CN112301147A - RPA primer probe combination, kit and detection method for detecting transgenic corn double antibody 12-6 - Google Patents
RPA primer probe combination, kit and detection method for detecting transgenic corn double antibody 12-6 Download PDFInfo
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Abstract
A transgenic corn double-antibody 12-6 RPA detection primer and probe combination, a kit and a detection method are disclosed, wherein a large number of RPA primers are designed according to the connection region of an exogenous inserted DNA sequence and a corn genome, and a pair of primer and probe combinations capable of quickly and effectively detecting the 12-6 components of the transgenic corn double-antibody are screened out. The transgenic corn double-antibody 12-6 genome DNA is used as a template, and the primer probe combination is utilized to carry out RPA amplification and real-time fluorescence detection, so that the method has the characteristics of high speed, good specificity and high sensitivity.
Description
Technical Field
The application relates to the technical field of molecular biology, in particular to an RPA primer probe combination, a kit and a detection method for detecting 12-6 of a transgenic corn double antibody.
Background
The transgenic corn double antibody 12-6 is a transgenic corn strain which is developed at Zhejiang university and is resistant to insects and glyphosate herbicide, and is a transgenic corn strain resistant to corn borers and glyphosate obtained by introducing cry1Ab/cry2Aj fusion gene and G10evo-epsps gene into corn by using a genetic engineering technology. The strain enters the stage of transgenic organism safety evaluation productivity test and has wide industrialization prospect. In the reported detection method of the transgenic corn double antibody 12-6, a PCR instrument is mainly used for carrying out conventional detection in a laboratory, but the PCR-based transgenic detection method needs professional equipment such as the PCR instrument and a gel imaging system, and the PCR amplification and product detection time is long (about 3-4 h), so that the aim of on-site rapid detection is difficult to achieve, and therefore, a novel more convenient and accurate transgenic detection technology is needed in actual work.
Recombinase polymerase isothermal amplification (RPA) belongs to one of nucleic acid isothermal amplification technologies. The isothermal nucleic acid amplification technology is a novel amplification technology, can perform reaction under the condition of constant temperature, has the advantages of simplicity, convenience, rapidness, sensitivity and the like, and can be used for field inspection. The RPA technique mimics the process of DNA replication in organisms, and recombinase proteins bind to single-stranded DNA (primers) in the presence of ATP to form DNA nucleoprotein filaments. The microwire involves surrounding DNA molecules, aligns the template DNA sequences and searches for sequences that match them, with the help of single-stranded binding proteins, the double-stranded template DNA unzips, the primer and template pair form the 3' hydroxyl terminus required for replication initiation, replication extension begins under the action of DNA polymerase, and new DNA is formed. The RPA fluorescence detection technology greatly shortens the reaction time. At present, no method for identifying the strain specificity of the transgenic corn double antibody 12-6 by utilizing the RPA technology exists.
Disclosure of Invention
The invention provides an RPA primer probe combination for detecting a transgenic corn double antibody 12-6, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
The invention also provides a transgenic corn double-antibody 12-6 RPA detection kit, which comprises the primer probe combination.
The invention also provides a transgenic corn double-antibody 12-6 RPA detection method, which comprises the following steps:
and (3) taking the genome DNA of a sample to be detected as a template, utilizing the primer probe combination to perform RPA amplification and perform fluorescence detection, and if an obvious amplification curve is obtained, proving that the detected sample contains 12-6 components of the transgenic corn double antibody.
Further, the RPA amplification reaction system included 20pmol each of the forward primer and the reverse primer, 5pmol each of the probe, and 50ng of the DNA template per 50. mu.l of the reaction system.
Furthermore, the RPA amplification reaction system also comprises 29.5 mul of rehydration buffer, 2.5 mul of 280 mu M magnesium acetate solution and the balance of water in each 50 mul of reaction system.
Further, the RPA amplification procedure was 39 ℃ for 20 minutes
The invention also provides application of the primer probe combination, the detection kit and/or the detection method in detection of the transgenic corn double-antibody 12-6 seed resource.
The invention provides a transgenic corn double-antibody 12-6 strain specific RPA detection method for the first time. According to the invention, a large number of RPA primers are designed according to the connection region of the exogenous inserted DNA sequence and the corn genome, and a pair of primer probe combinations capable of quickly and effectively detecting the 12-6 components of the transgenic corn double antibody is screened out. The transgenic corn double antibody 12-6 is used as a template, and the primer probe combination is used for fluorescence detection, so that an obvious amplification curve can be obtained. The RPA primer probe combination, the detection kit and the detection method have the characteristics of high speed, good specificity and high sensitivity.
