CN103773836A - Method for rapid detection of transgenic rice line TT51-1 - Google Patents
Method for rapid detection of transgenic rice line TT51-1 Download PDFInfo
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Abstract
The invention discloses a method for rapid detection of transgenic rice line TT51-1 and primers for use. The method belongs to the technical field of molecular biology, and relates to detection methods of transgenic products, the principle is as follows: using 6 specific primers and a DNA polymerase with chain displacement activity for amplification of a sample template DNA at 61 DEG C, amplified products can reach 109-1010 copies in a short period of time, result identification can be realized as follows: determining whether the DNA is amplified or not by direct observation of color change in a reaction tube by naked eyes, and the method has the characteristics of being high in efficiency, fast, simple in operation, high in specificity and the like, and provides a convenient means for the rapid detection of transgenic rice.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to the detection method of transgenic product, be the method for rapid detection transgenic paddy rice strain TT51-1 a kind of specifically, carry out whether to contain in judgement sample the composition of transgenic paddy rice TT51-1 by bore hole observing response pipe colour-change or observation agarose gel electrophoresis.
Background technology
Since first case commercialization genetically modified crops approval listing in 1994, transgenic technology is in the application and just steady-state growth always of development in Global Agriculture field.According to statistics, current global genetically modified crops cultivated area has continued to increase continuously for 16 years, and within 2011, global genetically modified crops cultivated area reaches 1.6 hundred million hectares, is 94 times in 1996, accounts for 10% of global available cultivated area.Between 15 years of 1996 to 2011, occupy 29 countries of the world's 60% population at plantation transgenic crop, exceed 100,000,000 person-times and planted genetically modified crops, accumulative total cultivated area has exceeded 12.5 hundred million hectares.
TT51-1 transgenic paddy rice, i.e. " extensive No. 1 of China " strain, is that its core knowledge property right all belongs to domestic research and development unit by the transgenic paddy rice of first acquisition production application safety certificate of Central China agricultural university independent research.2009, transgenic pest-resistant rice TT51-1 obtained the production application safety certificate that agricultural genetically modified organism security control office of the Ministry of Agriculture issues, and ratifies it and produces and apply in Hubei Province, and this indicates the beginning of China's transgenic paddy rice industrialization.TT51-1 acceptor kind used is Elite restorer line " bright extensive 63 ", anti insect gene Cry1Ab/Ac is the fusion gene that the Chinese Academy of Agricultural Sciences obtains patent, the method of cultivation of TT51-1 transgenic paddy rice has also obtained license in China, and " extensive No. 1 of China " is to relevant department's application new variety of plant power at present.
Along with the surge of genetically modified crops cultivated area and the acceleration of global transgenic plant research and development, also day by day receive much concern for the safety issue of genetically modified organism and products thereof composition.Genetically modified organism safety mainly comprises two aspects: on the one hand for edible safety, comprise the variation of nutritive ingredient and antinutritional factor in genetically modified crops and products thereof composition, the toxicity of specific expression protein and sensitization, and target gene drifts to potential risk causing after enteric microorganism or human body etc.; To pay close attention to by the impact on ecological environment security in genetically modified crops planting process in addition on the one hand, comprise agroecosystem and the outer biological multifarious impact of system, foreign gene is to non-transgenic plant and wild relatives lateral drift and the impact that may bring thereof, the impact of antibiont stress gene on non-target organism etc.
In the world genetically modified organism product is detected at present and food identity management aspect forming section common recognition property file, evaluation principle and examination criteria, but different countries and regions, because national conditions are different with policy guidance, cause there is larger difference in the management system such as threshold range, identification means of transgenic product label.For example, European Union and China enforce genetically modified food rules, and genetically modified organism is carried out to strict approval, mark and tracking requirement; And take loose policy as countries such as the U.S., carry out voluntary identity management.
