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Primer and probe for detecting fish anaphylactogen in food

An allergen and fish technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as high price and limited fish species to be detected

Inactive Publication Date: 2008-08-27
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are commercialized fish allergen PCR detection kits in foreign countries, but the price is expensive, and the fish species to be detected are limited. The present invention can make up for the above defects and complete the detection of more fish allergen proteins from different sources in food

Method used

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  • Primer and probe for detecting fish anaphylactogen in food
  • Primer and probe for detecting fish anaphylactogen in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Follow the procedure below for testing:

[0033] (1) Extraction of DNA from fish ball samples to be tested

[0034] A. Weigh 0.5g fish balls, cut them into pieces, and transfer them to a 1.5mL centrifuge tube. Add 500 μL extraction buffer lysate (0.05M Tris-HCL (trishydroxymethylaminomethane-hydrochloric acid), 0.1M EDTA (ethylenediaminetetraacetic acid), 0.2M sodium chloride, pH 7.6 sterilized double distilled water) , full oscillation;

[0035] B. Add 60 μL of proteinase K and 2 μL of 10% SDS, mix by inversion. Water bath at 57°C for 10 hours until the raw material is digested into a clear liquid, centrifuged at 4000 rpm for 2 minutes, and the supernatant is taken;

[0036] C. Add 500 μL equal volume of Tris-phenol, mix by inversion for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant;

[0037] D. Repeat the previous step;

[0038] E. Add Tris-phenol, chloroform and isoamyl alcohol (volume ratio is 25:24:1) to the supernatant, mix it u...

Embodiment 2

[0053] Follow the procedure below for testing:

[0054] (1) Extraction of DNA from fish floss samples to be tested

[0055] A. Weigh 0.5g fish floss, cut it into pieces, and transfer it to a 1.5mL centrifuge tube. Add 500 μL extraction buffer lysate (0.05M Tris-HCL (trishydroxymethylaminomethane-hydrochloric acid), 0.1M EDTA (ethylenediaminetetraacetic acid), 0.2M sodium chloride, pH 7.6 sterile double distilled water) , full oscillation;

[0056] B. Add 60 μL of proteinase K and 2 μL of 10% SDS, mix by inversion. Water bath at 57°C for 10 hours until the raw material is digested into a clear liquid, centrifuged at 4000 rpm for 2 minutes, and the supernatant is taken;

[0057] C. Add 500 μL equal volume of Tris-phenol, mix by inversion for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant;

[0058] D. Repeat the previous step;

[0059] E. Add Tris-phenol, chloroform and isoamyl alcohol (volume ratio is 25:24:1) to the supernatant, mix it upside ...

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PUM

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Abstract

The invention belongs to a detection technology of anaphylactogen in food, in particular to a primer / probe group which is used to detect fish anaphylactogen in food. The invention designs a group of specific primer and probe aiming to main fish anaphylactogen protein parvalbumin according to conservative sequence of gene thereof. Anaphylactogen protein of different fish in food can be fast detected in sensitivityand specificity through using the primer and probe and utilizing the real-time fluorescence PCR technology. The primer and probe can be provided together with other reagent in a reagent kit form, which is used to ribonucleotide amplification reaction. And the process is simply operated and has excellent repetitiveness.

Description

technical field [0001] The invention relates to a method for rapid detection of allergens by using nucleic acid amplification technology, in particular to a primer and probe sequence for real-time fluorescent PCR detection of fish allergens. Background technique [0002] Fish is one of the most common ingested allergens. It can cause different forms of clinical symptoms such as urticaria, runny nose, sneezing, asthma, dyspnea, vomiting, abdominal pain, and diarrhea in people with allergic constitution. In severe cases, death may result from bronchospasm, suffocation or anaphylactic shock. The most effective way to prevent and treat food allergies is to avoid contact with related foods. For this reason, the United States, Japan, Canada, the European Union, the United Kingdom, Italy, the Netherlands, New Zealand, Australia and other countries have successively formulated and implemented allergen labeling systems. In order to protect the food safety of consumers and maintain ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 孙敏梁成珠高宏伟刘彩霞贾俊涛
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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