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Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method

A lupin and allergen technology, applied in the fields of molecular biology and nucleic acid detection, can solve problems such as low equipment requirements

Inactive Publication Date: 2013-06-19
SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the detection and research of food allergen components in food, there are mainly protein-based methods (such as ELISA method) and gene-based methods (such as PCR method), both of which require high-end equipment and professional operation. personnel
At present, there is no reagent and method for the specific detection of food allergen lupine components with good detection effect, simple operation and low equipment requirements

Method used

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  • Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method
  • Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method
  • Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1. Preliminary screening and system optimization of food allergen lupine component-specific detection primers

[0106] Preliminary determination of 25 μL LAMP reaction system, its components include: 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3, 20 mM Tris-HCl (PH8.8), 10 mM KCl, 8 mM MgSO 4 , 10mM (NH 4 ) 2 SO 4 , 0.1%Tween20, 1M Betaine, 6mM MgSO 4 , 1.6mM dNTP, 8U Bst large fragment DNA polymerase, 2μl DNA to be tested. React at a constant temperature of 63°C for 90 minutes, then incubate at 80°C for 5 minutes to end the reaction, and preliminarily screen primers according to the peak time and the coincidence rate of negative and positive. 100% lupine standard was used as a template for the primary screening of primers, and negative DNA was used as a negative control. Experimental result shows, this primer begins to appear amplification in 30min ( figure 1 ).

[0107] Optimization of LAMP reaction conditions: selection of Mg 2+ Concentration o...

Embodiment 2

[0109] Example 2. Specific detection experiment of food allergen lupine component LAMP

[0110] Allergen primers were used to perform LAMP amplification on the 36 kinds of plant and animal materials tested. After the LAMP real-time turbidimeter detection, the yellow-flowered lupine and the white-flowered lupine showed obvious amplification curves, while the remaining 34 kinds of animal and plant samples There is no amplification curve in any of them, such as image 3 .

[0111] According to the real-time turbidity detection results of the specific detection of food allergen lupine component LAMP, the above-mentioned test materials were tested by chromogenic LAMP, and the results showed that the reaction liquid of yellow flower lupine and white flower lupine was green, and the DNA of other animal and plant materials The reaction solution is orange, such as Figure 4 .

[0112] The results of real-time turbidimeter LAMP detection and chromogenic LAMP detection showed that the...

Embodiment 3

[0113] Example 3. LAMP Detection Sensitivity of Allergen Lupine Components

[0114] Mix pure white lupine flour and wheat flour by weight to form a mixed sample with lupine content of 1%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001% and 0.0005%, and extract DNA for LAMP detection. Each concentration was repeated 3 times, and the results were recorded with a real-time turbidimeter. The results of real-time turbidity LAMP detection showed that 1%, 0.1%, 0.05%, 0.01%, 0.005%, and 0.001% lupine all had amplification curves, but 0.0005% lupine did not. As a result, the detection limit of this assay was 0.001%, as Figure 5 .

[0115] According to the real-time turbidity detection results of the sensitivity detection of food allergen lupine component LAMP, 0.1% lupine, 0.001% lupine, 0.0005% lupine and 0% lupine samples were detected by LAMP chromogenic method. The test results of LAMP colorimetric method showed that 0.1% lupine, 0.001% lupine had color reaction, and 0.0005% lupine had no ...

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Abstract

The invention relates to a food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method. The invention discloses a food allergen lupin component LAMP detection method for the first time. The invention uses lupin ITS1 gene as a target gene for identification, and designs LAMP amplification primers with favorable specificity. The method provided by the invention can be well used for identifying the food allergen lupin component, and has favorable repeatability and sensitivity.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and nucleic acid detection. Specifically, the present invention relates to a LAMP detection method and kit for food allergen lupine components in food. Background technique [0002] Food allergy is an adverse reaction of people to food, which is an allergic reaction of the body to foreign substances. At present, the internationally recognized eight major categories of allergic foods are: soybeans, peanuts, wheat, eggs, milk, fish, and nuts , Crustaceans. The general symptoms of food allergy include vomiting, abdominal pain, diarrhea, etc., as well as skin reactions and respiratory symptoms. In severe cases, systemic anaphylactic shock or death may even occur. There is no specific method for the treatment of food allergy, and strict avoidance of food containing allergen ingredients is the most effective therapy. Complete food taboos are difficult to achieve because food allergens may ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张舒亚欧阳可栋申恒于卓然
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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