CN101319996B - A kind of genetically modified soybean detection kit and detection method suitable for feed - Google Patents

Abstract

The invention relates to a transgenic crop, in particular to a transgenic soy detecting method and a detecting reagent box applied to feed. The reagent box is that: PCR reaction buffer solution 22.5:1, primers F2 and R2 are respectively 0.5:1 or F3 and R3 are respectively 0.5:1; Taq DNA polymerase 0.5 Mu1; masculine comparison DNA to 1:. The primer F2 is 5'-CATTTGGAGAGGACACGCTGACA-3'; R2 is 5'-CCGGAAAGGCCAGAGGATT-3'; F3 is 5'-TAACAACATGGCACAAGGGATACA-3'; R3 is 5'-CAGAGGATTTGCGGGCGGTTGC-3'. After the detection on the feed which is added with transgenic soybean meal with different contents, theresult shows that the method can successfully detect the external gene of transgenic soy in the feed. The invention is used for detecting various livestock and marine lives feed which are added with soybean meal and are sold in market. The result shows that 91 percent of matching feed comprises transgenic soy and the sensitivity of the transgenic crop is high.

Description

A kind of genetically engineered soybean detection kit and detection method that is applicable to feed

Technical field

The present invention relates to genetically modified crops, a kind of specifically genetically engineered soybean detection method and detection kit that is applicable to feed.

Background technology

Genetically modified crops be meant utilize recombinant DNA technology with exogenous origin gene integrator in the recipient plant genome, change the plant and the offspring thereof that produce after its genetic constitution, be also referred to as genetically modified organism, GMO (Genetically modified organisms, GMOs).Since nineteen eighty-three, the first genetically modified plants were come out, cultivated area and the sales revenue of global genetically modified crops all increased with multiple.But on serious scientific meaning, the biological safety of transgene agricultural product does not still have the conclusion of determining at present.When the mankind pay close attention to enetically modified food safety, also paying close attention to the safety of animal feed.Feed safety is an important step of food security, and the security of transgenosis feed detects also will provide strong evidence for the security of corresponding enetically modified food.The production of fodder of China has had considerable scale, and raw material commonly used in the feed has soybean, dregs of beans, the cottonseed dregs of rice, corn rapeseed dregs etc., and these may contain genetically modified crop has pair animal, human body and ecologic environment to have unknowable harm.Up to the present, Shang Weijian is about carrying out the patented technology that transgenosis detects to feed.

Summary of the invention

The object of the present invention is to provide a kind of genetically engineered soybean detection method and detection kit of being applicable to feed with high sensitivity.

For achieving the above object, the technical solution used in the present invention is:

Kit:

1) PCR reactant liquor system for the first time: PCR reaction buffer 22.5 μ l, primers F 2 0.5 μ l, primer R2 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l;

2) PCR reactant liquor system for the second time: PCR reaction buffer 22.5 μ l, primers F 30.5 μ l, primer R3 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l;

Wherein, the primer of PCR reaction for the first time is:

F2：5’-CATTTGGAGAGGACACGCTGACA-3’；R2：5’-CCGGAAAGGCCAGAGGATT-3’；

The primer of PCR reaction for the second time is:

F3：5’-TAACAACATGGCACAAGGGATACA-3’；R3：5’-CAGAGGATTTGCGGGCGGTTGC-3’；

For the first time PCR and for the second time in the PCR reactant liquor system PCR reaction buffer contain: 10mMTris-HCI, pH8.3; 50mM KCI, 1.5mM MgCI2, dNTP 0.2mM;

3) positive control dna 1 μ l.

Described positive control dna is a transgenic soybean DNA.