China announces and implements " agriculture genetically modified organism security control regulations " May 9 calendar year 2001, agriculture genetically modified organism safety evaluation, mark and three supporting management ways of import security management have been announced on January 5th, 2002, determine the agriculture genetically modified organism catalogue of first enforcement identity management, and formal enforcement in 20 days March in 2002.In September, 2007, for further standard transgenic plant safety evaluation content and method, agricultural genetically modified organism security control office of the Ministry of Agriculture has issued " transgenic plant safety evaluation guide ", and transgenic plant safety evaluation content has been done to further adjustment and expansion.
The testing requirement method of transgenic product is quick, accurate, sensitive, and must consider to adapt to the feature such as large sample amount, target gene kind be numerous.Therefore the target that, genetically modified crops detect is at present mainly exogenous protein (gene expression product) and foreign gene (DNA).In genetically modified crops, foreign protein can utilize the methods such as enzyme-linked immunosorbent assay (ELISA) based on immunology principle, ELISA test strip, Western hybridization to detect.The general matrix that contains target protein according to certain program extracting from testing sample, utilizes the characteristic of antibody and target protein (antigen) specific combination, produces detectable signal by the effect of coupling antibody and immune complex.The nucleic acid detection method of genetically modified crops mainly contains two kinds: hybridization (Sourthern blot) and polymerase chain reaction (Polymerase Chain Reaction, PCR).Wherein PCR detection method comprises qualitative PCR method, composite PCR method, nested PCR method, competitive quantifying PCR method, fluorescence quantifying PCR method etc.
At present, transgenosis detects main qualitative PCR and the real-time quantitative PCR detection method of using both at home and abroad.The ultimate principle of round pcr is similar to the natural reproduction process of DNA, and its specificity depends on the Oligonucleolide primers with the complementation of target sequence two ends, makes its object fragment that increases fast, specifically in vitro by polysaccharase.PCR detection method can be expanded to rapidly 1,000,000 times by trace, specific DNA fragmentation in several hours, and its amplified production is easy to observe after agarose gel electrophoresis, ethidium bromide staining, thereby possesses the advantages such as quick, sensitive.Although PCR detection method technology maturation is stable, reaction system and operating process are more numerous and diverse, need professional; Pcr amplification instrument is expensive, and testing environment is required to high and poor mobility, is not suitable for field quick detection; Qualitative PCR amplified reaction and electrophoresis detection are consuming time longer, and common dyes EB is strong carcinogen; The instrument of quantitative PCR and consumptive material price are higher, and need to connect the real-time monitoring and detection result of computer; Above factor all limits its application as field quick detection.
Summary of the invention
The object of the invention is to the method for open a kind of rapid detection transgenic paddy rice strain TT51-1 and design a set of primer pair according to foreign gene junction sequence that it increases, observe colour-change or judge amplification situation by observing agarose gel electrophoresis result by bore hole.
Technical scheme of the present invention is as follows:
The present invention is based on the border sequence information of transgenic paddy rice TT51-1, provide thereby design primer carries out ring-type isothermal duplication the method that detects transgenic paddy rice TT51-1.
For detection of the Auele Specific Primer of transgenic paddy rice TT51-1, it is characterized in that comprising:
Outer primer forward sequence: AATGACCGCTGTTATGCG;
Outer primer reverse sequence: TTTTGGAACATATGAGTGGTAG;
Inner primer forward sequence: CCTCTAGAGTCGACCTGCAGCCATTGATTTGTAGAGAGAGAC;
Inner primer reverse sequence: CGAGTGCTGGGGCAGATAAGCGTCCAGAAGGAAAAGGAAT;
Ring primer forward sequence: CATGCCCGCTGAAATCACC;
Ring primer reverse sequence: GTAGTGGTGGGGCTACGAAC.
