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Kit for detecting Listeria monocytogenes based on immune double-labeled colloidal gold probe and hybrid chain amplification, application and detection method

A technology of Listeria monocytogenes and hybridization chain is applied in the field of detection of Listeria monocytogenes based on double-labeled immunocolloidal gold probes and hybridization chain amplification, which can solve the problems such as the detection of Listeria monocytogenes that have not yet been found. , to achieve the effect of short detection time, good specificity and high stability

Active Publication Date: 2019-12-13
天津一安生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no method has been found to detect Listeria monocytogenes based on immunological double-labeled colloidal gold probes and hybridization chain amplification

Method used

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  • Kit for detecting Listeria monocytogenes based on immune double-labeled colloidal gold probe and hybrid chain amplification, application and detection method
  • Kit for detecting Listeria monocytogenes based on immune double-labeled colloidal gold probe and hybrid chain amplification, application and detection method
  • Kit for detecting Listeria monocytogenes based on immune double-labeled colloidal gold probe and hybrid chain amplification, application and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example is used to illustrate the preparation method of the double-labeled colloidal gold probe and hybridization chain amplification detection kit for Listeria monocytogenes.

[0058] 1) Preparation of pre-coated microwell plates

[0059] The microwell plate is made of black polystyrene and forms a detachable module with the bottom film of the 96-well plate, using carbonate buffer (1.59g Na 2 CO 3 +2.93g Na 2 CO 3 , pH 9.6 to 1L) Dilute the anti-Listeria monocytogenes monoclonal antibody 1000 times, mix well, add 100 μL monoclonal antibody diluent to each well, and place the microwell plate in an environment of 37°C to incubate the coating Incubate overnight at 2h or 4°C. After the coating is taken out, inject 200 μL of PBST washing solution (0.01M PBS+0.05% Tween 20v / v%) into each well, let it stand for 3 minutes, tap or blot the liquid in the well at one time, and repeat the washing step 3 times. Finally, pack it in a tin foil bag to evacuate it and store i...

Embodiment 2

[0071] This example is used to illustrate the detection method of the present invention. In the standard recovery experiment, different concentrations of Listeria monocytogenes were added to the sterilized milk and sterilized beef extract solution to simulate samples contaminated by Listeria monocytogenes, and the concentration of the target substance in the sample was determined.

[0072] Using the double-labeled colloidal gold probe prepared in Example 1 and the hybridization chain amplification detection kit for Listeria monocytogenes, the specific operations are as follows:

[0073] (1) Take out the kit from the refrigerator at 4°C, and equilibrate at room temperature for 10 minutes.

[0074] (2) Add different concentrations of Listeria monocytogenes standard substance (10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL,) Negative control group (PBS solution) and 50 μL of the sample to be tested after the pretreatment was completed, and 50 μL of double-la...

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Abstract

The invention belongs to the technical field of food safety detection immunoassay, and relates to a kit for detecting Listeria monocytogenes based on an immune double-labeled colloidal gold probe andhybrid chain amplification, application and a detection method. The kit comprises: (1) a microporous strip coated with an anti-Listeria monocytogenes monoclonal antibody; (2) a double-labeled colloidal gold probe, which is obtained by coating a sulfydryl-labeled single-stranded amplification initiation probe and an anti-Listeria monocytogenes polyclonal antibody with colloidal gold; (3) a DNA amplification probe; (4) a reaction buffer solution, which is a trihydroxyaminomethane hydrochloride buffer solution containing streptavidin labeled horse radish peroxidase; (5) a Listeria monocytogenes standard substance; (6) a micropore washing solution; and (7) a chemiluminescent substrate solution. Compared with a competitive detection method, the kit disclosed by the invention has the advantagesof higher sensitivity, wider detection linear range and lower detection cost by adopting a method of sandwiching a target object with the monoclonal antibody and the polyclonal antibody.

Description

technical field [0001] The invention belongs to the technical field of immunoassay for food safety detection, and more specifically relates to a kit and application for detecting Listeria monocytogenes based on immunological double-labeled colloidal gold probes and hybridization chain amplification and an immunoassay based on double-labeled A method for detecting Listeria monocytogenes with colloidal gold probes and hybridization chain amplification. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes) is a food-borne pathogenic bacteria with strong lethal ability, with a 25%-30% fatality rate among infected persons. Listeria monocytogenes widely exists in the natural environment, poultry and meat products, and 90% of the infection cases are caused by eating drinking water or food contaminated by Listeria monocytogenes. Due to its wide distribution and high pathogenicity, pose a serious threat to public health. Therefore, the development of simple,...

Claims

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Application Information

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IPC IPC(8): C12Q1/682C12Q1/6804C12Q1/689C12Q1/06C12R1/01
CPCC12Q1/682C12Q1/6804C12Q1/689C12Q2525/301C12Q2545/113C12Q2563/103
Inventor 范龙兴苑帅朱昱霖
Owner 天津一安生物技术有限公司
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