DNA metabarcoding detection target sequence, detection kit and detection method for screening animal provenance components in meat products

A detection kit and macro-barcode technology, which is applied in the field of DNA macro-barcode detection target sequence, can solve the problems of difficult design, poor specificity, and low sensitivity, so as to avoid false negative results, solve the difficulty of blind detection, and improve reliability Effect

Pending Publication Date: 2021-09-24
HUAZHONG AGRI UNIV
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Problems solved by technology

However, since there are more types to be detected, and the detection of deep-processed meat products has certain requirements on the sequence length, it is difficult to determine a marker with a suitable length and high amplification efficiency. Not advanced question
Aiming at the current problem of complex and diverse adulterated ingredients in meat products, common DNA barcodes need

Method used

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  • DNA metabarcoding detection target sequence, detection kit and detection method for screening animal provenance components in meat products
  • DNA metabarcoding detection target sequence, detection kit and detection method for screening animal provenance components in meat products
  • DNA metabarcoding detection target sequence, detection kit and detection method for screening animal provenance components in meat products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1.1. DNA metabarcode detection target sequence screening and universal primer design

[0044] Download 23 target species from NCBI's Nucleotide database (yak, common cattle, buffalo, goat, sheep, red deer, sika deer, pig, donkey, horse, dog, raccoon dog, fox, mink, cat, rabbit, guinea pig, mouse, Rat, hamster, chicken, duck, goose) target sequence, and use MEGA software for sequence alignment analysis, and design a pair of universal primers in the conserved region. The forward primer region contains three variant sites in the reference genomes of 23 species. The design uses degenerate bases when synthesizing the variant sites. The base M is synthesized according to the degenerate base A / C, and the base Y is synthesized according to the degenerate base A / C. The degenerate base C / T is synthesized, the base R is synthesized according to the degenerate base A / G, and the reverse primer region is completely conserved, and the amplified region has a high degree of interspecies...

Embodiment 2

[0062] Detection of target species-derived components in the sample of Example 2

[0063] 2.1. Sample collection and DNA extraction

[0064] Sample collection and DNA extraction were the same as in Example 1.

[0065] 2.2. Sample preparation

[0066] The DNA of 23 species was mixed in the same proportion, each species’ DNA content was 4.35%, and the final concentration was about 0.87 ng / μL. It was used as a test sample and stored at -20°C for future use.

[0067] 2.3.PCR amplification and product recovery and purification

[0068] PCR amplification is the same as in Example 1.

[0069] Purification of PCR products using Gel DNA Extraction Mini Kit kit or other recognized methods for purification of amplified products with the same efficacy.

[0070] 2.4. Next Generation Sequencing

[0071] Illumina library construction kit was used to construct the sequencing library of the purified amplified products, and the Illumina NovaSeq PE250 platform was used for next-generation...

Embodiment 3

[0084] Embodiment 3 sensitivity test

[0085] The DNA of 23 species is mixed in different proportions, and 14 food animals (yak, common cattle, buffalo, goat, sheep, red deer, sika deer, pig, donkey, horse, rabbit, chicken, duck, goose) are used as the main ingredients , the proportion of DNA in each species was 7.08%, and the final concentration was about 1.42ng / μL; 9 non-edible animals (dog, raccoon dog, fox, mink, cat, guinea pig, mouse, rat, hamster) were used as secondary Components, the proportion of DNA of each species is 0.10%, the final concentration is about 0.02ng / μL.

[0086] The PCR amplification is the same as in Example 1, and the recovery and purification of PCR amplification products, next-generation sequencing, and bioinformatics analysis are the same as in Example 2. The test results are shown in Table 3. The test results showed that after the sequencing data were analyzed by bioinformatics, all species components in the DNA mixed samples were identified. ...

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Abstract

The invention provides a DNA metabarcoding detection target sequence, a detection kit and a detection method for screening animal provenance components in meat products. The DNA metabarcoding detection target sequence is a fragment on a mitochondrial 16SrRNA gene, the length of the fragment in 23 target species is 368-384 bp, and the kit comprises a pair of universal primers and a reaction reagent. When the detection kit is used for screening animal provenance components in the meat products, the method comprises the following steps: carrying out PCR amplification on genome DNA of a detected sample by adopting the universal primer, and recovering and purifying the amplification product; carrying out next-generation sequencing on the purified amplification product; and performing comparison or bioinformatics analysis to determine the provenance components of the detected sample. A reliable means is provided for non-directional screening of 23 species (including 14 edible animals and 9 non-edible animals) in the meat products, and the detection method has the advantages of being non-directional, high in throughput, high in sensitivity and the like.

Description

technical field [0001] The invention belongs to the technical field, and in particular relates to a DNA macrobarcode detection target sequence, a detection kit and a detection method for screening animal provenance components in meat products. Background technique [0002] With the improvement of people's quality of life, the global demand for meat products is also increasing. The detection of animal provenance components in meat products has become an important task for food supervision and market management departments. The "Food Safety Law of the People's Republic of China", which came into effect on October 1, 2015, clearly prohibits the production and operation of food produced with non-food raw materials, and emphasizes that the label of prepackaged food should indicate its correct ingredients. Violators will be punished by local health administration Departments, food safety supervision and management departments will impose severe penalties. Subsequently, in 2017, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2531/113C12Q2535/122C12Q2537/165
Inventor 刘榜王心一周翔王文君吴清清
Owner HUAZHONG AGRI UNIV
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