CN101358189B - Method for extracting DNA for trangene ingredient detection from plant source edible oil - Google Patents
Method for extracting DNA for trangene ingredient detection from plant source edible oil Download PDFInfo
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- CN101358189B CN101358189B CN2008101675441A CN200810167544A CN101358189B CN 101358189 B CN101358189 B CN 101358189B CN 2008101675441 A CN2008101675441 A CN 2008101675441A CN 200810167544 A CN200810167544 A CN 200810167544A CN 101358189 B CN101358189 B CN 101358189B
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Abstract
The present invention relates to a method for extracting a DNA used for the detection of genetically modified components from edible vegetable oil. The method has the following characteristics: before the extraction of the DNA, the edible vegetable oil is emulsified by organic emulsifier for a long time; the extremely low-content DNA is sufficiently dissociated out, so that the first step of successful extraction is fulfilled; an independently developed unique buffer system (extraction buffer solution 00mM Tris.HCl, pH8.0; 20mM EDTA; 500mM NaCl; 1.5 percent of SDS; 4 percent of PVP) is utilized to sufficiently release and aggregate the DNA, which is different from the conventional CTAB cracking and phenol/chloroform extraction; in carrier RNA coprecipitated catalyst, isopropanol precipitation is carried out twice; the solution is transferred from a big centrifugal tube to a small centrifugal tube; the extremely low-content DNA obtained from the edible vegetable oil to the max is dissolved in 40Wu dissolving buffer solution; 9Wu1 is taken out to serve as a template which is used for the PCR reaction system of 20Wu1. The method, which has the advantages of rapidity, convenience, safety, high efficiency and good stability, is applicable to the detection of the genetically modified components of edible vegetable oil.
Description
Technical field
The invention belongs to the technology in nucleic acid extraction field, relate in particular to the method for extracting nucleic acid that plant-sourced edible oil transgene component detects usefulness.
Background technology
Since the seventies in 20th century, genetically modified organism and transgenic product were developed, transgenic product was always with swift and violent speed development.According to internal authority mechanism statistics, the whole world reaches nearly ten thousand kinds by the genetically modified food and the food ingredients of genetically modified crops processing.The soybean that processes raw material as edible oil wherein, rapes etc. occupy larger proportion, account for 63% of the total cultivated area of global genetically modified crops as soybean, and rape accounts for 5%.Because these transgenosis oil plant crop oil yield height, it is low to make oily cost, and numerous and confused import genetically engineered soybean of each developing country and rape are as the oily raw material of system.
At present, still lack scientific basis proof transgenosis edible oil to human health and environmentAL safety, but market survey has shown the concern of human consumer to this problem.Be assurance human consumer's the right of health, right to know and preference, national governments have stipulated that clearly edible oil must carry out the market sign.To methods for detecting transgenic foods, at present specific foreign genes that whether contain insertion in the PCR method detection food that adopt more.Therefore, the DNA extraction in the food is very crucial.Owing to treating processess such as process high temperature and high pressures in the edible fat production course of processing, DNA in the product is seriously damaged, it is shorter to be degraded to length, fragment not of uniform size, and be subjected to physics, and the influence of factors such as chemistry and biology, the quality of DNA also reduces greatly, therefore, the DNA extraction of edible oil is still one of difficult problem both domestic and external.
What the edible oil test kit was done on market at present is not a lot, and chief reason is more stable DNA extraction method or a test kit of domestic and international none.Our invention then can overcome the unsettled defective of said extracted.
Summary of the invention
The present invention has adopted special technique, is intended to set up method mature and stable, extract template DNA from the edible oil that the plant origin raw material processes, and the PCR that is used for transgene component detects.
Edible oil complete processing complexity is mostly passed through processes such as a series of pulverizing, high temperature, and DNA destroys very serious, its dna content is lower, dna fragmentation is less or the like, brings very large difficulty all for the edible oil DNA extraction, and the edible oil DNA extraction is one of domestic and international difficult problem at present.Purpose of the present invention is exactly a difficult problem that solves existing edible oil DNA extraction technology, provides a cover unique Laemmli buffer system Laemmli, makes to be suitable for extracting DNA from the plant-sourced edible oil, is used for the qualitative detection of transgene component, with stability and the reliability of guaranteeing to detect.
