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

A kit and dog-derived technology, which is applied in the field of kits for detecting dog-derived components in food, can solve the problems that the fluorescent PCR method for the detection of dog-derived components has not been reported, and achieve a fast and simple detection method that resists adulteration. False, good sensitivity effect

Inactive Publication Date: 2017-07-04
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 dog-derived components has not been reported yet.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Design of primers

[0025] After a large number of comparisons of the dog ND 2 gene in GenBank, the highly conserved and species-specific gene sequence of the ND 2 gene was selected as a template, and the dog ND 2-specific upstream and downstream primers and Taqman probes were designed. The lengths of the upstream and downstream primers were 19bp and 25bp nucleotides, the probe length is 28bp nucleotides, and the 5' end is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quencher group. The sequence is as follows:

[0026] Upstream primer: 5`-CTCCGGCCAATGGGTAATC-3`(SEQ ID NO.1)

[0027] Downstream primer: 5`-GAAGTGGAATGGAGATAGGCCTAGT-3`(SEQ ID NO.2)

[0028] Fluorescent probe: 5`-FAM-TCAAACCCCATCGCATCCATCATGATAA-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 dog 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 room te...

Embodiment 3

[0047] Embodiment 3 specificity test

[0048] In order to verify the specificity of this kit, the genomic DNA of domestic dogs was used as a positive control, and pigs, cattle, sheep, goats, chickens, ducks, pigeons, turkeys, quails, ostriches, gray geese, cats, mice, roe deer, Horse, donkey, fox, camel, salmon, perch, crucian carp, sika deer, rainbow trout, grass carp. A total of 24 species were used as negative controls, with dd H 2O is a blank control, and the fluorescent quantitative PCR detection system established in Example 2 is used to detect the above-mentioned 25 species. 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 , Table 1, the Ct value of the dog is 11.19, showing a positive result, and the CT values ​​of the remaining 24 species are all greater than 35, showing a negative result. ...

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Abstract

The invention provides a kit for detecting dog-source ingredients in food and application thereof and belongs to the technical field of food detection. The kit adopts a primer shown in SEQ ID NO.1-2 and a fluorescent probe shown in SEQ ID No.3 to perform fluorescence quantitative PCR detection on food to be detected, thereby judging whether dog-source ingredients exist in the food according to Ct value. The kit has the advantages of accurate detection, high sensitivity and strong specificity, is simple, convenient and quick, has the minimum limit of detection of 100fg, has favorable capability of detecting dog-source ingredients in food, can effectively prevent meat product adulteration, and do more to protect consumers' benefits.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a kit for detecting dog-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 credibility ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2537/165C12Q2545/114C12Q2563/107
Inventor 杨昕霆薛晨玉赵琳娜王丹黄华暴瑞玲胡智恺宋丽萍毛婷杜建平
Owner 北京市食品安全监控和风险评估中心
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