CN102191326A - Nucleic acid hybrid film strip for detecting exogenous gene and use thereof - Google Patents
Nucleic acid hybrid film strip for detecting exogenous gene and use thereof Download PDFInfo
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- CN102191326A CN102191326A CN2011100960341A CN201110096034A CN102191326A CN 102191326 A CN102191326 A CN 102191326A CN 2011100960341 A CN2011100960341 A CN 2011100960341A CN 201110096034 A CN201110096034 A CN 201110096034A CN 102191326 A CN102191326 A CN 102191326A
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Abstract
The invention relates to a nucleic acid hybrid film strip for detecting an exogenous gene and use thereof and belongs to the field of bioengineering. Each specific capture probe on the film strip has 30 to 37 bases, and for an exogenous gene transplanted into a plant, oligo(dt)n is added at the 5' end of a probe and is marked by an amino. The detection of the plant exogenous gene by using the system provided by invention is implemented by the following steps: designing the probe; synthesizing the probe; preparing the nucleic acid hybrid film strip; fixing the probe; extracting DNA from a sample; marking the DNA from the sample; crossing; and developing. In a plant exogenous gene detection process, radioisotope are avoided, the crossing, developing and washing steps are performed in a closed disposable microtube, the detection result can be observed by bare eyes, and the detection process is convenient and safe. The nucleic acid hybrid film strip can be used in plant seeds, foods and processing processes thereof and exogenous gene detection of plant seed resources and products such as brood bodies of ornamental plants.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of nucleic acid hybridization film bar and application thereof that detects foreign gene, the foreign gene that can be used for plant germplasm resource such as plant seed, food and the course of processing thereof, ornamental type propagulum and product detects.
Background technology
Existing at present more than ten countries are extensive plantation genetically modified crops in the global range, and cultivated area reaches up to ten million hectares, relate to crop and reach tens of kinds, reach up to ten thousand kinds especially with the food of these genetically modified crops process for processing.Transgenic plant have output height, resistance height, quality better, characteristic such as disease-resistant, pest-resistant, and products such as the transgenic Fructus Lycopersici esculenti of now having developed, Insect Resistant Cotton are respond well, are subjected to the attention of peasant household and have further strengthened cultivated area.Simultaneously, because country has strengthened transgenic product research dynamics, new technology of transgenic plant and product innovation are also in continuous generation.
But, occur from transgenic technology, countries in the world, UNO (United Nations Organization) and other non governmental organizations pay close attention to the security of transgenic product always, especially pay close attention to genetically modified food to the influence to ecology of people's health and genetically modified crops, change over to bacillus thuringiensis (
Bacillus thuringiensis, be called for short
Bt) transgenic plant that contain toxalbumin such as gene endure dispute especially to the fullest extent.Therefore, this type of transgenic plant product is not used in human food prods's processing at present, but as industrial or agricultural starting material and animal-feed.
Owing to also do not have clear and definite transgenic technology whether can produce harmful effect to human health and ecotope at present, national governments have all taked careful attitude to transgenic product." Biosafety Protocol " specified in more detail that United Nations in 2000 passes through genetically modified organism about passing by, examine, detect and responsibility such as management, prevent that risky transgenic product from spreading arbitrarily.Calendar year 2001, European Union required genetically modified food is forced identity management.Therefore, related products is carried out transgenosis detect, identify, ensure that human consumer's interests are very necessary strengthening the management of transgenic product.Judge that whether plant germplasm resource and product are the percentage composition of transgenic product, transgenic product and whether be that the transgenic product of the security of generally acknowledging in the world is that transgenic plant and products thereof are carried out the basis that the safety evaluation and the flow direction and purposes are implemented supervision.
Detection to transgene component mainly is that foreign gene (DNA) and exogenous gene expression product (RNA and protein) are detected at present.Wherein, DNA detection applied widely, the accuracy height is convenient to qualitative and detection by quantitative.
The main detection method of DNA comprises Southern hybrid method, gene chip detection method, the PCR detection method is relevant with other improves one's methods.The Southern hybrid method requires lower to appointed condition, but complex operation, easily pollutes; The PCR detection method is a detection method commonly used now, but equipment price and higher to Test Condition Requirements; It is few that chip technology has amount of samples, and the advantage that the one-time detection target gene is many, but highdensity gene chip cost of manufacture height need the instrument price of detection signal high.
Film bar hybrid method is a kind of gene chip detection method in essence, is that oligonucleotide is fixed on the nylon membrane according to addressable mode, forms array.Simultaneously, according to promotor (as the CaMV35S promotor) commonly used in the transgenic engineering, terminator (NOS terminator), screening-gene and target gene commonly used (as pest-resistant, the herbicide-resistant gene) etc. sequences Design capture probe is made the film bar, just can catch the target gene in the sample, thereby finish detection.The a plurality of target genes of film bar hybridization energy one-time detection, and cost of manufacture is low, and signal detection does not need high end instrument, therefore equips simple laboratory and can use.
