CN106929584A - A kind of RPA primers of detection Camv35S promoters, kit and detection method - Google Patents
A kind of RPA primers of detection Camv35S promoters, kit and detection method Download PDFInfo
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- CN106929584A CN106929584A CN201710203520.6A CN201710203520A CN106929584A CN 106929584 A CN106929584 A CN 106929584A CN 201710203520 A CN201710203520 A CN 201710203520A CN 106929584 A CN106929584 A CN 106929584A
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- 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 of detection Camv35S promoters, kit and detection method, the RPA primers have high specificity, with biotin, after digoxin or FITC are marked respectively, in RPA amplified reactions, probe need not be introduced, after RPA amplifications terminate, using double labelling nucleic acid product test strip, only need 5~10 minutes, just whether can contain transgene component in the tested product of quick detection, testing result is accurately and reliably, sensitivity is high, intuitive is strong, the step of eliminating electrophoresis, without the fluorescent dye ethidium bromide for using high carcinogenic, substantially reduce experiment process, simplify experimental procedure, significant raising has been obtained in terms of accuracy and quick detection.
Description
Technical field
The invention belongs to Transgene-safty biological technical field, and in particular to a kind of RPA of detection Camv35S promoters draws
Thing, kit and detection method.
Background technology
Transgenic technology is fast-developing, at present, has had multiple genetically modified crops strains to enter safety evaluation rank in China
Section, applies for commercial growth.
At the same time, the extensive concern of the public is received because of the safety issue that genetically modified crops diffusion causes.It is protection
The right to know and right to choose of consumer, implement transgenic product mark system, prevent genetically modified crops from entering without safety approval
Production intermediate links, in the urgent need to setting up fast and convenient nucleic acid detection method, for market surpervision and routine monitor.
And in detection GMOs, at present, there is cumbersome, high cost, unicity etc. and ask in Standard PCR detection method
Topic, it is impossible to adapt to the fast-developing and frequently trade contacts of modern economy.
Recombinase polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA), with regular-PCR
Mechanism is replicated in difference, its parody, reaction does not need annealing process, entirely reacts simple and quick, can enter in 15 minutes
Monomolecular nucleic acid detection under row normal temperature.The detection technique system set up using the method, is suitable for large-scale transgenosis and planted
The detection of thing, can greatly improve scene detection efficiency, and for China ranks among generation in terms of the scene speed survey of transgenic product
Boundary prostatitis lays a good foundation.
The design that it is critical only that amplimer and probe of RPA methods.PCR primer is inapplicable mostly, because,
RPA primers are more long than general PCR primer, it usually needs reach 30-38 base, primer is too short to reduce recombination fraction, influence amplification
Speed and detection sensitivity.Therefore, the design of primers difficulty of RPA is larger.The shortcoming of sonde method is then upper difficult probe design.
In addition, sonde method needs to read instrument with fluorescence after amplified reaction is finished and can just complete last result and show.
Also, it is necessary to use the fluorescence of high carcinogenic when currently the detection display result of transgenic product is using electrophoresis
Dye ethidium bromide, not only endangers the health of operator, and cannot exclude the problem of false positive, can not carry out showing for field
Field speed is surveyed, and this severely limits the detection efficiency of transgenic product, the safety management to transgenic product is maximum restriction.
At present, in the transgenic product commercially produced in the world, such as paddy rice, corn, rape, soybean seed are produced
Product and deep-processed food based on this, product, many strains are to use Camv35S promoters, i.e. P-Camv35S, often
The detection means of rule needs for test sample to deliver to laboratory and is detected, wastes time and energy.
The content of the invention
It is an object of the invention to provide a kind of RPA primers of detection Camv35S promoters, kit and detection method,
Without probe, mark the forward and reverse primers of RPA respectively using biotin, digoxin and FITC 5 ' are held, can the tested system of quick detection
Whether contain transgene component in product, with high specificity, sensitivity is high, detection speed is fast, and intuitive is strong.
In order to achieve the above object, the present invention provides following technical scheme：
One group of RPA primer of detection Camv35S promoters, including：
RPA-35S-F：5'-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
RPA-35S-R：5'-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'.