The present invention adopts above-mentioned a set of primer to carry out the method for rapid detection transgenic paddy rice strain TT51-1, it is characterized in that comprising the steps:
(1) adopt 6 special primers claimed in claim 1 and a kind of archaeal dna polymerase with strand displacement activity, add template DNA, 61 ℃ of incubation 60min, then at 80 ℃ of lasting 2min, can make amplified production reach 10
9-10
10individual copy;
Wherein the cumulative volume of amplified reaction is 25 μ L, and its various compositions are respectively: 10 × Buffer, 2.5 μ L, 10mM dNTPs 2 μ L, 50mM MgSO
42 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mM MnCl
21 μ L, primer mixing solutions 1 μ L, 8000U/ml Bst archaeal dna polymerase large fragment 1 μ L, template DNA 1-5 μ L, with sterilizing deionized water polishing to 25 μ L;
(2) amplified reaction finishes, and extracts reaction solution 3-25 μ L, adopts different methods judgement whether to increase, and comprising: directly have or not amplified reaction by observing colour-change judgement; Or judge amplification by observing agarose gel electrophoresis band.For example can pass through bore hole observing response pipe colour-change judged result, orange negative, green positive; Or by agarose gel electrophoresis judged result, negative without gradient band, there is gradient band positive.
Primer mixing solutions of the present invention refer to by synthetic primer dry powder respectively compound concentration be the mother liquor of 100 μ M, then get the each 1 μ L of outer primer F3, B3, the each 8 μ L of inner primer FIP, BIP, the each LF of ring primer, the each 1 μ L of LB, fully mix, make primer mixing solutions.
Template DNA of the present invention refers to and adopts CTAB method to extract the genomic dna that purification of samples obtains.
For measuring method of the present invention can be more clearly described, below test method of the present invention is done with detailed explanation.
1, principle
Present method is applied novel nucleic acid amplification method, its principle is to adopt 6 special primers and a kind of archaeal dna polymerase with strand displacement activity, adds template DNA, at 61 ℃ of reaction 60min, and after 80 ℃ of lasting 2min, stop, in the short period of time, amplified production can reach 10
9-10
10individual copy.There is the features such as high specific, high efficiency, quick, easy, easy detection.
2, design of primers
This research according to transgenic paddy rice TT51-1 foreign gene and native gene junction sequences Design 6 primers, be respectively forward outer primer F3, oppositely outer primer B3, forward inner primer FIP, oppositely inner primer BIP, forward ring primer LF and oppositely encircle primer LB.Inner primer length exceedes 40 bases conventionally, is about 20 bases outward.Except the length of primer, based composition, also to consider the textural factor of primer and target sequence, make primer can better be combined and form ring-type amplification structure with template goal gene sequence.Primer is synthesized by Shanghai bio-engineering corporation.
Table 1 primer sequence table is as follows:
3, reaction conditions
Reaction reagent needs strand displacement type archaeal dna polymerase, dNTPs, transgenic paddy rice TT51-1 special primer, BSA, MgSO
4, fluorexon, MnCl
2and reaction buffer.Reaction is carried out under constant temperature, the reaction times because of the amplification efficiency of primer and the quality of template DNA different, be generally 1h or still less.Add template DNA, at 61 ℃ of reaction 60min, and stop after 80 ℃ of lasting 2min.The advantage of this technology is exactly not need thermal cycling.Because amplified reaction carries out under constant temperature, therefore only need thermostat water bath or METAL HEATING PROCESS piece can maintain temperature of reaction, do not need the expensive instruments such as PCR instrument.
Materials and methods:
(1) Bst archaeal dna polymerase large fragment and 10 times of Thermal Buffer solution that reagent: BioLabs (NEW ENGLAND) produces; DNTPs; MgSO
4solution; BSA; Fluorexon; MnCl
2solution; TT51-1 strain specificity primer; Deionized water.
(2) amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, and its various compositions and consumption are respectively: 10 × Buffer2.5 μ L, 10mM dNTPs 2 μ L, 50mM MgSO
42 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mMMnCl
21 μ L, primer mixing solutions 1 μ L, 8000U/ml BstDNA polysaccharase large fragment 1 μ L, template DNA 1-5 μ L, with sterilizing deionized water polishing to 25 μ L.
