CN112176087A - RPA detection primer and probe combination, kit and detection method of yellow 6106 in transgenic soybean - Google Patents
RPA detection primer and probe combination, kit and detection method of yellow 6106 in transgenic soybean Download PDFInfo
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Abstract
A primer and probe combination, a kit and a detection method for detecting the yellow 6106 in transgenic soybeans are disclosed, wherein a large number of RPA primers are designed according to the connection region of an exogenous inserted DNA sequence and a soybean genome, and a pair of primer and probe combination capable of quickly and effectively detecting the yellow 6106 in the transgenic soybeans is screened out. The transgenic soybean Zhonghuang 6106 genome DNA is taken as a template, and the pair of primers and the probe are utilized to carry out RPA amplification and real-time fluorescence detection, thereby having 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 detection primer and probe combination, a kit and a detection method for yellow 6106 in transgenic soybeans.
Background
Transgenic soybean medium yellow 6106(ZH10-6) is a new strain of soybean with herbicide tolerance of G2-EPSPS gene and GAT gene, which is developed by the research institute of crop science of Chinese academy of agricultural sciences. The strain has obtained agricultural transgenic organism safety certificate (production application) in 6 months in 2020, and has wide industrial application prospect. The currently reported detection method for soybean medium yellow 6106 is mainly to use a PCR instrument to carry out conventional detection in a laboratory, but the PCR-based transgenic detection method needs professional instruments such as a PCR instrument and a gel imaging system, and the 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 transgenic detection technology which is more convenient and accurate and is suitable for on-site operation 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 yellow 6106(ZH10-6) in transgenic soybeans by using an RPA technology exists.
Disclosure of Invention
The invention provides an RPA detection primer and probe combination of yellow 6106 in transgenic soybean, the primer comprises a forward primer and a reverse primer, 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 an RPA detection kit of yellow 6106 in transgenic soybeans, which comprises the primer and the probe combination.
The invention also provides an RPA detection method of yellow 6106 in transgenic soybean, which comprises the following steps:
and (3) taking the genome DNA of a sample to be detected as a template, and carrying out RPA amplification and real-time fluorescence detection by utilizing the primer and probe combination. If an obvious amplification curve is obtained, the detected sample is proved to contain yellow 6106 components in the transgenic soybean.
Further, the RPA amplification 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 total RPA amplification system is 50. mu.l, the rehydration buffer is 29.5. mu.l, the 280. mu. m M magnesium acetate solution is 2.5. mu.l, and the rest is water.
Further, the RPA amplification procedure was 39 ℃ for 20 minutes
The invention also provides application of the detection primer and probe combination, the detection kit and/or the detection method in detection of the seed resource of yellow 6106 in transgenic soybeans.
The invention provides a method for detecting the specific RPA of yellow 6106 strain in transgenic soybean 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 soybean genome, and a pair of primers and probe combinations capable of quickly and effectively detecting the yellow 6106 component in the transgenic soybean are screened out. The yellow 6106 genome DNA in the transgenic soybean is taken as a template, and the pair of primers and the probe are utilized to carry out RPA amplification and real-time fluorescence detection, so that an obvious amplification curve can be obtained. The RPA detection primer and probe combination, the kit and the detection method have the characteristics of high speed, good specificity and high sensitivity.