After present method is processed through the rigor condition of High Temperature High Pressure at the plant-sourced edible oil, the characteristics that wherein water miscible dna molecular content is extremely low, degraded is serious, when carrying out DNA extraction, the water soluble component that content is extremely low is fully separated from fat-soluble grease earlier, and this is a key extracting DNA from grease; Adopt unique extraction damping fluid (extraction damping fluid: 100mM Tris.HCl, pH8.0 then; 20mMEDTA; 500mM NaCl; 1.5%SDS (W/V); 4%PVP (W/V)) and sodium acetate buffer (sodium acetate buffer: 3M sodium acetate; 11.5% glacial acetic acid (V/V)), auxiliary down at carrier RNA, through isopropanol precipitating be used for the precipitation second time of enrichment DNA, be transferred to little centrifuge tube through big centrifuge tube, be another key of further concentration of DNA molecule.Present method is different from traditional CTAB cracking, phenol/chloroform extracting needs several days time, quick, convenient, the safety of this operation compared to the traditional method single job, efficient height, good stability are suitable for the qualitative detection of the transgene component of plant-sourced edible oil.
Technological line of the present invention is summarized as follows:
(1) sample pretreatment: get edible oil 30ml, add the equal-volume emulsifying agent, on magnetic stirring apparatus, stirred 2-3 hour;
(2) learn from else's experience pretreated solution 10ml in big centrifuge tube, add 5ml and extract damping fluid, the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes;
(3) add the 2ml sodium acetate buffer, abundant mixing, the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes;
Centrifugal 10 minutes of (4) 10,000rpm, with the lower layer of water phase transition to the new big centrifuge tube;
(5) Virahol of adding 10 μ l Carrier RNA and 0.7 times of volume in aqueous phase solution, fully mixing.(for example the aqueous phase solution of 5ml adds the 3.5ml Virahol), 10, centrifugal 10 minutes of 000rpm abandons supernatant, keeps precipitation;
(6) add 700 μ l ultrapure waters in big centrifuge tube, vortex vibration 15 seconds adds 700 μ l Virahols, and vortex vibration 15 seconds is briefly centrifugal, and solution is transferred in the new little centrifuge tube;
(7) 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant;
(8) add 700 μ l70% ethanol, vortex vibration 5 seconds, 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant;
(9) inversion of uncapping, room temperature is placed 10min, thoroughly dries remaining ethanol;
(10) add 40 μ l elution buffer TE, vortex vibration 1 minute finally obtains the edible oil dna solution.
According to method set forth in the present invention, used emulsifying agent is a normal hexane.
According to method set forth in the present invention, used extraction damping fluid consists of: 100mMTris.HCl, pH8.0; 20mM EDTA; 500mM NaCl; 1.5%SDS (W/V); 4%PVP (W/V).
According to method set forth in the present invention, used sodium acetate buffer consists of: the 3M sodium acetate; 11.5% glacial acetic acid (V/V).
According to method set forth in the present invention, solution is transferred in the new 1.5ml centrifuge tube behind the isopropanol precipitating for the second time.
The invention has the beneficial effects as follows: a kind of effective ways stable, extract minim DNA from edible oil are provided, the qualitative detection that is used for the edible oil transgene component, this method is quick, convenient, safety, efficient height, good stability, the transgene component qualitative detection of suitable plant origin edible oil.
Description of drawings
The pcr amplification result of the native gene Lectin of Fig. 1 one-level soybean oil DNA
(1.DNA Marker; 2. good fortune one-level soybean oil near the house; 3. golden imperial fish one-level soybean oil; 4. soybean seeds contrast; 5. clear water contrast)
The pcr amplification result of the foreign gene CaMV35S of Fig. 2 one-level soybean oil DNA
(1.DNA Marker; 2. good fortune one-level soybean oil near the house; 3. golden imperial fish one-level soybean oil; 4. soybean seeds contrast; 5. clear water contrast)
The pcr amplification result of native gene PE3-PEPcase of Fig. 3 rapeseed oil DNA
(1.DNA Marker; 2. green precious rapeseed oil; 3. Semen Brassicae campestris contrast; 4. clear water contrast)
Fig. 4. the pcr amplification result of the native gene Zein of Semen Maydis oil DNA
(1.DNA Marker; 2. good fortune Fructus Maydis oil near the house; 3. corn seed contrast; 4. clear water contrast)
Embodiment
1. material and reagent
1) extract reagent:
Extract damping fluid: 50mM Tris.HCl, 10mM EDTA, 100mM NaCl, 1%SDS (W/V), 2%PVP (W/V), autoclaving;
Sodium acetate buffer: 3M sodium acetate, 11.5% acetate (V/V), autoclaving;
TE damping fluid: 10mM Tris.HCl, 1mM NaEDTA, pH8.0, autoclaving;
Normal hexane, carrier RNA, 70% ethanol (V/V), Virahol, ultrapure water.
2.DNA extraction flow process:
(1) sample pretreatment: get edible oil 30ml, add the equal-volume normal hexane, on magnetic stirring apparatus, stirred 2-3 hour.
(2) the pretreated solution 10ml that learns from else's experience adds 5ml and extracts damping fluid, and the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes.
