Multi-animal-derived adulteration identification method based on whole genome sequencing technology

A whole-genome sequencing, animal-derived technology, applied in the field of biological analysis, can solve the problems of amplification bias, increase the complexity of data integration, and the difficulty of direct quantification of template DNA molecules, and achieve the effect of wide application prospects.

Active Publication Date: 2020-04-10
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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Problems solved by technology

However, the main method for meta-DNA barcoding analysis is still the standard PCR-amplification-dependent method, which has the limitation of requiring universal primers for specific markers, which are often lacking in all taxa even for the same marker.
Use of different universal markers and primers increases the complexity of data integration when different markers are used in different settings, or even when different primer pairs are used for the same marker
Second, even in the presence of universal primers, template DNA molecules of different sequences have different melting temperatures, which will lead to amplification bias
Therefore, template DNA molecules of different sequences are difficult to quantify directly

Method used

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  • Multi-animal-derived adulteration identification method based on whole genome sequencing technology
  • Multi-animal-derived adulteration identification method based on whole genome sequencing technology
  • Multi-animal-derived adulteration identification method based on whole genome sequencing technology

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Experimental program
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Effect test

Embodiment 1

[0037] 1. Mock sample preparation, library construction and next-generation DNA sequencing

[0038] The simulated samples were prepared from the meat of 15 kinds of animals, including duck, cow, camel, dog, horse, chicken, mouse, ferret, nutria, raccoon dog, rabbit, sheep, rat, pig and fox, among which Duck, beef, chicken and pork were purchased from local markets in Beijing, China, and other meats were collected and identified by the Xiamen Customs Technology Center from January 2017 to December 2018. The meat samples of the above species were selected mainly considering that some of them have great economic significance, and some of them often appear in adulterated foods.

[0039] Fresh meat samples were immediately frozen and stored in a -80°C freezer until use. The samples were pooled in two ways, a pooled sample containing an equal mass pool from 15 meats, hereinafter referred to as "M15"; M15 had three replicates, labeled "R1", "R2" and "R3". Another mixed sample conta...

Embodiment 2

[0117] The quantitative analysis of two kinds of mixtures of embodiment 2

[0118] The analysis in Example 1 shows that WGS plus mitochondrial genomes can qualitatively identify taxon components in mixed samples. To determine how quantitative the method is, a series of mock samples were prepared using different proportions of porcine and chicken material:

[0119] The M2 mixed sample in Example 1 was used for processing, and the DNA extraction, library construction, DNA sequencing and DNA analysis methods were the same as those of the M15 sample, and the DNA sequencing results are shown in Table 1. Such as Figure 5 As shown, the proportion of the unique sequence compared to the pig reassembled mitochondrial genome in the mixed sample NGS data (raw sequencing sequence) (3 groups mean) and the weight ratio of the mixed sample ( Figure 5 The correlation coefficient between A) is R 2 = 0.978. Similarly, the correlation coefficient between the proportion of the unique sequenc...

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Abstract

The invention discloses a multi-animal-derived adulteration identification method based on a whole genome sequencing technology. The multi-animal-derived adulteration identification method comprises the following steps: 1) constructing a mitochondrial genome database, comparing sequencing data with the mitochondrial genome database, and extracting mitochondrial sequences obtained by comparison; 2)reassembling mitochondrial genomes of various species in the mitochondrial sequences extracted in the step 1); 3) comparing the mitochondrial sequences extracted in the step 1) to a recombined mitochondrial genome in the step 2), and extracting the sequences compared to the recombined mitochondrial genome in the step 2); and 4) dividing the sequences compared to the recombined mitochondrial genome in the step 3) into two classes, extracting the sequences compared to the recombined mitochondrial genome of a single species, and analyzing the species composition of a mixture according to the number of the sequences. The method provided by the invention can qualitatively and quantitatively determine the content of various biological components in a complex meat sample, and has extensive application prospect in food and pharmaceutical industries.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a method for qualitative and quantitative analysis of mixed meat samples. Background technique [0002] Meat is an important part of people's daily consumption. However, many merchants make high profits by mixing expensive beef and mutton with cheap chicken, duck, mink or other animal meat, which damages the rights and interests of consumers. disrupted the market order. Therefore, it is very important to identify adulterated ingredients in meat and meat products. At present, real-time PCR technology is the mainstream technology for meat identification, but it can only detect a single variety, that is, detect the presence or absence of the tested variety, and cannot simultaneously determine multiple biological components of mixed meat samples qualitatively and quantitatively. Components, MTCs) source. [0003] With the widespread use of next-generation DNA sequencin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2537/165C12Q2545/113
Inventor 刘昶姜梅张慧孔凡德唐泰山
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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