Detection method:

1) extract template DNA:

Take by weighing the raw material of 1-10g genetically engineered soybean, add the extraction damping fluid mixing of 10-100ml precooling; The lysis buffer mixing that adds 60-70 ℃ of preheating; 60-70 ℃ of incubation 30-60min; The cooling back adds the mixed liquor mixing of 5-50ml chloroform, isoamylol and ethanol, and room temperature was placed 3-10 minute; Then at the centrifugal 8-15min of 10000-13000rpm, get supernatant and add 5-50ml phenol and chloroform mixed liquor mixing in supernatant, room temperature was placed 3-10 minute; Get supernatant with the centrifugal 8-15min of 10000-13000rpm again; The pre-cold isopropanol that adds its volume 2/3 in the supernatant is placed 20-30min for-20--40 ℃, and the centrifugal 10-15min of 10000-13000rpm promptly obtains extracting template DNA, and is stand-by;

2) carrying out transgenosis detects:

The dna profiling liquid that step 1) obtains is got 1 μ l and is added the PCR reaction buffer, and adding primers F 2 and R2 each 0.5 μ l and TaqDNA polymerase 0.5 μ l, mix, then at 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 35 circulations, last 72 ℃ are extended 10min; Obtain pcr amplification product for the first time, get 1 μ l for the first time pcr amplification product add aseptic double-distilled water and carry out 50-100 and doubly dilute, then getting 1 μ l dilution adds in the PCR reactant liquor damping fluid, and adding primers F 3 and 3 each 0.5 μ l and TaqDNA polymerase 0.5 μ l, mix, carry out second with above-mentioned PCR reaction conditions and take turns amplification, obtain amplified production;

F2：5’-CATTTGGAGAGGACACGCTGACA-3’；R2：5’-CCGGAAAGGCCAGAGGATT-3’；

F3：5’-TAACAACATGGCACAAGGGATACA-3’；R3：5’-CAGAGGATTTGCGGGCGGTTGC-3’；

The product of amplification is carried out agarose gel electrophoresis and ultraviolet light testing result,, prove in the raw material that is detected and contain genetically engineered soybean if there is the DNA band of 250bp.

It is to wash 1-2 time in 70% ethanol that the template DNA that extracts in the described step 1) is deposited in volumetric concentration, after drying, is dissolved in the 500-1000 μ l distilled water, stand-by.The volume ratio of isoamylol, ethanol and chloroform is 4: 20: 76 in the described step 1); Phenol and chloroform volume ratio are 1: 1; The extraction damping fluid is 0.8MNaCI; 50M Tris-CI, 10mM EDTA; PH8.0; Lysis buffer is 0.8M NaCI; 50MTris-CI, 10mM EDTA; 20%CTAB; PH8.0.The isopropyl alcohol precooling temperature of described precooling is 4 ℃.

What the present invention had has a few:

The present invention has been by having set up the transgenosis detection technique with high sensitivity at various livestock and poultry and aquatic feeds, and extracts high-quality DNA and adopt highly sensitive detection technique that its foreign gene is detected.

It is the shortcoming that is subjected to the DNA of havoc that the present invention overcomes the transgenosis dregs of beans DNA that adds in the mixed feed, dregs of beans is that genetically engineered soybean is produced the secondary product that produces in the soya-bean oil process, its genomic DNA has produced violent destruction in the process of squeezing, pulverize in the feed process, high temperature such as granulation and slaking, problems such as high shear force, also further destroyed its genomic DNA, the detection that obtains foreign gene in the transgenosis feed thus has higher difficulty, and can extract high-quality DNA and adopt highly sensitive detection technique that its foreign gene is detected by kit of the present invention.

The present invention adopts the CTAB method to extract the genome DNA of mixed feed, and 2 pairs of primers have been designed according to the genetically engineered soybean exogenous gene sequence of logining among the GenBank, adopt these two pairs of primers to containing 2%, 5%, 20%, the feed of the transgenosis dregs of beans of 30%, 50% content carries out nest-type PRC and detects, and the result shows the transgene component that can successfully detect wherein.Utilize the present invention to 32 parts of special exogenous gene sequences that detected genetically engineered soybean are arranged in commercially available 35 parts of feeds, find that wherein 91% feed has adopted the transgenosis dregs of beans.The present invention has very high sensitivity.

Description of drawings

Fig. 1 is the DNA electrophoresis pattern in the feed of the present invention.

Fig. 2 is the electrophoresis pattern of the present invention with the nest-type PRC amplification of the prawn feed of soybean meal content, wherein: according to M be the DL2000 molecular weight standard; 1 is, negative control (non-transgenic feed); 2-7, the prawn feed of different soybean meal content (percentage by weight is respectively 50%, 30%, 20%, 5%, 2%, 0%); 8 are, positive control dna (genetically engineered soybean)

Embodiment

Be described in further detail the present invention below.