(3) amplified reaction process: at 61 ℃ of reaction 60min, and at 80 ℃ of lasting 2min, 4 ℃ of preservations;
(4) amplified reaction finishes, and extracts reaction solution 3-25 μ L, judges and whether increases by different detection methods.
4, amplification is observed
There are two kinds of observational techniques, are applicable to carrying out under different situations:
1) detect and can directly observe colour-change by bore hole and whether judge reaction.Before reaction, Mn
2+ion is preferentially combined with fluorexon, aobvious orange; In reaction, because nucleic acid synthesizes the mn ion (Mn in pyrophosphate ion and the reaction soln producing in a large number
2+) combination, produce manganese pyrophosphate precipitation; Magnesium ion (Mg simultaneously
2+) be combined with fluorexon, present fluorescent green; Therefore, without amplified reaction be orange, what have amplified reaction will become green.The results are shown in Figure 1.
2) detect whether to judge reaction by agarose gel electrophoresis.Use 2% sepharose, add EB staining agent, 100V electrophoresis 50min observes under ultraviolet lamp.Reaction can produce the amplified production that possesses loop-stem structure of multiple different lengths, therefore in electrophoretogram, shows as stair-stepping band below point sample hole.Because EB dyestuff is strong carcinogen, therefore the method application is not general.The results are shown in Figure 2.
The amplification method that the present invention detects for transgenic paddy rice strain TT51-1, has the following advantages:
1) easy and simple to handle: not need complicated instrument, do not need special reagent, also do not need to carry out in advance the sex change of double-stranded DNA, only need a steady temperature just can react.
2) high specific: this technology, by 6 sections of 6 primer amplification target sequences, therefore has high degree of specificity.
3) rapidly and efficiently: detection method of the present invention is quick, accurate, sensitive, whole amplification procedure can complete less than 1h, and output can reach 10
9-10
10individual copy.
4) highly sensitive: it is only 20 copies gene orders even still less that amplified reaction can detect.
5) identify easy: can whether increase by colour-change judgement in the direct observing response pipe of bore hole.
Accompanying drawing explanation
Fig. 1 is amplified production result figure.Be followed successively by from left to right negative control, positive control, negative sample 1, negative sample 2, positive 1, positive 2, each sample repeats 2 times.
Fig. 2 is the electrophoretic analysis collection of illustrative plates of amplified production.Be followed successively by from left to right Marker, negative control, positive control, negative sample 1, negative sample 2, positive 1, positive 2, each sample repeats 2 times.
Fig. 3 is amplified production result figure.Be followed successively by from left to right negative control, positive control, testing sample 1, testing sample 2, testing sample 3, testing sample 4.
Fig. 4 is the electrophoretic analysis collection of illustrative plates of amplified production.Be followed successively by from left to right Marker, negative control, positive control, testing sample 1, testing sample 2, testing sample 3, testing sample 4.
Fig. 5 is embodiment 3 the inventive method amplified production result figure.From left to right: blank, 10ng, 1ng, 100pg, 50pg, 10pg, 5pg, 1pg.
Fig. 6 is the agarose gel electrophoresis analysis of embodiment 3 the inventive method amplified productions, observations under ultraviolet lamp, and wherein M is DL2000DNA Marker; 1, blank; 2,10ng; 3,1ng; 4,100pg; 5,50pg; 6,10pg; 7,5pg; 8,1pg.
Fig. 7 is the agarose gel electrophoresis analysis of the conventional qualitative PCR method of embodiment 3 amplified production, observations under ultraviolet lamp, and wherein M is DL2000DNA Marker; 1, blank; 2,10ng; 3,1ng; 4,100pg; 5,50pg; 6,10pg; 7,5pg; 8,1pg.
For method of the present invention can be more clearly described, below test method of the present invention is done with detailed explanation, need be illustrated at this:
(1) primer sequence of the present invention is in table 1.
