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Identification method of Tan sheep-derived components

A source ingredient, Tan sheep technology, applied in the fields of molecular biology and food testing, to achieve the effect of enhancing the overall brand value, promoting the healthy and rapid development of the industry, and reducing the number of non-coding areas

Active Publication Date: 2022-08-02
CHINA MEAT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The adulteration of meat and meat products in the market is the main problem in food processing, circulation and catering. The benefits brought by price differences drive some unscrupulous traders and enterprises to shoddy and adulterate high-priced meat products such as Tan mutton. some nontan lamb

Method used

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  • Identification method of Tan sheep-derived components
  • Identification method of Tan sheep-derived components
  • Identification method of Tan sheep-derived components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 SNP site combination ability to distinguish Tan sheep breeds

[0056] The genomic DNA of 100 Tan sheep and 44 non-Tan sheep was extracted by CTAB method, and the quality was checked by agarose gel electrophoresis. figure 1 ). The DNA that passed the quality inspection was amplified and sequenced by PCR, and a total of 144 mitochondrial gene sequences of sheep were obtained. At the same time, 19 non-Tan sheep mitochondrial genes were downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and a total of 163 mitochondrial DNA sequences of Tan sheep and non-Tan sheep samples were obtained.

[0057] The collected 163 mitochondrial gene information was processed by Python program and Mega software to construct the Tan sheep identification method. The loci of SNP loci that are conserved in Tan sheep species and with high polymorphism in other breeds were selected, and 35 loci were selected from 399 loci as characteristic markers for identifying Tan sheep...

Embodiment 2 3

[0060] Example 2 Establishment and specificity of triple and quadruple RT-PCR reaction systems

[0061] 1. SNP site primer design

[0062] Use Oligo 7.0 software to design PCR amplification primers for the SNP site to be tested (Table 1), and hand them over to Yingweijieji Company for synthesis.

[0063] 2. Establishment of RT-PCR reaction system

[0064] Combine primers according to reaction system A: 12.5 μL of Premix master mix, 0.3-1.5 μL of 5 μM Primer 1F and Primer 1R each, 0.3-1.5 μL of 5 μM Primer 2F and Primer 2R each, 0.3-1.5 μL of 5 μM Primer 3F and Primer 3R each, 5μM Primer 4F and Primer 4R each 0.3-1.5μL, 0.5ng / μL template DNA 1μL, deionized water to make up to 25μL;

[0065] Reaction system B: 12.5μL of Premix master mix, 0.3-1.5μL each of 5μM Primer 5F and Primer 5R, 5μM Primer 6F and Primer 6R each 0.3-1.5μL, 5μM Primer 7F and Primer 7R each 0.3-1.5μL, 0.5ng / μL template DNA 1 μL, deionized water to make up to 25 μL.

[0066] Under the condition of ensuring s...

Embodiment 3

[0074] Example 3 Sample Detection and Method Accuracy Investigation

[0075] 1. Collect samples and prepare templates

[0076] 60 DNA samples from Tan sheep and 60 non-Tan sheep samples were extracted by CTAB method. According to the DNA extraction method in Reference Example 1, the DNA of Tan sheep and non-Tan sheep were extracted respectively, and the DNA concentration and purity were calculated. The concentration of DNA samples was diluted to 0.5ng / μL and used as detection template.

[0077] 2. Validation of triple and quadruple RT-PCR reactions

[0078] Referring to the RT-PCR reaction process in Example 2, 120 samples of Tan sheep and non-Tan sheep DNA templates were simultaneously or separately subjected to triple and quadruple RT-PCR melting curve analysis in two reaction wells, and Tm values ​​were calculated and counted. The number of melting curve peaks.

[0079] 3. Sample verification

[0080] Determination criteria for Tan sheep: 60 Tan sheep and 60 non-Tan sh...

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Abstract

The invention discloses a method for identifying Tan sheep-derived components. The invention identifies and distinguishes Tan sheep from other sheep species based on 35 mitochondrial SNP site combinations or 14 core mitochondrial SNP site combinations. The 14 mitochondrial SNP loci of Tan sheep with high classification efficiency were obtained through the inter-species mitochondrial DNA sequence alignment and intelligent algorithm screening, which can effectively distinguish Tan sheep from non-Tan sheep. The multiplex RT-PCR melting curve method can simultaneously identify the genotypes of the above 14 loci of the sample to be tested, and then complete the identification of the authenticity of the sample Tan sheep or Tan mutton. The method has the advantages of quick and simple operation, intuitive and reliable results, and low cost, and is of great significance for preventing adulteration and counterfeiting, enhancing the overall brand value of the Tan sheep industry, and promoting the healthy and rapid development of the industry.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and food detection, in particular to a method for identifying Tan sheep-derived components. Background technique [0002] Tan sheep is a unique local breed developed through long-term natural selection and artificial selection in my country. Tan sheep meat is tender, nutritious, and not greasy. It is also an extremely rare breed of sheep for fur in the world, with high economic value. Tan sheep has entered the list of the first batch of national livestock and poultry genetic resources gene banks in China. [0003] The adulteration of meat and meat products in the market is a major problem in food processing, distribution and catering. The benefits brought by price differences drive some unscrupulous traders and enterprises to shoddy and adulterate high-priced meat products such as Tan mutton. Some non-tan lamb. Therefore, establishing a fast, effective and low-cost method to identify T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2600/166C12Q2600/172C12Q2531/113C12Q2527/107C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 李家鹏熊苏玥乔晓玲陈曦李金春许随根戚彪韦忆萱曲超许典
Owner CHINA MEAT RES CENT
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