Chinese patent application CN01214517 has proposed a kind of transgenic plant genes identified detection chip that is used for, on the plane of flush type branching carrier, be coated with the rete of positively charged material, be placed with the foreign gene probe of judging usefulness for transgenic plant in the microarray mode on the surface of this rete; Chinese patent application CN101717825A discloses a kind of method of rapid detection copy number of foreign gene of transgenic tobacco, with a pair of SSR special primer transgenic plant is carried out PCR and detects, copy number that just can the rapid detection transgenic line; Chinese patent application CN1718742A discloses a kind of diagnosing beta-thalassemic nucleic acid hybridization film bar and test kit that be used for, its nucleic acid hybridization film bar comprises substrate and is fixed in suprabasil specific probe, at the beta-globin gene locus of a sudden change, each bar mutant probe comprises the sequence of 14-20 base to the described specific probe of each bar respectively.This nucleic acid hybridization film bar normal, heterozygous mutation, sudden change homozygote all are easy to judge, and signal specificity is very good, diagnosis efficiency and diagnostic accuracy height, can reduce cost again; Chinese patent application CN101545011 discloses a kind of drug resistant mutant genes of mycobacterium tuberculosis and has detected film bar and test kit, the specific oligonucleotide probe that detects the Mycobacterium tuberculosis drug resistant mutant genes is fixed in the substrate, test kit can detect multiple common medicament-resistant mutation type on rpoB, katG, inhA, embB, five kinds of genes of rpsL simultaneously, has detection and fast, accurately reaches advantages such as flux is big; Chinese patent application CN2758268 discloses a kind of keloid p53 gene test film bar, and this film bar is a strip, is divided into three lattice, and wherein lattice at edge are the blank lattice, are respectively equipped with allele specific oligonucleotide in other two lattice
ProProbe and allele specific oligonucleotide Arg probe have detected result resolving power height, easy, quick, the reliable advantage of technology.
Summary of the invention
Technical purpose of the present invention is to provide a kind of nucleic acid hybridization film bar that detects foreign gene; Another technical purpose of the present invention is to provide a kind of application method of this nucleic acid hybridization film bar.
In order to realize technical purpose of the present invention, technical program of the present invention lies in:
One, a kind of nucleic acid hybridization film bar that detects foreign gene is characterized in that making by the following method:
At first design the coupling probe according to the foreign gene correspondence in the transgenic plant: the special capture probe of the conservative region of the foreign gene in transgenic plant always, and at 5 of probe ' add oligo (dt) n; Wherein, oligo (dt) n is a 5-15 oligonucleotide; Carry out amido modified to 5 of probe ' end;
Secondly, with the probe stationary that obtains on the nucleic acid hybridization film bar substrate diaphragm: draw lattice on nucleic acid hybridization film bar substrate diaphragm, the grid size is 0.1-1.0 cm, and compartment is arranged; The substrate diaphragm is behind EDC activation treatment 20-40 min, its surperficial pendant carboxylic group can have amino oligonucleotide probe with end and react, generate amido linkage, thereby the probe that will add oligo (dt) n and amido modified mistake is fixed on firmly on the substrate diaphragm; By spot sample device, the probe that will add oligo (dt) n and amido modified mistake on the substrate diaphragm, is made nucleic acid hybridization film bar through hydration after the fixing and drying treatment according to matrix rule point sample, and is dry then, obtains nucleic acid hybridization film bar of the present invention.
Wherein, oligo of the present invention (dt) n is a 10-12 oligonucleotide.
Grid size on the nucleic acid hybridization film bar substrate diaphragm of the present invention is 0.3-0.5 cm.
The method of catching, preparing and modify of probe of the present invention is interpreted as that the probe of any routine of the prior art is caught, preparation and modifying method.The special capture probe of each bar of the present invention comprises 30-37 base not to be waited.The sequence of special capture probe can make probe better combine with goal gene from the conservative region of foreign gene like this, improves detection efficiency.
Wherein, the foreign gene in the described transgenic plant of this step is interpreted as any foreign gene in the prior art, imports used promoter sequence of these foreign genes and terminator sequence.These target genes mainly are one or more dna sequence dnas and the combinations thereof that openly is selected from plant, microorganism or the animal gene, its effect mainly be pest-resistant, antiweed, disease-resistant, degeneration-resistant, improve quality, but be not limited to these effects.The foreign gene that relates in the present invention includes but not limited to CaMV35S, Nos, Npt II, Aad, aadA, Ahas, ALS, AMY797E, APH4, Aph II, Bar, Barstar, Bla, Cordap A, CP, Cry1A (b), Cry2Ab, Cry1As, cry2c, cry3A, Epsps, PAT, PG.The effect of these expression of exogenous gene in transgenic plant is as described in Table 1.