Detect Camv35S promoters RPA labeled primer groups, its by forward primer RPA-35S-F- and reverse primer
RPA-35S-R-FITC is constituted, or is made up of forward primer RPA-35S-F- lifes and reverse primer RPA-35S-R-FITC, or by
Forward primer RPA-35S-F- gives birth to and reverse primer RPA-35S-R- ground composition；Wherein, the forward primer be through biotin or
The labeled primer that digoxin is marked to the 5' ends of RPA primers RPA-35S-F, specially：
RPA-35S-F- ground：
5'-Dig-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
RPA-35S-F- gives birth to：
5'-Biotin-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
The reverse primer is the labeled primer marked to the 5' ends of RPA primers RPA-35S-R with FITC or digoxin, tool
Body is：
RPA-35S-R-FITC：
5'-6-FAM-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'；
RPA-35S-R- ground：
5'-Dig-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'。
A kind of RPA detection kits of detection Camv35S promoters, it contains described RPA primers or described RPA marks
Note primer sets.
A kind of RPA detection methods of detection Camv35S promoters, comprise the following steps：
1) DNA of testing sample is extracted；
2) RPA amplifications
Using the DNA of testing sample as template, described RPA labeled primer groups are added in RPA amplification reaction systems,
RPA amplifications are carried out, the nucleic acid constant-temperature amplification product of double labelling is obtained；
3) amplified production detection
The nucleic acid detection test strip inserting step of biotin antibody and fluorescein isothiocynate (FITC) antibody will be adsorbed
2) in amplified production, lateral chromatography 5~10 minutes is judged according to colour developing band：Test strips are in detection line and control line
There is purple band at place, then it represents that contain Camv35S promoters in testing sample；If only there is purple band at control line,
Camv35S promoters are not contained in expression testing sample, if without purple band at control line, then it represents that without amplification or test strips
It is invalid.
Further, in described RPA reaction systems, final concentration of 0.5~2ng/ μ l of DNA profiling, forward and reverse mark draws
The final concentration of thing is respectively 0.3~0.6 μm of ol/L.
Preferably, the RPA reaction systems cumulative volume is 50 μ l, wherein, concentration is that the forward and reverse mark of 10 μm of ol/L draws
Thing respectively adds 2.4 μ l, and concentration adds 2 μ l, RPA reaction buffer 29.5 μ l, ddH for the DNA profiling of 20ng/ μ l2O complements to 50
μl。
Further, step 2) in, the RPA amplified reactions program is：37-39 DEG C of constant temperature, 14~20 minutes.
Preferably, the step 2) in, RPA amplified reaction programs are：Isothermal reaction 37 DEG C 4 minutes, takes out, and mixing is equal
It is even, then expand 15 minutes.
The present invention is using the polymerase-mediated isothermal amplification technique (RecombinasePolymerase of recombinase
Amplification, RPA) quick detection Camv35S promoters, the DNA sequence dna of controlling element is inserted according to foreign gene, if
Substantial amounts of RPA specific primers are counted, one group of RPA primer has therefrom been filtered out, has recycled biotin, digoxin and isothiocyanic acid glimmering
Light element FITC carries out RPA amplified reactions, in reaction, fluorescein isothiocynate after marking the 5 ' of forward and reverse primer to hold respectively
(FITC) the loop-mediated isothermal amplification technique amplified production specific hybrid of the probe of mark and biotin labeling, and and collaurum
The antibody of the anti-fluorescein isothiocynate of mark combines to form ternary complex and combines has life in lateral flow Lateral Flow Strip
In the detection line of thing element antibody；Non-hybridized marked by fluorescein isothiocyanate probe and the anti-isosulfocyanic acid fluorescence of colloid gold label
The antibody of element forms the two-spot compound without biotin；By detection line, with reference on the control line, can quick effective detection
Whether contain Camv35S promoters in transgenic product and product.