(2) extracting method of sample DNA template: conventional CTAB method is extracted (seeing appendix)
Embodiment
(1) BstDNA polysaccharase large fragment and 10 times of Thermal Buffer solution that reagent: BioLabs (NEWENGLAND) produces; TT51-1 Auele Specific Primer mixed solution; Fluorexon solution, manganese chloride solution; Adlerika; DNTPs; BSA; Deionized water.DNA profiling comprises negative control, positive control, negative sample 1, negative sample 2, positive 1, positive 2.
(2) amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, and its various compositions and consumption are respectively: 10 × Buffer2.5 μ L, 10mM dNTPs 2 μ L, 50mM MgSO
42 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mMMnCl
21 μ L, primer mixing solutions 1 μ L, 8000U/ml Bst archaeal dna polymerase large fragment 1 μ L, template DNA 1 μ L, with sterilizing deionized water polishing to 25 μ L, mixes rear centrifugal;
(3) amplified reaction program: 61 ℃ of incubation 60min, 80 ℃ keep 2min, 4 ℃ of preservations afterwards;
(4), after amplified reaction finishes, can pass through the direct observations of bore hole.Be yellow without the pipe of amplified reaction, have the pipe of amplified reaction will become green.The results are shown in Figure 1.Be followed successively by from left to right negative control, positive control, negative sample 1, negative sample 2, positive 1, positive 2, each sample repeats 2 times.Also can pass through detected through gel electrophoresis result.Extract reaction solution 5 μ L, through 2% agarose gel electrophoresis analysis, observations under ultraviolet lamp.Without obvious band, there is the pipe of amplified reaction to occur stepped band without the pipe of amplified reaction.The results are shown in Figure 2.
(1) Bst archaeal dna polymerase large fragment and 10 times of Thermal Buffer solution that reagent: BioLabs (NEW ENGLAND) produces; TT51-1 Auele Specific Primer mixed solution; Fluorexon solution, manganese chloride solution; Adlerika; DNTPs; BSA; Deionized water.DNA profiling comprises negative control, positive control, testing sample 1, testing sample 2, testing sample 3, testing sample 4.
(2) amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, and its various compositions and consumption are respectively: 10 × Buffer2.5 μ L, 10mM dNTPs 2 μ L, 50mM MgSO
42 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mMMnCl
21 μ L, primer mixing solutions 1 μ L, 8000U/ml BstDNA polysaccharase large fragment 1 μ L, template DNA 2 μ L, with sterilizing deionized water polishing to 25 μ L, mix rear centrifugal;
(3) amplified reaction program: 61 ℃ of incubation 60min, 80 ℃ keep 2min, 4 ℃ of preservations afterwards;
(4), after amplified reaction finishes, can pass through the direct observations of bore hole.Be yellow without the pipe of amplified reaction, have the pipe of amplified reaction will become green.The results are shown in Figure 3.From left to right: negative control, positive control, testing sample 1, testing sample 2, testing sample 3, testing sample 4.Also can pass through detected through gel electrophoresis result.Extract reaction solution 5 μ L, through 2% agarose gel electrophoresis analysis, observations under ultraviolet lamp.Without obvious band, there is the pipe of amplified reaction to occur stepped band without the pipe of amplified reaction.The results are shown in Figure 4.From result, testing sample 1 and testing sample 4 all contain transgenic paddy rice TT51-1 composition.
Contrast experiment: the measuring method of transgenic paddy rice TT51-1 and the contrast of measuring method of the present invention at present.
(1) present method reagent: BioLabs (NEW ENGLAND) produces Bst archaeal dna polymerase large fragment and 10 times of Buffer solution; TT51-1 Auele Specific Primer mixed solution; Fluorexon solution, manganese chloride solution; Adlerika; DNTPs; BSA; Deionized water.DNA profiling comprises the sample that contains transgenic paddy rice TT51-1 composition 10ng (approximately 20000 copies), 1ng (approximately 2000 copies), 100pg (approximately 200 copies), 50pg (approximately 100 copies), 10pg (approximately 20 copies), 5pg (approximately 10 copies), 1pg (approximately 2 copies).