Common foreign gene and effect thereof in table 1 transgenic plant of the present invention
| The gene title | The effect summary | The gene title | The effect summary |
| CaMV35S | Promotor | Bla | Selected gene |
| Nos | Terminator | Cordap A | Promote Methionin synthetic, improve quality |
| Npt II | Selected gene | CP | Antiviral |
| Aad | Herbicide tolerant | Cry1A(b) | Pest-resistant |
| aadA | Herbicide tolerant | Cry2Ab | Pest-resistant |
| Ahas | Herbicide tolerant | Cry1As | Pest-resistant |
| ALS | Herbicide tolerant | cry2c | Pest-resistant |
| AMY797E | Promote amylolysis | cry3A | Pest-resistant |
| APH4 | Selected gene | Epsps | Herbicide tolerant |
| Aph II | Selected gene | PAT | Herbicide tolerant |
| Bar | Herbicide tolerant | PG | Delayed maturity |
| Barstar | Male sterile is used for breeding | ? | ? |
Utilize the coupling probe such as the table 2 of the common foreign gene that the described method of step (1) obtains, the foreign gene of these probes in can specific aim Identification Lists 1.The probe positive control adopts the λ dna fragmentation.Its principle is:
Wherein, the principle of design of coupling probe of the present invention is: 1) probe can mate with corresponding goal gene; 2) reduce the secondary structure that self forms; 3) length is controlled at about 25mer; 4) the Tm value is between 65-75 ℃, and GC% is controlled at 40%-60%; 5) in order to reduce the steric effect of film, at 5 of probe ' add oligo (dt) n to probe.Wherein, oligo (dt) n is a 5-15 oligonucleotide, preferred 10-12 Nucleotide; 6) 5 of probe ' end carries out amido modified.As shown in table 2 according to above principle designed probe.
The coupling probe of table 2 common foreign gene correspondence of the present invention (oligo (dt) n is a 5-15 oligonucleotide)
| The gene title | The probe title | Gene order number | Sequence (5 '-3 ') |
| CaMV35S | CaMV35S-p | SEQ NO:1 | TTTTTTTTTTCTCCACTGACGTAAGGATGACGCA |
| Nos | Nos-p | SEQ NO:2 | TTTTTTTTTTGCCGGTCTTGCGATTATCATA |
| Npt II | Npt II-p | SEQ NO:3 | TTTTTTTTTTCACGAACTCGCTGAGCAGGA |
| Aad | Aad-p | SEQ NO:4 | TTTTTTTTTTCTCCGATACCCAAGCGGCAGCAACTA |
| aadA | aadA-p | SEQ NO:5 | TTTTTTTTTTCCGACTACCTTGGTGTTCTCGCCTTT |
| Ahas | Ahas-p | SEQ NO:6 | TTTTTTTTTTATCGCAGCAGGAAGTCCAAATCCCATA |
| ALS | ALS-p | SEQ NO:7 | TTTTTACCAGCGGCGAGGGTGACAAAG |
| AMY797E | AMY797E-p | SEQ NO:8 | TTTTTTTTTTTCGCCGAGGTCAAAGTAATCATAGGGA |
| APH4 | APH4-p | SEQ NO:9 | TTTTTTTCGCCGATAGTGGAAACCGACGCTC |
| Aph II | Aph II-p | SEQ NO:10 | TTTTTTTTTTCGGTCAGCCCATTCGCCGCAAAAC |
| Bar | Bar-p | SEQ NO:11 | TTTTTTTTTTCGAGCCGCTGGTGCTGGTGGGAGACAT |
| Barstar | Barstar-p | SEQ NO:12 | TTTTTTTTTTCGCAGCCTTCCGCTTTCGCTTCAC |
| Bla | Bla-p | SEQ NO:13 | TTTTTTTTTTTTCCAGCATTTCCCAGCCCAGACCCT |
| Cordap A | Cordap A-p | SEQ NO:14 | TTTTTTTTTTCCGCCCAAAGCAAGCCAAACAAGG |
| CP | CP-p | SEQ NO:15 | TTTTTTTTTTTCCATCCATCATAACCCAAACTCCG |
| Cry1A(b) | Cry1A(b) -p | SEQ NO:16 | TTTTTTTTTTCCACGCTGTCCACGATGAATGT |
| Cry2Ab | Cry2Ab-p | SEQ NO:17 | TTTTTTTTTTACCATCAACAGCCGCTAGAACGACC |
| Cry1As | Cry1As-p | SEQ NO:18 | TTTTTTTTTTCGTTCATCACTGAGTCGCTTCGC |
| cry2c | cry2c-p | SEQ NO:19 | TTTTTTTTTTTTTTTCACGAACTCGCTGAGCAGGA |
| cry3A | cry3A-p | SEQ NO:20 | TTTTTTTTTTTGTGACGCCTATTTGTAAGTTATTCCC |
| Epsps | Epsps-p | SEQ NO:21 | TTTTTTTTTTGCCACCAGGTAGCCCTCCGATTCCA |
| PAT | PAT-p | SEQ NO:22 | TTTTTTTTTTGGTTCACGCTCGGGTCGTTGGGCAGT |
| PG | PG-p | SEQ NO:23 | TTTTTTTTTTACCCAGAACCACCCTGCCAAGTC |
Of the present invention to be used for preparing nucleic acid hybridization film bar substrate diaphragm-operated can be any material that can the immobilized oligonucleotide probe of prior art, as nylon membrane, nitrocellulose filter, aldehyde radical sheet glass, amination glass or magnetic bead etc.
Two, the application of nucleic acid hybridization film bar of the present invention in the foreign gene that detects plant germplasm resource and product.
