid semi-quantitative detection of Listeria monocytogenes.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and specifically discloses a method for rapidly detecting Listeria monocytogenes by nano-immunomagnetic bead technology combined with colloidal gold chromatography, which is suitable for the detection of the pathogenic Listeria monocytogenes in food. Background technique [0002] Listeria monocytogenes ( Listeria monocytogenes , referred to as Listeria monocytogenes), is a zoonotic pathogen and one of the common food-borne pathogens, widely present in meat, eggs, seafood, dairy products, vegetables and other foods , the bacterium can still grow at 4°C, and there have been many incidents of Listeria monocytogenes contamination and poisoning. It can cause clinical symptoms such as meningitis, gastroenteritis, septicemia, and miscarriage, especially for newborns, pregnant women, and the elderly. It is very harmful to people with low immunity and has a high fatality rate, which become...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 张红星齐颖颖段慧霞谢远红刘慧张帅
Owner BEIJING UNIV OF AGRI
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