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Dual digital PCR (Polymerase Chain Reaction) method for quantitatively detecting turkey derived ingredients

A source component, quantitative detection technology, applied in the field of molecular biology detection, can solve problems such as unseen quantitative detection methods

Active Publication Date: 2018-05-29
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 turkey-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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  • Dual digital PCR (Polymerase Chain Reaction) method for quantitatively detecting turkey derived ingredients
  • Dual digital PCR (Polymerase Chain Reaction) method for quantitatively detecting turkey derived ingredients
  • Dual digital PCR (Polymerase Chain Reaction) method for quantitatively detecting turkey derived ingredients

Examples

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 products, 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, co...

Embodiment 1

[0102] Example 1 Verification of the relative qualitative detection limit of the copy number concentration of turkey-derived components

[0103] Samples for testing: using genomic DNA of pigs, cattle, sheep, and chickens as substrates, turkey genomic DNA is mixed according to the copy number percentage to form a test DNA sample whose copy number percentage of turkey genomic DNA is 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 turkey-derived components was 0.01%.

Embodiment 2

[0104] Example 2 Verification of the relative quantitative detection limit of the copy number concentration of turkey-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 turkey 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 .

[0106] For the turkey genomic DNA samples whose copy number percentages were 0.1%, 1%, 10% and 100%, the detection results on the ddPCR platform were 0.100%, 1.016%, 10.60% and 95.35%, respectively, between the three parallel The RSD value was between 1.11% and 12.88%, and the recovery rate was between 95.35% and 100.2%; the detection results on the cdPCR platform were 0.100%, 0.972%, 10.43% and 96.09%, respectively, an...

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Abstract

The invention provides a dual digital PCR (Polymerase Chain Reaction) method for quantitatively detecting turkey derived ingredients. The dual digital PCR method comprises the following steps: simultaneously detecting two fluorescence signals of a turkey specific species gene and a higher animal specific gene by adopting a dual-channel detection method and utilizing a digital PCR system, respectively marking probes for sequence detection of the turkey specific species gene and the higher animal specific gene as FAM and VIC, and calculating relative content of the turkey derived ingredients inhigh animal derived ingredients through copy numbers, which are detected in the same PCR system, of sequences of the turkey specific species gene and the higher animal specific gene. According to thedual digital PCR method provided by the invention, a ratio of the copy number of the turkey derived ingredients in total meat derived ingredients of meat food can be relatively quantified.

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 turkey-derived components. 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. When our country investigated ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6888
CPCC12Q1/6851C12Q1/6888C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/159
Inventor 高东微刘津易敏英董洁李伟琦李志勇
Owner TECH CENT OF GUANGZHOU CUSTOMS
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