CN108034757A - A kind of RPA primers and detection method for detecting transgenic corns Bt11 strains - Google Patents
A kind of RPA primers and detection method for detecting transgenic corns Bt11 strains Download PDFInfo
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- CN108034757A CN108034757A CN201810061953.7A CN201810061953A CN108034757A CN 108034757 A CN108034757 A CN 108034757A CN 201810061953 A CN201810061953 A CN 201810061953A CN 108034757 A CN108034757 A CN 108034757A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
A kind of RPA primers and detection method for detecting transgenic corns Bt11 strains, according to the sequence of transgenic corns Bt11 foreign genes glufosinate acetyl transferase gene PAT and Maize genome join domain, devise RPA primers, the RPA primers include forward primer F and reverse primer R, particular sequence are：F：5’‑CTGGGAGGCCAAGGTATCTAATCAGCCATCCC‑3’；R：5 ' CTGCTGTAGCTGGCCTAATCTCAACTGGTCTCC 3 ', establish the quick determination method based on recombinase polymerase isothermal amplification technique, Rapid identification is carried out to transgenic corns Bt11 strains, high specificity, high sensitivity, can realize the exponential amplification to purpose fragment in the short time, environmental unit be required low, it is easy to operate, there is wide popularizing application prospect in transgenic product composition detection field.
Description
Technical field
The invention belongs to field of plant molecular biology, and in particular to a kind of RPA for detecting transgenic corns Bt11 strains
Primer and detection method.
Background technology
With the fast development of transgenic technology research and application, the research of GMO detection technology becomes the whole world
The important component of GMO bio-safety management.Round pcr has a wide range of applications in detection GMOs, still, often
The PCR detections of rule need accurate thermal cycler and the experimental arrangement of complexity, it is difficult to meet transgenosis under non-lab environment
Nucleic acid compositions rapid screening and the demand of detection.
In recent years, nucleic acid isothermal amplification technology has obtained faster development, compared with Standard PCR, nucleic acid isothermal amplification skill
Art no longer needs thermal cycler instrument, can go out purpose fragment in rapid amplifying under constant temperature, quick, easy, sensitive.At present, it is main
The isothermal amplification technique wanted have loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification,
LAMP), rolling circle amplification (Rolling circle amplification, RCA) and recombinase polymeric enzymatic amplification technology
(Recombinase ploymerase amplification, RPA) etc..
RPA technical principles be simulation organism in DNA replication dna, by recombinase-mediated, can be to target fragment at 37 DEG C
Isothermal duplication is carried out, realizes that monomolecular nucleic acid detects.Restructuring zymoprotein can combine single stranded DNA (primer) in the presence of ATP
And form DNA nucleoprotein microfilaments.Nucleoprotein microfilament both ends can extend, and can extend the side shortened rear end by front end
Normal direction double chain DNA molecule moves, and microfilament can catch hold of the DNA molecular of surrounding, by single-stranded oligonucleotide sequence and DNA sequence dna into
Row compares；When finding matched sequence, two molecules will be closely adhered to ensures that restructuring is ensued together, in single-stranded knot
With the help of hop protein, template DNA starts to unwind, and primer invasion double-stranded DNA simultaneously starts free 3 ' needed for pairing formation duplication
C-terminal, carries out duplication extension under the action of polymerase, forms new DNA complementary strand reaction products.
The characteristics of RPA is maximum is need not to realize nucleic and melting and annealing by high and low temperature circulation, it is only necessary to which 1 pair is drawn
Thing can in 37 DEG C of constant temperature, the amplification of template nucleic acid can be completed within half an hour, have compared with other isothermal amplification techniques
Clear superiority.Requirement of the technology to hardware device is very low, particularly suitable for in-vitro diagnosis, food security, bio-safety, agriculture
The fields such as industry, the technology is studied also seldom in transgene agricultural product detection field at present, but great development potentiality.
The content of the invention
It is an object of the invention to provide a kind of RPA primers and detection method for detecting transgenic corns Bt11 strains, use
In carrying out Rapid identification to transgenic corns Bt11 strains, high specificity, high sensitivity, can realize to purpose piece in 20min
The exponential amplification of section, detection process are low to environmental unit requirement, easy to operate, have in transgenic product composition detection field wide
Wealthy popularizing application prospect.