Drawings
FIG. 1 is a Basic electrophoresis diagram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F1 and diabody 12-6-R1, 3: diabody 12-6-F1 and diabody 12-6-R2, 4: diabody 12-6-F1 and diabody 12-6-R3, 5: diabody 12-6-F1 and diabody 12-6-R4, 6: diabody 12-6-F1 and diabody 12-6-R5, 7: diabody 12-6-F1 and diabody 12-6-R6, 8: diabody 12-6-F1 and diabody 12-6-R7, 9: diabodies 12-6-F1 and diabodies 12-6-R8;
FIG. 2 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F2 and diabody 12-6-R1, 3: diabody 12-6-F2 and diabody 12-6-R2, 4: diabody 12-6-F2 and diabody 12-6-R3, 5: diabody 12-6-F2 and diabody 12-6-R4, 6: diabody 12-6-F2 and diabody 12-6-R5, 7: diabody 12-6-F2 and diabody 12-6-R6, 8: diabody 12-6-F2 and diabody 12-6-R7, 9: diabodies 12-6-F2 and diabodies 12-6-R8;
FIG. 3 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F3 and diabody 12-6-R1, 3: diabody 12-6-F3 and diabody 12-6-R2, 4: diabody 12-6-F3 and diabody 12-6-R3, 5: diabody 12-6-F3 and diabody 12-6-R4, 6: diabody 12-6-F3 and diabody 12-6-R5, 7: diabody 12-6-F3 and diabody 12-6-R6, 8: diabody 12-6-F3 and diabody 12-6-R7, 9: diabodies 12-6-F3 and diabodies 12-6-R8;
FIG. 4 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F4 and diabody 12-6-R1, 3: diabody 12-6-F4 and diabody 12-6-R2, 4: diabody 12-6-F4 and diabody 12-6-R3, 5: diabody 12-6-F4 and diabody 12-6-R4, 6: diabody 12-6-F4 and diabody 12-6-R5, 7: diabody 12-6-F4 and diabody 12-6-R6, 8: diabody 12-6-F4 and diabody 12-6-R7, 9: diabodies 12-6-F4 and diabodies 12-6-R8;
FIG. 5 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F5 and diabody 12-6-R1, 3: diabody 12-6-F5 and diabody 12-6-R2, 4: diabody 12-6-F5 and diabody 12-6-R3, 5: diabody 12-6-F5 and diabody 12-6-R4, 6: diabody 12-6-F5 and diabody 12-6-R5, 7: diabody 12-6-F5 and diabody 12-6-R6, 8: diabody 12-6-F5 and diabody 12-6-R7, 9: diabodies 12-6-F5 and diabodies 12-6-R8;
FIG. 6 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F6 and diabody 12-6-R1, 3: diabody 12-6-F6 and diabody 12-6-R2, 4: diabody 12-6-F6 and diabody 12-6-R3, 5: diabody 12-6-F6 and diabody 12-6-R4, 6: diabody 12-6-F6 and diabody 12-6-R5, 7: diabody 12-6-F6 and diabody 12-6-R6, 8: diabody 12-6-F6 and diabody 12-6-R7, 9: diabodies 12-6-F6 and diabodies 12-6-R8;
FIG. 7 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F7 and diabody 12-6-R1, 3: diabody 12-6-F7 and diabody 12-6-R2, 4: diabody 12-6-F7 and diabody 12-6-R3, 5: diabody 12-6-F7 and diabody 12-6-R4, 6: diabody 12-6-F7 and diabody 12-6-R5, 7: diabody 12-6-F7 and diabody 12-6-R6, 8: diabody 12-6-F7 and diabody 12-6-R7, 9: diabodies 12-6-F7 and diabodies 12-6-R8;
FIG. 8 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F8 and diabody 12-6-R1, 3: diabody 12-6-F8 and diabody 12-6-R2, 4: diabody 12-6-F8 and diabody 12-6-R3, 5: diabody 12-6-F8 and diabody 12-6-R4, 6: diabody 12-6-F8 and diabody 12-6-R5, 7: diabody 12-6-F8 and diabody 12-6-R6, 8: diabody 12-6-F8 and diabody 12-6-R7, 9: diabodies 12-6-F8 and diabodies 12-6-R8;
FIG. 9 is the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F9 and diabody 12-6-R1, 3: diabody 12-6-F9 and diabody 12-6-R2, 4: diabody 12-6-F9 and diabody 12-6-R3, 5: diabody 12-6-F9 and diabody 12-6-R4, 6: diabody 12-6-F9 and diabody 12-6-R5, 7: diabody 12-6-F9 and diabody 12-6-R6, 8: diabody 12-6-F9 and diabody 12-6-R7, 9: diabodies 12-6-F9 and diabodies 12-6-R8;
FIG. 10 shows the Basic electrophoretogram of double-antibody 12-6 primer screening, wherein, 1: 100bp Maker, 2: diabody 12-6-F10 and diabody 12-6-R1, 3: diabody 12-6-F10 and diabody 12-6-R2, 4: diabody 12-6-F10 and diabody 12-6-R3, 5: diabody 12-6-F10 and diabody 12-6-R4, 6: diabody 12-6-F10 and diabody 12-6-R5, 7: diabody 12-6-F10 and diabody 12-6-R6, 8: diabody 12-6-F10 and diabody 12-6-R7, 9: diabodies 12-6-F10 and diabodies 12-6-R8;
FIG. 11 is a real-time fluorescence detection map of double-antibody 12-6 primer screening, wherein, 1: diabody 12-6-F6 and diabody 12-6-R1, 2: diabody 12-6-F1 and diabody 12-6-R1, 3: diabody 12-6-F3 and diabody 12-6-R1, 4: diabody 12-6-F6 and diabody 12-6-R2, 5: diabody 12-6-F1 and diabody 12-6-R2, 6: diabody 12-6-F3 and diabody 12-6-R2, 7: diabody 12-6-F7 and diabody 12-6-R8, 8: blank control;