(3) add the 2ml sodium acetate buffer, abundant mixing, the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes.
Centrifugal 10 minutes of (4) 10,000rpm, with the lower layer of water phase transition to the new centrifuge tube.
(5) Virahol of adding 10 μ l Carrier RNA and 0.7 times of volume in aqueous phase solution, fully mixing.(for example the aqueous phase solution of 5ml adds the 3.5ml Virahol), 10, centrifugal 10 minutes of 000rpm abandons supernatant, keeps precipitation.
(6) add 700 μ l ultrapure waters in centrifuge tube, vortex vibration 15 seconds adds 700 μ l Virahols, and vortex vibration 15 seconds is briefly centrifugal, and solution is transferred in the new 1.5ml centrifuge tube.
(7) 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant.
(8) add 700 μ l70% ethanol, vortex vibration 5 seconds, 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant.
(9) inversion of uncapping, room temperature is placed 10min, thoroughly dries remaining ethanol.
(10) add 40 μ l elution buffer TE, vortex vibration 1 minute finally obtains the edible oil dna solution.
Adopt gene specific primer, template DNA is carried out qualitative PCR detect.Experimental technique is as follows:
1) gene specific primer sequence (seeing Table 1)
The PCR sequence of table 1 gene specific primer
2) the PCR reaction system of specific gene is formed (seeing Table 2)
(catalog number (Cat.No.): KT201) detect, each reaction system is provided with two parallel pipes to the 2*Taq PCR MasterMix of the template DNA employing Tiangen company that the present invention extracts, and soybean oil, Semen Maydis oil, rapeseed oil are all right.
Table 2PCR detection reaction system (20 μ l)
* the positive control system adopts 2 μ l templates, and clear water is supplied.
3) the pcr amplification condition of specific gene (seeing Table 3)
The pcr amplification condition of table 3 specific gene
4) deposition condition
With the sepharose of 0.5 * TBE electrophoretic buffer preparation 3% (W/V), it is the bromination second pyridine of 0.5/ml that gel melts back adding content.With PCR reaction product and the sample-loading buffer uniform mixing of 8 μ l, join then in the gel pore in proportion, carry out electrophoresis with the voltage of 5V/cm, electrophoresis time is 40-60 minute.After electrophoresis finishes gel placed and observe on the gel imaging instrument and take a picture.
The invention will be further described below in conjunction with specific embodiment.
Embodiment 1:
With the imperial fish genetically engineered soybean of commercially available gold oil, good fortune near the house genetically engineered soybean oil be material, adopt following method to extract DNA,
(1) gets soybean oil 30ml, add the equal-volume normal hexane, on magnetic stirring apparatus, stirred 2-3 hour.
(2) the pretreated solution 10ml that learns from else's experience adds 5ml and extracts damping fluid, vortex vibration 1 minute.Room temperature left standstill 10 minutes.
(3) add the 2ml sodium acetate buffer, abundant mixing, vortex vibration 1 minute.Room temperature left standstill 10 minutes.
Centrifugal 10 minutes of (4) 10,000rpm, with the lower layer of water phase transition to the new centrifuge tube.
(5) Virahol of adding 10 μ l Carrier RNA and 0.7 times of volume in aqueous phase solution, fully mixing.(for example the aqueous phase solution of 5ml adds the 3.5ml Virahol), 10, centrifugal 10 minutes of 000rpm abandons supernatant, keeps precipitation.
(6) add 700 μ l ultrapure waters in centrifuge tube, vortex vibration 15 seconds adds 700 μ l Virahols, and vortex vibration 15 seconds is briefly centrifugal, and solution is transferred in the new 1.5ml centrifuge tube.
(7) 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant.
(8) add 700 μ l70% ethanol, vortex vibration 5 seconds, 12,000rpm (～13,400 * g) centrifugal 5 minutes, abandon supernatant.
(9) inversion of uncapping, room temperature is placed 10min, thoroughly dries remaining ethanol.
(10) add 40 μ l elution buffer TE, vortex vibration 1 minute finally obtains the edible oil dna solution.
The soybean seeds DNA that extracts with the product DP321 of Tiangen company test kit in contrast.With the Auele Specific Primer of soybean native gene Lectin, the DNA that extracts with present method is a template, detects the DNA that is extracted by pcr amplification and whether satisfies the requirement that transgene component detects, and the results are shown in Figure 1.
As seen from Figure 1, the DNA that near the house extracts the genetically engineered soybean oil from the imperial fish genetically engineered soybean of gold oil and good fortune, after utilizing the Lectin gene specific primer to carry out pcr amplification, the amplified fragments that obtains is consistent with the amplified fragments size of contrast soybean seeds, and the clear water contrast there is no amplification, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
Embodiment 2:
Material, step are with embodiment 1.