Embodiment 1 is applicable to the genetically engineered soybean detection kit of feed

1) PCR reactant liquor system for the first time: PCR reaction buffer 22.5 μ l, primers F 2 and R2 (each 0.5 μ l), Taq archaeal dna polymerase 0.5 μ l.

2) PCR reactant liquor system for the second time: PCR reaction buffer 22.5 μ l, primers F 3 and R3 (each 0.5 μ l), Taq archaeal dna polymerase 0.5 μ l.

Wherein, the primer of PCR reaction for the first time is:

F2：5’-CATTTGGAGAGGACACGCTGACA-3’；R2：5’-CCGGAAAGGCCAGAGGATT-3’；

The primer of PCR reaction for the second time is:

F3：5’-TAACAACATGGCACAAGGGATACA-3’；R3：5’-CAGAGGATTTGCGGGCGGTTGC-3’；

For the first time PCR and for the second time in the PCR reactant liquor system PCR reaction buffer contain: 10mMTris-HCI, pH8.3; 50mM KCI, 1.5mM MgCI2, dNTP 0.2mM.

3) positive control dna 1 μ l.

Described positive control dna is a transgenic soybean DNA.

Embodiment 2 is applicable to the detection method of the genetically engineered soybean of feed

1) extract template DNA:

The preparation method who contains the prawn mixed feed of transgenosis dregs of beans different quality number percent: shown in the prescription of the mixed feed table composed as follows:

Soybean meal content (%)	Fish meal (%)	Brewer's yeast (%)	Shrimp shell meal (%)	Squid viscera powder (%)	Strong flour (%)	Lecithin (%)	Ocean fish oil (%)	Mineral matter (%)	Vitamin (%)	50% choline (%)	VC (％)
												50	6	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03
30	26	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03
												20	36	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03
5	51	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03
												2	54	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03
0	56	4	5	5	21.97	1.5	2	4	0.3	0.2	0.03

Table mineral prescription is: calcium dihydrogen phosphate, 23.5g/kg, sodium chloride, 1g/kg; Potassium dihydrogen phosphate, 10g/kg; Epsom salt, 4g/kg; Green-vitriol 502.8mg/kg; Manganese sulfate monohydrate 62.1mg/kg; White vitriol 442mg/kg; Potassium iodide (1%) 100mg/kg, sodium selenite (1%) 100mg/kg.Cross 60 mesh sieves after the various raw material pulverizing, micro-adding ingredient takes step by step that enlargement method mixes, mixing granulation, 90 ℃ of oven dry of pellet 30 minutes, the air-dry freezing genetically engineered soybean composition detection that is used for.

Take by weighing the prawn mixed feed powder of the transgenosis soybean meal content 2% of the above-mentioned preparation of 1g; Powder is changed in the 50ml centrifuge tube, add the extraction damping fluid of 10ml, 4 ℃ of precoolings, mixing; The lysis buffer that adds 65 ℃ of preheatings, mixing; 65 ℃ of incubation 30min; The cooling back adds the mixed liquor of 5ml chloroform, isoamylol and ethanol, wherein isoamylol: ethanol: chloroform (V/V)=4: 20: 76, put upside down mixing, and room temperature was placed 8 minutes, made its natural layering; Then at the centrifugal 8min of 10000rpm, get supernatant and in supernatant, add 5ml phenol and chloroform (V/V=1: 1) mixed liquor, put upside down mixing, room temperature was placed 7 minutes, made its natural layering; Get supernatant with the centrifugal 8min of 10000rpm again; The isopropyl alcohol that adds 4 ℃ of precoolings of its volume 2/3 in the supernatant, careful mixing is placed 20min for-20 ℃, the centrifugal 10min of 10000g, (referring to Fig. 1, the DNA in the visible feed has been subjected to violent destruction promptly to obtain template DNA.)。DNA is deposited in 70% ethanol and washs once, after drying slightly, is dissolved in the 500 μ l distilled waters, and stand-by, the prawn mixed feed of other content extracts by this step.

Wherein: the extraction damping fluid of precooling is: 0.8M NaCI, 50M Tris-CI, 10mM EDTA, pH8.0; The lysis buffer of preheating is: 0.8M NaCI, 50M Tris-CI, 10mM EDTA, 20%CTAB, pH8.0.