In order to achieve the above object, the present invention provides following technical solution：
A kind of RPA primers for detecting transgenic corns Bt11 strains, it includes forward primer F and reverse primer R, specific sequence
Row are as follows：
F：5’-CTGGGAGGCCAAGGTATCTAATCAGCCATCCC-3’；
R：5’-CTGCTGTAGCTGGCCTAATCTCAACTGGTCTCC-3’.
A kind of RPA detection kits for being used to detect transgenic corns Bt11 strains, it includes the RPA primers.
Further, the detection kit includes RPA reaction systems, in the RPA reaction systems, the final concentration of each component
For：Forward primer, the reverse primer of RPA primers are respectively 0.4-1.0 μm of ol/L, and, forward primer, the ratio of reverse primer are
0.5-2:1, DNA profiling 100-1000ng/ μ L, magnesium acetate 10-16mmol/L.
A kind of RPA detection methods for detecting transgenic corns Bt11 strains, including：
1) DNA of sample to be tested is extracted as template；
2) the RPA primer pairs of claim 1 are added in RPA amplification reaction systems, carry out RPA amplified reactions, amplification
32-42 DEG C of temperature, 15~25 minutes reaction time；
3) and then by agarose gel electrophoresis, if obtaining the band that clip size is 188bp, prove in sample to be tested
Contain transgenic corns Bt11 strains.
Further, when carrying out RPA amplified reactions, in the RPA reaction systems, each component it is final concentration of：RPA primers
Forward and reverse primer is respectively 0.4-1.0 μm of ol/L, and, forward primer, the ratio of reverse primer are 0.5-2：1, DNA profiling 100-
1000ng/ μ L, magnesium acetate 10-16mmol/L.
Preferably, the RPA reaction systems cumulative volume is 50 μ L, wherein, wherein, concentration is the RPA primers of 10 μm of ol/L
Forward primer, reverse primer respectively add 2.4 μ L, the DNA profiling that concentration is 100ng/ μ L adds 1 μ L, TwistAmpTM
29.5 μ L of Rehydration buffer, concentration are 280mmol/L magnesium acetate solutions 2.5 μ L, ddH2O complements to 50 μ L.
Preferably, the RPA amplified reactions program is：35-40 DEG C of temperature.
It is highly preferred that in the step 2), RPA amplified reaction programs are：37 DEG C of isothermal reactions 20 minutes.
The present invention is according to transgenic corns Bt11 foreign gene glufosinate acetyl transferase genes (phosphinothricin
Acetyl transferase gene, PAT) and Maize genome join domain sequence, design RPA primers, and establish base
In the specific quick determination method of recombinase polymerase isothermal amplification technique (RPA).
Compared with normal PCR primer, RPA primers have longer length, if length is between 30-35bp, amplified fragments
100-200bp is preferably controlled in, other rules are similar to Standard PCR design of primers, as G/C content draws in 40-60%, upstream and downstream
The G/C content of thing is suitable；To ensure the primer of design as far as possible, its own does not form ring-type hairpin structure；Avoid between primer, draw
Thing itself forms the continuous pairing of 4 bp or more.
In the design of RPA primers of the present invention, according to transgenic corns Bt11 foreign genes PAT and the company with Maize genome
Connect region design primer, not only can identify whether belong to transgenosis, can also unique identification it is special to strain Bt11, this primer pair
Property high, high sensitivity, amplification efficiency is high.
The present invention explores suitable RPA reaction systems, which is located at 15- according to the RPA primers obtained
25min, when the reaction time is less than 15min, the band expanded is not clear enough, when reacting 15min, obvious purpose has occurred
Band；When reacting 20min, purpose band is brighter, and when reacting the longer time, brightness is similar to 20min, shows that RPA reactions exist
During 20min, just largely expand, the efficiency of reaction is very high, and the reaction time is more than 25min, and band brightness does not increase, from fast
Fast testing requirements are set out, and the 15-25min reaction time is enough.
The present invention sets the RPA reaction time in 15-25min, shortens the RPA reaction time, and then shorten what is entirely tested
Time make it that detection process is faster, and amplified band becomes apparent from, and is more conducive to subsequent detection.
The RPA reactions of the present invention, temperature are set in 32-42 DEG C, and when reaction temperature is less than 32 DEG C, temperature is too low, non-template
The suitable temperature combined with primer, no amplification or only faint amplification；When reaction temperature is higher than 42 DEG C, temperature is excessive, uncomfortable
Combined in template with primer, no amplification or only faint amplification.