(2) present method amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, and its various compositions and consumption are respectively: 10 × Buffer, 2.5 μ L, 10mM dNTPs 2 μ L, 50mM MgSO
42 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mM MnCl
21 μ L, primer mixing solutions 1 μ L, 8000U/ml Bst archaeal dna polymerase large fragment 1 μ L, template DNA 1 μ L, with sterilizing deionized water polishing to 25 μ L, mixes rear centrifugal;
(3) present method amplified reaction program: 61 ℃ of incubation 60min, 80 ℃ keep 2min, 4 ℃ of preservations afterwards;
(4), after amplified reaction finishes, can pass through the direct observations of bore hole.Be yellow without the pipe of amplified reaction, have the pipe of amplified reaction will become green.The results are shown in Figure 5.From left to right: blank, 10ng, 1ng, 100pg, 50pg, 10pg, 5pg, 1pg.Also can pass through detected through gel electrophoresis result.Extract reaction solution 5 μ L, through 2% agarose gel electrophoresis analysis, observations under ultraviolet lamp.Without obvious band, there is the pipe of amplified reaction to occur stepped band without the pipe of amplified reaction.The results are shown in Figure 6.M is DL2000DNA Marker; 1, blank; 2,10ng; 3,1ng; 4,100pg; 5,50pg; 6,10pg; 7,5pg; 8,1pg.
(5) PCR reaction primer adopts the primer using in No. 1193 bulletins-3 of the Ministry of Agriculture, and this cover primer is that the qualitative PCR of the current TT51-1 generally using detects primer.PCR reaction is 25 μ L systems, 10 × PCR buffer (Promega), 2.5 μ L, 10mMdNTPs (Promega) 0.5 μ L, the each 0.5 μ L of upstream and downstream primer (10mM), Taq enzyme (5U/ μ L, Promega) 0.5 μ L, DNA profiling 1 μ L, sterilizing deionized water 19.5 μ L.Response procedures is 95 ℃ of denaturation 2min, 95 ℃ of sex change 30s, and 56 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations, 72 ℃ are extended 7min.PCR product is got 10 μ L in 2% agarose gel electrophoresis, and 40min under 100V voltage, by gel image analyser observations.The results are shown in Figure 7.M is DL2000DNAMarker; 1, blank; 2,10ng; 3,1ng; 4,100pg; 5,50pg; 6,10pg; 7,5pg; 8,1pg.
Comparative result by two kinds of methods can find out, method sensitivity of the present invention, apparently higher than the susceptibility of PCR method, can detect the sample that TT51-1 content is lower.
Annex: conventional CTAB method is extracted the method for transgenic paddy rice TT51-1 genomic dna
(1) seed of getting appropriate trans-genetic hybrid rice TT51-1 is put into mortar, adds a small amount of liquid nitrogen to grind rapidly, and liquid nitrogen adds 3～4 times repeatedly, till being milled to powder.
(2) get 50～100mg powder in 2ml centrifuge tube, add the CTAB Extraction buffer of 65 ℃ of preheatings of 1.5mL, fully mix, 65 ℃ of incubation 30min, do not stop to put upside down to mix during this time.
(3) the about centrifugal 10min of 12000 × g; Shift the new centrifuge tube of supernatant to, add the phenol of 1 times of volume: chloroform: primary isoamyl alcohol (25: 24: 1), fully mixes; The approximately centrifugal 15min of 12000 × g; Shift in the new centrifuge tube of supernatant to.
(4) add the chloroform of 1 times of volume: primary isoamyl alcohol (24: 1), fully mixes; The approximately centrifugal 15min of 12000 × g; Shift in the new centrifuge tube of supernatant to.
(5) add 2 times of Volume CT AB precipitation buffering liquid, room temperature leaves standstill 60min; The centrifugal 15min of 12000 × g, abandons supernatant; Add 350 μ L sodium chloride solution dissolution precipitations; The centrifugal 10min of 12000 × g, shifts the new centrifuge tube of supernatant to.
