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Primer and probe for detecting racoon dog components in foods and feeds

A probe and primer sequence technology, applied in the field of rapid detection of animal-derived ingredients, can solve problems related to religious beliefs, endangering human health, and harming consumers' interests

Inactive Publication Date: 2014-04-30
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

This kind of behavior of producing and selling counterfeit products may not only endanger human health, but also involves religious beliefs, which seriously damages the interests of consumers.

Method used

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  • Primer and probe for detecting racoon dog components in foods and feeds
  • Primer and probe for detecting racoon dog components in foods and feeds
  • Primer and probe for detecting racoon dog components in foods and feeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Extraction of DNA from non-raccoon samples

[0042] A. Weigh 0.1 g of sample and add it to a 2 mL centrifuge tube. Add 600 μL of preheated CTAB lysis buffer and 40 μL of proteinase K, mix gently, and incubate in a 65°C water bath for 90 min;

[0043]B. Centrifuge at 2 000 g for 5 min, take the supernatant into another clean 2 mL centrifuge tube, add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), shake and mix ;

[0044] C. Centrifuge at 12 000 g for 10 min, take the supernatant into a clean 2 mL centrifuge tube, add an equal volume of isopropanol, and invert to mix;

[0045] D. Centrifuge at 12 000 g for 10 min, discard the supernatant, and dissolve the precipitate with TE buffer preheated to 65°C;

[0046] E. Add 5 μL RNase solution and incubate at 37 °C for 30 min;

[0047] F. Add 200 μL of chloroform:isoamyl alcohol (24:1), shake vigorously;

[0048] G. Centrifuge at 12 000 g for 10 min, take the supernatant into a clean 2 ...

Embodiment 2

[0060] (1) Extraction of DNA from raccoon samples with different contents

[0061] A. Add liquid nitrogen to raccoon meat and mutton, grind them into powder, weigh 0.1 g each, and add them to a 2 mL centrifuge tube. Add 600 μL of preheated CTAB lysis buffer and 40 μL of proteinase K to each tube, mix gently, and incubate in a 65°C water bath for 90 minutes;

[0062] B. Centrifuge at 2 000 g for 5 min, take the supernatant into another clean 2 mL centrifuge tube;

[0063] C. Mix the DNA extraction buffers of raccoon meat and mutton samples in proportion (100%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%);

[0064] D. Add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), shake and mix;

[0065] E. Centrifuge at 12 000 g for 10 min, take the supernatant into a clean 2 mL centrifuge tube, add an equal volume of isopropanol, and invert to mix;

[0066] F. Centrifuge at 12 000 g for 10 min, discard the supernatant, and dissolve the precipitate ...

Embodiment 3

[0082] (1) Extraction of DNA from the meat roll sample to be tested

[0083] A. Weigh 0.3 g of meat roll sample and add it to a 2 mL centrifuge tube. Add 600 μL of preheated CTAB lysis buffer and 40 μL of proteinase K, mix gently, and incubate in a 65°C water bath for 90 min;

[0084] B. Centrifuge at 2 000 g for 5 min, take the supernatant into another clean 2 mL centrifuge tube, add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), shake and mix ;

[0085] C. Centrifuge at 12 000 g for 10 min, take the supernatant into a clean 2 mL centrifuge tube, add an equal volume of isopropanol, and invert to mix;

[0086] D. Centrifuge at 12 000 g for 10 min, discard the supernatant, and dissolve the precipitate with TE buffer preheated to 65°C;

[0087] E. Add 5 μL RNase solution and incubate at 37 °C for 30 min;

[0088] F. Add 200 μL of chloroform:isoamyl alcohol (24:1), shake vigorously;

[0089] G. Centrifuge at 12 000 g for 10 min, take the su...

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PUM

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Abstract

The invention discloses a primer and a probe for detecting racoon dog components in foods and feeds and belongs to a qualitative detection technology of animal origin components in the foods and the feeds. According to the primer and the probe, a set of a specific primer and a probe are designed by selecting mitochondria cell pigment C oxidordeuctase I subunit gene sequence; the primer and the probe are used and a real-time fluorescence PCR (Polymerase Chain Reaction) technology is adopted so as to rapidly, sensitively and specifically detect the racoon dog components in the foods and the feeds. The primer and the probe can be provided in a form of a kit with other reagents and are used for a nucleic acid amplification reaction. The primer and the probe are simple and convenient to operate and have good repeatability.

Description

technical field [0001] The invention relates to a method for rapid detection of animal-derived components using nucleic acid amplification technology, in particular to a primer and probe sequence for real-time fluorescent PCR detection of raccoon dog components. Background technique [0002] Raccoon dog, also known as raccoon dog, belongs to Mammalia, Carnivora, Canidae (Canidae) and raccoon dog ( Nyctereutes ). It is mainly distributed in the countries of the former Soviet Union, China, North Korea, Japan, Mongolia, Finland and other countries, and it is distributed in almost every province in my country. Raccoon is a precious fur animal. Raccoon fur is a large capillary skin, which has the advantages of toughness, wear resistance, lightness, softness, beauty and heat preservation. It is a high-quality raw material for making fur products such as coats, fur collars, hats and fur mattresses. The guard hair and tail hair of raccoon dogs can be used to make advanced cosmetic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/101C12Q2561/113
Inventor 孙敏邓明俊吴兴海高宏伟
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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