FIG. 12 is a real-time fluorescence detection map of double-antibody 12-6 primer screening, wherein, 1: diabody 12-6-F6 and diabody 12-6-R4, 2: diabody 12-6-F9 and diabody 12-6-R1, 3: diabody 12-6-F10 and diabody 12-6-R8, 4: diabody 12-6-F9 and diabody 12-6-R4, 5: diabody 12-6-F9 and diabody 12-6-R2, 6: diabody 12-6-F10 and diabody 12-6-R1, 7: diabody 12-6-F6 and diabody 12-6-R6, 8: blank control;
FIG. 13 is a graph showing the specific detection of the primers, namely, double antibody 12-6-F6 and double antibody 12-6-R4, wherein the ratio of 1: transgenic maize double antibody 12-6, 2: other transgenic corn mixes, 3: transgenic rice mixed sample, 4: transgenic soybean mixed sample, 5: transgenic cotton blend, 6: transgenic canola blendstock, 7: transgenic maize dual-antibody 12-6 receptor, 8: blank control;
FIG. 14 is a graph showing the specific detection of the primers, namely, double antibody 12-6-F6 and double antibody 12-6-R4, wherein the ratio of 1: transgenic maize double antibody 12-6, 2: non-transgenic corn blendstock, 3: non-transgenic rice pool, 4: non-transgenic soybean pool, 5: non-transgenic cotton mixed sample, 6: non-transgenic canola blendstock, 7: transgenic maize dual-antibody 12-6 receptor, 8: blank control;
FIG. 15 is a graph showing the sensitivity detection of the primers, double antibody 12-6-F6 and double antibody 12-6-R4, wherein the DNA template copy number of samples 1-8 is 20000 in sequence; 2000; 1000, parts by weight; 200 of a carrier; 100, respectively; 50; 10; 0.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1 design and screening of primer and Probe combinations
Primers and probes are designed according to the specific region of the transgenic corn double-antibody 12-6 transformant. The method is characterized in that a secondary structure is prevented from being formed between front and rear primers and a repeated sequence is prevented from appearing between the primers when the primers are designed, two T bases are selected at the middle rear part of the probe and are respectively marked with a fluorescent group (FAM and BHQ1), a abasic site (THF) is arranged between the two bases and is recognized and cut by exonuclease in the reaction process, so that the two fluorescent groups are separated to generate fluorescent signals, and the specific probe can monitor the result of fluorescent detection in real time. The length of the primer is about 35nt, multiple pairs of primers need to be designed from two ends of a target sequence for optimization and screening in an RPA experiment, and the increase, decrease or replacement of individual bases can generate important influence on the experimental result. The target sequence used for amplification by the RPA method in the invention is a 3' end part sequence of a maize genome and exogenous inserted DNA. Aiming at the specific sequence of the transformant, an RPA probe (numbered as double-antibody 12-6-P) is designed, and 10 forward primers (numbered as double-antibody 12-6-F1 to double-antibody 12-6-F10) and 10 reverse primers (numbered as double-antibody 12-6-R1 to double-antibody 12-6-R10) are respectively designed at the upstream and the downstream of the probe. In the screening process, firstly, the RPA-Basic kit is used for amplification and electrophoresis, and a primer pair capable of amplifying a correct band is screened, wherein the specific electrophoresis result is shown in FIGS. 1-10. The results of specific real-time fluorescence screening using the RPA-EXO kit are shown in FIGS. 11-12, in which the takeoff time and amplification effect of line 1 in FIG. 12 are better, and the primer pairs are the same as those of the 5-channel primer pair in FIG. 6 of Basic experiment. Therefore, the primary screening primer pair of the double-resistant 12-6-F6 and the double-resistant 12-6-R4 is used for the strain specificity detection of the double-resistant 12-6 of the transgenic corn. The sequences of the primer and probe combinations are shown in Table 1.
