Constant temperature amplification detection kit and method for detecting food allergen crustacean gene

A technology of constant temperature amplification detection and crustacean, which is applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of unsuitability for rapid detection and wide application, high price, false positives, etc., to meet the needs of high-throughput and low-throughput sample detection, fast response, and simple steps

Inactive Publication Date: 2010-12-01
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, the application of polymerase chain reaction (Polymerase Chain Reaction, PCR) technology in crustacean gene detection is currently the main method for the detection of crustacean food allergen genes. Food allergen gene detection kit, but its detection requires a large number of expensive instruments, especially not suitable for rapid on-site detection and wide application in grassroots units; and it is easy to cause false positives and other problems

Method used

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  • Constant temperature amplification detection kit and method for detecting food allergen crustacean gene
  • Constant temperature amplification detection kit and method for detecting food allergen crustacean gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Composition and preparation of the constant temperature amplification detection kit for food allergen crustacean nucleic acid

[0037] 1. Kit composition:

[0038] a) DNA extraction reagent (lysate): 10mmol / L Tris-HCl (pH8.0), which contains chelex resin with a mass concentration of 1%;

[0039] b) Reaction solution: two peripheral primers (0.05 μmol), two probes (0.5 μmol), and one cross primer (0.5 μmol), 1×Thermol buffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol), Bst DNA polymerase (10U) and sterile double distilled water.

[0040] The two peripheral primers include:

[0041] The forward peripheral primer sequence is 5'-GATTAAGTTACTTTAG-3',

[0042] The reverse peripheral primer sequence was 5'-GATTTAAAGGTCGAACAG-3'.

[0043] Cross primer: 5'-TTCAACATCGAGGTCGCTTTAGGGATAACAGCGT-3'.

[0044] The sequences of the two probes are "5'-TTCAACATCGAGGTCGC-3'" and "5'-TGTCGATATGAACTCTC-3'" respectively, one of which is labeled with biotin at the 5' end and the o...

Embodiment 2

[0050] Embodiment 2 uses kit of the present invention to detect the specific method of food allergen crustacean nucleic acid

[0051]a) Use DNA lysate to extract DNA from the specimen to be tested: add 40 μL of DNA extraction reagent to the specimen test tube, then add 0.5-0.1 g of shrimp chip specimen to the specimen test tube, directly in a boiling water bath for 10 minutes, and centrifuge at 10,000 g for 5 minutes. The supernatant is sample DNA.

[0052] b) Take the sample DNA as a template and add it to the PCR tube containing the reaction solution, and carry out the amplification reaction at 58°C for 80 minutes, including 4 μl of sample DNA and 16 μl of the reaction solution; add the positive control template and negative control template respectively to the control PCR tube.

[0053] c) Put the reacted PCR tube into the nucleic acid anti-pollution detection device for detection, and interpret the result after 15 minutes. When the sample contains food allergen crustacean...

Embodiment 3

[0055] Embodiment 3 detects the specificity of food allergen crustacean nucleic acid with kit of the present invention

[0056] According to the method of Example 2, it is detected whether hairtail, jumping fish, water toad, herring, grass carp, lobster, small shrimp, clam, rice shrimp, blue crab, river prawn, giant giant rosenbergii, prawn, and river crab contain food allergens of crustaceans nucleic acid. The results are as follows:

[0057] serial number

name

Test results

1

Hairtail

-

2

jumping fish

-

3

water toad

-

4

herring

-

5

grass carp

-

6

lobster

+

7

Shrimp

-

8

clams

+

9

rice shrimp

+

10

blue crab

-

11

river prawns

+

12

Macrobrachium rosenbergii

+

13

prawns

+

14

river crab

+

[0058] Note: "-" means negative, ...

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Abstract

The invention provides a constant temperature amplification detection kit and a method for detecting a food allergen crustacean gene. The kit comprises DNA lysis solution and constant temperature amplification reaction liquid, wherein the constant temperature amplification reaction liquid comprises a forward peripheral primer, a backward peripheral primer, two probes, a cross-species amplification primer, 1*Thermol buffer, MgSO4, dNTPs, Bst DNA polymerase and sterile double-distilled water. The invention also provides the method for detecting the food allergen crustacean gene by using the kit. The method comprises the steps of extracting DNA from a sample to be detected with the DNA lysis solution, performing amplification reaction and detecting. The method for detecting the food allergen crustacean gene has the advantages of accuracy, flexibility and capability of rapidly detecting the food allergen crustacean gene and is suitable for field rapid detection.

Description

technical field [0001] The invention relates to a rapid detection technology of food allergen crustacean gene, which is suitable for qualitative detection of food allergen crustacean gene components. Background technique [0002] Food allergy is an abnormal reaction of the body to the immune system caused by food. It is mostly caused by the body's overreaction to certain foreign food ingredients or the lack of digestion ability of certain proteins and certain food ingredients. Common food allergies are associated with immunoglobulin E; allergens are certain proteins. There is currently no good treatment for food allergies. Common allergic foods include: crustaceans, peanuts, tree nuts, eggs, milk, wheat products, soy products, etc. Among them, crustacean seafood is one of the most serious allergens. [0003] In recent years, the application of polymerase chain reaction (Polymerase Chain Reaction, PCR) technology in crustacean gene detection is currently the main method for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 祝长青陈颖胡林石建华徐高连蒋原吴斌张睿沈崇钰袁飞邵景东吴亚君尤其敏杨海荣
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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