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Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method

A real-time fluorescence quantitative and fox animal technology, applied in the field of molecular biology, can solve the problems of inaccurate detection results, rising prices, and reductions, and achieve stable, easy-to-operate, high-precision, and high-sensitivity effects

Active Publication Date: 2015-07-15
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Donkey meat has always been hailed as a delicious and healthy traditional food. However, in recent years, due to the greatly reduced breeding volume, the supply of donkey meat has decreased year by year, which cannot meet the hot market demand at all, and the price has continued to rise
The international retail giant Wal-Mart’s “Fake Donkey Meat” incident after New Year’s Day in 2014 is not a special case. Driven by profit, some restaurants and unscrupulous merchants bought problematic donkey meat and mixed it with cheap horse meat in order to seek power and profit. , fox meat and other strategies to deceive consumers, this cheap raw material has been deeply processed, and its composition can no longer be identified from the appearance
[0005] In addition, the current fluorescent quantitative PCR detection system for animal origin lacks an internal standard (IAC) to monitor the reaction system, which cannot avoid false negative results caused by reaction inhibitory factors, resulting in inaccurate test results

Method used

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  • Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method
  • Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method
  • Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Universal primer design: According to the mitochondrial 16SrRNA gene sequences of donkey, horse and fox downloaded from the NCBI database, the homology analysis tool DNAMAN software was used to compare their homology, such as figure 1 As shown, a pair of general PCR primers were designed by using the primer design software Primer5.0 according to the similar sequences at both ends of the screened core fragments.

[0059] 2. Design and construct a recombinant plasmid containing internal standard DNA: Use DNA random generation software to generate a DNA sequence so that no homologous DNA fragments appear after Blast in NCBI, and connect the donkey upstream and downstream of this random DNA sequence The general primer sequence of 3 species, horse and fox, thus forming a 100bp internal standard DNA sequence. This internal standard sequence was entrusted with artificial gene synthesis, the synthesized fragment was connected to the carrier PMD18-T, transformed into competen...

Embodiment 2

[0067] Using the kit of the present invention and the multiple real-time fluorescent quantitative PCR detection method to detect the animal origin of meat products, the method steps are as follows, and whether various meat samples to be tested contain donkey, horse, and fox animal origin components can be detected:

[0068] 1. DNA extraction: Take 50g of the meat sample to be tested, grind it thoroughly and mix well, and take 50mg from it for DNA extraction. DNA can be extracted with an animal tissue extraction kit, or the classic hand-held method (refer to the DNA extraction method of animal tissue in the Molecular Cloning Manual). Use the Nanodrop nucleic acid detector to detect the concentration and purity of the DNA sample, which is required to be 1-20ng / μl, and the OD value is between 1.7-1.8.

[0069] 2. Real-time fluorescent PCR amplification of the sample DNA to be tested

[0070] Add the reaction system shown in Table 1 into the PCR reaction tube, put the PCR reaction...

Embodiment 3

[0098] Application of embodiment 3 detection method

[0099] Select the actual sample to be detected, the sample is the meat products purchased from the major supermarkets in Jinan City, farmer's market, etc., adopt the test kit of the present invention to detect the authenticity of donkey meat according to the method described in Example 2, to verify the authenticity of the method use value.

[0100] Proceed as follows:

[0101] 1. DNA extraction: Extract DNA from the sample to be tested, and use a Nanodrop nucleic acid detector to detect that the DNA sample concentration is 20ng / μl, and the OD value is between 1.7-1.8.

[0102] 2. Real-time fluorescent PCR amplification of the sample DNA to be tested

[0103] Add the PCR reaction system shown in the following table into the PCR reaction tube, put the PCR reaction tube into the fluorescent quantitative PCR instrument, and complete the PCR amplification according to the following reaction conditions: Amplification program: 9...

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Abstract

The invention discloses a primer probe composition for identifying sources of animals including donkeys, horses and foxes. The primer probe composition comprises a pair of general primers, three special TaqMan probes, a competitive type interior label and a special TaqMan probe of the competitive type interior label. The invention further discloses a kit comprising the primer probe composition and a multiplex real-time fluorescence quantitative PCR detecting method. The primer probes are high in specificity and sensitivity, whether source ingredients of three animals including the donkeys, the horses and the foxes are contained in a sample or not can be detected, false negative can be effectively indicated, the detection accuracy is improved, and a new way is provided for authentication searching of the source ingredients of the multiple animals in skin, meat and processed products of the skin and the meat.

Description

technical field [0001] The invention relates to a primer probe composition, a kit and a multiplex real-time quantitative PCR detection method for quickly identifying three animal-derived components of donkey, horse and fox, belonging to the technical field of molecular biology. Background technique [0002] "Dragon meat in the sky, donkey meat in the ground" is the highest praise people give to donkey meat. From the perspective of nutrition and food science, donkey meat has better taste and higher nutrition than beef and pork. The amino acid composition in donkey meat is very comprehensive, and the content of 8 kinds of essential amino acids and 10 kinds of non-essential amino acids is very rich. The content of unsaturated fatty acids in donkey meat, especially the content of linoleic acid and linolenic acid with extremely high biological value, is much higher than that of pork and beef. Donkey meat has always been hailed as a delicious and healthy traditional food. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2600/166C12Q2537/143C12Q2561/101C12Q2545/101
Inventor 步迅张全芳刘艳艳范阳阳
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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