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High-throughput sequencing-based animal-derived ingredient identification method and application thereof

An animal component, high-throughput technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of effective identification of animal source components that cannot be mixed and unknown animal source components, and achieve flexible and changeable classification methods , Improve the speed of information acquisition and broad application prospects

Inactive Publication Date: 2017-06-09
SHANGHAI INST OF QUALITY INSPECTION & TECHN RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the need to establish a fingerprint library in advance, this method cannot give accurate results in time for uncommon meat ingredients.
Therefore, the current traditional PCR amplification method cannot effectively identify mixed animal-derived components and unknown animal-derived components

Method used

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  • High-throughput sequencing-based animal-derived ingredient identification method and application thereof
  • High-throughput sequencing-based animal-derived ingredient identification method and application thereof
  • High-throughput sequencing-based animal-derived ingredient identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Weigh 2 g of fresh mutton, homogenize, add CTAB lysate and protease at 50°C, incubate for 2 h, take 1 ml of lysate, centrifuge at 12000 g for 10 min, take supernatant, add an equal volume (chloroform: isoamyl alcohol: saturated phenol) ( 24:1:25), mix upside down, centrifuge at 12000g for 10min, take the supernatant, add (chloroform:isoamyl alcohol) (24:1), mix upside down, centrifuge at 12000g for 10min, take the supernatant, add 2 times ice-free Water ethanol, or 0.6 times isopropanol, place for 30min, centrifuge at 12000g for 10min, wash twice with 70% ethanol, air dry at room temperature, add ddH 2 O 100uL. Using Nanodrop to analyze the concentration and quality of extracted DNA, DNA (ng / μL): 356.05, OD (260 / 280): 1.88, OD (260 / 230): 2.17.

[0039] The sample was taken and diluted with pure water, the DNA was ultrasonically disrupted using Covaris S220, and a 200bp fragment was recovered by electrophoresis. The TruSeqDNASample Preparation kit was used to build a li...

Embodiment 2

[0047] Weigh 2 g of homogenized meat samples from pigs (commercially available), cattle (commercially available), sheep (commercially available), chicken (commercially available), rabbits (commercially available), and rats (laboratory source) respectively, add SDS to crack solution and protease at 55°C, incubate for 2 hours, take 1ml of the lysate, centrifuge at 12000g for 10min, take the supernatant, add an equal volume (chloroform: isoamyl alcohol: saturated phenol) (24:1:25), mix upside down, 12000g Centrifuge for 10 minutes, take the supernatant, add (chloroform:isoamyl alcohol) (24:1), mix upside down, centrifuge at 12000g for 10 minutes, take the supernatant, add 2 times ice-free ethanol, or 0.6 times isopropanol, and let stand for 30 minutes , centrifuged at 12000g for 10min, washed twice with 70% ethanol, air-dried at room temperature, added ddH 2 O 100uL. Extracted DNA concentration and quality were analyzed using Nanodrop (Table 3).

[0048] Table 3 DNA concentrati...

Embodiment 3

[0058] Weigh pig (commercially available) 1.05g, cattle (commercially available) 10.12g, sheep (commercially available) 20.36g, chicken (commercially available) 5.29g, after homogeneous mixing, weigh the mixed sample 5g, add SDS The lysate and protease were incubated overnight at 55°C, 1ml of the lysate was taken, centrifuged at 12000g for 10min, the supernatant was taken, and an equal volume (chloroform: isoamyl alcohol: saturated phenol) (24:1:25) was added, and mixed evenly by inversion. Centrifuge at 12000g for 10min, take the supernatant, add (chloroform:isoamyl alcohol) (24:1), mix upside down, centrifuge at 12000g for 10min, take the supernatant, add 2 times of ice absolute ethanol, or 0.6 times of isopropanol, and place 30min, centrifuge at 12000g for 10min, wash twice with 70% ethanol, air dry at room temperature, add ddH 2 O 100uL. Use Nanodrop to analyze the concentration and quality of extracted DNA DNA (ng / μL): 541.78; OD (260 / 280): 1.78; OD (260 / 230): 2.02. Thi...

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Abstract

The invention provides a high-throughput sequencing-based animal-derived ingredient identification method and application thereof. The method comprises the following steps: taking a deoxyribonucleic acid (DNA) sequence, obtained by high-throughput sequencing, as an analysis object, taking a public DNA sequence database as a reference, and investigating the pairing number of the analysis object and the reference sequence data, so as to reflect the existence or number of specific categories. The method is simple in operation steps and high in accuracy. The detection limit of specific types of animal-derived ingredients reaches 1%, and the species identification types of the method theoretically cover all species. Therefore, when being used for identifying the animal-derived ingredients in a sample, the method can analyze the animal-derived ingredients in the sample with unknown composition, and simplifies the operation process, so that the method has wide application prospect in the aspect of animal-derived ingredient identification.

Description

technical field [0001] The present invention relates to a method for the identification of ingredients of animal origin and its use. Background technique [0002] Meat food is the main channel for people to obtain high-quality protein, and the quality of meat food is closely related to human health. According to reports, in 2012, the annual consumption of meat products by human beings in the world was about 284 million tons, and the annual meat consumption in my country accounted for about a quarter of the world's consumption, of which processed and semi-processed meat products accounted for 4%, and These figures show a trend of substantial increase year by year, and people's demand for meat products is increasing day by day. In recent years, in domestic and international trade, beef and mutton have been mixed with cheap pork, and shoddy deceitful behaviors are common. Adding pork to halal meat instead of beef and mutton has seriously offended the majority of Muslims. Consu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2535/122C12Q2537/165
Inventor 张奕南段文锋陶飞曲勤凤徐琼顾文佳胡雪莲赵燕张清平
Owner SHANGHAI INST OF QUALITY INSPECTION & TECHN RES
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