Compared with prior art, the present invention has the advantages that：
The RPA primers of the present invention, have high degree of specificity to Bt11 strains, when detecting transgenic corns, only turn base
Because there is purpose band in the RPA reaction systems of corn Bt11, other Transgenic corn lines, as GA21, Bt176, NK603 are equal
Without amplified band, illustrate that primer pair Bt11 has high specificity.
RPA reactions are carried out using the RPA systems of the present invention, the reaction time is short, and range of reaction temperature is wider, between 32-42 DEG C
Amplification can all be realized by being reacted, and reaction efficiency is high, when reacting 15min, obvious purpose band can occur, can be in 20min
The interior specific detection for completing transgenic corns Bt11, accelerates detection progress, for wanting the shorter experiment of seeking time, 15min is
Can reaction was completed, detection is more convenient.
When detecting transgenic corns Bt11 strains using the RPA primers and reaction system of the present invention, specific good, sensitivity
Height, the copy number of its detectable template DNA illustrate its absolute sensitivity height, can accurately detect and turn in 100 copies/more than μ L
Sample of the gene corn Bt11 strain contents more than 0.1%, illustrates its relative sensitivity height.
Brief description of the drawings
Fig. 1 is using Bt11 as template in the embodiment of the present invention 1, and the agarose after 6 pairs of RPA primers of design expand coagulates
Gel electrophoresis result.
Fig. 2 is that different Transgenic corn lines carry out the agarose gel electrophoresis knot after RPA reactions in the embodiment of the present invention 2
Fruit.
Fig. 3 is the Ago-Gel for the amplified production verified in the 2 transgenosis Bt11RPA reaction time of the embodiment of the present invention
Electrophoresis result.
Fig. 4 is the Ago-Gel for the amplified production that 2 transgenosis Bt11RPA reaction temperatures of the embodiment of the present invention are verified
Electrophoresis result.
Carry out what RPA reacted after the template-setup copy number gradient that Fig. 5 is 2 transgenic Bt11 of the embodiment of the present invention
The agarose gel electrophoresis result of amplified production.
RPA is carried out after the template-setup percentage composition gradient that Fig. 6 is 2 transgenic Bt11 of the embodiment of the present invention to react to obtain
Amplified production agarose gel electrophoresis result.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
A kind of acquisition for detecting transgenic corns Bt11 strain RPA primers of embodiment 1
1. design primer
According to transgenic corns Bt11 foreign gene glufosinate acetyl transferase genes (phosphinothricin
Acetyl transferase gene, PAT) and Maize genome join domain sequence, devise 6 pairs of RPA primers, specifically
Sequence is referring to table 1.
Table 1
2. screen primer
1) DNA of transgenic corns Bt11 is extracted, method is as follows：
About 20mg transgenic corns Bt11 seed powders are added into EP pipes, add health as (health is century biotechnology
Co., Ltd (Beijing)) Buffer GF1 (400 μ L) and RNase A (4 μ L) in kit, vibrates, about 1 point of time together
Clock, 65 DEG C of water-baths 10 minutes, during which carry out the 2-3 mixing that is vortexed；130 μ L Buffer GF2 are added, are mixed, are incubated 5 points on ice
Clock；14000rpm (~20000 × g) is centrifuged 5 minutes, should be noted：Buffer GF1 and RNase A please don't be mixed before use
Close.
Gained liquid is transferred in the Filter column for having been charged into collecting pipe, 12000rpm centrifugation 2mins, collect filtrate, need
Pay attention to：For convenience of sampling, pipette tips tip can be cut in the liquid in taking centrifuge tube, Filter column can remove most debris,
A little debris but can be also flowed out, precipitation can be formed after centrifugation, not be drawn onto precipitation later.
The filtrate being collected into collecting pipe is transferred in a new centrifuge tube, is careful not to be drawn onto precipitation, discards collection
Pipe；750uL Buffer GF3 are added, fully mixes, should be noted：To be mixed immediately after adding Buffer GF3.
Resulting solution and precipitation are added in the adsorption column equipped with collecting pipe together, can be with if cannot once add
Several times plus；12000rpm, 1min are centrifuged, waste liquid is outwelled, adsorption column is reloaded into collecting pipe.
