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Fluorogenic quantitative PCR detection kit and detection method for detecting mass percent of beef component in mixed meat product

A technology of mass percentage and detection kit, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficult to determine illegal manufacturers, quantitative detection standards of beef component content, etc.

Inactive Publication Date: 2015-10-28
SHANGHAI FOOD RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing testing standards are mainly for qualitative testing of bovine-derived components in food, but currently there is no quantitative testing standard for beef component content (w / w) in meat products, making it difficult for regulatory authorities to determine the responsibility of illegal manufacturers

Method used

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  • Fluorogenic quantitative PCR detection kit and detection method for detecting mass percent of beef component in mixed meat product
  • Fluorogenic quantitative PCR detection kit and detection method for detecting mass percent of beef component in mixed meat product
  • Fluorogenic quantitative PCR detection kit and detection method for detecting mass percent of beef component in mixed meat product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 kit composition

[0049]The kit of the present invention comprises: total DNA extraction reagent, DNA purification reagent, 18S fluorescent quantitative PCR premix reagent, Beef fluorescent quantitative PCR premix reagent, and standard products. 18S fluorescent quantitative PCR premix reagent is the amplification reaction solution for eukaryotic general gene detection, which includes the upstream primer and downstream primer of eukaryotic general oligonucleotide, the nucleotide sequence of the upstream primer is as SEQ ID NO:1 As shown, specifically: 5'-CTGCCCTATCAACTTTCGATGG-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, specifically: 5'-TAATTTGCGCGCCTGCTG-3'; Beef fluorescence quantitative PCR premix reagent is bovine The amplification reaction solution for specific gene detection, which includes an upstream primer and a downstream primer of a bovine specific oligonucleotide, the nucleotide sequence of the upstream primer i...

Embodiment 2

[0059] Example 2 Identification of multi-species fluorescent PCR amplification curves of eukaryotic primers and bovine specific primers in the kit of the present invention

[0060] Main equipment:

[0061] Fluorescence quantitative PCR instrument (ABI 7900HT, the United States), refrigerated small centrifuge (Eppendorf 5418R, Germany), high-throughput tissue grinder (Wanbai, Shanghai), nucleic acid and protein analyzer (Thermo NanoDrop 2000, United States), 5mL syringe Wait.

[0062] Reagent test kit:

[0063] With embodiment 1.

[0064] Reagent:

[0065] PBS buffer, isopropanol solution, and TE buffer were purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0066] Samples to be tested: pure meat samples of cattle, pigs, goats, chickens, ducks, donkeys, dogs, silver carp, and bighead carp.

[0067] 1) Pretreatment of samples to be tested:

[0068] Weigh 2g of the sample to be tested into a 50mL centrifuge tube, add 20mL of PBS buffer, and grind for 80s with a grind...

Embodiment 3

[0072] Embodiment 3 adopts kit and detection method provided by the present invention to detect pure beef sample

[0073] The main instruments and equipment, kits and reagents are the same as in Example 2.

[0074] Sample to be tested: pure beef sample.

[0075] Carry out the same pretreatment, DNA extraction and purification, and fluorescent quantitative PCR reaction as in Example 2.

[0076] The similarity was compared by the amplification curves, in which the eukaryotic general gene amplification curve and the bovine specific gene amplification curve had the same figure 1 , figure 2 have similar amplification curves. By comparing the similarity of the melting curve, the melting curve of eukaryotic universal oligonucleotide primers to pure beef DNA is as follows: image 3 , the peak melting curve of eukaryotic general gene is at 85.5±0.5℃. The melting curve of bovine specific oligonucleotide primers on pure beef DNA is as follows: Figure 4 As shown, the peak of the m...

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Abstract

The invention belongs to the technical field of food quality and safety detection, and particularly discloses a fluorogenic quantitative PCR detection kit and detection method for detecting the mass percent of the beef component in a mixed meat product. The kit comprises a total DNA extraction reagent, a DNA purification reagent, a 18S fluorogenic quantitative PCR premixed liquid reagent, a Beef fluorogenic quantitative PCR premixed liquid reagent and a standard substance. The detection method includes the steps of pretreatment on a sample to be detected, DNA extraction and purification, fluorogenic quantitative PCR reactions and detection. The kit can be used for quantitatively determining the mass percent of the beef component in the mixed meat product and has the advantages of being convenient to use, low in cost and high in specificity and sensitivity, the detection time is short, and the extraction reagent is safe.

Description

technical field [0001] The invention belongs to the technical field of food quality and safety detection, and in particular relates to a real-time fluorescence quantitative PCR detection kit and a detection method for detecting the mass percentage of beef components in mixed meat products. Background technique [0002] The quality and safety of food has always been the focus of social attention. With the improvement of people's living standards, the consumption of meat products has become the mainstream of food consumption. To ensure the vital interests of consumers, it is necessary to ensure the quality of various meat products. At present, the use of adulterated meat products to deceive consumers and make huge profits has occurred frequently. Moreover, if pork is mixed with halal food, it will involve religious and ethnic issues, which is not conducive to social harmony and stability. The "adulteration and deception" of meat products not only seriously disrupts the normal ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/16C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 林露严维凌沈菊泉谢志镭
Owner SHANGHAI FOOD RES INST
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