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

An allergen, wheat technology, applied in the field of molecular biology and nucleic acid detection, can solve the problem of 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

[0004] 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 operators
At present, there is no reagent and method for specific detection of food allergen wheat components with good detection effect, simple operation and low equipment requirements

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1. Preliminary screening of primers for specific detection of food allergen wheat components and system optimization

[0107] 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, 0.8 μM each of LF and LB, 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. After mixing other reagents except the template into a mixed solution, divide 23 μL into each tube, add 20 μL of sealing solution, and then add 2 μL of template DNA or negative and positive control templates, mix well and centrifuge, then spot 1 μL of chromogenic solution on the tube In the middle of the cap, carefully close the tube cap tightly to prevent the chromogenic solution from falling into the reaction solution. The mixture was placed in the reaction well, and reacted at a constant tempera...

Embodiment 2

[0110] Example 2. Specific detection experiment of food allergen wheat component LAMP

[0111] Allergen primers were used to perform LAMP amplification on 36 kinds of plant and animal materials tested. After the LAMP real-time turbidimeter test, wheat showed an obvious amplification curve, while no amplification appeared in the remaining 35 animal and plant samples. curves, such as image 3 .

[0112] According to the real-time turbidity detection results of the specific detection of food allergen wheat component LAMP, the above-mentioned test materials were tested by chromogenic LAMP, and the results showed that the reaction liquid of wheat was green, and the DNA reaction liquid of other animal and plant materials was orange, such as Figure 4 .

[0113] The results of real-time turbidity LAMP detection and chromogenic LAMP detection showed that the LAMP primers and reaction system were species-specific.

Embodiment 3

[0114] Example 3, LAMP detection sensitivity of allergen wheat components

[0115] Mix pure wheat flour and corn flour by weight to form mixed samples with wheat content of 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% and 0.001%, and extract DNA for LAMP detection. Each concentration Repeat 3 times and record the results with a real-time turbidimeter. The real-time turbidity LAMP test results show that 1%, 0.5%, 0.1%, 0.05%, 0.01% wheat all have amplification curves, 0.005% wheat and 0.001% wheat have no amplification curves, and the detection limit of this detection method is 0.01 %,Such as Figure 5 .

[0116] According to the real-time turbidity detection results of the sensitivity detection of food allergen wheat component LAMP, 0.05% wheat, 0.01% wheat, 0.005% wheat and 0% wheat samples were detected by LAMP chromogenic method. The test results of LAMP colorimetric method show that 0.05% wheat, 0.01% wheat have color reaction, and 0.005% wheat has no color reaction. The detect...

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Abstract

The invention relates to a food allergen wheat component LAMP (loop-mediated isothermal amplification) field quick detection method. The invention discloses a food allergen wheat component LAMP detection method for the first time. The invention uses wheat GAG56D 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 wheat 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 wheat components in food or feed. 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 be hidd...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张舒亚欧阳可栋宋青李富威
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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