The Buffer GW that 500 μ L have been added to ethanol are added in adsorption column, 12000rpm, 1min centrifugation, will be useless after centrifugation
Liquid is outwelled, which comes again.
Adsorption column is placed to a few minutes at room temperature to dry.Adsorption column is put into a new centrifuge tube, to adsorption column plus
Enter Buffer GE30 μ L, stand 5min, centrifugation, 12000rpm, 1min, are repeated once 30 μ L elutions, obtain DNA solution.
2) RPA amplifications and electrophoresis
Using TwistAmp RPA gel detection kits, using Bt11 as template DNA, drawn respectively with 6 couples of RPA in table 1
Thing carries out RPA amplifications, and in 37 DEG C of metal baths, isothermal reaction 20min, reaction product is taken after amplification, through phenol/chloroform
After purification, identified on 2% Ago-Gel with 120V constant pressures electrophoresis 25 minutes using gel imaging system.
Wherein, the total system of RPA reactions is 50 μ L, including：ddH212.2 μ L, Rehydration Buffer of O, 29.5 μ
L, concentration be 10 μm of ol/L each 2.4 μ L of forward primer, reverse primer, concentration be 100ng/ μ L 1 μ L of template DNA, kit
Carry the 2.5 μ L of magnesium acetate solution that concentration is 280mmol/L, the lyophilized enzyme powder that kit carries.
Amplification referring to Fig. 1, wherein, 1-6 swimming lanes are respectively primer pair F1/R1, F2/R2, F3/R3, F4/R4, F5/
The amplified production of R5, F6/R6, the results show：Primer pair F1/R1, F2/R2 and F3/R3 do not amplify purpose band；F4/R4
The band of amplification is indefinite；There is purpose band in F5/R5, but amplification efficiency is low；There is clear and definite purpose band in F6/R6, and expands
Increasing Efficiency is higher, it is RPA primer pairs F/R of the invention.
A kind of method validation for detecting transgenic corns Bt11 strains of embodiment 2
1. specificity verification
It is that century genome DNA extracting reagent kit (health is century bio tech ltd (Beijing)) extraction turns base with health
Because of the genomic DNA of corn GA21 seed powders, Bt176 seed powders and NK603 seed powders, specific extraction experimental procedure is same
Embodiment 1.
Respectively using the genomic DNA of transgenic corns Bt11, GA21, Bt176 and NK603 as template DNA, with the present invention's
RPA primer pairs are primer, carry out RPA reactions.
Wherein, the total system of RPA reactions is 50 μ L, including：ddH210.0 μ L, Rehydration Buffer of O, 29.5 μ
L, concentration be 10 μm of ol/L each 3.0 μ L of forward primer, reverse primer, concentration be 100ng/ μ L 2.0 μ L of template DNA, reagent
The 2.5 μ L of magnesium acetate solution that box carries, the lyophilized enzyme powder that kit carries.
To the RPA reaction products of transgenic corns Bt11, GA21, Bt176 and NK603 into row agarose gel electrophoresis, knot
Fruit sees Fig. 2, wherein, 1-6 swimming lanes are respectively：Blank (ddH2O), negative control (non-transgenic corn), transgenic corns GA21,
Transgenic corns Bt176, transgenic corns NK603, transgenic corns Bt11.
The results show：Only there are 188bp purpose bands in the reaction system of the Bt11 of corn containing GM, the blank without template
With without amplified band, show that experimentation is pollution-free in negative control (non-transgenic corn), other GM corn strains GA21,
Bt176, NK603 are without amplified band.
As it can be seen that the RPA primer pair transgenic corns Bt11 evidences of the present invention have high specificity.
2. transgenic corns Bt11RPA reaction time and temperature conditionss verification
1) the transgenic corns Bt11RPA reaction time is verified
Using transgenic corns Bt11 genomic DNAs template, RPA reaction systems are configured, are reacted in 37 DEG C of metal baths, controlled
Reaction time processed is respectively 5min, 10min, 15min, 20min, 30min, 40min, and reaction terminates, and adds phenol/chloroform immediately
Purifying, takes supernatant to run electrophoresis, and RPA reaction systems are referring to embodiment 1, the result is shown in Fig. 3, wherein, 1-6 swimming lanes are respectively：During reaction
Between 5min, 10min, 15min, 20min, 30min, 40min electrophoresis result.