2) carrying out transgenosis detects:

Get 1 μ l transgenosis soybean meal content respectively and be respectively 2%, 5%, 20%, 30%, 50% prawn mixed feed DNA liquid mixes with 24 μ l PCR reactant liquor first time system respectively, then at 94 ℃ of pre-sex change 2min as template; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 35 circulations; Last 72 ℃ are extended 10min, obtain containing the pcr amplification product first time of the transgenosis dregs of beans of different content respectively, get the 1 μ l above-mentioned first time of pcr amplification product adding distilled water respectively and carry out 100 times of dilutions, then mix with 24 μ l PCR reactant liquor second time system, carry out second with above-mentioned PCR reaction conditions again and take turns amplification, obtain amplified production.

Wherein, PCR reactant liquor system for the first time: PCR reaction buffer 22.5 μ l, each 0.5 μ l of primers F 2 and R2, Taq archaeal dna polymerase 0.5 μ l;

F2：5’-CATTTGGAGAGGACACGCTGACA-3’；R2：5’-CCGGAAAGGCCAGAGGATT-3’；

PCR reactant liquor system for the second time: PCR reaction buffer 22.5 μ l, each 0.5 μ l of primers F 3 and R3, Taq archaeal dna polymerase 0.5 μ l.

F3：5’-TAACAACATGGCACAAGGGATACA-3’；R3：5’-CAGAGGATTTGCGGGCGGTTGC-3’；

Wherein, for the first time with second time PCR reactant liquor system in the PCR reaction buffer contain 10mMTris-HCI, pH8.3; 50mM KCI; 1.5mM MgCI2; DNTP 0.2mM

Will be through the product of secondary amplification, the agarose gel electrophoresis 20-30 through 0.8% minute (80-100V/cm) gets agarose electrophoresis and detects collection of illustrative plates (referring to Fig. 2).The ultraviolet light testing result if there is the DNA band of 250bp, proves in the raw material that is detected and contains genetically engineered soybean.

Embodiment 3

Difference from Example 2 is:

1) extract template DNA:

Take by weighing the raw material of 10g genetically engineered soybean, add the extraction damping fluid mixing of 100ml precooling; The lysis buffer mixing that adds 70 ℃ of preheatings; 70 ℃ of incubation 60min; The cooling back adds the mixed liquor mixing of 50ml chloroform, isoamylol and ethanol, and room temperature was placed 3 minutes; Then at the centrifugal 15min of 13000rpm, get supernatant and add 50ml phenol and chloroform mixed liquor mixing in supernatant, room temperature was placed 3 minutes; Get supernatant with the centrifugal 15min of 13000rpm again; The pre-cold isopropanol that adds its volume 2/3 in the supernatant is placed 20min for-40 ℃, and 10000rpm is centrifugal; 15min promptly obtains extracting template DNA, and it is to wash 2 times in 70% ethanol that the template DNA of extraction is deposited in volumetric concentration, after drying, is dissolved in the 1000 μ l distilled waters, stand-by.2) carrying out transgenosis detects.

Embodiment 4

Difference from Example 2 is:

1) extract template DNA:

Take by weighing the raw material of 5g genetically engineered soybean, add the extraction damping fluid mixing of 60ml precooling; The lysis buffer mixing that adds 60 ℃ of preheatings; 60 ℃ of incubation 450min; The cooling back adds the mixed liquor mixing of 25ml chloroform, isoamylol and ethanol, and room temperature was placed 5 minutes; Then at the centrifugal 12min of 12000rpm, get supernatant and add 25ml phenol and chloroform mixed liquor mixing in supernatant, room temperature was placed 8 minutes; Get supernatant with the centrifugal 12min of 12000rpm again; The pre-cold isopropanol that adds its volume 2/3 in the supernatant is placed 20min for-30 ℃, and 10000rpm is centrifugal; 15min promptly obtains extracting template DNA, and it is to wash 2 times in 70% ethanol that the template DNA of extraction is deposited in volumetric concentration, after drying, is dissolved in 700 μ, 1 distilled water, stand-by.2) carrying out transgenosis detects.