Drawings
FIG. 1 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F1 and ZH10-6-R1, 3: ZH10-6-F1 and ZH10-6-R2, 4: ZH10-6-F1 and ZH10-6-R3, 5: ZH10-6-F1 and ZH10-6-R4, 6: ZH10-6-F1 and ZH10-6-R5, 7: ZH10-6-F1 and ZH10-6-R6, 8: ZH10-6-F1 and ZH10-6-R7, 9: ZH10-6-F1 and ZH 10-6-R8;
FIG. 2 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F2 and ZH10-6-R1, 3: ZH10-6-F2 and ZH10-6-R2, 4: ZH10-6-F2 and ZH10-6-R3, 5: ZH10-6-F2 and ZH10-6-R4, 6: ZH10-6-F2 and ZH10-6-R5, 7: ZH10-6-F2 and ZH10-6-R6, 8: ZH10-6-F2 and ZH10-6-R7, 9: ZH10-6-F2 and ZH 10-6-R8;
FIG. 3 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F3 and ZH10-6-R1, 3: ZH10-6-F3 and ZH10-6-R2, 4: ZH10-6-F3 and ZH10-6-R3, 5: ZH10-6-F3 and ZH10-6-R4, 6: ZH10-6-F3 and ZH10-6-R5, 7: ZH10-6-F3 and ZH10-6-R6, 8: ZH10-6-F3 and ZH10-6-R7, 9: ZH10-6-F3 and ZH 10-6-R8;
FIG. 4 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F4 and ZH10-6-R1, 3: ZH10-6-F4 and ZH10-6-R2, 4: ZH10-6-F4 and ZH10-6-R3, 5: ZH10-6-F4 and ZH10-6-R4, 6: ZH10-6-F4 and ZH10-6-R5, 7: ZH10-6-F4 and ZH10-6-R6, 8: ZH10-6-F4 and ZH10-6-R7, 9: ZH10-6-F4 and ZH 10-6-R8;
FIG. 5 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F5 and ZH10-6-R1, 3: ZH10-6-F5 and ZH10-6-R2, 4: ZH10-6-F5 and ZH10-6-R3, 5: ZH10-6-F5 and ZH10-6-R4, 6: ZH10-6-F5 and ZH10-6-R5, 7: ZH10-6-F5 and ZH10-6-R6, 8: ZH10-6-F5 and ZH10-6-R7, 9: ZH10-6-F5 and ZH 10-6-R8;
FIG. 6 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F6 and ZH10-6-R1, 3: ZH10-6-F6 and ZH10-6-R2, 4: ZH10-6-F6 and ZH10-6-R3, 5: ZH10-6-F6 and ZH10-6-R4, 6: ZH10-6-F6 and ZH10-6-R5, 7: ZH10-6-F6 and ZH10-6-R6, 8: ZH10-6-F6 and ZH10-6-R7, 9: ZH10-6-F6 and ZH 10-6-R8;
FIG. 7 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F7 and ZH10-6-R1, 3: ZH10-6-F7 and ZH10-6-R2, 4: ZH10-6-F7 and ZH10-6-R3, 5: ZH10-6-F7 and ZH10-6-R4, 6: ZH10-6-F7 and ZH10-6-R5, 7: ZH10-6-F7 and ZH10-6-R6, 8: ZH10-6-F7 and ZH10-6-R7, 9: ZH10-6-F7 and ZH 10-6-R8;
FIG. 8 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F8 and ZH10-6-R1, 3: ZH10-6-F8 and ZH10-6-R2, 4: ZH10-6-F8 and ZH10-6-R3, 5: ZH10-6-F8 and ZH10-6-R4, 6: ZH10-6-F8 and ZH10-6-R5, 7: ZH10-6-F8 and ZH10-6-R6, 8: ZH10-6-F8 and ZH10-6-R7, 9: ZH10-6-F8 and ZH 10-6-R8;
FIG. 9 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F9 and ZH10-6-R1, 3: ZH10-6-F9 and ZH10-6-R2, 4: ZH10-6-F9 and ZH10-6-R3, 5: ZH10-6-F9 and ZH10-6-R4, 6: ZH10-6-F9 and ZH10-6-R5, 7: ZH10-6-F9 and ZH10-6-R6, 8: ZH10-6-F9 and ZH10-6-R7, 9: ZH10-6-F9 and ZH 10-6-R8;
FIG. 10 shows the Basic electrophoretogram of Zhonghuang 6106(ZH10-6) primer screening, wherein, 1: 100bp Maker, 2: ZH10-6-F10 and ZH10-6-R1, 3: ZH10-6-F10 and ZH10-6-R2, 4: ZH10-6-F10 and ZH10-6-R3, 5: ZH10-6-F10 and ZH10-6-R4, 6: ZH10-6-F10 and ZH10-6-R5, 7: ZH10-6-F10 and ZH10-6-R6, 8: ZH10-6-F10 and ZH10-6-R7, 9: ZH10-6-F10 and ZH 10-6-R8;
figure 11 is a real-time fluorescence assay of zhonghuang 6106(ZH10-6) primer screen, wherein, 1: ZH10-6-F6 and ZH10-6-R3, 2: ZH10-6-F6 and ZH10-6-R8, 3: ZH10-6-F6 and ZH10-6-R2, 4: ZH10-6-F6 and ZH10-6-R5, 5: ZH10-6-F6 and ZH10-6-R7, 6: ZH10-6-F6 and ZH10-6-R9, 7: ZH10-6-F6 and ZH10-6-R10, 8: blank control;
FIG. 12 is a specific assay for primers ZH10-6-F6 and ZH10-6-R3, wherein, 1: transgenic soybean ZH10-6, 2: other transgenic soybean mixes, 3: transgenic rice mixed sample, 4: transgenic corn blendstock, 5: transgenic cotton blend, 6: transgenic canola blendstock, 7: transgenic soybean ZH10-6 receptor, 8: blank control;
FIG. 13 is a specific assay for primers ZH10-6-F6 and ZH10-6-R3, wherein 1: transgenic soybean ZH10-6, 2: non-transgenic soybean pool, 3: non-transgenic rice pool, 4: non-transgenic corn blendstock, 5: non-transgenic cotton mixed sample, 6: non-transgenic canola blendstock, 7: transgenic soybean ZH10-6 receptor, 8: blank control;
FIG. 14 is a sensitivity test chart of primers ZH10-6-F6 and ZH10-6-R3, wherein the template copy number of samples 1-8 is 20000 in order; 2000; 1000, parts by weight; 500, a step of; 200 of a carrier; 100, respectively; 50; 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 yellow 6106 transformant in the transgenic soybean. 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. Aiming at the specific sequence of the transformant, an RPA probe (numbered as ZH10-6-P) is designed, and 10 forward primers (numbered as ZH10-6-F1 to ZH10-6-F10) and 10 reverse primers (numbered as ZH10-6-R1 to ZH10-6-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 by using the RPA-EXO kit are shown in FIG. 11, wherein the takeoff time of line No.1 and the amplification effect are better in FIG. 11, and the primer pair (numbered ZH10-6-F6+ ZH10-6-R3) is the same as the 4-channel primer pair in FIG. 6 of the Basic experiment.