As seen from Figure 3, as the increase in reaction time, purpose band brightness change in gradient, the blank reaction time is
40min；When reacting 5min, band is unintelligible；When reacting 10min, band is not clear and definite enough；When reacting 15min, occur obvious
Purpose band；When reacting 20min, purpose band is brighter；30min and 40min is reacted, brightness is similar to 20min, shows that RPA is anti-
Just should largely it be expanded in 20min, the efficiency of reaction is very high.
2) transgenic corns Bt11RPA reaction temperatures are verified
Using transgenic corns Bt11 genomic DNAs template, RPA reaction systems are configured, RPA reaction systems are referring to embodiment
1, it is 20min to control the reaction time, and reaction temperature is with 37 DEG C of expansion, with 5 DEG C for gradient, be respectively set to 17 DEG C, 22 DEG C, 27
DEG C, 32 DEG C, 37 DEG C, 42 DEG C, 47 DEG C, 52 DEG C, reaction terminates, and immediately plus phenol/chloroform purifying, takes supernatant to run electrophoresis, as a result
See Fig. 4, wherein, 1-8 swimming lanes are respectively：17 DEG C of reaction temperature, 22 DEG C, 27 DEG C, 32 DEG C, 37 DEG C, 42 DEG C, 47 DEG C, 52 DEG C of electricity
Swimming result.
From fig. 4, it can be seen that reaction temperature 17 DEG C, 22 DEG C with 52 DEG C when, no amplification；At 27 DEG C and 47 DEG C, there is faint expansion
Increase；There is amplification when 32 DEG C, 37 DEG C are with 42 DEG C, it is 37 DEG C higher with amplification efficiency at 42 DEG C.
3. sensitivity is verified
1) absolute sensitivity is tested
Use ddH2The GM corn Bt11 genomic DNAs of measured concentration are diluted by O, and it is respectively 1000 to copy to obtain concentration
The DNA solution of shellfish/μ L, 500 copies/μ L, 200 copies/μ L, 100 copies/μ L, 50 copies/μ L and 10 copies/μ L, as template
RPA reactions are carried out, and carry out 2% agarose gel electrophoresis, referring to embodiment 1, electrophoresis result is shown in for reaction condition and reaction system
Fig. 5, wherein, 1-7 swimming lanes are respectively：Transgenic corns Bt11DNA template concentrations 1000 copy/μ L, 500 copies/μ L, 200 copy
Shellfish/μ L, 100 copies/μ L, 50 copies/μ L, 10 copies/μ L and blank ddH2The electrophoresis result of O controls.
The results show：When DNA content is 1000 copies/μ L, 500 copies/μ L, 200 copies/μ L, 100 copies/μ L,
There is the purpose band of 188bp.When DNA content is 50 copies/μ L and 10 copies/μ L, no purpose band.Show what this experiment was established
The absolute sense limit of RPA method specific detections Bt11 about 100 copies.
2) relative sensitivity is tested
Transgenic corns Bt11 contents 5%, content 1%, the DNA of content 0.1%, with the non-GM maize dnas of same concentrations
The Bt11 maize dnas of content 0.1% are diluted to the DNA sample that Bt11 contents are 0.05%, 0.01%, obtain 5%, 1%,
0.1%th, the template of 0.05%, 0.01% 5 concentration gradients, carries out RPA reactions, and carries out 2% agarose gel electrophoresis, instead
Answering condition and reaction system, electrophoresis result is shown in Fig. 6 referring to embodiment 1, wherein, 1-6 swimming lanes are respectively：Transgenic corns Bt11 contains
Measure 5%, 1%, 0.1%, 0.05%, 0.01% and blank ddH2The electrophoresis result of O controls.
The results show：When the content of Bt11 is 5%, 1%, 0.1%, there is the purpose band of 188bp；When Bt11 contents
For 0.05%, 0.01% when, no purpose band, the opposite test limit of RPA method specific detections Bt11 for showing to establish is about
0.1%.
Claims (8)
1. a kind of RPA primers for detecting transgenic corns Bt11 strains, it includes forward primer F and reverse primer R, particular sequence
It is as follows：
F：5’-CTGGGAGGCCAAGGTATCTAATCAGCCATCCC-3’；
R：5’-CTGCTGTAGCTGGCCTAATCTCAACTGGTCTCC-3’.
