CN1597982A - Process of extracting DNA for testing transgene component from food oil of soybean - Google Patents
Process of extracting DNA for testing transgene component from food oil of soybean Download PDFInfo
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- CN1597982A CN1597982A CN 200410020176 CN200410020176A CN1597982A CN 1597982 A CN1597982 A CN 1597982A CN 200410020176 CN200410020176 CN 200410020176 CN 200410020176 A CN200410020176 A CN 200410020176A CN 1597982 A CN1597982 A CN 1597982A
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Abstract
The invention relates to a method of distilling the DNA from the edible oil which takes the soybean as the material. It is used to detecting the transgene. The invention has following features: during distilling the DNA, separate water soluble components (including the partial dissociative DNA) with extremely low content from the fat soluble lipin; release the DNA molecule completely from the cells with the help of the extracting solution of the bromohexadecane trimethylamine at the room temperature; deposit it through the isopropyl alcohol with the same volume and the acetic acid with 1/10 volume to make the micromolecule DNA molecule; resolve the DNA solution into the 30mul deionized water, getting 15mul for once PCR reaction to increase the density of the DNA cyclostyle in the reaction system. The method is featured by high speed, convenience, strong specificity and high efficiency and suitable for the qualitative detection of the transgene in ketchup and other deep processing food.
Description
Technical field
Present method relates to the detection technique of transgene component in the deep processing food, especially from being to extract the method that is used for the template DNA that transgene component detects the edible oil of raw material with the soybean.
Background technology
Fast development along with genetic engineering technique, a large amount of is that the genetically modified food that processes raw material enters the commercialization production and selling with genetically modified crops, add up according to internal authority mechanism, kind surplus the whole world reaches 4000 by the genetically modified food of genetically modified crops processing and food ingredient (referring to: U.S.Food and Drug Administration, Office of Food AdditiveSafety.List of Completed Consultations on Bioengineering Food[DB/OL] .http: //www.fda-cfsan/biotechnology.2002-10.).The transgenic crop of the main plantation in the whole world is a soybean, and cultivated area reaches 3,330 ten thousand hectares, accounts for 63% of the total cultivated area of genetically modified crops.Because genetically engineered soybean oil yield height, it is low to make oily cost, and the numerous and confused import genetically engineered soybean of each developing country is as the oily raw material of system.At present still disputable to the security of genetically modified food, its potential risks cause that countries in the world government greatly pays close attention to, and therefore launch respectively the tag control system and the entry and exit product reporting system of genetically modified food.The requirement of United Nations and European Union is more strict, the regulation genetically modified food all needs to detect monitoring from studying, produce, transport, store, be sold to all links such as import and export, therefore an important process that becomes each main trading country check is identified in the detection of transgenic product, and the relevant stdn detection architecture of foundation, (referring to Zhu Guangfu, countries in the world are to the attitude and the management of genetically modified food, oil and foodstuffs science and technology to be adapted to various methods for detecting transgenic foods, 2001,3:1-5).
Because edible fat production needs through 240 ℃ for the treatment of processess such as high temperature and high pressure, the nucleic acid component in the product (DNA) is degraded to short, the not of uniform size dna fragmentation of length, and this causes very big difficulty to DNA extraction.And the DNA extraction technology remains the bottleneck based on the transgenosis detection technique of DNA cloning, be the key point that transgenosis detects success or not, therefore exploitation is important assurance that realization edible oil import and export check and the market access at the template DNA extractive technique that is the soybean oil transgenosis detection of raw material with the soybean.
Present domestic ten minutes lacks the template DNA extractive technique of the extremely low salad oil of targeted and exercisable food oils, particularly dna content, has seriously restricted the accuracy and the reliability of the transgene component detected result of these products.The present invention has now solved this detection technique difficult problem.
Summary of the invention
It is to extract template DNA the edible oil that processes of raw material that present method is intended to set up from genetically engineered soybean, and the PCR that is used for transgene component detects.
The objective of the invention is to solve the difficult problem in the existing detection technique, highly sensitive exercisable a kind of method of extracting the DNA that is used for the transgene component detection from the extremely low soybean salad oil of dna content is provided, so that set up quick, easy, reliable, high-level efficiency, be the template DNA extracting method of the edible oil of raw material with the soybean cheaply, the qualitative detection that is used for transgene component is with the reliability of guaranteeing to detect.