Sequence table

SEQUENCE LISTING

＜110〉Institute of Oceanology of the Chinese Academy of Sciences

＜120〉a kind of genetically engineered soybean detection kit and detection method that is applicable to feed

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Claims (4)

1. A genetically modified soybean detection kit suitable for feed, characterized in that: 1) The first PCR reaction solution system: PCR reaction buffer 22.5 μl, primer F20.5 μl, primer R2 0.5 μl, Taq DNA polymerase 0.5 μl; 2) The second PCR reaction solution system: PCR reaction buffer 22.5 μl, primer F30.5 μl, primer R3 0.5 μl, Taq DNA polymerase 0.5 μl; Wherein, the first PCR reaction primers are: F2: 5'-CATTTGGAGAGGACACGCTGACA-3'; R2: 5'-CCGGAAAGGCCAGAGGATT-3'; The primers for the second PCR reaction are: F3: 5'-TAACAACATGGCACAAGGGATACA-3'; R3: 5'-CAGAGGATTTGCGGGCGGTTGC-3'; The PCR reaction buffer in the first PCR and the second PCR reaction solution system contains: 10mM Tris-HCl, pH8.3; 50mM KCI, 1.5mM MgCI2, dNTP 0.2mM; 3) Positive control DNA 1 μl; The positive control DNA is transgenic soybean DNA. 2. a detection method applicable to the transgenic soybean detection kit for feed according to claim 1, characterized in that: 1) Extract template DNA: Weigh 1-10g of transgenic soybean raw material, add 10-100ml pre-cooled extraction buffer and mix well; add preheated 60-70℃ lysis buffer and mix well; incubate at 60-70℃ for 30-60min; cool and add Mix 5-50ml of chloroform, isoamyl alcohol and ethanol, and let it stand at room temperature for 3-10 minutes; then centrifuge at 10000-13000rpm for 8-15min, take the supernatant and add 5-50ml of phenol and chloroform to the supernatant to mix Mix the solution and place it at room temperature for 3-10 minutes; then centrifuge at 10000-13000rpm for 8-15 minutes to take the supernatant; -30min, centrifuge at 10000-13000rpm for 10-15min to obtain the extracted template DNA, ready to use; 2) Conduct genetically modified testing: Step 1) Take 1 μl of the obtained DNA template solution and add it to the PCR reaction buffer, add 0.5 μl each of primers F2 and R2, and 0.5 μl of TaqDNA polymerase, mix well, and then pre-denature at 94°C for 2 minutes, denature at 94°C for 30 seconds, and 55°C Anneal for 30 s, extend at 72°C for 45 s, a total of 35 cycles, and finally extend at 72°C for 10 min; to obtain the first PCR amplification product, take 1 μl of the first PCR amplification product and add sterile double distilled water for 50-100 times dilution. Then take 1 μl of the diluted solution and add it to the PCR reaction buffer, add 0.5 μl each of primers F3 and R3 and 0.5 μl of TaqDNA polymerase, mix well, and perform the second round of amplification under the above PCR reaction conditions to obtain the amplified product; F2: 5'-CATTTGGAGAGGACACGCTGACA-3'; R2: 5'-CCGGAAAGGCCAGAGGATT-3'; F3: 5'-TAACAACATGGCACAAGGGATACA-3'; R3: 5'-CAGAGGATTTGCGGGCGGTTGC-3'; Perform agarose gel electrophoresis and ultraviolet light detection on the amplified product. If there is a 250bp DNA band, it proves that the detected raw material contains transgenic soybeans; The template DNA precipitate extracted in the step 1) is washed in 70% ethanol for 1-2 times, dried, dissolved in 500-1000 μl of double distilled water, and set aside. 3. by the detection method of the transgenic soybean detection kit that is applicable to feed according to claim 2, it is characterized in that: the volume ratio of isoamyl alcohol, ethanol and chloroform is 4: 20: 76 in the described step 1); The volume ratio of chloroform and chloroform is 1:1; the extraction buffer is 0.8M NaCI; 50M Tris-CI, 10mM EDTA; pH8.0; the lysis buffer is 0.8M NaCI; 50M Tris-CI, 10mM EDTA; 20% CTAB; pH8 .0. 4. The detection method of the transgenic soybean detection kit suitable for feed according to claim 2, characterized in that: the precooling temperature of the precooled isopropanol is 4°C.