TABLE 1
Note that FAM: a luminescent group; THF: abasic sites; BHQ 1: a quencher group; block: blocking group, carbonation of this use
Example 2 analysis of specificity and sensitivity of the detection of transgenic maize double antibody 12-6 Using the primer and Probe combination selected in example 1
1. Experimental Material
1.1 plant Material
12-6 transgenic corn double antibody, 12-6 transgenic corn double antibody receptor material, other transgenic corn mixed sample, transgenic rice mixed sample, transgenic soybean mixed sample, transgenic cotton mixed sample, transgenic rape mixed sample, non-transgenic corn mixed sample, non-transgenic rice mixed sample, non-transgenic soybean mixed sample, non-transgenic cotton mixed sample, non-transgenic rape mixed sample.
1.2 enzymes and reagents
The molecular biological reagents, twist Amp DNA amplification Exo Kits, are purchased from twist DX, twist Amp DNA amplification Basic Kits, are purchased from twist DX, and other biochemical reagents are imported for split charging or domestic analysis. The primer probe combination sequences were synthesized by Beijing Biotechnology Ltd, as shown in Table 1 of example 1.
1.3 Experimental instruments
DNA processing apparatus: low temperature ball mill instrument μ M400(Retsch)
A fluorescence detector: RPA amplification detector (twist)
Other instruments include: a constant temperature water bath, an electronic balance, a centrifuge, a water purifier and the like.
2. Experimental methods and procedures
2.1 extraction of genomic DNA
The extraction of the DNA from the Plant material was carried out according to the manual of the TianGen Plant Genomic DNA Kit. The method comprises the following specific steps:
taking fully-milled plant material seed powder 150mg, adding 800 μ l of buffer solution GP1 preheated at 65 ℃, and carrying out water bath at 65 ℃ for 60min, wherein the centrifuge tubes are inverted for several times during the process to mix the sample.
Adding 1: 1 was extracted with phenol/chloroform and centrifuged at 12000rpm for 10 min.
And thirdly, taking the supernatant, adding 800 mu l of chloroform, fully and uniformly mixing, and centrifuging at 12000rpm for 10 min.
And fourthly, taking the supernatant, adding 800 mu l of buffer solution GP2, and fully mixing.
Fifthly, transferring the uniformly mixed liquid into an adsorption column CB3, centrifuging at 12000rpm for 30sec, and discarding the waste liquid
Sixthly, adding 600 mu l of buffer GD into the adsorption column, centrifuging at 12000rpm for 30sec, and discarding waste liquid
Adding 800 μ l of rinsing solution PW into adsorption column, centrifuging at 12000rpm for 30sec, discarding waste solution
Adding 600 μ l of rinsing solution PW to the adsorption column, centrifuging at 12000rpm for 30sec, and discarding the waste solution
Putting absorption column CB3 into the collection tube, idling at 12000rpm for 3min, discarding the waste liquid, standing at room temperature for 10min
Transfer the adsorption column CB3 into a clean collecting tube, drop 50 mul of water, stand for 10min at room temperature, and collect into a centrifuge tube at 12000 rpm.
2.2 DNA concentration and purity determination
The purity and concentration of DNA was determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and adjusted to 25 ng/. mu.l with deionized double distilled water.
2.3 RPA reaction System
The optimized RPA amplification system is obtained by experiments, the total system is 50 mu l, the Rehydration Buffer (Rehydration Buffer) is 29.5 mu l, the 280 mu m Magnesium acetate solution (Magnesium acetate) is 2.5 mu l, the primers are respectively 2.4 mu l (10uM), the plant material DNA is 2 mu l (25 ng/mu l), and the rest is made up by water.