After present method is processed through the rigor condition of High Temperature High Pressure at edible oil, the characteristics that wherein water miscible dna molecular content is extremely low, degraded is serious, when entering DNA extraction, water soluble component that must content is extremely low (comprising the dna molecular that part has dissociated out) fully dissociates out from fat-soluble grease, utilize bromohexadecane base Trimethylamine 99 extracting solution in room temperature dna molecular fully to be discharged from cell again, this is first key of extracting DNA from grease; And the sodium acetate that utilizes equal-volume Virahol and 1/10 volume fully precipitates the extremely low small molecule DNA of content through long-time precipitation, is second key of further concentration of DNA molecule.This method is quick, easy, high specificity, and efficient height, cost are low, and being fit to the soybean is the qualitative detection of transgene component in the deep processing edible oil of raw material.
Technical scheme of the present invention is summarized as follows:
From being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is made up of following step:
1. get the 20-30mL sample, add 20-30mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 40-60mL TE damping fluid, continue on magnetic stirring apparatus, to stir 6-15 hour,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 3～6 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 10%, mixing, 20-25 ℃ left standstill 6-15 hour, 12000 rev/mins, centrifugal 20 minutes, stayed precipitation.
5. add 1mL TE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
6. add the 3mol/L sodium acetate of 1/10 volume and the Virahol of isopyknic 4 ℃ of precoolings, slow mixing, 4 ℃ left standstill 6-15 hour.
7. in 4 ℃, 14000 rev/mins, centrifugal 15 minutes, abandon supernatant.
8. with the 70% washing with alcohol precipitation of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
9. add the DNA precipitation that the deionized water dissolving of 25-35 μ L through sterilizing extracts from edible oil.
Preferably, among the present invention from being that to extract the method that is used for the DNA that transgene component detects the edible oil of raw material as follows with the soybean:
1. get the 25mL sample, add 25mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 50mL TE damping fluid, continue on magnetic stirring apparatus, to stir 12 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 6 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 1/10 volume, mixing, 20-25 ℃ left standstill 12 hours, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
5. add 1mL TE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
6. the 3mol/L sodium acetate that adds isopyknic Virahol and 1/10 volume, mixing, 20-25 ℃ left standstill 12 hours, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
7. in 4 ℃, 14000 rev/mins, centrifugal 15 minutes, abandon supernatant, stay precipitation.
8. with the 70% washing with alcohol precipitation of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
9. add the DNA precipitation that the deionized water dissolving of 30 μ L through sterilizing extracts from edible oil.
The invention has the beneficial effects as follows: provide a kind of from the extremely low edible oil of dna molecular content the qualitative checking method of transgene component, this method is quick, easy, high specificity, efficient height, cost are low, and being fit to the soybean is the qualitative detection of transgene component in the deep processing edible oil of raw material.
Description of drawings
Fig. 1 good fortune is the endogenous special pcr amplification result with reference to the Lectin gene of soybean salad oil DNA near the house
(1.DNA Marker; 2. soybean seeds contrast; 3. good fortune soybean salad oil near the house)
The endogenous special pcr amplification result of the imperial fish soybean salad oil DNA of Fig. 2 gold with reference to the Lectin gene
(1.DNA Marker; 2. soybean seeds contrast; 3. golden imperial fish soybean salad oil)
Embodiment
1. material and reagent
1) material: the soybean oil sample is available from the supermarket, contrasts to be soybean seeds.
2) extract reagent:
DNA extraction damping fluid (1L): dissolving bromohexadecane base Trimethylamine 99 20g in the 1000ml water, Tris 12.114g, NaCl 81.816g, Na
2EDTA 7.444g, polyvinylpyrrolidone 20g, autoclaving;
Sodium acetate solution (3mol/L): dissolving 408.1g sodium acetate trihydrate in the 800ml water, transfer pH to 5.2 with glacial acetic acid, add water and be settled to 1L, autoclaving;
TE damping fluid: Tris 0.01mol/L, Na
2EDTA 1.0mmol/L transfers pH to 8.0, autoclaving with hydrochloric acid;
Normal hexane; Sherwood oil; Trichloromethane: primary isoamyl alcohol (24: 1); 70% ethanol; Virahol; The deionization sterilized water.
