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A kit for detecting roe deer-derived components in food and its application

A technology of source components and kits, which is used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem that the fluorescent PCR method for the detection of roe deer-derived components has not been reported yet, and achieves accuracy. High, increase protection, resist the effect of adulteration

Inactive Publication Date: 2017-06-30
北京市食品安全监控和风险评估中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there have been many reports on the design of species-specific primers based on the differences in mitochondrial genomic DNA sequences, and the establishment of PCR and real-time fluorescent PCR methods. However, the fluorescent PCR method for the detection of roe deer-derived components has not been reported yet

Method used

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  • A kit for detecting roe deer-derived components in food and its application
  • A kit for detecting roe deer-derived components in food and its application
  • A kit for detecting roe deer-derived components in food and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Design of primers

[0025] After a large number of comparisons of CYTB genes in GenBank, the highly conserved and species-specific gene sequence of CYTB gene was selected as a template, and the roe deer CYTB-specific primer pair and Taqman probe were designed, which were named roe deer CYTB-F(P1) and roe deer CYTB-F(P1) respectively. R(P2), roe deer CYTB-Probe(Probe-P3), the primers and probe sequences for Taqman real-time fluorescent PCR amplification are as follows:

[0026] Roe deer CYTB-F: 5`-AGCAATACATTACACATCCGACACA-3`(SEQ ID NO.1)

[0027] Roe deer CYTB-R: 5`-AAATATTGATGCTCCGTTTGCA-3`(SEQ ID NO.2)

[0028] Roe deer CYTB-P3: 5`-FAM-AGCATTTCTCCTCTGTTACTCACATCTGCCG-TAMRA-3`(SEQ ID NO.3)

Embodiment 2

[0029] Example 2 Establishment of Fluorescent Quantitative PCR Detection Method

[0030] 1. Extract sample DNA

[0031] (1) Take 0.2g of roe deer meat sample and cut it into pieces as much as possible. Place in a 1.5ml centrifuge tube, add 1ml of cell lysis buffer, 20μl proteinase K (500μg / ml), and mix well. Water-bath in a constant temperature water bath at 65°C for 30 minutes, and shake the centrifuge tube several times intermittently. Centrifuge at 12,000 rpm for 5 minutes in a tabletop centrifuge, and transfer the supernatant to another centrifuge tube.

[0032] (2) Add an equal volume of phenol:chloroform mixture (1:1), shake and mix, and centrifuge at 12,000rpm for 10min.

[0033] (3) Take the supernatant to another tube, add an equal volume of chloroform, shake and mix, and centrifuge at 12,000 rpm for 10 min.

[0034] (4) Take the supernatant to another tube, add 1 / 10 volume of 3mol / L sodium acetate and 2 times the volume of absolute ethanol, mix well, settle at ro...

Embodiment 3

[0046] Embodiment 3 specificity test

[0047] In order to verify the specificity of this kit, roe deer genomic DNA was used as a positive control, and pigs, cattle, sheep, goats, chickens, ducks, pigeons, quails, turkeys, ostriches, gray geese, cats, mice, domestic dogs, Rabbit, horse, fox, mink, camel, deer, salmon, rainbow trout, perch, crucian carp, grass carp Genomic DNA is the detection object, and the fluorescent quantitative PCR method established in Example 2 is used to verify the specificity of the method of the present invention.

[0048]After the amplification was completed, the same threshold was taken to analyze the data after deducting the background fluorescence signal, and the Ct value of each sample was determined. See the experimental results figure 1 , the results showed that the Ct value of roe deer was 16.34, and the CT values ​​of the other 25 species were all greater than 35. This experiment proved that this kit has good species specificity.

[0049] T...

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Abstract

The invention provides a kit for detecting roe deer original component in food and application of the kit, and belongs to the technical field of food detection. The kit adopts a premier represented by the SEQ ID NO.1-2 and a fluorescence probe represented by the SEQ ID NO.3 to perform fluorescence quantitative PCR detection on to-be-detected food, and judges whether the food contains the roe deer original component according to a Ct value. The kit provided by the invention has the advantages of being accurate in detection, high in sensitivity, strong in specific, simple and fast, and the lowest detection limit is 100fg; the kit has good capacity for detecting the roe deer original component in the food, and is capable of effectively resisting meat product adulteration, and increasing the protection degree to the consumer benefit.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a kit for detecting roe deer-derived components in food and its application. Background technique [0002] The proportion of animal-derived food in people's daily diet is gradually increasing, and the proportion of consumption of various cold fresh meat, intensively processed semi-finished meat, cooked meat products, etc. is increasing year by year. However, there are great differences in the quality and price of meat of different species, which creates great profit margins for adulteration. In order to seek economic benefits, some unscrupulous companies and traders use low-cost meat instead of high-priced meat in meat products. have happened. This not only seriously violates the health and rights of consumers, but also involves religious beliefs, leading to ethnic issues, and directly affects the image of the country, the harmonious development of society and the credibi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2537/143C12Q2545/114C12Q2561/113C12Q2561/101
Inventor 薛晨玉杨昕霆赵琳娜王丹韩月贝郭淼毛婷杜建平陶庆会宋丽萍
Owner 北京市食品安全监控和风险评估中心
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