Its concrete application method may further comprise the steps:
(1) extraction of plant sample DNA and mark:
Adopt test kit method or CTAB method to carry out the plant sample DNA extraction; Adopt the UTP of nontoxic, "dead" vitamin H or digoxin to come the mark sample DNA, and, add the EDTA of the 0.2-0.5 M of 1-2 μ L after finishing, termination reaction under pH 8.0 conditions ,-20 ℃ of preservations 37 ℃ of following incubation 3-10 h reactions;
(2) hybridization
Hybridization step of the present invention is interpreted as in the prior art arbitrarily the method that the plant sample DNA that will extract and the probe on the nucleic acid hybridization film bar are hybridized; In the present invention, concrete method is:
1) get the salmon sperm dna solution of 5 mg/mL, behind water-bath 10 min under 95 ℃ of conditions, cooled on ice 5 min finish the sex change of salmon sperm dna rapidly; The salmon sperm dna of sex change is joined in the hybridization solution, and making sex change salmon sperm dna hybridization solution concentration is 50 μ g/mL, is prepared into the hybridization solution I; Wherein 10mL hybridization solution prescription is: 20 * SSC, 5 mL; 100 * Denhart ' s, 1 mL; Dd H-
2O 3 mL; 10%SDS 1 mL;
2) nucleic acid hybridization film bar is inserted in the disposable 2 mL centrifuge tube I of sealing, in this pipe, adds 1.5 mL hybridization solution I then, and in 69 ℃ metal bath, hybridize 4 h;
3) sample DNA with vitamin H or digoxigenin labeled reacts 10 min in 100 ℃ hot water bath, rapid then cooled on ice 5 min; Sample DNA after the sex change is added in the hybridization solution I, be prepared into the hybridization solution II;
4) with process 2) the film bar that obtains is put in the disposable 2 mL centrifuge tube II of sealing, and the hybridization solution II of adding 1.5 mL, hybridization 20 h in 69 ℃ of metal baths then;
5) after hybridization was finished, interpolation concentration was 0.1% SSC+SDS mixing, washing liquid in the centrifuge tube II, washs 3 times; Wash conditions is: soak 20 min under 65 ℃ of conditions.
(3) colour developing judges whether to exist foreign gene:
After hybridization is finished, the sample DNA that vitamin H or digoxigenin labeled are crossed is fixed on the probe, utilize corresponding vitamin H detection kit or digoxin detection kit to finish color reaction, whole process color is to carry out in the disposable microtubule of sealing, and the color reaction time is 0.5-6 h; After colour developing was finished, whether each grid formed even trace on the visual inspection diaphragm, and there is the probe of even blotting membrane lattice in comparison, can directly judge the foreign gene that it is corresponding.Simultaneously, also can take pictures saving result with camera.
Beneficial effect of the present invention is:
Based on prior art, the contriver studies by experiment, and in conjunction with forefathers at the foreign gene of gene chip at plant germplasm resource and product, the particularly research in the transgenic plant foreign gene, invent a nucleic acid hybridization film bar that detects the plant foreign gene, whether the present invention can pollute by transgenosis in the simple and rapid test sample of dna level.The present invention utilizes any material that can the immobilized oligonucleotide probe such as nylon membrane, nitrocellulose filter, aldehyde radical sheet glass, amination sheet glass, magnetic bead to be substrate, adopt vitamin H or digoxigenin labeled, finish color reaction by enzymatic reaction, experimental implementation is easy to be reliable, to not injury of experimenter, and can directly arrive the result, not need to add expensive detection equipment by naked-eye observation.In addition, hybridization of the present invention, colour developing and washing process are to carry out in the disposable microtubule of sealing, reduce pollution, the flow process that simplifies the operation, improve conventional efficient.
Embodiment
Enumerate embodiment below the present invention is further specified, but therefore do not limit content of the present invention.
The detection of foreign gene in embodiment 1 transgene cotton
1. probe design
The doubtful foreign gene that carries is CaMV35S, Nos, Cry2c, Cry1A (b), Cry2Ab, the Cry1As in the table 1 in the cotton sample.
Therefore, its coupling probe and corresponding gene order thereof are the CaMV35S-p(SEQ NO:1 in the table 2), Nos-p(SEQ NO:2), Cry2c-p(SEQ NO:19), Cry1A (b)-p(SEQ NO:16), Cry2Ab-p(SEQ NO:17), Cry1As-p(SEQ NO:18).
2. probe is synthetic
The special capture probe of each bar comprises 30-37 base respectively.
3. nucleic acid hybridization film bar prepares and probe stationary
Nylon membrane is through drawing the lattice point sample on diaphragm after the cutting, the grid size is 0.3 cm, and compartment is arranged.Nylon membrane is behind EDC activation treatment 30 min, and the pendant carboxylic group on surface can react with the amino that end has an amino oligonucleotide probe, generates amido linkage, thereby probe is fixed on the nylon membrane surface firmly.
By spot sample device, with probe according to matrix rule point sample on nylon membrane, make nucleic acid hybridization film bar through hydration after the fixing and drying treatment, kept dry is standby then.
