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Method for detecting content of bovine derived components in meat product based on digital PCR (Polymerase Chain Reaction) method

A bovine-derived, content-based technology, applied in the field of molecular biology analysis, can solve problems such as time-consuming, inability to quantify the content of heterologous animal components, and cumbersome operations

Inactive Publication Date: 2018-09-07
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the shortcomings of traditional meat identification methods such as long time-consuming, cumbersome operation, and inability to quantify the content of heterologous animal components in samples, and discloses a method for quickly detecting the content of bovine-derived components in meat products using internal reference genes

Method used

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  • Method for detecting content of bovine derived components in meat product based on digital PCR (Polymerase Chain Reaction) method
  • Method for detecting content of bovine derived components in meat product based on digital PCR (Polymerase Chain Reaction) method
  • Method for detecting content of bovine derived components in meat product based on digital PCR (Polymerase Chain Reaction) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Sample S1: a self-made simulated mixed sample with 70% bovine-derived ingredients, the mixing method is 70g beef+30g horsemeat.

[0041] (2) Reagents: ddPCR Supermix for probes master mix produced by Bio-Rad; internal reference genes and bovine species-specific gene amplification primers and probes synthesized by Shanghai Sangon.

[0042] (3) Amplification reaction system and amplification program: the total volume of the amplification reaction is 20 μL, and its various components are: 10 μL of 2×ddPCR Supermix for probes premix, 1.0 μL of upstream and downstream primers (concentration of 10 μmol / L ), probe primer 1.0 μL (concentration is 10 μmol / L), template DNA 1.0 μL (concentration is 30 / μL), make up to 20 μL with sterilized deionized water, mix well and centrifuge; droplet generation: 20 μL reaction Carefully transfer the system to the micro-card generator, avoiding the generation of air bubbles. At the same time, add 70 μL of heavy oil to the corresponding posi...

Embodiment 2

[0053] (1) Sample S2: a self-made simulated mixed sample with 30% bovine ingredients, the mixing method is 30g beef+70g chicken.

[0054] (2) Reagents: ddPCR Supermix for probes master mix produced by Bio-Rad; internal reference genes and bovine species-specific gene amplification primers and probes synthesized by Shanghai Sangon.

[0055] (3) Amplification reaction system and amplification program: the total volume of the amplification reaction is 20 μL, and its various components are: 10 μL of 2×ddPCR Supermix for probes premix, 1.0 μL of upstream and downstream primers (concentration of 10 μmol / L ), probe primer 1.0 μL (concentration is 10 μmol / L), template DNA 1.0 μL (concentration is 30 / μL), make up to 20 μL with sterilized deionized water, mix well and centrifuge; droplet generation: 20 μL reaction Carefully transfer the system to the micro-card generator, avoiding the generation of air bubbles. At the same time, add 70 μL of heavy oil to the corresponding position of th...

Embodiment 3

[0066] (1) Sample S3: a self-made simulated mixed sample with 30% bovine-derived ingredients, the mixing method is 30g beef+70g pork.

[0067] (2) Reagents: ddPCR Supermix for probes master mix produced by Bio-Rad; internal reference genes and bovine species-specific gene amplification primers and probes synthesized by Shanghai Sangon.

[0068] (3) Amplification reaction system and amplification program: the total volume of the amplification reaction is 20 μL, and its various components are: 10 μL of 2×ddPCR Supermix for probes premix, 1.0 μL of upstream and downstream primers (concentration of 10 μmol / L ), probe primer 1.0 μL (concentration is 10 μmol / L), template DNA 1.0 μL (concentration is 30 / μL), make up to 20 μL with sterilized deionized water, mix well and centrifuge; droplet generation: 20 μL reaction Carefully transfer the system to the micro-card generator, avoiding the generation of air bubbles. At the same time, add 70 μL of heavy oil to the corresponding position ...

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Abstract

The invention discloses a method for detecting the content of bovine derived components in a meat product based on a digital PCR (Polymerase Chain Reaction) method. The method is characterized by screening single-copy gene sequences from genomic sequences of 15 kinds of animals by applying a bioinformatics means, and designing an internal reference primer and a bovine derived species specific primer. According to the method disclosed by the invention, the content of the bovine derived components in the meat product is absolutely and quantitatively analyzed based on a droplet digital PCR technology, standard products are not required, a standard curve is not required to be established, and the defect that a part of standard products cannot be easily obtained is remedied; meanwhile, magnitude deviation caused by technological difference between the standard products and samples is also effectively avoided, so that internal reference genes are applied to correct the percentage content ofthe bovine derived species specific primer, and true and believable food adulteration numerical value can be obtained; the method is capable of completing identification work on the content of the bovine derived components within 3 hours and has the characteristics of simpleness, practicability, high efficiency, sensitivity and the like, and a wide prospect is opened up for identifying quantitative adulteration of animal derived components.

Description

technical field [0001] The invention belongs to the field of molecular biology analysis, and in particular relates to an identification method for the content of bovine-derived components in meat products, which is a method for correcting bovine-derived species-specific genes for bovine-derived components on the basis of real-time fluorescence quantitative PCR technology. methods for quantitative testing. Background technique [0002] The variety of meat from multiple sources is an important part of people's increasingly rich table culture. However, as the price of beef in the market continues to rise sharply, some unscrupulous companies and traders, in order to maximize their own economic interests, use high-cost beef or Beef is mixed with other low-cost meat varieties, or processed by soaking in butter, adding seasonings, etc., and then sold as beef. [0003] Therefore, it is necessary to vigorously improve the level of market supervision, cooperate with the smooth implem...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6888C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/159C12Q2545/101
Inventor 赵新兰青阔陈锐王永朱珠
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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