Kit for detecting rabbit-derived components in food and application of kit

A rabbit-derived, kit-based technology, 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 rabbit-derived components has not yet been reported, and achieve good detection. The effect of the ability, sensitivity and accuracy of rabbit-derived ingredients in food

Inactive Publication Date: 2015-04-22
北京市食品安全监控和风险评估中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 rabbit-derived components has not been reported

Method used

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  • Kit for detecting rabbit-derived components in food and application of kit
  • Kit for detecting rabbit-derived components in food and application of kit
  • Kit for detecting rabbit-derived components in food and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The design of embodiment 1 primer

[0024] After a large number of comparisons of the COX 1 gene in GenBank, the highly conserved and species-specific gene sequence of the COX 1 gene was selected as a template, and the upstream and downstream primers and probes specific to rabbit COX 1 were designed. The main feature is that the length of the upstream and downstream primers is 17bp And 24bp nucleotides, the probe length is 26bp 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:

[0025] Upstream primer: 5`-CTGCATGGCGGCAACAT-3`(SEQ ID NO.1)

[0026] Downstream primer: 5`-AATTCCTGTAAGACCGCCTACTGT-3`(SEQ ID NO.2)

[0027] Fluorescent probe: 5`-FAM-AAATGATCCCCCGCTATGCTCTGAGC-TAMRA-3`(SEQ ID NO.3)

Embodiment 2

[0028] The establishment of embodiment 2 fluorescence quantitative PCR detection method

[0029] 1. Extract sample DNA

[0030] (1) Take 0.2g of rabbit 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.

[0031] (2) Add an equal volume of phenol:chloroform mixture (1:1), shake and mix, and centrifuge at 12,000 rpm for 10 min.

[0032] (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.

[0033] (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, sett...

Embodiment 3

[0045] Embodiment 3 specificity test

[0046] In order to verify the specificity of this kit, the genomic DNA of rabbits was used as a positive control, the DNA of 15 species such as pigs, cattle, and sheep were used as detection objects, and dd H 2 O is a blank control, and the fluorescent quantitative PCR detection system established in Example 2 is used to detect the above 16 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. The experimental results are shown in Table 1. The Ct value of the rabbit is 15.31, showing a positive result, while the CT values ​​of the other 15 species are all greater than 35, showing a negative result. This experiment proves that the kit has good species specificity.

[0047] Table 1 The inventive method is to the detection result of 16 kinds of animal DNAs

[0048] species

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Abstract

The invention provides a kit for detecting rabbit-derived components in a food and an application of the kit and belongs to the technical field of food detection. According to the kit provided by the invention, fluorescent quantitative PCR detection is carried out on a to-be-detected food by using primers shown in SEQ ID NO: 1-2 and a fluorescent probe shown in SEQ ID NO. 3, and whether the food contains the rabbit-derived components or not is judged according to a Ct value. The kit provided by the invention has the advantages of accurate detection, high sensitivity, strong specificity and simplicity and rapidness. The lowest detection limit is 100fg. The kit has a good ability of detecting the rabbit-derived components in the food and can be used for effectively resisting adulteration of meat products so as to enlarge the protective force 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 rabbit-derived components in food and an application thereof. 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 cr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2545/114C12Q2561/101
Inventor 王丹杨昕霆薛晨玉赵琳娜杨红莲李赫婧肖辉武艳茹陶庆会王猛
Owner 北京市食品安全监控和风险评估中心
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