2.DNA the flow process of extracting:
(1) get the 25mL sample, add 25mL normal hexane or sherwood oil, mixing, room temperature stirred on magnetic stirring apparatus 2～3 hours.
(2) add 50mL TE damping fluid, continuation is stirred on magnetic stirring apparatus in room temperature and is spent the night, and then in 4 ℃, 12000 rev/mins, centrifugal 20 minutes, carefully removes oil phase.
(3) add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing, room temperature was placed 6 hours, 12000 rev/mins, centrifugal 20 minutes, got supernatant.
(4) add the 3mol/L sodium acetate of isopyknic Virahol and 10%, mixing, room temperature was placed 12 hours, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
(5) add 1mL TE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
(6) add the 3mol/L sodium acetate of 1/10 volume and the Virahol of isopyknic 4 ℃ of precoolings, slow mixing, 4 ℃ left standstill 12 hours.
(7) in 4 ℃ 14000 rev/mins, centrifugal 15 minutes, abandon supernatant, stay precipitation.
(8) with 70% the washing with alcohol precipitation of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
(9) add the DNA precipitation that the deionized water dissolving of 30 μ L through sterilizing extracts from edible oil, get the template of 15 μ L as the PCR reaction; DNA extraction thing with the soybean seeds that compares dissolves with 100 μ L TE, and it is standby to put-20 ℃ of preservations.
(10) whether extract the DNA that can be used for the PCR detection, adopt the qualitative TRAP of special endogenous reference gene PCR to identify.Experimental technique and condition are as follows:
1) the PCR primer sequence of special endogenous reference gene (seeing Table 1)
The PCR primer sequence of the special endogenous reference gene of table 1
Detect gene primer sequence amplification fragment length raw material crop
Just: 5 '-gcc ctc tac tcc acc ccc atc c-3 '
Lectin 118bp soybean
Instead: 5 '-gcc cat ctg caa gcc ttt ttg tg-3 '
2) the PCR reaction system of special endogenous reference gene is formed (seeing Table 2)
The amount of each reagent is suitably adjusted according to the cumulative volume of reaction system in the reaction system, and each reaction system should be provided with two parallel pipes.The reaction system that soybean oil DNA quality examination is adopted is identical.
Table 2 PCR detects reference reaction system (25 μ L system)
Reagent name adds the amount of PCR reaction system
10 * PCR reaction buffer (contains Mg
2+) 2.5 μ L
DNTP solution (every kind is 10mmol/L) 0.5 μ L
Forward primer (10pmol/ μ L) 2.5 μ L
Reverse primer (10pmol/ μ L) 2.5 μ L
Taq enzyme (2U/ μ L) 2.0 μ L
*Template DNA 10～15 μ L
Water is supplied reaction system, and making cumulative volume is 25 μ L
*: the dna profiling consumption of positive control sample is 1 μ L in the table.
3) the PCR reaction conditions of special endogenous reference gene (seeing Table 3)
The PCR of the special endogenous reference gene of table 3 detects the reference reaction condition
The gene sex change amplification cycles number of times that is amplified extends at last
94℃，30s
62℃，30s
Lectin 96℃，2min 40 72℃，3?min
72℃，25s
4) deposition condition
Sepharose (gel thawing back room temperature adds ethidium bromide when being placed into 60 ℃ of left and right sides, and content is 0.5g/mL) with 1 * TAE electrophoretic buffer preparation 2%.With PCR product and the sample-loading buffer uniform mixing of 10 μ L, join then in the gel pore in proportion, (3V/cm～5V/cm) carry out electrophoresis, electrophoresis time is 40～60 minutes to select suitable voltage.Place sepharose on the gel imaging instrument or observation and photograph on the ultraviolet transilluminator.
Below in conjunction with specific embodiment the present invention is further described.
Embodiment 1:
The good fortune of producing with commercially available Baihai Grain-Oil Industry Co., Ltd., Tianjin soybean salad oil near the house is a material, adopts following method to extract wherein DNA,
1. get the 25mL sample, add 25mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 50mL TE damping fluid, continue on magnetic stirring apparatus, to stir 12 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 5 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 10%, mixing was placed 12 hours for 20-25 ℃, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
5. add 1mL TE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
6. add the 3mol/L sodium acetate of 1/10 volume and the Virahol of isopyknic 4 ℃ of precoolings, slow mixing, 4 ℃ left standstill 12 hours.