2. a kind of RPA detection kits for being used to detect transgenic corns Bt11 strains, it is characterised in that including claim 1
The RPA primers.
3. RPA detection kits according to claim 2, it is characterised in that it includes RPA reaction systems, RPA reactions
In system, each component it is final concentration of：Forward primer, the reverse primer of RPA primers are respectively 0.4-1.0 μm of ol/L, and, forward direction is drawn
Thing, the ratio of reverse primer are 0.5-2：1, DNA profiling 100-1000ng/ μ L, magnesium acetate 10-16mmol/L.
4. a kind of RPA detection methods for detecting transgenic corns Bt11 strains, including：
1) DNA of sample to be tested is extracted as template；
2) the RPA primer pairs of claim 1 are added in RPA amplification reaction systems, carry out RPA amplified reactions, expand temperature
32-42 DEG C, 15~25 minutes reaction time；
3) and then by agarose gel electrophoresis, if obtaining the band that clip size is 188bp, prove to contain in sample to be tested
Transgenic corns Bt11 strains.
5. the RPA detection methods of transgenic corns Bt11 strains are detected according to claim 4, it is characterised in that carry out RPA
During amplified reaction, in the RPA reaction systems, each component it is final concentration of：Forward primer, the reverse primer of RPA primers be respectively
0.4-1.0 μm of ol/L, and, forward primer, the ratio of reverse primer are 0.5-2：1, DNA profiling 100-1000ng/ μ L, magnesium acetate
10-16mmol/L。
6. the RPA detection methods of transgenic corns Bt11 strains are detected according to claim 4, it is characterised in that the RPA
Reaction system cumulative volume is 50 μ L, wherein, concentration respectively adds 2.4 for forward primer, the reverse primer of the RPA primers of 10 μm of ol/L
μ L, the DNA profiling that concentration is 100ng/ μ L add 1 μ L, TwistAmpTM29.5 μ L of Rehydration buffer, concentration are
280mmol/L magnesium acetate solutions 2.5 μ L, ddH2O complements to 50 μ L.
7. the RPA detection methods of transgenic corns Bt11 strains are detected according to claim 4, it is characterised in that the step
2) in, the RPA amplified reactions program is：Expand 35-40 DEG C of temperature.
8. the RPA detection methods of transgenic corns Bt11 strains are detected according to claim 4, it is characterised in that the step
It is rapid 2) in, RPA amplified reaction programs are：37 DEG C expand 20 minutes.
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| CN112301145A (en) * | 2020-10-13 | 2021-02-02 | 中国农业科学院生物技术研究所 | RPA detection primer and probe combination of transgenic rice EB7001S-6, kit and detection method |
| CN112301146A (en) * | 2020-10-13 | 2021-02-02 | 中国农业科学院生物技术研究所 | RPA detection primer and probe combination, kit and detection method for transgenic rice B2A68-1 |
| CN112813189A (en) * | 2021-03-12 | 2021-05-18 | 浙江经贸职业技术学院 | Method for rapidly identifying transgenic corn strain by utilizing quadruple real-time fluorescent PCR (polymerase chain reaction) |
| CN113913501A (en) * | 2021-11-18 | 2022-01-11 | 吉林农业科技学院 | Method for identifying trans Bt gene based on recombinase polymerase isothermal amplification method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112301144A (en) * | 2020-10-13 | 2021-02-02 | 中国农业科学院生物技术研究所 | RPA primer probe combination, kit and detection method for detecting transgenic corn DBN9958 |
| CN112301145A (en) * | 2020-10-13 | 2021-02-02 | 中国农业科学院生物技术研究所 | RPA detection primer and probe combination of transgenic rice EB7001S-6, kit and detection method |
| CN112301146A (en) * | 2020-10-13 | 2021-02-02 | 中国农业科学院生物技术研究所 | RPA detection primer and probe combination, kit and detection method for transgenic rice B2A68-1 |
| CN112813189A (en) * | 2021-03-12 | 2021-05-18 | 浙江经贸职业技术学院 | Method for rapidly identifying transgenic corn strain by utilizing quadruple real-time fluorescent PCR (polymerase chain reaction) |
| CN113913501A (en) * | 2021-11-18 | 2022-01-11 | 吉林农业科技学院 | Method for identifying trans Bt gene based on recombinase polymerase isothermal amplification method |
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