4. cotton DNA extracts
The DNA extraction test kit that adopts Axygen company to produce extracts sample DNA according to its schedule of operation.
5. cotton DNA mark
Sample thief DNA utilizes the Klenow fragment in the biotin labeling test kit, exo as dna profiling
–(5U) cotton genomic dna is carried out random amplification and mark with random primer; 37 ℃ following incubation 3-10 hour.
EDTA (pH 8.0) termination reaction that adds 1 μ L, 0.5 M then.By this step reaction, be mixed with Biotin-11-dUTP in the amplified fragments of goal gene.
6. hybridization
1) get the salmon sperm dna solution of 5 mg/mL, behind water-bath 10 min under 95 ℃ of conditions, cooled on ice 5 min finish the sex change of salmon sperm dna rapidly; The salmon sperm dna of sex change is joined in the hybridization solution, and making sex change salmon sperm dna hybridization solution concentration is 50 ug/mL, is prepared into the hybridization solution I.10 mL hybridization solution prescriptions are: 20 * SSC, 5 mL; 100 * Denhart ' s, 1 mL; Dd H-
2O 3 mL; 10%SDS 1 mL.
2) nucleic acid hybridization film bar is inserted in the disposable 2 mL centrifuge tube I of sealing, in this pipe, adds 1.5 mL hybridization solution I then, and in 69 ℃ metal bath, hybridize 4 h.
3) biotin labeled sample DNA is reacted 10 min in 100 ℃ hot water bath, rapid then cooled on ice 5 min.Sample DNA after the sex change is added in the hybridization solution I, be prepared into the hybridization solution II.
4) with crossover process 2) the film bar that obtains is put in the disposable 2 mL centrifuge tube II of sealing, and the hybridization solution II of adding 1.5 mL, hybridization 20 h in 69 ℃ of metal baths then.
5) after hybridization was finished, interpolation concentration was 0.1% SSC+SDS mixing, washing liquid in the centrifuge tube II, washs 3 times.Wash conditions is: soak 20 min under 65 ℃ of conditions.
7. colour developing
After hybridization was finished, the sample DNA that biotin labeling is crossed was fixed on the probe, utilizes the vitamin H detection kit to finish color reaction, and whole process color is to carry out in the disposable microtubule of sealing.The color reaction time is 1 h.
8. observe
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of CaMV35S-p, NOS-p and cry1 (A) b-p probe.Show that contain CaMV35S, NOS and cry1 (A) b foreign gene in this cotton test sample, promptly this cotton test sample is carried exogenous promoter, terminator and anti insect gene.
The detection of foreign gene in embodiment 2 transgenic corn seeds
1. probe design
The doubtful foreign gene that carries is CaMV35S, Nos, Epsps, Npt II, CP, the Cry1As in the table 1 in the corn seed sample.
Therefore, its coupling probe and corresponding gene order thereof are the CaMV35S-p(SEQ NO:1 in the table 2), Nos-p(SEQ NO:2), Epsps-p(SEQ NO:21), Npt II-p(SEQ NO:3), CP-p(SEQ NO:15), Cry1As-p(SEQ NO:18).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Substrate diaphragm-operated material is a nitrocellulose filter, and diaphragm grid size is 0.5 cm.Preparation of film bar and probe stationary method are with embodiment 1.
4. corn seed DNA extraction
Carrying out sample DNA with the CTAB method extracts.
5. corn seed dna marker
Sample thief DNA utilizes digoxigenin labeled test kit and random primer that the corn seed genomic dna is carried out random amplification and mark as dna profiling; 37 ℃ following incubation 3-10 hour.
The EDTA(pH 8.0 that adds 2 μ L, 0.2 M then) termination reaction.By this step reaction, be mixed with Digoxigenin-11-dUTP in the amplified fragments of goal gene.
6. hybridization
1) get the salmon sperm dna solution of 5 mg/mL, behind water-bath 10 min under 95 ℃ of conditions, cooled on ice 5 min finish the sex change of salmon sperm dna rapidly; The salmon sperm dna of sex change is joined in the hybridization solution (with embodiment 1), and making sex change salmon sperm dna hybridization solution concentration is 50 ug/mL, is prepared into the hybridization solution I.
2) nucleic acid hybridization film bar is inserted in the disposable 2 mL centrifuge tube I of sealing, in this pipe, adds 1.5mL hybridization solution I then, and in 69 ℃ metal bath, hybridize 4 h.
3) sample DNA with digoxigenin labeled reacts 10 min in 100 ℃ hot water bath, rapid then cooled on ice 5 min.Sample DNA after the sex change is added in the hybridization solution I, be prepared into the hybridization solution II.
4) with crossover process 2) the film bar that obtains is put in the disposable 2mL centrifuge tube II of sealing, and the hybridization solution II of adding 1.5 mL, hybridization 20 h in 69 ℃ of metal baths then.
5) after hybridization was finished, interpolation concentration was 0.1% SSC+SDS mixing, washing liquid in the centrifuge tube II, washs 3 times.Wash conditions is: soak 20 min under 65 ℃ of conditions.
