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A dual digital PCR method for the quantitative detection of fox-derived components

A digital, fox-sourced technology, applied in the field of dual digital PCR, can solve the problems of unseen quantitative detection methods

Active Publication Date: 2021-07-20
TECH CENT OF GUANGZHOU CUSTOMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of fox-derived components in food and feed is limited to molecular biology detection techniques such as real-time fluorescent PCR, common PCR, and agarose gel electrophoresis, and there is no quantitative detection method that can meet the needs of the industry

Method used

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  • A dual digital PCR method for the quantitative detection of fox-derived components
  • A dual digital PCR method for the quantitative detection of fox-derived components
  • A dual digital PCR method for the quantitative detection of fox-derived components

Examples

Experimental program
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Effect test

preparation example Construction

[0065] 1. Sample preparation and DNA template extraction: Take 25-30 g of the sample (meat, meat products or feed, etc.) and cut it into pieces, then use a tissue grinder to crush it. The crushing condition is 1800 rpm for 3 minutes. Weigh 20mg~50mg of the prepared sample into a 1.5mL centrifuge tube, and use the kit method to extract the sample DNA. The kit can be used: Animal Tissue Genomic DNA Extraction Kit (KuraboQuickGene DNA Extraction Kit DT-S), Wizard Genomic DNA purification kit ( Promega, A1120), PSS nucleic acid automatic extractor and other DNA extraction methods.

[0066] 2. Preparation and dispersion of reaction system

[0067] (1) The reaction system of ddPCR digital PCR is:

[0068] ddPCR (Droplet digital PCR, Droplet digital PCR) reaction system is 20 μL, and the components are as follows: 10 μL of 2×ddPCRTM master mix; 0.8 μL of each primer with a concentration of 10 μmol / μL, and a probe with a concentration of 10 μmol / μL 0.4 μL each, 2 μL DNA template, an...

Embodiment 1

[0102] Example 1 Verification of the Relative Qualitative Detection Limit of the Copy Number Concentration of Fox-derived Components

[0103] Samples for testing: Using genomic DNA from pigs, cattle, sheep, and chickens as a substrate, mix fox genomic DNA according to the copy number percentage to form a test DNA sample with a fox genomic DNA copy number percentage of 0.01%. Three parallel ddPCR and cdPCR experiments were carried out, and the obtained data are shown in figure 1 and figure 2 . The results showed that both ddPCR and cdPCR could be detected when the content of fox-derived components was 0.01%.

Embodiment 2

[0104] Example 2 Verification of the Relative Quantitative Detection Limit of the Copy Number Concentration of Fox-derived Components

[0105] Samples to be tested: In order to verify the quantitative detection limit of this method, pig, cattle, sheep, and chicken genomic DNA were used as substrates, and fox genomic DNA with copy number percentages of 0.1%, 1%, 10%, and 100% were mixed into it . Three parallel ddPCR and cdPCR experiments were carried out respectively, and the obtained experimental results are shown in image 3 and Figure 4 .

[0106] For fox genomic DNA samples with copy number percentages of 0.1%, 1%, 10% and 100%, the detection results on the ddPCR platform were 0.099%, 1.006%, 9.700% and 103.41%, respectively, and the three parallel The RSD value was between 1.07% and 6.28%, and the recovery rate was between 97.03% and 103.41%; the detection results on the cdPCR platform were 0.10%, 0.96%, 10.06% and 101.39%, respectively, and the RSD among the three pa...

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Abstract

The invention provides a double digital PCR method for the quantitative detection of fox-derived components, which adopts a dual-channel detection method, uses a digital PCR system to simultaneously detect two fluorescent signals of fox-specific species genes and higher animal-specific genes, and converts fox-specific The probes for the detection of sex species genes and higher animal-specific gene sequences are labeled as FAM and VIC respectively, and the fox The relative content of ingredients derived from higher animal origin. The method can perform relative quantification of the copy number ratio of the fox-derived components in the meat-based food and / or feed to the total meat-derived components.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a double digital PCR method for quantitative detection of fox-derived components. Background technique [0002] The "horsemeat incident" that swept across Europe in early 2013 brought the adulteration of animal-derived ingredients in food to the forefront, and economically motivated adulteration (EMA) has become a hot issue in food safety that is commonly faced by the world. Meat-based foods containing meat ingredients without labels on the ingredient list, especially non-edible meat ingredients, have become one of the main types of EMA. In the "horsemeat scandal", beef products from 16 EU countries including France, Germany, and Italy all contain unlabeled horsemeat ingredients. Official investigations in South Africa found that some beef and mutton products include buffalo meat, donkey meat, and even kangaroo meat, giraffe meat, and zebra meat. When our country inve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6888
CPCC12Q1/6851C12Q1/6888C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/159
Inventor 李志勇高东微刘津李伟琦李婷
Owner TECH CENT OF GUANGZHOU CUSTOMS