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Kit for detecting ostrich-derived component in food and application of kit

A technology of ostrich-derived components and kits, which is applied in the field of kits for detecting ostrich-derived components in food, can solve the problems that the fluorescent PCR method for detecting ostrich-derived components has not been reported, and achieve accurate and rapid identification and detection, and increase High protection and high accuracy

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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  • 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, there have been no reports on fluorescent PCR methods for the detection of ostrich-derived components.

Method used

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  • Kit for detecting ostrich-derived component in food and application of kit
  • Kit for detecting ostrich-derived component in food and application of kit
  • Kit for detecting ostrich-derived component in food and application of kit

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 COX 1 genes in GenBank, the highly conserved and species-specific gene sequence of COX 1 was selected as a template, and the upstream and downstream primers and probes specific to ostrich COX 1 were designed. The main feature is that the length of the upstream and downstream primers is 25bp And 23bp nucleotides, the probe length is 22bp 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`-ATCACAAAGACATTGGCACCCTATA-3`(SEQ ID NO.1)

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

[0028] Fluorescent probe: 5`-FAM-AGTGGGCACAGCCCTCAGCCTG-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 ostrich 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 12,000rpm for 10min.

[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 temper...

Embodiment 3

[0044] Embodiment 3 specificity test

[0045] In order to verify the specificity of this kit, ostrich 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, Roe deer, horse, fox, mink, camel, deer, salmon, rainbow trout, perch, crucian carp, grass carp, a total of 25 species as negative controls, with 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-mentioned 26 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 ostrich is 14.89, showing a positive result, and the CT values ​​of the remaining 25 species are all greater than 35, showing a negative result. This experiment...

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Abstract

The invention provides a kit for detecting an ostrich-derived component in a food and application of the kit, belonging to the technical field of food detection. The kit provided by the invention is used for carrying out fluorescent quantitative PCR detection on a food to be detected by using primers as shown in SEQ ID NO.1-2 and a fluorescent probe as shown in SEQ ID NO.3, and then, whether the food contains the ostrich-derived component is judged according to a Ct value. The kit provided by the invention has the advantages of detection accuracy, high sensitivity, strong specificity, simplicity, convenience and high speed, has the minimum detection limit of less than 100fg, and is capable of favorably detecting the ostrich-derived component in the food, effectively preventing a meat product from being adulterated and increasing the protection power for the consumer benefit.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/6851
Inventor 赵琳娜杨昕霆薛晨玉王丹黄华暴瑞玲郭淼李赫婧胡智恺王猛
Owner 北京市食品安全监控和风险评估中心
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