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Double digital PCR (polymerase chain reaction) method of African struthio camelus derived ingredient quantitative detection

A technology for quantitative detection of source components, applied in the field of molecular biology detection, can solve problems such as no quantitative detection methods, save reagents and time costs, and avoid systematic errors.

Active Publication Date: 2018-05-22
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 ostrich-derived components in food 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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  • Double digital PCR (polymerase chain reaction) method of African struthio camelus derived ingredient quantitative detection
  • Double digital PCR (polymerase chain reaction) method of African struthio camelus derived ingredient quantitative detection
  • Double digital PCR (polymerase chain reaction) method of African struthio camelus derived ingredient quantitative detection

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Experimental program
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Effect test

preparation example Construction

[0065] 1. Preparation of samples and extraction of DNA templates: Take 25-30 g of samples (meat or meat-based foods, etc.) and cut them into pieces, then use a tissue grinder to crush them. The crushing conditions are 1800 rpm for 3 minutes. Weigh 20mg~50mg of the prepared sample into a 1.5mL centrifuge tube, and extract the DNA of the sample using the kit method. 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] The ddPCR (Droplet digital PCR) reaction system is 20 μL, and the components are as follows: 2×ddPCR TM 10 μL of master mix; 0.8 μL of each primer with a concentration of 10 μmol / μL, 0.4 μL of each probe with a concentration of 10 μmol / μL, 2 μL of DNA template, and replenish water to 20 μL.

[0069] Add 20 μL of the reaction system and 70 μL of droplet generation oil into the droplet generation card slot,...

Embodiment 1

[0103] Example 1 Verification of the Relative Qualitative Detection Limit of the African Ostrich-derived Component Copy Number Concentration

[0104] Samples to be tested: Genomic DNA from pigs, cattle, sheep, and chickens is used as a matrix, and African ostrich genomic DNA is mixed according to the copy number percentage to form a test DNA sample with a copy number percentage of African ostrich genomic DNA 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 African ostrich-derived components was 0.01%.

Embodiment 2

[0105] Example 2 Verification of the Relative Quantitative Detection Limit of the Copy Number Concentration of African Ostrich-derived Components

[0106] Samples to be tested: In order to verify the quantitative detection limit of this method, pig, cow, sheep, and chicken genomic DNA were used as substrates, and African ostrich genomes with copy number percentages of 0.1%, 1%, 10%, and 100% were incorporated into them DNA. Three parallel ddPCR and cdPCR experiments were carried out respectively, and the obtained experimental results are shown in image 3 and Figure 4 .

[0107] For African ostrich genomic DNA samples with copy number percentages of 0.1%, 1%, 10% and 100%, the detection results on the ddPCR platform were 0.098%, 1.062%, 9.697% and 101.34%, respectively, and the three parallel The RSD value was between 1.03% and 5.34%, and the recovery rate was between 96.97% and 101.34%. The RSD value is between 3.08% and 14.11%, and the recovery rate is between 97.91% an...

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Abstract

The invention provides a double digital PCR (polymerase chain reaction) method of African struthio camelus derived ingredient quantitative detection. A two-channel detection method is used; a digitalPCR system is used; meanwhile, two kinds of fluorescence signals of African struthio camelus specific species gene and higher animal specific gene are detected; the sequence detection probes of the African struthio camelus specific species gene and the higher animal specific gene are respectively marked as FAM and VIC; the sequence copy number of the African struthio camelus specific species geneand the higher animal specific gene is measured through the same PCR system; the relative content of the African struthio camelus derived ingredients accounting for higher animal derived ingredients is obtained through calculation. The method has the advantage that the relative quantification on the copy number proportion of the African struthio camelus derived ingredients accounting for the totalmeat derived ingredients in meat food can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a double digital PCR method for quantitative detection of components derived from African ostriches. Background technique [0002] The "horsemeat scandal" 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 the world is facing. 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. Wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686
CPCC12Q1/686C12Q1/6888C12Q2600/158C12Q2537/143C12Q2563/159C12Q2537/16
Inventor 刘津李志勇高东微易敏英李婷凌莉李伟琦
Owner TECH CENT OF GUANGZHOU CUSTOMS
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