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RT-PCR-based method for determining the content of specific meat components in mixed meat products

A technology for RT-PCR and meat products, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the difference between the total amount of DNA extraction and the total amount of amplified target sequence templates, nucleic acid components Quantitative detection technology research lag, standard sample selection and preparation difficulties and other issues

Active Publication Date: 2011-12-28
CHINA MEAT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • RT-PCR-based method for determining the content of specific meat components in mixed meat products
  • RT-PCR-based method for determining the content of specific meat components in mixed meat products
  • RT-PCR-based method for determining the content of specific meat components in mixed meat products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Identification of the specificity of the pig-specific primers and probe system of the present invention and the versatility of the vertebrate universal primer-probe system

[0080] Main equipment:

[0081] Fluorescence quantitative PCR instrument (Roche480 II, Switzerland), high-speed desktop centrifuge (Eppendorf5417R, Germany), micropipettes (10 μl, 100 μl, 1000 μl), homogenizer (ACE, Japan), ultraviolet-visible spectrophotometer (UNIC 2800A, Shanghai), etc.

[0082] Main reagents:

[0083] Chloroform, isopropanol, EDTA, Tris, purchased from Sinopharm, TE buffer (Tris-HCl, EDTA buffer): 10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTA (pH 8.0, 1% CTAB Lysis solution (0.05mol / L Tris-HCl (pH 8.0), 0.7mol / L NaCl, 0.01mol / L EDTA (pH 8.0)), CTAB precipitation solution (chloroform + isoamyl alcohol (24:1)) were all Commonly used reagents in laboratories; Premix Ex Taq TM The master mix was purchased from Treasure Bioengineering (Dalian) Co., Ltd.; primers and probes w...

Embodiment 2

[0109] Embodiment 2 The linear relationship of detection method system of the present invention

[0110] Main equipment:

[0111] Fluorescent quantitative PCR instrument (Roche480 II, Switzerland), high-speed desktop centrifuge (Eppendorf5417R, Germany), micropipettes (10 μl, 100 μl, 1000 μl), homogenizer (ACE, Japan), ultraviolet-visible spectrophotometer (UNIC 2800A, Shanghai), etc.

[0112] Main reagents:

[0113] Chloroform, isopropanol, EDTA, Tris, purchased from Sinopharm, TE buffer (Tris-HCl, EDTA buffer): 10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTA (pH 8.0, 1% CTAB Lysis solution (0.05mol / L Tris-HCl (pH 8.0), 0.7mol / L NaCl, 0.01mol / L EDTA (pH 8.0)), CTAB precipitation solution (chloroform + isoamyl alcohol (24:1)) were all Commonly used reagents in laboratories; Premix Ex Taq TM The master mix was purchased from Treasure Bioengineering (Dalian) Co., Ltd.; primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0114] Detection steps:

[0...

Embodiment 3

[0131] Example 3 Use the method system provided by the present invention to detect the content of pork components in mixed raw meat samples with known pork content to test the accuracy of the method system

[0132] Main equipment:

[0133] Fluorescent quantitative PCR instrument (Roche480 II, Switzerland), high-speed desktop centrifuge (Eppendorf5417R, Germany), micropipettes (10 μl, 100 μl, 1000 μl), homogenizer (ACE, Japan), ultraviolet-visible spectrophotometer (UNIC 2800A, Shanghai), etc.

[0134] Main reagents:

[0135] Chloroform, isopropanol, EDTA, Tris, purchased from Sinopharm, TE buffer (Tris-HCl, EDTA buffer): 10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTA (pH 8.0, 1% CTAB Lysis solution (0.05mol / L Tris-HCl (pH 8.0), 0.7mol / L NaCl, 0.01mol / L EDTA (pH 8.0)), CTAB precipitation solution (chloroform + isoamyl alcohol (24:1)) were all Commonly used reagents in laboratories; Premix Ex Taq TM The master mix was purchased from Treasure Bioengineering (Dalian) Co., Ltd.; prim...

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Abstract

The invention relates to a RT-PCR based method for determining specific meats in mixed meat products, especially to a method for determining the component content of pork. The invention provides real time fluorescence PCR porcine specific oligonucleotide primers and probe for determining the component content of pork in mixed meat products as well as vertebrate general oligonucleotide primers and probe. Furthermore, the invention also provides a detection kit containing the above porcine specific primers and probes as well as the vertebrate general primers and probe. The method provided by the invention can be adopted to simply, rapidly and accurately determine the porcine-derived DNA content in raw meat or a cooked sample. Based on the correlation between a porcine specific primer system and a general primer system amplification curve, the method provided by the invention has a high accuracy and an anti-interference capability. In addition, the invention has such a strong portability that the component contents of other species in the mixed meat sample can be determined by replacing the specific primer system.

Description

technical field [0001] The invention relates to the fields of food inspection and biotechnology, in particular to a method for measuring the content of specific meat components in mixed meat products based on RT-PCR. Background technique [0002] Food "adulterated to make fake" 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. 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 identif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李家鹏乔晓玲陈文华田寒友杨君娜周彤曲超申思
Owner CHINA MEAT RES CENT
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