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Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey

A dual fluorescence, donkey-derived technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems that have not been reported, achieve good sensitivity, increase protection, and resist adulteration fake effect

Inactive Publication Date: 2015-04-29
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 dual fluorescent PCR method for the simultaneous detection of animal-derived components in horses and donkeys has not been reported.

Method used

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  • Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey
  • Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey
  • Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The design of embodiment 1 primer

[0028] 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 horse COX 1 / donkey COX 1-specific primer pair and Taqman MGB probe were designed, which were named horse COX 1 -F(P1), Horse COX 1-R(P2), Donkey COX 1-F(P3), Donkey COX 1-R(P4), Horse COX 1-FAM-MGB-Probe(Probe-P5), Donkey COX 1-VIC-MGB-Probe (Probe-P6), the primer and probe sequences for dual Taqman MGB real-time fluorescent PCR amplification are as follows:

[0029] P1: 5`-AACCCCCCCTATTCGTTTGATCT-3`

[0030] P2: 5`-ACGGTCTGTGAGAAGCATGGT-3`

[0031] P3: 5`-AGCCTCCTAATCCGTGCTGAA-3`

[0032] P4: 5`-ATGCATGGGCAGTTACAATAACA-3`

[0033] P5: 5`-FAM-AGCCCCCCGGTCC-MGB-3`

[0034] P6: 5`-VIC-ACCCTGCTGGGAGAT-MGB-3`

Embodiment 2

[0035] The establishment of embodiment 2 fluorescence quantitative PCR detection method

[0036] 1. Extract sample DNA

[0037] (1) Take 0.2g of horse meat or donkey meat samples and cut them 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.

[0038] (2) Add an equal volume of phenol:chloroform mixture (1:1), shake and mix, and centrifuge at 12,000rpm for 10min.

[0039] (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.

[0040] (4) Take the supernatant to another tube, add 1 / 10 volume of 3mol / L sodium acetate and 2 times the volume of absolute ethanol,...

Embodiment 3

[0055] Embodiment 3 specificity test

[0056] In order to verify the specificity of this kit, horse and donkey genomic DNA were used as positive controls, and pigs, cattle, sheep, goats, chickens, ducks, pigeons, quails, turkeys, ostriches, gray geese, cats, mice, domestic Dog, rabbit, roe deer, fox, mink, camel, deer, salmon, rainbow trout, perch, crucian carp, grass carp. A total of 25 species were detected, 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 , figure 2 .

[0057] 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 ...

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Abstract

The invention provides a dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey, and belongs to the technical field of food detection. The method comprises the following steps: performing fluorescence quantitative PCR detection on a to-be-detected sample by using a primer of SEQ ID NO.1-6, judging whether the food contains original components of the horse and the donkey according to a Ct value. The detection method provided by the invention can fast identify and detect the original components of the horse and the donkey simultaneously within one reaction, the operation can be finished within 1-2 hours, and the method has the advantages of being accurate in detection, high in flexibility, strong in specificity, simple and fast, the lowest detection limit is 100fg, the requirements of fast identifying and detecting the original components of the horse and the donkey in large volumes can be satisfied, and the meat product adulteration can be effectively resisted, the protection on the consumer benefit is increased, and good social benefit is provided.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a double fluorescent PCR detection method for detecting components derived from horses and donkeys in food. Background technique [0002] In recent years, the proportion of animal-derived food in people's daily diet has gradually increased, and the proportion of consumption of various chilled fresh meat, intensively processed semi-finished meat, and cooked meat products has increased year by year. However, the quality and price of meat from different species vary greatly, creating huge 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 from time to time. 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 developm...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 杨昕霆赵琳娜薛晨玉王丹杨红莲韩月贝黄华暴瑞玲武艳茹肖辉
Owner 北京市食品安全监控和风险评估中心
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