7. in 4 ℃, 14000 rev/mins, centrifugal 15 minutes, abandon supernatant, stay precipitation.
8. with the 70% washing with alcohol precipitation of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
9. add the DNA precipitation that the deionized water dissolving of 30 μ L through sterilizing extracts from edible oil.
With traditional method for extracting soybean seeds DNA in contrast.With the Auele Specific Primer of the special native gene lectin of soybean, the DNA that extracts with present method is a template, identifies that by pcr amplification whether the DNA that extracts by present method can satisfy the requirement that transgene component detects, and the results are shown in Figure 1.
As seen from Figure 1, the DNA that from salad oil, extracts, after the special primer that utilizes the lectin gene carried out pcr amplification, the amplified fragments that obtains was in full accord with the amplified fragments size of contrast soybean seeds, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
The good fortune of producing with commercially available Baihai Grain-Oil Industry Co., Ltd., Tianjin soybean salad oil near the house is a material, adopts following method to extract wherein DNA,
1. get the 20mL sample, add 20mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 40mL TE damping fluid, continue to stir 6 hours on magnetic stirring apparatus at 20-25 ℃, then in 4 ℃, 12000 commentaries on classics/powder clocks centrifugal 15-20 minute, are carefully removed oil phase.
All the other steps and process are with embodiment 1.
1. get the 30mL sample, add 30mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 60mL TE damping fluid, continue on magnetic stirring apparatus, to stir 15 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
All the other steps and process are with embodiment 1.
Embodiment 4
1. get the 25mL sample, add 25mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 50mL TE damping fluid, continue on magnetic stirring apparatus, to stir 10 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 12 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
The 4-8 step is with embodiment 1.
9. add the DNA precipitation that the deionized water dissolving of 25 μ L through sterilizing extracts from edible oil.
All the other processes are with embodiment 1.
Embodiment 5
1. get the 25mL sample, add 25mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 50mL TE damping fluid, continue on magnetic stirring apparatus, to stir 15 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 3 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 1/10 volume, mixing was placed 15 hours for 20-25 ℃, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
All the other steps and process are with embodiment 1.
Embodiment 6
The Golden Elephant soybean salad oil of producing with Industrial Co., Ltd. of Zhengda Group (Tianjin) is a material, adopts following method extraction DNA wherein:
1. get the 25mL sample, add 25mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 50mL TE damping fluid, continue on magnetic stirring apparatus, to stir 12 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 5 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 10%, mixing was placed 12 hours for 20-25 ℃, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
5. add 1mL TE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant.
6. add the 3mol/L sodium acetate of 1/10 volume and the Virahol of isopyknic 4 ℃ of precoolings, slow mixing, 4 ℃ left standstill 12 hours.
7. in 4 ℃ 14000 rev/mins, centrifugal 15 minutes, abandon supernatant, stay precipitation.
8. with the 70% washing with alcohol precipitation of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation.
9. add the DNA precipitation that the deionized water dissolving of 30 μ L through sterilizing extracts from edible oil.
With traditional method for extracting soybean seeds DNA in contrast.With the Auele Specific Primer of the special native gene lectin of soybean, the DNA that extracts with present method is a template, identifies that by pcr amplification whether the DNA that extracts by present method can satisfy the requirement that transgene component detects, and the results are shown in Figure 2.
As seen from Figure 2, the DNA that from salad oil, extracts, after the special primer that utilizes the lectin gene carried out pcr amplification, the amplified fragments that obtains was in full accord with the amplified fragments size of contrast soybean seeds, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
Embodiment 7 detects
1. get the 30mL sample, add 30mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 60mL TE damping fluid, continue on magnetic stirring apparatus, to stir 15 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
All the other steps and process are with embodiment 6.
Embodiment 8
The 1-2 step is same as embodiment 6.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 5 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
The 4-8 step is same as embodiment 6.
9. add the DNA precipitation that the deionized water dissolving of 35 μ L through sterilizing extracts from edible oil.
All the other processes are with embodiment 6.
Embodiment 9
1. get the 20mL sample, add 20mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours.
