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Identification method of Qingyu pig-derived components

A pig-derived, non-Qingyu technology, applied in the fields of molecular biology and food testing, can solve the problems of ineffective clustering of varieties, large sample size required for modeling, and large number of SNP sites, etc., to avoid counterfeit raw materials and products Harm to industry, enhancement of black pork value, effect of high sensitivity

Active Publication Date: 2022-07-01
CHINA MEAT RES CENT
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The polymorphism of chromosomal SNP is high, and non-linkage disequilibrium loci are easy to find. However, due to its high mutation rate and diverse genotypes, it also requires a large sample size for modeling, and the clus

Method used

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  • Identification method of Qingyu pig-derived components
  • Identification method of Qingyu pig-derived components
  • Identification method of Qingyu pig-derived components

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0054] The preparation method of the template is as follows: mixing meat or meat products with sterilized deionized double-distilled water in a ratio of 1:3 to 1:5, and homogenizing it with a tissue homogenizer at 11000 to 13000 r / min for 6 to 10 minutes to make a uniform tissue. slurry. The total genomic DNA of the samples was extracted using a tissue DNA extraction kit.

[0055] Further, the 25 μL reaction system in the triple RT-PCR method is shown in Table 2:

[0056] Table 2

[0057]

[0058] RT-PCR reaction conditions are: (1) Pre-denaturation: 95°C, 3min; (2) Denaturation: 95°C, 10-15s; Annealing and extension: 58-62°C, 20-45s, 30 cycles, (3) Melting curve preparation: 95°C, 1min, 70°C, 1min, heating to 95°C at a rate of 0.02°C / s, while continuously detecting the fluorescence intensity.

[0059] Considering the migration law of RT-PCR melting curve and the detection error of the instrument, a Tm value range is given for each primer to determine the result. Among ...

Embodiment 1

[0066] Example 1 The ability of SNP site combination to distinguish pig breeds

[0067] 1. Obtaining mitochondrial DNA sequence information

[0068] A total of 258 porcine mitochondrial genes were obtained by downloading from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and sample sequencing, which were used to construct the identification method for Qingyu pigs. There were 172 pig mitochondrial genes, and 88 mitochondrial genes were obtained by DNA sequencing of pork samples, involving a total of 52 domestic and foreign pig breeds. For purchased pork samples, DNA was extracted using the DNeasy Animal Tissue DNA extraction kit. The extracted DNA was measured by fluorescence microplate reader at 260nm and 280nm, and the DNA concentration and purity were calculated. Then, mitochondrial gene information was obtained by PCR amplification and product sequencing.

[0069] 2. SNP site screening and theoretical accuracy

[0070] The collected 258 mitochondrial gene information wa...

Embodiment 2 3

[0073] Example 2 Establishment and specificity of triple RT-PCR reaction system

[0074] 1. Sample processing method

[0075] Weigh 25mg Qingyu pig and 14 other common breed pork samples into centrifuge tubes respectively.

[0076] 2. DNA extraction method

[0077] Use the DNeasy animal tissue DNA extraction kit to extract DNA from each species, and operate according to the kit instructions, or other recognized extraction methods with the same efficacy. The extracted DNA was measured by fluorescence microplate reader at 260nm and 280nm, and the DNA concentration and purity were calculated.

[0078] 3. Primer sequence

[0079] A total of 9 SNP sites from SNP site 1 to SNP site 9 on the mitochondrial DNA of Qingyu pigs were used as the 3'-end targets of primers. A total of 36 pairs of primers were designed, and 6 pairs of specific primers were screened out: QY1-1F / R, QY1-2F / R, QY1-3F / R, QY2-1F / R, QY2-2F / R, QY2-3F / R. To improve the resolution and accuracy of the method, two ...

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Abstract

The invention discloses a method for identifying Qingyu pig-derived components. The invention identifies and distinguishes Qingyu pigs from other pig breeds based on 19 mitochondrial SNP site combinations or 9 core mitochondrial SNP site combinations. The above SNP loci were obtained through a large sample of pig mitochondrial DNA sequence alignment and intelligent algorithm screening, which can effectively distinguish Qingyu pigs from non-Qingyu pigs. The multiple RT-PCR melting curve method can simultaneously identify the genotypes of the 9 core loci in the above-mentioned locus combination of the tested product, and then complete the identification of the authenticity of the sample Qingyu pig or Qingyu pork. The method solves the problem of identification of local characteristic breeds in the field of meat breed identification, has the advantages of accurate, rapid and low-cost detection, and is of great significance for protecting high-quality local black pig brands and promoting the healthy and rapid development of the black pig 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 Qingyu pig-derived components. Background technique [0002] Qingyu pig, as a typical representative of Huchuan mountain pig, is one of the excellent local pig breeds in my country. Qingyu pig has the advantages of strong adaptability, good roughness resistance, fast growth and development, strong disease resistance, rich in snowflake meat, bright red meat, fragrant meat, and tender meat, and has high nutritional value and economic value. [0003] However, the frequent occurrence of low-priced ordinary pork masquerading as high-priced local specialty black pork has not only seriously infringed on the interests of consumers, but also hindered the overall structural upgrading of the pork industry. [0004] At present, there is no effective method for accurate breed identification of Qingyu pigs. The identification of livestock ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2600/166C12Q2600/172C12Q2531/113C12Q2527/107C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 李家鹏李金春王守伟许随根刘睿茜韦忆萱杨君娜曲超郭雅
Owner CHINA MEAT RES CENT
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