(6) add 0.6 times of volume Virahol, be inverted centrifuge tube and softly mix, room temperature is placed 20min; The centrifugal 15min of 12000 × g, abandons supernatant; Add 500 μ L70% ethanolic solns, put upside down centrifuge tube for several times; The centrifugal 10min of 12000 × g, abandons supernatant.
(7) dry DNA precipitation, adds 100 μ L water or TE damping fluid dissolving DNA.
Sequence table
Claims (4)
1. for detection of an Auele Specific Primer of transgenic paddy rice TT51-1, it is characterized in that, by outer primer forward sequence: 5 '-AATGACCGCTGTTATGCG-3 '; Outer primer reverse sequence: 5 '-TTTTGGAACATATGAGTGGTAG-3 '; Inner primer forward sequence: 5 '-CCTCTAGAGTCGACCTGCAGCCATTGATTTGTAGAGAGAGAC-3 '; Inner primer reverse sequence: 5 '-CGAGTGCTGGGGCAGATAAGCGTCCAGAAGGAAAAGGAAT-3 '; Ring primer forward sequence: 5 '-CATGCCCGCTGAAATCACC-3 '; Ring primer reverse sequence: 5 '-GTAGTGGTGGGGCTACGAAC-3 ' composition.
2. a method that adopts Auele Specific Primer rapid detection transgenic paddy rice TT51-1 described in claim 1, is characterized in that comprising the steps:
(1) adopt 6 special primers claimed in claim 1 and a kind of archaeal dna polymerase with strand displacement activity, when adding after DNA profiling the constant-temperature amplification that carries out 60min at 61 ℃, at 80 ℃ of lasting 2min, can make amplified production reach 109-1010 copy afterwards;
Wherein the cumulative volume of amplified reaction is 25 μ L, its various compositions are respectively: 10 × Buffer, 2.5 μ L, 10mMdNTPs 2 μ L, 50mM MgSO4 2 μ L, 12.5 × BSA, 2 μ L, 625 μ M Calcein 2 μ L, 12.5mMMnCl2 1 μ L, primer mixing solutions 1 μ L, 8000U/ml Bst archaeal dna polymerase large fragment 1 μ L, template DNA 1-5 μ L, with sterilizing deionized water polishing to 25 μ L;
(2) after amplified reaction finishes, extract reaction solution 3-25 μ L, adopt different methods judgement whether to increase, comprising: directly observe colour-change judgement by bore hole and have or not amplified reaction; Or judge amplification by observing agarose gel electrophoresis band.
3. detection method as claimed in claim 2, wherein said primer mixing solutions refers to synthetic primer dry powder is mixed with respectively to the mother liquor that concentration is 100 μ M, then get the each 1 μ L of outer primer F3, B3, the each 8 μ L of inner primer FIP, BIP, ring primer LF, the each 1 μ L of LB, fully mix, make primer mixing solutions.
4. detection method as claimed in claim 2, wherein said template DNA refers to and adopts CTAB method to extract the DNA that purification of samples obtains, and volume is 1-5 μ L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636360A (en) * | 2016-11-18 | 2017-05-10 | 浙江省检验检疫科学技术研究院 | Microdroplet type digital PCR (polymerase chain reaction) detection method and kit for transgenic rice Bt63 |
| CN107312820A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for genetically modified rice TT51-1 strain-specific gene compositions |
| CN108277293A (en) * | 2018-03-22 | 2018-07-13 | 上海交通大学 | Transgenic paddy rice G6H1 strain specificity LAMP detection methods |
| CN109136339A (en) * | 2018-10-19 | 2019-01-04 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice TT51-1 |
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| CN108277293A (en) * | 2018-03-22 | 2018-07-13 | 上海交通大学 | Transgenic paddy rice G6H1 strain specificity LAMP detection methods |
| CN109136339A (en) * | 2018-10-19 | 2019-01-04 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice TT51-1 |
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