7. colour developing
After hybridization was finished, the sample DNA that digoxigenin labeled is crossed was fixed on the probe, utilizes the digoxin detection kit to finish color reaction, and whole process color is to carry out in the disposable microtubule of sealing.The color reaction time is 1.5 h.
8. observe
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of CaMV35S-p, NOS-p, Epsps-p and Npt II-p probe.Show that contain foreign gene CaMV35S, NOS, Epsps and Npt II in this corn seed test sample, promptly this corn seed test sample is carried exogenous promoter, terminator, herbicide tolerant and selected gene.
10 kinds of foreign gene synchronous detection in 3 100 parts of doubtful transgene cottons of embodiment
1. probe design
The doubtful foreign gene that carries is CaMV35S, Nos, Cry2c, Cry1A (b), Cry2Ab, Cry1As, Epsps, Npt II, Barstar, the Bla in the table 1 in 100 parts of cotton samples.
Therefore, its coupling probe and corresponding gene order thereof are the CaMV35S-p(SEQ NO:1 in the table 2), Nos-p(SEQ NO:2), Cry2c-p(SEQ NO:19), Cry1A (b)-p(SEQ NO:16), Cry2Ab-p(SEQ NO:17), Cry1As-p(SEQ NO:18), Epsps-p(SEQ NO:21), Npt II-p(SEQ NO:3), Barstar-p(SEQ NO:12), Bla-p(SEQ NO:13).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Substrate diaphragm-operated material is the aldehyde radical sheet glass, and diaphragm grid size is 0.4 cm.Preparation of film bar and probe stationary method are with embodiment 1.
Cotton DNA extraction, cotton DNA mark, hybridization, coloration method are all with embodiment 1.Developing time is 6 h.
After colour developing is finished, observe in the corresponding film lattice of test sample CaMV35S-p, NOS-p probe of 14 portions of cottons and even circular trace occurs; Even circular trace appears in the corresponding film lattice of the test sample CaMV35S-p of 28 portions of cottons, NOS-p and Npt II-p probe; Even circular trace appears in the corresponding film lattice of the test sample NOS-p of 36 portions of cottons, Epsps-p and Npt II-p probe; All the other product corresponding film lattice of test sample that grow cotton do not have trace.Show that it is transgenic product that 78 parts of cotton samples are arranged in this batch test sample.
The detection of foreign gene in the embodiment 4 doubtful transgenic Fructus Lycopersici esculentis
1. probe design
The doubtful foreign gene that carries is CP in the table 1 in the corn seed sample.
Therefore, its coupling probe and corresponding gene order thereof are the CP-p(SEQ NO:15 in the table 2).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Substrate diaphragm-operated material is the amination sheet glass, and diaphragm grid size is 0.1 cm.Preparation of film bar and probe stationary method are with embodiment 1.
Tomato DNA extraction, tomato dna marker, hybridization, coloration method are all with embodiment 1.Developing time is 0.5 h.
After colour developing is finished, observe in the corresponding film lattice of CP-p probe and even circular trace do not occur.Show, do not contain external source antiviral gene CP in this tomato test sample.
The detection of foreign gene in the embodiment 5 doubtful genetically engineered soybean oil
Although the food that with the transgene agricultural product is raw material production does not emerge as yet in a large number, commercially available various edible oils all have transgenic product to exist at present, and especially genetically engineered soybean oil accounts for ninety percent of soybean oil total amount on sale.99.9% is lipid in the soybean oil, dna content seldom, but DNA almost is present in all soybean oil products.
1. probe design
The doubtful foreign gene that carries is CaMV35S, Nos, Epsps, the Ahas in the table 1 in the soybean oil sample.
Therefore, its coupling probe and corresponding gene order thereof are the CaMV35S-p(SEQ NO:1 in the table 2), Nos-p(SEQ NO:2), Epsps-p(SEQ NO:21), Ahas-p(SEQ NO:6).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a nylon membrane, and diaphragm grid size is 1.0 cm.Preparation of film bar and probe stationary method are with embodiment 1.
4. the extraction of DNA in the soybean oil
Because the dna content in the soybean oil is less, so the soybean oil consumption during extraction DNA is 500 g.Adopt the grease DNA extraction test kit of Axygen company.
Soybean oil dna marker, hybridization, colour developing are with embodiment 2.
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of 35s-p, nos-p and Epsps-p probe.Show, contain foreign gene CaMV35S, NOS and Epsps in this soybean oil test sample, promptly also have exogenous promoter, terminator and herbicide tolerant gene, illustrate that this soybean oil sample is a genetically engineered soybean oil.
The detection of foreign gene in embodiment 6 transgenic Rhizoma Solani tuber osis
1. probe design
The doubtful foreign gene that carries is cry3A, Epsps, PAT, the PG in the table 1 in the potato sample.
Therefore, its coupling probe and corresponding gene order thereof are the cry3A-p(SEQ NO:20 in the table 2), Epsps-p(SEQ NO:21), PAT-p(SEQ NO:22), PG-p(SEQ NO:23).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a magnetic bead, and diaphragm grid size is 0.5 cm.Preparation of film bar and probe stationary method are with embodiment 1.