The present invention is only needed in the reaction solution after test strips insertion amplification, you can naked eyes sentence read result, concise, used
Lateral flow ELISA test strip technology, be that the 5 ' of forward and reverse primer ends are marked into upper fluorescein isothiocynate respectively
(FITC) primer, biotin or digoxin, the nucleic acid constant-temperature amplification product of double labelling is obtained by RPA reactions, will adsorb raw
The nucleic acid detection test strip of thing element antibody and fluorescein isothiocynate antibody is inserted into the amplified production, and it can be with collaurum mark
Its specific antibody of note is combined, and is shown at detection line, conversely, non-hybridized primer can be special with its colloid gold label
Property antibody be combined, and at control line show, naked eyes it is observed that display result, substantially reduce experiment process, simplify
Experimental procedure, has obtained significant raising in terms of accuracy and quick detection, and this detection method can save the step of electrophoresis
Suddenly, without introducing probe, without the fluorescent dye ethidium bromide for using high carcinogenic.
It is compared with the prior art, the present invention has the advantages that：
RPA primers of the invention, with high specificity, after being marked respectively with biotin, digoxin and FITC,
In RPA amplified reactions, without introducing probe, just whether can contain transgene component in the tested product of quick detection, sensitivity is high,
Detection speed is fast, and intuitive is strong.
Using detection method of the invention, without introducing probe, using double labelling nucleic acid product test strip, it is only necessary to 5
~10 minutes, it is possible to observe testing result, the step of eliminate electrophoresis, without the fluorescent dye bromination for using high carcinogenic
Second ingot, substantially reduces experiment process, simplifies experimental procedure, has obtained significantly carrying in terms of accuracy and quick detection
Height, can carry out field examination to the transgenic product containing Camv35S promoters, and quick detection Camv35S promoters have
Nothing, greatlys save testing cost.
Brief description of the drawings
Fig. 1 is amplification electricity of the RPA labeled primers for RPA-35S-F- with RPA-35S-R-FITC in the embodiment of the present invention 2
Swimming collection of illustrative plates；
Wherein, M：DL2000Marker；1：RRS：Genetically engineered soybean；2：Bt11：Transgenic corns；3：Bt176：Transgenosis
Corn；4：KF：Transgenic paddy rice；5：KMD：Transgenic paddy rice；6：MON531：Transgene cotton；7：MON863：Transgenosis is beautiful
Rice；8：NTC：Blank.
Fig. 2 be the embodiment of the present invention 2 in RPA labeled primers be RPA-35S-F- life and RPA-35S-R- ground amplification electrophoresis
Collection of illustrative plates；
Wherein, M：DL2000Marker；1：NTC：Blank；2：RRS：Genetically engineered soybean；3：Bt11：Transgenosis is beautiful
Rice；4：Bt176：Transgenic corns；5：KF：Transgenic paddy rice；6：KMD：Transgenic paddy rice；7：MON531：Transgene cotton；8：
MON863：Transgenic corns.
Fig. 3 be the embodiment of the present invention 2 in RPA labeled primers be RPA-35S-F- life and RPA-35S-R- ground sensitivity expand
Increase electrophoresis pattern；
Wherein, M：DL2000Marker；DNA use RRS (genetically engineered soybean) difference gradient dilution for：1：0copies；2：
20copies；3：50copies；4：100copies；5：200copies；6：500copies；7：1000copies；8：
2000copies。
Fig. 4 is cloning and sequencing result comparison result in the embodiment of the present invention 2；
Wherein, RPA-35S-F：Forward primer；Camv35S：P-Camv35S complete sequences；35S-KF：Transgenic paddy rice；
35S-KMD：Transgenic paddy rice；35S-M531：Transgene cotton；35S-MON863：Transgenic corns；35S-RRS：Transgenosis is big
Beans；35S-Bt11：Transgenic corns；35S-Bt176：Transgenic corns；35S-RPA-35S-123-F：Reverse primer.
Fig. 5-6 is result display result after test strips insertion in the embodiment of the present invention；
Wherein, 1：NTC：Blank；2：RRS：Genetically engineered soybean；3：Bt11：Transgenic corns；4：Bt176：Transgenosis
Corn；5：KF：Transgenic paddy rice；6：KMD：Transgenic paddy rice；7：MON531：Transgene cotton；8：MON863：Transgenosis is beautiful
Rice.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
A kind of acquisition of the RPA primers of detection Camv35S promoters of embodiment 1
By consulting literatures and use BLAST software analysis, using the genetically engineered soybean GTS40-3-2 strains of 10ng/ μ L as
Template optimizes screening, filters out the gene nucleic acid sequence of Camv35S promoters, and carries out the design and screening of primer.
