Method for quantitatively detecting bovine-derived materials in livestock and poultry meat based on QPCR (quantitative polymerase chain reaction)

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

Inactive Publication Date: 2019-10-18
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] 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 quanti

Method used

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  • Method for quantitatively detecting bovine-derived materials in livestock and poultry meat based on QPCR (quantitative polymerase chain reaction)
  • Method for quantitatively detecting bovine-derived materials in livestock and poultry meat based on QPCR (quantitative polymerase chain reaction)
  • Method for quantitatively detecting bovine-derived materials in livestock and poultry meat based on QPCR (quantitative polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Sample S1: a homemade simulated mixed sample with 30% bovine-derived components, in a mixing method of 30g beef + 70g pork.

[0045] (2) Reagents: Premix Ex Taq (Probe qPCR) premix solution produced by TaKaRa company; internal reference gene and bovine species-specific gene amplification primers and probes synthesized by Shanghai Shenggong.

[0046] (3) Sample DNA extraction: SDS-silica gel column modified method extraction (see attachment).

[0047] (4) Amplification reaction system and amplification procedure: The total volume of the amplification reaction is 20 μL, and its various components are: 20 μL of 2×Premix Ex Taq (Probe qPCR) premix, 1 μL of 10 μmol / L upstream and downstream primers, 10 μmol / L probe primer 0.4 μL, template DNA 1 μL (template DNA concentration 50 ng / μL), make up to 20 μL with sterilized deionized water; Reaction program: denaturation at 95 °C for 5 min; carry out 45 cycles of amplification reaction (95 °C Denaturation for 15s, annealing a...

Embodiment 2

[0059] (1) Sample S2: a homemade simulated mixed sample with 70% bovine-derived components, in a mixing method of 70g beef + 30g chicken.

[0060] (2) Reagents: Premix Ex Taq (Probe qPCR) premix solution produced by TaKaRa company; internal reference gene and exogenous species-specific gene amplification primers and probes synthesized by Shanghai Shenggong.

[0061] (3) Sample DNA extraction: SDS-silica gel column modified method extraction (see attachment).

[0062] (4) Amplification reaction system and amplification procedure: The total volume of the amplification reaction is 20 μL, and its various components are: 20 μL of 2×Premix Ex Taq (Probe qPCR) premix, 1 μL of 10 μmol / L upstream and downstream primers, 10 μmol / L probe primer 0.4 μL, template DNA 1 μL (template DNA concentration 50 ng / μL), make up to 20 μL with sterilized deionized water; Reaction program: denaturation at 95 °C for 5 min; carry out 45 cycles of amplification reaction (95 °C Denaturation for 15s, annea...

Embodiment 3

[0074] (1) Sample S3: a homemade simulated mixed sample with 70% bovine-derived components, in a mixing method of 70g beef + 30g duck.

[0075] (2) Reagents: Premix Ex Taq (Probe qPCR) premix solution produced by TaKaRa company; internal reference gene and exogenous species-specific gene amplification primers and probes synthesized by Shanghai Shenggong.

[0076] (3) Sample DNA extraction: SDS-silica gel column modified method extraction (see attachment).

[0077] (4) Amplification reaction system and amplification procedure: The total volume of the amplification reaction is 20 μL, and its various components are: 20 μL of 2×Premix Ex Taq (Probe qPCR) premix, 1 μL of 10 μmol / L upstream and downstream primers, 10 μmol / L probe primer 0.4 μL, template DNA 1 μL (template DNA concentration 50 ng / μL), make up to 20 μL with sterilized deionized water; Reaction program: denaturation at 95 °C for 5 min; carry out 45 cycles of amplification reaction (95 °C Denaturation for 15s, annealin...

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Abstract

The invention discloses a method for quantitatively detecting bovine-derived materials in livestock and poultry meat based on QPCR (quantitative polymerase chain reaction). By the method, the contentof bovine-derived materials in meat products is quantitatively analyzed by real-time fluorescent PCR technology; 15 animal genome sequences are analyzed by applying a bioinformatical method; universalprimers and specific primers are designed by screening single-copy genes; the percentage of bovine-derived species specific genes is corrected by an internal reference gene; and the deviation of quantity value results is eliminated, thereby obtaining the authentic and reliable food adulteration value, and effectively improving the sensitivity and accuracy of the detection method. The detection method disclosed by the invention can complete the identification work of the bovine-derived materials within 3 hours, has the characteristics of simple and easy operation, high sensitivity, rapidity, low cost, convenient promotion, and the like, and opens up a broad prospect for quantized adulteration identification of animal-derived materials.

Description

technical field [0001] The invention belongs to the field of molecular biology analysis, and in particular relates to a method for identifying the content of bovine-derived components in meat products, which is a method for correcting the content of bovine-derived components by using an internal reference gene on the basis of real-time fluorescence quantitative PCR technology method for quantitative detection. Background technique [0002] Diversified meat varieties are an important part of people's increasingly rich dining table culture. However, with the continuous sharp rise of beef prices in the market, some unscrupulous enterprises and traders, in order to maximize their own economic interests, are using high-cost beef or beef. Beef is mixed with other low-cost meat varieties, or it is sold as beef after soaking in butter and adding seasonings. [0003] Therefore, vigorously improve the level of market supervision, cooperate with the smooth implementation of the nutrit...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2545/101C12Q2561/101
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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