Potato DNA extraction, potato dna marker, hybridization, colour developing are with embodiment 1.
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of cry3A-p probe.Show, contain external source anti insect gene cry3A in this potato test sample.
The detection of foreign gene in embodiment 7 transgenic paddy rice seeds
1. probe design
The doubtful foreign gene that carries is Barstar, Bla, Cordap A, AMY797E, the Cry1A (b) in the table 1 in the rice paddy seed sample.
Therefore, its coupling probe and corresponding gene order thereof are the Barstar-p(SEQ NO:12 in the table 2), Bla-p(SEQ NO:13), Cordap A-p(SEQ NO:14), AMY797E-p(SEQ NO:8), Cry1A (b)-p(SEQ NO:16).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a magnetic bead, and diaphragm grid size is 0.4 cm.Preparation of film bar and probe stationary method are with embodiment 1.
4. the extraction of rice paddy seed DNA
Will carry out accelerating germination of rice seed before the rice paddy seed DNA extraction, utilize seedling to carry out DNA extraction, extraction step is with embodiment 1.
Rice paddy seed dna marker, hybridization, colour developing are with embodiment 1.
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of Bla-p and Cry1A (b)-p probe.Show, contain external source selected gene Bla and external source anti insect gene Cry1A (b) in this rice paddy seed sample.
The detection of foreign gene in the embodiment 8 doubtful transgenic Fructus Lycopersici esculenti sauce
1. probe design
The doubtful foreign gene that carries is CP, Cry2Ab, Cry1As, PG in the table 1 in the tomato-sauce sample.
Therefore, its coupling probe and corresponding gene order thereof are the CP-p(SEQ NO:15 in the table 2), Cry2Ab-p(SEQ NO:17), Cry1As-p(SEQ NO:18), PG-p(SEQ NO:23).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a nylon membrane, and diaphragm grid size is 0.4 cm.Preparation of film bar and probe stationary method are with embodiment 1.
4. the extraction of DNA in the tomato-sauce
In the process of manufacture of tomato-sauce, the palliating degradation degree of tomato DNA composition is more serious, therefore when extracting tomato-sauce DNA, needs with 100 g tomato-sauce.Extracting method is with embodiment 1.
Tomato-sauce dna marker, hybridization, colour developing are with embodiment 1.
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of PG-p and CP-p probe.Show, contain external source delayed maturity gene PG and external source antiviral gene CP in this tomato-sauce sample.
The detection of transgene component in the Fructus Hordei Germinatus of embodiment 9 beer production
1. probe design
The doubtful foreign gene that carries is Cry2Ab, Cry1As, Epsps, aadA in the table 1 in the Fructus Hordei Germinatus sample.
Therefore, its coupling probe and corresponding gene order thereof are the Cry2Ab-p(SEQ NO:17 in the table 2), Cry1As-p(SEQ NO:18), Epsps-p(SEQ NO:21), aadA-p(SEQ NO:5).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a nylon membrane, and diaphragm grid size is 0.4 cm.Preparation of film bar and probe stationary method are with embodiment 1.
4. the extraction of DNA in the Fructus Hordei Germinatus
With embodiment 1.
Fructus Hordei Germinatus dna marker, hybridization, colour developing are with embodiment 1.
After colour developing is finished, do not observe in the corresponding film lattice of Cry2Ab-p, Cry1As-p, Epsps-p and aadA-p probe and even circular trace do not occur.Show, do not contain transgene component in this Fructus Hordei Germinatus sample.
The detection of foreign gene in the embodiment 10 transgenic rapeseed dregs of rice
The Ministry of Agriculture of the People's Republic of China, MOA is in issue on July 11 calendar year 2001 " agriculture genetically modified organism identity management way ", regulation is implemented annotation management to the 17 kinds of agricultural-food and the converted products thereof that comprise the Semen Brassicae campestris dregs of rice, must be labeled as " transgenosis XX processed goods (finished product) " or " process raw material and be transgenosis XX " during sale.
1. probe design
The doubtful foreign gene that carries is CaMV35S, the Nos in the table 1 in the Semen Brassicae campestris dregs of rice sample.
Therefore, its coupling probe and corresponding gene order thereof are the CaMV35S-p(SEQ NO:1 in the table 2), Nos-p(SEQ NO:2).
2. probe is synthetic
With embodiment 1.
3. nucleic acid hybridization film bar prepares and probe stationary
Basilar membrane sheet material is a magnetic bead, and diaphragm grid size is 0.4 cm.Preparation of film bar and probe stationary method are with embodiment 1.
Semen Brassicae campestris dregs of rice DNA extraction, Semen Brassicae campestris dregs of rice dna marker, hybridization, colour developing are with embodiment 2.
After colour developing is finished, observe the even circular trace of appearance in the corresponding film lattice of 35s-p and nos-p probe.Show, contain exogenous promoter CaMV35S and external source terminator NOS in this Semen Brassicae campestris dregs of rice sample.