Following main points are considered in RPA design of primers：(1) length of the selection RPA primers of primer length is general
It is 30 to 35 nucleotides；(2) primer G/C content is between 40%~60%, base random distribution, the 3-5 nucleotides at 5' ends
Poly- guanine should be avoided；(3) tried one's best during design of primers and avoid easily forming secondary structure, primer-primer interaction, hairpin structure
Sequence, reduce primer dimer formation；(4) selection of being tried one's best when designing primer is specific in the structure of Camv35S promoters
Inside, to increase the versatility of screening element.
A pair of RPA primers are obtained after screening, particular sequence is as follows：
RPA-35S-F：5'-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
RPA-35S-R：5'-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'.
A kind of RPA detection methods checking of the detection Camv35S promoters of embodiment 2
Using common transgenic product soybean, corn, cotton, paddy rice, rape etc. as object, amplified reaction is carried out,
And detected using test strips, verified by contrast of electrophoresis.
1) testing sample DNA (RRS are extracted respectively：Genetically engineered soybean；Bt11：Transgenic corns；Bt176：Transgenic corns
KF：Transgenic paddy rice；KMD：Transgenic paddy rice；MON531：Transgene cotton；MON863：Transgenic corns)；
2) RPA primers are marked
To the RPA primers obtained in embodiment 1, biotin, digoxin and FITC mark are carried out at 5 ' ends respectively, specifically such as
Table 1.
Table 1
| Primer | Primer sequence |
| RPA-35S-F- ground | 5’-Dig-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3’ |
| RPA-35S-R-FITC | 5’-6-FAM-CCCTTACGTCAGTGGAGATATCACATCAATCC-3’ |
| RPA-35S-F- gives birth to | 5’—Biotin-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3’ |
| RPA-35S-R- ground | 5’-Dig-CCCTTACGTCAGTGGAGATATCACATCAATCC-3’ |
3) constant-temperature amplification
Primer in table 1 is carried out into recombinase polymerase isothermal duplication using the RPA kits that TwistDx companies develop, is expanded
Increase reaction cumulative volume be 50 μ l, concentration be 10 μm of forward primer RPA-35S-F- of ol/L and reverse primer RPA-35S-R-
FITC (or forward primer RPA-35S-F- life and reverse primer RPA-35S-R-FITC or forward primer RPA-35S-F- life and
Reverse primer RPA-35S-R- ground) 2.4 μ l of each addition, concentration is DNA profiling 2 μ l, the RPA reaction buffers of addition of 20ng/ μ l
29.5 μ l, ddH2O adds 11.2 μ l to complement to the μ l of cumulative volume 50.
Amplified reaction program is：37 DEG C of temperature of amplification, reaction time point two parts：It is put into PCR instrument device or constant-temperature amplification instrument
Middle reaction 4 minutes, takes out and turns upside down 10 times, then expand 15 minutes.
4) product detection
Product detection is verified by two methods：
(1) electrophoresis：By step 3) isothermal duplication product to carry out phenol chloroform according to the explanation of TwistDx companies kits pure
Change rear electrophoresis analysis, electrophoresis pattern as shown in Figure 1-2, Tu1-2Zhong, have amplified band be then containing Camv35S promoters turn
Gene prod, conversely, then representing that the product is the transgenic product for not containing Camv35S promoters without band.
Sensitive amplification checking is carried out to primer, the fragment containing purpose band is cloned on plasmid, with plasmid as mould
Plate, 2 × 1037 gradients are set between the copy of copy -20 and enters line sensitivity checking, as a result referring to Fig. 3, from the figure 3, it may be seen that 100
Band more than copy can be amplified, 50,20 copies are then without amplification.
After above-mentioned amplified production is carried out into cloning and sequencing, comparison result is as shown in figure 4, by Fig. 4 it can be shown that amplified production
It is Camv35S promoters.
(2) test strips method：The double labelling nucleic acid constant-temperature that biotin antibody and fluorescein isothiocynate antibody will be fixed with expands
Increase test strip and be inserted into step 3) amplification reaction system in, in 5-10 minute, display result.
Test strip has band at detection line and control line, then it represents that the product is to contain Camv35S promoters
Transgenic product, conversely, only there is band at control line, then it represents that the product be do not contain Camv35S promoters turn base
It is specific such as Fig. 5-6 because of product, if without band at detection line and control line, then it represents that invalid without amplification or test strips.
