Method for detecting bovine-derived materials in meat and meat products

A cattle-derived, meat product technology, applied in DNA/RNA fragmentation, recombinant DNA technology, etc., can solve the difficulty of standard sample selection and preparation, the research lag of quantitative detection technology of nucleic acid components, the total amount of DNA extracted and the amplification target Problems such as the difference in the total amount of sequence templates

Inactive Publication Date: 2013-03-27
CHINA MEAT RES CENT
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Problems solved by technology

[0004] However, most of the existing research focuses on the qualitative detection of animal-derived ingredients such as pigs, cattle, and sheep in feed, while the research on the identification of food adulteration, especially the quantitative detection technology related to nucleic acid components, is lagging behind.
Pure qualitative testing can no longer meet the needs. For example, in the case of meat, milk and other food "adulterated and faked" cases, how to determine whether the adulterated ingredients are caused by addition or pollution, and how to determine the severity of adulteration requires reliable quantitative relying on technology
The complexity of biological samples, such as differences in animal species, age, sex, organs, muscle types, etc., leads to differences in the total amount of DNA extraction and the total amount of amplified target sequence templates, making it difficult to select and prepare standard samples

Method used

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  • Method for detecting bovine-derived materials in meat and meat products
  • Method for detecting bovine-derived materials in meat and meat products
  • Method for detecting bovine-derived materials in meat and meat products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1 bovine specific primer of the present invention, the specificity of probe system

[0099] 1. Primer and probe sequence

[0100] Bovine-specific oligonucleotide primers and probes,

[0101] Upstream primer: 5'-ATCTGAATCAATGCAACAGTACAT-3';

[0102] Downstream primer: 5'-TGATAGTGAGTCGGAGAAGAATGT-3';

[0103] Probe: 5'-FAM-TCTCCTCATTAGCCTGATCAGTCTATCCC-TAMRA-3'.

[0104] 2. Sample processing method

[0105] Weigh 0.2g of 13 common meat samples of pig, cattle, sheep, chicken, duck, donkey, rabbit, deer, dog and fish into 1.5ml centrifuge tubes.

[0106] 3. DNA extraction method

[0107] Add 800 μL of lysate to the 1.5ml centrifuge tube from which the sample was taken in step 2, and incubate at 65°C for 30 minutes, shaking and mixing from time to time. Centrifuge at 12000g for 5min, transfer 600μL supernatant to a clean centrifuge tube, add 400μL CTAB protein sedimentation solution, shake and mix well, centrifuge at 12000g for 5min, take 400μL supernatant i...

Embodiment 2

[0119] Embodiment 2 The linear relationship of detection method system of the present invention

[0120] 1. Primer and probe sequence

[0121] Bovine-specific oligonucleotide primers and probes,

[0122] The upstream primer is: 5'-AATATAATTTGGGTTAACTCCACAGC-3';

[0123] The downstream primer is: 5'-GCCATATGGTTAAAATTAGTAGTGGAGTG-3';

[0124] The probe is: 5'-FAM-CACAGCCTTCTAATTAGCTTTACAAGCCTCC–TAMRA-3'.

[0125] Vertebrate Universal Oligonucleotide Primers and Probes

[0126] Upstream primer: 5'-TACGACCTCGATGTTGGATCA-3';

[0127] Downstream primer: 5'-AGATAGAAACCGACCTGGAT-3';

[0128] Probe: 5'-FAM-CCGGTCTGAACTCAGATCACGTAGGA-TAMRA-3' (for singleplex PCR);

[0129] Probe: 5'-HEX-CCGGTCTGAACTCAGATCACGTAGGA-TAMRA-3' (for duplex PCR);

[0130] 2. Processing of pure beef samples and preparation of beef / sheep / pig mixed meat samples containing series of concentrations of beef

[0131] Weigh 30g of beef, mutton and pork respectively into 3 clean homogenization cups, add 120g of...

Embodiment 3

[0149] Embodiment 3 The accuracy and precision of detection method system of the present invention

[0150] 1. Preparation of samples to be tested

[0151] Weigh 30g of beef, mutton, pork, chicken and duck into 5 clean homogenization cups, add 120g of deionized double-distilled water, and homogenize at 12000r / min for 8min to obtain 5 homogenates. Use an analytical balance (accurate to 0.0001g) to accurately weigh each homogenate according to the proportions in Table 6, and obtain beef contents of 0.1%, 1.9%, 10.0%, 30.0%, 60.0%, 98.0%, 5.83%, 32.27%, 42.87%, 22.33% beef / sheep / pig mixed meat samples 3g were placed in 5ml centrifuge tubes, each group of samples was set up in three parallels, vortexed for 1min, and 50μl homogenate was pipetted into 1.5ml centrifuge with a 100μl micropipette tube, pending further processing.

[0152] Table 8 The ratio of each component of the mixed sample

[0153] Sample serial number

Cattle

sheep

pig

chicken

d...

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Abstract

The invention relates to a method for detecting bovine-derived materials in meat and meat products. The method comprises a pair of cattle specific primers: an upstream primer 5'-AATATAATTTGGGTTAACTCCACAGC-3' and a downstream primer 5'-GCCATATGGTTAAAATTAGTGGAGTG-3', a probe 5'-F-G-CACAGCCTTCTAATTAGCTTTACAAGCCTCC-M-3' matched with the primers and a kit containing the primers and the probe. The method disclosed by the invention furthest reduces quantitative errors, has higher accuracy and anti-jamming capability and can accurately and conveniently detect the percentage of bovine-derived materials in meat and meat products. The invention provides a single-PCR mode and a double-PCR mode, which are respectively suitable for different requirements.

Description

technical field [0001] The invention relates to the fields of food inspection and biotechnology, in particular to a method for detecting bovine-derived components in meat and meat products. Background technique [0002] "Fake food adulteration" has always been one of the focus of consumers' attention. In order to reduce costs, some unscrupulous enterprises and merchants use pork as beef and mutton in the process of meat processing, or replace high-priced meat with other low-priced meat instead of stated on the label. This not only seriously infringes on the interests of consumers, but also involves issues such as ethnicity and religion if halal food is mixed with pork ingredients, causing bad social impact. In addition, epidemiological studies have demonstrated that animal-derived ingredients in feed are a major factor in the spread of neurological diseases such as "mad cow disease". [0003] At present, most of the technologies used for the identification of meat species ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李家鹏周彤田寒友杨君娜邹浩乔晓玲陈文华曲超
Owner CHINA MEAT RES CENT
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