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Meat identification positive reference product as well as preparation method and application thereof

A positive control, edible meat technology, applied in the field of DNA identification, can solve the problems of difficult preservation, troublesome time-consuming, inapplicability, etc., and achieves the effect of clear DNA information, rapid method, and easy preservation.

Inactive Publication Date: 2018-12-11
BEIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, real-time fluorescent PCR technology is mostly used in the authenticity identification of common edible meat in national standards, but real-time fluorescent PCR technology is costly, troublesome and time-consuming, and is not suitable for local general inspection and quarantine departments
Moreover, when PCR is used for identification, the positive control is not easy to obtain, and the conventional way is to purchase it from authoritative institutions such as quality inspection institutes and drug inspection institutes.
However, there are also risks in whether such a positive control can be recognized abroad, and there are also defects such as difficult storage and poor stability.

Method used

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  • Meat identification positive reference product as well as preparation method and application thereof
  • Meat identification positive reference product as well as preparation method and application thereof
  • Meat identification positive reference product as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] A positive control substance for edible meat identification is prepared by the following method:

[0077] 1. Amplification.

[0078] 1. Pretreatment.

[0079] Take beef, sheep, pig, chicken, duck and donkey meat (fresh products) provided and identified by Changchun Food and Drug Inspection Center, put 50mg each in 6 small beakers, add about 10mL cold saline solution to soak for 20min , after removing connective tissue with a scalpel in a plate, mince the tissue to about 1mm 3 .

[0080] 2. Mitochondrial DNA extraction (using a commercially available animal-derived tissue genomic DNA extraction kit).

[0081] (1) Lysis: Add 500 μL of P1 lysis buffer, 30 μL of P2 solution, and 15 μL of P3 solution to the pretreated sample, mix well, and shake in a water bath at 56°C for 1 hour.

[0082] (2) Precipitation: Add 500 μL of P4 solution, shake for 10 min, centrifuge at 11000 r / min at 4 °C for 10 min; take the supernatant, add an equal volume of P5 solution, mix well, place ...

Embodiment 2

[0141] A method for identifying edible meat, comprising the following steps:

[0142] 1. Pre-treatment of test samples.

[0143] The test samples are all fresh products, 1-2 are donkey samples, 3-4 beef samples, 5-6 mutton samples, 7-8 pork samples, 9-10 chicken samples, 11-12 duck samples are all provided by Changchun Food Provided and identified by the Drug Testing Center, take 50 mg of each sample to be tested (fresh product) and put it in a small beaker, add about 10 mL of cold physiological saline solution and soak for 20 minutes, remove the connective tissue with a scalpel in a plate, and chop the tissue until about 1mm 3 .

[0144] 2. Mitochondrial DNA extraction.

[0145] (1) Lysis: Add 500 μL of P1 lysis buffer, 30 μL of P2 solution, and 15 μL of P3 solution to the pretreated sample, mix well, and shake in a water bath at 56°C for 1 hour.

[0146] (2) Precipitation: Add 500 μL of P4 solution, shake for 10 min, centrifuge at 11000 r / min at 4 °C for 10 min; take the...

Embodiment 3

[0160] Identification of six common edible meat samples available in the market

[0161] 1. Materials.

[0162] Commercially available cattle, sheep, pigs, chickens, ducks and donkey meat samples (purchased from Dafuyuan Supermarket in Jilin City). After gene cloning and sequencing, plasmids were extracted from cattle, sheep, pigs, chickens, ducks and donkeys as positive controls.

[0163] 2. Pre-treatment of test samples.

[0164] Take 50 mg of the sample to be tested (fresh product) in a centrifuge tube, add about 10 mL of cold saline solution to a small beaker and soak for 20 minutes, remove the connective tissue with a scalpel in a plate, and chop the tissue to about 1mm3.

[0165] 3. Mitochondrial DNA extraction.

[0166] Refer to the mitochondrial DNA extraction step in Example 2.

[0167] 4. PCR reaction

[0168] Add corresponding primer pairs to each sample, refer to the PCR reaction steps in Example 2.

[0169] 5. Result judgment

[0170] Prepare 1.5% agarose g...

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Abstract

The invention relates to a meat identification positive reference product as well as a preparation method and application thereof, which belongs to the technical field of PCR identification. The preparation method of the reference product comprises the following steps: S1, performing amplification: collecting at least one of identified beef, mutton, pork, chicken, duck and ass meat, performing PCRamplification for the meat by using a corresponding primer pair, analyzing an amplification result by virtue of agarose gel electrophoresis, and separately recycling a specific DNA band; S2, purifying: purifying the specific DNA band by using an absorption column; S3, connecting: connecting the purified DNA product and a vector; S4, transforming: separately transforming a connection product to escherichia coli competent cells, and obtaining transformation bacteria; and S5, culturing: culturing the transformation bacteria on a plate, screening positive colonies, selecting the positive coloniesto be amplified, thus obtaining a positive reference product. Compared with the identification positive reference product for identifying the conventional meat, the meat identification positive reference product has the advantages of definite DNA information, stability and convenience in storage, and can be used as a standard substance.

Description

technical field [0001] The invention relates to the technical field of DNA identification, in particular to a positive control substance for identification of edible meat and its preparation method and application. Background technique [0002] Since mad cow disease first occurred in the United Kingdom in 1987, countries and regions around the world have enacted laws and regulations to control the use of animal-derived ingredients. At the beginning of 2013, after some European countries were involved in the "horse meat scandal" scandal, it aroused the attention of various countries on the adulteration of meat and meat products. [0003] At present, there are relatively serious meat adulteration phenomena in the domestic market, among which the adulteration of beef and mutton is the most prominent. It is common to adulterate beef and mutton with pork, and mutton with duck and beef. The huge price difference is the economic motivation for unscrupulous traders to adulterate. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6806
CPCC12Q1/6806C12Q1/6888C12Q2600/166C12Q2531/113
Inventor 艾金霞孙丽媛高丽君李明成周亭亭王艳双张丽华段思琪刘玟妍
Owner BEIHUA UNIV