2. add 40mL TE damping fluid, continue on magnetic stirring apparatus, to stir 6 hours,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃.
3. add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 3 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant for 20-25 ℃.
4. the 3mol/L sodium acetate that adds isopyknic Virahol and 1/10 volume, mixing was placed 6 hours for 20-25 ℃, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant, stay precipitation.
All the other steps and process are with embodiment 6.
Embodiment 10
The 1-8 step is with embodiment 6.
9. add the DNA precipitation that the deionized water dissolving of 25 μ L through sterilizing extracts from edible oil.
All the other processes are with embodiment 6.
Claims (7)
1. from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is made up of following step:
(1) get the 20-30mL sample, add 20-30mL normal hexane or sherwood oil, 20-25 ℃ was stirred on magnetic stirring apparatus 2～3 hours;
(2) add 40-60mL TE damping fluid, continue on magnetic stirring apparatus, to stir 6-15 hour,, 12000 rev/mins, centrifugal 15-20 minute, carefully remove oil phase then in 4 ℃ at 20-25 ℃;
(3) add isopyknic bromohexadecane base Trimethylamine 99 and extract damping fluid, mixing was placed 3～6 hours, and 12000 rev/mins, centrifugal 20 minutes, was got supernatant liquor for 20-25 ℃;
(4) the 3mol/L sodium acetate of adding and the isopyknic Virahol of supernatant liquor and supernatant liquor 1/10 volume, mixing, 20-25 ℃ left standstill 6-15 hour, and 12000 rev/mins, centrifugal 20 minutes, abandon supernatant liquor, stay precipitation;
(5) add 1mLTE damping fluid dissolution precipitation, add isopyknic volume ratio and be 24: 1 trichloromethane: primary isoamyl alcohol, slowly put upside down and shake up 5 minutes, 12000 rev/mins, centrifugal 10 minutes, get supernatant liquor;
(6) add the 3mol/L sodium acetate of 1/10 volume and the Virahol of isopyknic 4 ℃ of precoolings, slow mixing, 4 ℃ left standstill 6-15 hour;
(7), 14000 rev/mins, centrifugal 15 minutes, abandon supernatant liquor in 4 ℃;
(8) wash precipitation with 70% ethanol of 4 ℃ of precoolings of 400 μ L, remove residual ethanol, drying precipitated, obtain the DNA precipitation;
(9) add the deionized water that 25-35 μ L sterilizes, the dissolving DNA precipitation.
2. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: described step (1) normal hexane consumption is 1 times of sample volume.
3. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: used TE damping fluid is 2 times of sample volumes in the described step (2).
4. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: described step (2) churning time is 12 hours.
5. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: be 5 hours the storage period in the described step (3).
6. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: time of repose is 12 hours in the described step (4).
7. according to claim 1 from being to extract the method that is used for the DNA that transgene component detects the edible oil of raw material with the soybean, it is characterized in that: the sterilization deionized water volume in the described step (9) is 30 μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277426A (en) * | 2011-07-25 | 2011-12-14 | 南京农业大学 | Detection kit of transgenic component in soybean edible oil and its deeply-processed product and detection method thereof |
| CN105603056A (en) * | 2014-11-21 | 2016-05-25 | 丰益(上海)生物技术研发中心有限公司 | Method for detecting doped impurity in vegetable fat |
| CN110656107A (en) * | 2019-10-24 | 2020-01-07 | 九三集团哈尔滨惠康食品有限公司 | Method for extracting transgenic components in deep-processed vegetable oil |
-
2004
- 2004-07-28 CN CN 200410020176 patent/CN1597982A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277426A (en) * | 2011-07-25 | 2011-12-14 | 南京农业大学 | Detection kit of transgenic component in soybean edible oil and its deeply-processed product and detection method thereof |
| CN105603056A (en) * | 2014-11-21 | 2016-05-25 | 丰益(上海)生物技术研发中心有限公司 | Method for detecting doped impurity in vegetable fat |
| CN105603056B (en) * | 2014-11-21 | 2020-09-25 | 丰益(上海)生物技术研发中心有限公司 | Vegetable oil doping detection method |
| CN110656107A (en) * | 2019-10-24 | 2020-01-07 | 九三集团哈尔滨惠康食品有限公司 | Method for extracting transgenic components in deep-processed vegetable oil |
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