By Fig. 5-6 as can be seen that utilizing RPA methods of the invention, the degree of accuracy is high, is matched with electrophoresis result, makes up
The deficiency of electrophoresis, application is more extensive.
Claims (8)
1. one group of RPA primer of detection Camv35S promoters, including：
RPA-35S-F：5'-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
RPA-35S-R：5'-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'.
2. detect Camv35S promoters RPA labeled primer groups, its by forward primer RPA-35S-F- and reverse primer RPA-
35S-R-FITC is constituted, or is made up of forward primer RPA-35S-F- lifes and reverse primer RPA-35S-R-FITC, or by forward direction
Primer RPA-35S-F- gives birth to and reverse primer RPA-35S-R- ground composition；
Wherein, the forward primer is that the 5' ends of RPA-35S-F primers described in claim 1 are marked through biotin or digoxin
Labeled primer, specially：
RPA-35S-F- ground：
5'-Dig-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
RPA-35S-F- gives birth to：
5'-Biotin-CCTCTGCCGACAGTGGTCCCAAAGATGGACC-3'；
The reverse primer is the mark marked to the 5' ends of RPA-35S-R primers described in claim 1 with FITC or digoxin
Primer, specially：
RPA-35S-R-FITC：
5'-6-FAM-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'；
RPA-35S-R- ground：
5'-Dig-CCCTTACGTCAGTGGAGATATCACATCAATCC-3'。
3. a kind of RPA detection kits of detection Camv35S promoters, it contains RPA primers or the right described in claim 1
It is required that the RPA labeled primer groups described in 2.
4. a kind of RPA detection methods of detection Camv35S promoters, comprise the following steps：
1) DNA of testing sample is extracted；
2) RPA amplifications
Using the DNA of testing sample as template, the RPA labeled primer groups described in claim 2 are added to RPA amplified reaction bodies
In system, RPA amplifications are carried out, obtain the nucleic acid constant-temperature amplification product of double labelling；
3) amplified production detection
The nucleic acid detection test strip inserting step 2 of biotin antibody and fluorescein isothiocynate antibody will be adsorbed) amplification produce
In thing, lateral chromatography 5~15 minutes；
Judged according to colour developing band：Test strips have purple band at detection line and control line, then it represents that testing sample
In contain Camv35S promoters；If only there is purple band at control line, then it represents that Camv35S is not contained in testing sample and is opened
Mover, if without purple band at control line, then it represents that invalid without amplification or test strips.
5. the RPA detection methods of Camv35S promoters are detected according to claim 4, it is characterised in that described RPA is anti-
In answering system, final concentration of 0.5~2ng/ μ l of DNA profiling, the final concentration of forward primer and reverse primer is respectively 0.3~0.6 μ
mol/L。
6. RPA detection methods of detection Camv35S promoters according to claim 4, it is characterised in that the RPA is anti-
System cumulative volume is answered for 50 μ l, wherein, concentration is that 10 μm of forward and reverse labeled primers of ol/L respectively add 2.4 μ l, and concentration is
The DNA profiling of 20ng/ μ l adds 2 μ l, RPA reaction buffer 29.5 μ l, ddH2O complements to 50 μ l.
7. the RPA detection methods of Camv35S promoters are detected according to claim any one of 4-6, it is characterised in that step
2) in, RPA amplified reaction programs are：37-39 DEG C of constant temperature, 14~20 minutes.
8. RPA detection methods of the detection Camv35S promoters according to claim any one of 4-6, it is characterised in that step
It is rapid 2) in, RPA amplified reaction programs are：In 37-39 DEG C of isothermal reaction 4 minutes, take out, be well mixed, then expand 15 minutes.
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| CN109234448A (en) * | 2018-11-27 | 2019-01-18 | 中国农业大学 | A kind of visualization Cascaded amplification functional nucleic acid sensor of the quantitative detection for transgenosis CaMV35S promoter |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109234448A (en) * | 2018-11-27 | 2019-01-18 | 中国农业大学 | A kind of visualization Cascaded amplification functional nucleic acid sensor of the quantitative detection for transgenosis CaMV35S promoter |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
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