A combination of primers for the simultaneous detection of 14 animal-derived components in meat or meat products and its application

A technology of animal-derived components and primer combinations, which is applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of single-plex PCR detection of few species, limited popularization and application, and low detection efficiency. , to achieve the effect of strengthening meat market supervision, improving detection efficiency, and accurate inspection results

Active Publication Date: 2021-08-17
CHINA MEAT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Single-plex PCR detects few species, low detection efficiency, and high detection cost, so multiplex PCR technology has gradually become the focus of research
At present, based on multiplex PCR technology, it mainly relies on agarose gel electrophoresis to distinguish PCR amplification products of different lengths. This method has disadvantages and limitations such as complicated process and environmental pollution.
Although the next-generation sequencing technology is gradually applied to the detection of animal-derived components, its popularization and application is limited due to the high cost of instruments and reagents.

Method used

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  • A combination of primers for the simultaneous detection of 14 animal-derived components in meat or meat products and its application
  • A combination of primers for the simultaneous detection of 14 animal-derived components in meat or meat products and its application
  • A combination of primers for the simultaneous detection of 14 animal-derived components in meat or meat products and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 5 heavy PCR reaction systems and detection results of the present invention

[0069] 1. Sample processing method

[0070] Weigh 25 mg of 14 common meat samples of pig, cow, sheep, goat, chicken, duck, dog, fox, raccoon dog, deer, cat, mouse, horse, and donkey into centrifuge tubes.

[0071] 2. DNA extraction method

[0072] Use DNeasy Animal Tissue DNA Extraction Kit to extract the DNA of each species, operate according to the kit instructions, or other recognized extraction methods with the same efficacy. The extracted DNA was measured with a fluorescent microplate reader at 260nm and 280nm absorbance, and the DNA concentration and purity were calculated.

[0073] 3. Primer sequence

[0074] A reaction well:

[0075] Specific primers required for amplification of bovine components are:

[0076] Bovine F: 5'-GTTCTTCACGACACATACTACGTT-3' (SEQ ID NO.1)

[0077] Bovine R: 5'-GCAAATACAGCTCCTATTGATAAA-3' (SEQ ID NO.2)

[0078] Specific primers required for...

Embodiment 2

[0114] Embodiment 2 The specificity of 10 pairs of primer systems of 5 times PCR of the present invention

[0115] 1. Template preparation method

[0116] Referring to the DNA extraction method in Example 1, the DNA of 16 species of pigs, cattle, sheep, goats, chickens, ducks, dogs, foxes, raccoon dogs, cats, mice, donkeys, deer, horses, fish, and mink were extracted respectively, and calculated DNA concentration and purity. Dilute the DNA samples of 16 species to 1ng / μL for later use.

[0117] 2. 5-fold PCR reaction

[0118] 2.1A reaction well

[0119] With reference to the 5-fold PCR reaction in Example 1, the mixed primers in reaction well A were mixed with pigs, cattle, sheep, goats, chickens, ducks, dogs, foxes, raccoon dogs, cats, mice, donkeys, deer, horses, fish, and mink respectively. 16 templates were amplified.

[0120] 2.2B reaction well

[0121] With reference to the 5-fold PCR reaction in Example 1, the mixed primers in reaction wells B were mixed with pigs...

Embodiment 3

[0124] Example 3 The detection limit of the method for identifying animal-derived components in meat according to the present invention

[0125] 1. Template preparation method

[0126] 1.1A reaction well

[0127] Referring to the DNA extraction method in Example 1, cow, donkey, fox, deer, and horse DNA were mixed in equal proportions as the initial template, and the concentration of each component was 1 ng / μL, and then the initial template was diluted 10 times to 10 -3 ,spare. The amount of template added was 2 μL.

[0128] 1.2B reaction well

[0129] Referring to the DNA extraction method in Example 1, cat, pig, mouse, sheep, and duck DNA were mixed in equal proportions as the initial template, and the concentration of each component was 1 ng / μL, and then the initial template was diluted 10 times to 10 -3 ,spare. The amount of template added was 2 μL.

[0130] 2. 5-fold PCR reaction

[0131] The reaction was carried out with reference to the 5-fold PCR system in Exampl...

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Abstract

The invention relates to the technical field of food detection, and discloses a primer combination, a method and a kit for synchronously detecting 14 kinds of animal-derived components in meat or meat products. The present invention uses 10 pairs of specific primers to identify whether pigs, cattle, sheep, goats, chickens, ducks, dogs, foxes, raccoon dogs, cats, and mice are contained in meat or meat products based on two 5-fold PCR reactions and microchip electrophoresis technology. , donkey, deer and horse 14 animal-derived components, the sequences of the 10 pairs of specific primers are shown in SEQ ID NO.1-20. The method of the present invention has the advantages of accurate detection, high sensitivity, strong specificity, simplicity, rapidity and high efficiency, and can satisfy large-scale and rapid identification of whether pigs, cattle, sheep, goats, chickens, ducks, dogs, foxes, etc. are contained in meat or meat products. Requirements for ingredients derived from raccoon dogs, cats, rats, donkeys, deer and horses can effectively resist the adulteration of meat products, effectively protect the interests of consumers, and have good social benefits.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a primer combination, a detection method and a kit for identifying 14 kinds of animal-derived components in meat or meat products. Background technique [0002] Meat adulteration has always been a quality problem in the fields of food processing, distribution, and catering. Due to the temptation of interests brought about by price differences, some unscrupulous traders and companies have been driven to adulterate pigs, ducks, and other high-priced meat products such as cattle, sheep, and deer. , chicken and other low-priced meat raw materials. The outbreak of the "horse meat scandal" in Europe in 2013 pushed people's attention to meat adulteration to a climax. This not only seriously violated the interests of consumers, but also caused great harm to the entire meat industry, and more seriously, it caused adverse social impacts. [0003] The detection technology of animal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 李金春周彤李家鹏许随根杨君娜黄鑫米瑞芳熊苏玥乔晓玲
Owner CHINA MEAT RES CENT
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