Claims (8)
1. nucleic acid hybridization film bar that detects foreign gene is characterized in that making by the following method:
At first design the coupling probe according to the foreign gene correspondence in the transgenic plant: the special capture probe of the conservative region of the foreign gene in transgenic plant always, and at 5 of probe ' add oligo (dt) n; Wherein, oligo (dt) n is a 5-15 oligonucleotide; Carry out amido modified to 5 of probe ' end;
Secondly, with the probe stationary that obtains on the nucleic acid hybridization film bar substrate diaphragm: draw lattice on nucleic acid hybridization film bar substrate diaphragm, the grid size is 0.1-1.0 cm, and compartment is arranged; The substrate diaphragm is through EDC activation treatment 20-40 min; By spot sample device, the probe that will add oligo (dt) n and amido modified mistake on the substrate diaphragm, is made nucleic acid hybridization film bar through hydration after the fixing and drying treatment according to matrix rule point sample, and is dry then, obtains nucleic acid hybridization film bar.
2. the nucleic acid hybridization film bar of detection foreign gene according to claim 1 is characterized in that described oligo (dt) n is a 10-12 oligonucleotide.
3. the nucleic acid hybridization film bar of detection foreign gene according to claim 1 is characterized in that the grid size on the described nucleic acid hybridization film bar substrate diaphragm is 0.3-0.5 cm.
4. the nucleic acid hybridization film bar of detection foreign gene according to claim 1 is characterized in that described foreign gene is CaMV35S, Nos, Npt II, Aad, aadA, Ahas, ALS, AMY797E, APH4, Aph II, Bar, Barstar, Bla, Cordap A, CP, Cry1A (b), Cry2Ab, Cry1As, cry2c, cry3A, Epsps, PAT, PG.
5. the nucleic acid hybridization film bar of detection foreign gene according to claim 1 is characterized in that described to be used to prepare nucleic acid hybridization film bar substrate diaphragm be nylon membrane, nitrocellulose filter, aldehyde radical sheet glass, amination sheet glass or magnetic bead.
6. the application of the nucleic acid hybridization film bar of the described detection foreign gene of claim 1 in the foreign gene that detects plant germplasm resource and product.
7. application according to claim 6 is characterized in that may further comprise the steps:
(1) extraction of plant sample DNA and mark:
Adopt test kit method or CTAB method to carry out the plant sample DNA extraction; Adopt the UTP of nontoxic, "dead" vitamin H or digoxin to come the mark sample DNA, and, add the EDTA of the 0.2-0.5 M of 1-2 μ L after finishing, termination reaction under pH 8.0 conditions ,-20 ℃ of preservations 37 ℃ of following incubation 3-10 h reactions;
(2) hybridization
The hybridization that the plant sample DNA that extracts and the probe on the nucleic acid hybridization film bar are carried out;
(3) colour developing judges whether to exist foreign gene:
After hybridization is finished, the plant sample DNA that vitamin H or digoxigenin labeled are crossed is fixed on the probe, utilize corresponding vitamin H detection kit or digoxin detection kit to finish color reaction, whole process color is to carry out in the disposable microtubule of sealing, and the color reaction time is 0.5-6 h; After colour developing was finished, whether each grid formed even trace on the visual inspection diaphragm, and there is the probe of even blotting membrane lattice in comparison, can directly judge the foreign gene that it is corresponding.
8. application according to claim 7 is characterized in that the method for the hybridization of described step (2) is:
(1) get the salmon sperm dna solution of 5 mg/mL, behind water-bath 10 min under 95 ℃ of conditions, cooled on ice 5 min finish the sex change of salmon sperm dna rapidly; The salmon sperm dna of sex change is joined in the hybridization solution, and making sex change salmon sperm dna hybridization solution concentration is 50 μ g/mL, is prepared into the hybridization solution I; Wherein 10mL hybridization solution prescription is: 20 * SSC, 5 mL; 100 * Denhart ' s, 1 mL; Dd H-
2O 3 mL; 10%SDS 1 mL;
(2) nucleic acid hybridization film bar is inserted in the disposable 2 mL centrifuge tube I of sealing, in this pipe, adds 1.5 mL hybridization solution I then, and in 69 ℃ metal bath, hybridize 4 h;
(3) sample DNA with vitamin H or digoxigenin labeled reacts 10 min in 100 ℃ hot water bath, rapid then cooled on ice 5 min; Sample DNA after the sex change is added in the hybridization solution I, be prepared into the hybridization solution II;
(4) with process 2) the film bar that obtains is put in the disposable 2 mL centrifuge tube II of sealing, and the hybridization solution II of adding 1.5 mL, hybridization 20 h in 69 ℃ of metal baths then;
(5) after hybridization was finished, interpolation concentration was 0.1% SSC+SDS mixing, washing liquid in the centrifuge tube II, washs 3 times; Wash conditions is: soak 20 min under 65 ℃ of conditions.
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| CN112662661A (en) * | 2020-12-30 | 2021-04-16 | 华南理工大学 | Genome DNA room temperature preservation card and manufacturing method and application thereof |
| CN112662661B (en) * | 2020-12-30 | 2023-11-03 | 华南理工大学 | Genomic DNA room temperature preservation card and manufacturing method and application thereof |
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