Optimized RPA amplification procedure: the RPA amplification detector was allowed to react at 39 ℃ for 30 minutes.
2.4 specific detection
Carrying out an RPA-EXO experiment on DNA samples of the transgenic corn double antibody 12-6, the transgenic corn double antibody 12-6 receptor material, other transgenic corn mixed samples, transgenic rice mixed samples, transgenic soybean mixed samples, transgenic cotton mixed samples, transgenic rape mixed samples, non-transgenic corn mixed samples, non-transgenic rice mixed samples, non-transgenic soybean mixed samples, non-transgenic cotton mixed samples and non-transgenic rape mixed samples according to the reaction system in the step 2.3 to test the specificity.
2.5 sensitivity detection
The double-antibody 12-6 genome DNA is respectively diluted by the following copy numbers: 20000, 2000, 1000, 200, 100, 50, 10. And (3) carrying out an RPA-EXO experiment according to the reaction system in the step 2.3 to test the sensitivity, and simultaneously carrying out negative control.
3. Results of the experiment
The specific detection results are shown in FIGS. 13-14, and an obvious amplification curve can be obtained by using the transgenic corn double antibody 12-6 as a template and adopting the combination of the double antibody 12-6-F6 and the double antibody 12-6-R9 primer probe. The other transgenic corn mixed samples, the transgenic rice mixed samples, the transgenic soybean mixed samples, the transgenic cotton mixed samples, the transgenic rape mixed samples, the non-transgenic corn mixed samples, the non-transgenic rice mixed samples, the non-transgenic soybean mixed samples, the non-transgenic cotton mixed samples and the genome DNA of the non-transgenic rape mixed samples are taken as templates, and no amplification curve is generated. The specification shows that the primer probe combination and the detection method have strong specificity for identifying the transgenic corn double antibody 12-6.
The sensitivity detection result is shown in FIG. 15, when the double antibody 12-6-F6+ the double antibody 12-6-R9 is adopted, under the premise that the negative material does not take off, amplification curves exist when the copy number of the template DNA is 20000, 2000, 1000, 200, 100 and 50 copies, only 10 copies are not amplified, and the sensitivity of the primer probe combination and the detection method for identifying the double antibody 12-6 of the transgenic corn is higher and can reach 50 copies.
As shown in FIGS. 13-15, the amplification curve can exceed the threshold take-off in about 10 minutes, so that the primer and the method can be used for rapidly identifying the transgenic corn double antibody 12-6, and the identification time is only about 10 minutes.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
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Claims (7)
1. An RPA primer probe combination for detecting a transgenic corn double antibody 12-6 is disclosed, wherein the primer comprises a forward primer and a reverse primer, and is characterized in that the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. A transgenic corn double antibody 12-6 RPA detection kit, characterized in that, the kit comprises the primer probe combination of claim 1.
3. A transgenic corn double-antibody 12-6 RPA detection method is characterized by comprising the following steps: using the genomic DNA of a sample to be tested as a template, performing RPA amplification by using the primer-probe combination of claim 1, and performing fluorescence detection.
4. The method for detecting the RPA of transgenic maize double antibody 12-6 as claimed in claim 3, wherein said RPA amplification reaction system comprises 20pmol each of said forward primer and reverse primer, 5pmol probe and 50ng DNA template in each 50 μ l reaction system.
5. The RPA detection method of transgenic corn double antibody 12-6 as claimed in claim 4, wherein the RPA amplification system is 50 μ l total system, rehydration buffer 29.5 μ l, 280 μm M magnesium acetate solution 2.5 μ l, and the rest is water.
6. The method for detecting the RPA of transgenic corn double antibody 12-6 according to any one of claims 3-5, wherein the RPA amplification program is 39 ℃ reaction for 20 minutes.
7. Use of the primer probe combination of claim 1, the detection kit of claim 2 and/or the detection method of any one of claims 3 to 6 for detecting the seed resource of transgenic corn double antibody 12-6.
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| CN112063748A (en) * | 2020-10-13 | 2020-12-11 | 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 G1105E-823C |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755488A (en) * | 2017-01-22 | 2017-05-31 | 中国农业科学院生物技术研究所 | Transgenic corn BT 11 strain specificity is identified using RPA technologies |
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| CN106755488A (en) * | 2017-01-22 | 2017-05-31 | 中国农业科学院生物技术研究所 | Transgenic corn BT 11 strain specificity is identified using RPA technologies |
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| CN112063748A (en) * | 2020-10-13 | 2020-12-11 | 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 G1105E-823C |
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