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Method for detecting allergen filbert component in foods by fluorescent PCR technology

A technical detection and allergen technology, applied in the field of allergen detection, can solve problems such as undetectable primer sequences with no clear specificity, save time, avoid complex processing procedures, and achieve the effect of less interference

Inactive Publication Date: 2010-02-10
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the protein in the product is damaged, the ELISA method cannot detect it
DNA is more stable than protein, and PCR method is more accurate than ELISA method, and avoids the need for a large number of antibodies. EU, Japan and AOAC all recommend using PCR method for confirmation, but there is no specific primer sequence

Method used

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  • Method for detecting allergen filbert component in foods by fluorescent PCR technology
  • Method for detecting allergen filbert component in foods by fluorescent PCR technology
  • Method for detecting allergen filbert component in foods by fluorescent PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Sample: Hazelnuts.

[0041] 1. Sample Processing

[0042] (1) Take 300mg samples and grind them into powder, put them into a 1.5mL centrifuge tube, add 600μL CTAB buffer solution (CTAB55mmol / L, EDTA 20mmol / L, Tris 100mmol / L, 10% hydrochloric acid to adjust the pH value to 8.0), 15μL (20mg / ml) proteinase K, incubate at 65°C for 30min; add 500μL phenol: chloroform: isoamyl alcohol (25:24:1) mixture, shake vigorously, centrifuge at 12000rpm for 15min; absorb the supernatant and add an equal volume of isopropanol After strong shaking, centrifuge at 12000rpm for 10min, discard the supernatant; dissolve with 200μL TE (the amount of TE depends on the amount of DNA precipitated); add an equal volume of chloroform: isoamyl alcohol (24:1) mixture, shake vigorously, and centrifuge at 12000rpm for 15min; Aspirate the supernatant and add an equal volume of isopropanol, shake vigorously and centrifuge at 12,000 rpm for 10 min, discard the supernatant; dissolve with 200 μL TE. The d...

Embodiment 2

[0069] Sample: Chocolate without hazelnut ingredients as an added base. Take 1g of hazelnuts ground into a paste and add it to 99g of chocolate in a 40°C water bath to melt, stir for 30min, and mix well. Take 3g of the above-mentioned chocolate with hazelnuts and add it to 27g of chocolate, so as to continuously dilute, and finally make chocolates containing 1000mg / kg, 100mg / kg, 30mg / kg, 20mg / kg, 10mg / kg, 5mg / kg of hazelnuts.

[0070] 1. Sample Processing

[0071] (1) Take 300mg samples respectively, put them into 1.5mL centrifuge tubes, add 600μL CTAB buffer solution (CTAB 55mmol / L, EDTA 20mmol / L, Tris 100mmol / L, adjust the pH value to 8.0 with 10% hydrochloric acid), 15μL (20mg / ml) proteinase K, incubate at 65°C for 30 minutes; add 500 μL of phenol: chloroform: isoamyl alcohol (25:24:1) mixture and shake vigorously, centrifuge at 12000 rpm for 15 minutes; absorb the supernatant and add an equal volume of isopropanol, shake vigorously Centrifuge at 12000rpm for 10min, disc...

Embodiment 3

[0100] Samples: hazelnuts, wheat flour, peanuts, chestnuts, pine nuts, summer berries, walnuts.

[0101] 1. Sample Processing

[0102] (1) Take 300mg samples and grind them into powder, put them into a 1.5mL centrifuge tube, add 600μL CTAB buffer solution (CTAB55mmol / L, EDTA 20mmol / L, Tris 100mmol / L, 10% hydrochloric acid to adjust the pH value to 8.0), 15μL (20mg / ml) proteinase K, incubate at 65°C for 30min; add 500μL phenol: chloroform: isoamyl alcohol (25:24:1) mixture, shake vigorously, centrifuge at 12000rpm for 15min; absorb the supernatant and add an equal volume of isopropanol After strong shaking, centrifuge at 12000rpm for 10min, discard the supernatant; dissolve with 200μL TE (the amount of TE depends on the amount of DNA precipitated); add an equal volume of chloroform: isoamyl alcohol (24:1) mixture, shake vigorously, and centrifuge at 12000rpm for 15min; Aspirate the supernatant and add an equal volume of isopropanol, shake vigorously and centrifuge at 12,000 rp...

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Abstract

The invention discloses a method for detecting an allergen filbert component in foods by a fluorescent PCR technology, belonging to allergen detecting technologies, in particular a method for detecting an allergen filbert component in foods by an exonuclease probe fluorescent PCR technology (TaqMan). Aiming at an allergen almond component rCor a 1.0401 DNA sequence a primer and a TaqMan probe aredesigned, the fluorescent PCR detecting method is established. The method includes the designed primer and the probe of the filbert component specificity, and the fluorescent PCR reaction condition matched with the primer and the probe. The method has no cross reaction with peanuts, buckwheat, chestnuts, pine nuts, macadamia nuts, walnuts, and the like and has favorable specificity; and the detecting sensitivity can reach 5mg / kg. The method can be used for detecting the allergen in the foods and preventing anaphylactic reaction caused by the foods and has practical meanings.

Description

technical field [0001] The invention relates to allergen detection technology, in particular to the technology of using fluorescent PCR reaction to detect allergens, especially the method of allergen hazelnut ingredients in food. Background technique [0002] Food allergy is an adverse reaction that people have to food, and it belongs to an allergic reaction of the body to exogenous substances. In the past two decades, food allergy has gradually been paid attention to and has been considered as a serious public health problem, and WHO predicts that food allergy may become one of the most common epidemics in the future. According to the US FDA statistics, in the United States, 2% of adults and 5% of infants suffer from food allergy, about 30,000 people need clinical emergency treatment every year, and 150 people die from food-induced allergic reactions. Peanut and tree nut allergies account for 10-47% of all allergic reactions. [0003] FDA issued the "Food Allergen Labelin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 高旗利张霞陈颖张海滨刘培张海英王乃福吴冬雪
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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