The soybean seeds DNA that extracts with the product DP321 of Tiangen company test kit in contrast.With the Auele Specific Primer of genetically engineered soybean promotor CaMV35S, the DNA that extracts with present method is a template, detects the DNA that is extracted by pcr amplification and whether satisfies the requirement that transgene component detects, and the results are shown in Figure 1.
As seen from Figure 2, the DNA that near the house extracts the genetically engineered soybean oil from the imperial fish genetically engineered soybean of gold oil and good fortune, after utilizing CaMV35S promotor special primer to carry out pcr amplification, the amplified fragments that obtains is consistent with the amplified fragments size of contrast soybean seeds, and the clear water contrast there is no amplification, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
With commercially available green precious rapeseed oil is material, adopts following method to extract DNA,
(1) gets rapeseed oil 30ml, add the equal-volume normal hexane, on magnetic stirring apparatus, stirred 2-3 hour.
All the other steps are with embodiment 1.
The Semen Brassicae campestris DNA that extracts with the product DP321 of Tiangen company test kit in contrast.With the Auele Specific Primer of rape native gene PE3PEPcase, the DNA that extracts with present method is a template, detects the DNA that is extracted by pcr amplification and whether satisfies the requirement that transgene component detects, and the results are shown in Figure 1.
As seen from Figure 2, the DNA that from rapeseed oil, extracts, after the special primer that utilizes the PE3PEPcase gene carries out pcr amplification, the amplified fragments that obtains is consistent with the amplified fragments size of contrast Semen Brassicae campestris, and the clear water contrast there is no amplification, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
With commercially available good fortune near the house Fructus Maydis oil be material, adopt following method to extract DNA,
(1) gets Semen Maydis oil 30ml, add the equal-volume normal hexane, on magnetic stirring apparatus, stirred 2-3 hour.
All the other steps are with embodiment 1.
The corn grain DNA that extracts with the product DP321 of Tiangen company test kit in contrast.With the Auele Specific Primer of corn native gene Zein, the DNA that extracts with present method is a template, detects the DNA that is extracted by pcr amplification and whether satisfies the requirement that transgene component detects, and the results are shown in Figure 1.
As seen from Figure 2, the DNA that from Semen Maydis oil, extracts, after the special primer that utilizes the Zein gene carries out pcr amplification, the amplified fragments that obtains is consistent with the amplified fragments size of contrast corn grain, and the clear water contrast there is no amplification, so the DNA that extracts with present method can satisfy the requirement that transgene component detects fully.
Claims (2)
1. from being the method for extracting the DNA that detects in order to transgene component the edible oil of raw material with the plant, form by following step:
(1) sample pretreatment: get edible oil 30ml, add the equal-volume emulsifying agent, on magnetic stirring apparatus, stirred 2-3 hour; Wherein, emulsifying agent is a normal hexane;
(2) learn from else's experience pretreated solution 10ml in big centrifuge tube, add 5ml and extract damping fluid, the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes; Wherein, extracting damping fluid consists of: the Tris.HCl of 100mM pH8.0,20mM EDTA, 500mM NaCl, 1.5%SDS and 4%PVP;
(3) add the 2ml sodium acetate buffer, abundant mixing, the vortex vibration is after 1 minute, and room temperature left standstill 10 minutes; Wherein, sodium acetate buffer consists of: 3M sodium acetate and 11.5% glacial acetic acid;
Centrifugal 10 minutes of (4) 10,000rpm, with the lower layer of water phase transition to the new big centrifuge tube;
(5) Virahol of adding 10 μ l Carrier RNA and 0.7 times of volume in aqueous phase solution, abundant mixing, 10, centrifugal 10 minutes of 000rpm abandons supernatant, keeps precipitation;
(6) add 700 μ l ultrapure waters in big centrifuge tube, vortex vibration 15 seconds adds 700 μ l Virahols, and vortex vibration 15 seconds is briefly centrifugal, and solution is transferred in the new little centrifuge tube;
Centrifugal 5 minutes of (7) 12,000rpm abandon supernatant;
(8) add 700 μ l, 70% ethanol, vortex vibration 5 seconds, 12, centrifugal 5 minutes of 000rpm abandons supernatant;
(9) inversion of uncapping, room temperature is placed 10min, thoroughly dries remaining ethanol;
(10) add 40 μ l elution buffer TE, vortex vibration 1 minute finally obtains the edible oil dna solution.
2. according to claim 1 from being the method for extracting the DNA that detects in order to transgene component the edible oil of raw material with the plant, it is characterized in that: described step (6) is transferred to solution the little centrifuge tube of 1.5ml from the big centrifuge tube of 50ml.
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