Finally, a pair of RPA primers ZH10-6-F6+ ZH10-6-R3 obtained by screening in the application is used for detecting the strain specificity of yellow 6106 in the transgenic soybeans, and specific primer and probe sequences 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 detection of yellow 6106 in transgenic Soybean Using the primer and Probe combination selected in example 1
1. Experimental Material
1.1 plant Material
Transgenic soybean medium yellow 6106(ZH10-6), transgenic soybean medium yellow 6106 receptor material, other transgenic soybean blendstocks, transgenic rice blendstocks, transgenic corn blendstocks, transgenic cotton blendstocks, transgenic canola blendstocks, non-transgenic soybean blendstocks, non-transgenic rice blendstocks, non-transgenic corn blendstocks, non-transgenic cotton blendstocks, non-transgenic canola blendstocks.
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 and probe 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 total RPA amplification system is 50. mu.l, the rehydration buffer is 29.5. mu.l, the 280. mu. m M magnesium acetate solution is 2.5. mu.l, and the rest is 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 soybean medium yellow 6106, the transgenic soybean medium yellow 6106 receptor material, other transgenic soybean mixed samples, transgenic rice mixed samples, transgenic corn mixed samples, transgenic cotton mixed samples, transgenic rape mixed samples, non-transgenic soybean mixed samples, non-transgenic rice mixed samples, non-transgenic corn mixed samples, non-transgenic cotton mixed samples and non-transgenic rape mixed samples according to the reaction system of the step 2.3 to test the specificity.
2.5 sensitivity detection
Yellow 6106 genomic DNA was diluted individually in copy numbers as follows: 20000, 2000, 1000, 500, 200, 100, 50. 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 results of the specificity detection are shown in fig. 12-13, and obvious amplification curves can be obtained by using transgenic soybean medium yellow 6106 as a template, and no amplification curve can be generated by using other transgenic soybean mixed samples, transgenic rice mixed samples, transgenic corn mixed samples, transgenic cotton mixed samples, transgenic rape mixed samples, non-transgenic soybean mixed samples, non-transgenic rice mixed samples, non-transgenic corn mixed samples, non-transgenic cotton mixed samples, and non-transgenic rape mixed samples as templates.
The sensitivity detection result is shown in fig. 14, and 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, 500, 200, 100 and 50 copies, which indicates that the sensitivity of identifying the yellow 6106 in the transgenic soybean can reach 50 copies by the primer and the method designed by the invention.
As shown in FIGS. 12-14, the amplification curves can exceed the threshold take-off within 10 minutes, and thus it can be concluded that the primers and the method of the present invention can rapidly identify yellow 6106 in transgenic soybeans, and the identification time is only about 10 minutes.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> RPA detection primer and probe combination, kit and detection method of yellow 6106 in transgenic soybean
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Claims (7)
1. An RPA detection primer and a probe combination of yellow 6106 in transgenic soybean, 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. An RPA detection kit of yellow 6106 in transgenic soybean comprising the primer and probe combination of claim 1.
3. A method for detecting the RPA of yellow 6106 in transgenic soybeans is characterized by comprising the following steps:
using the genomic DNA of a sample to be tested as a template, performing RPA amplification using the primer and probe combination of claim 1, and performing fluorescence detection.
4. The method for detecting RPA of yellow 6106 in transgenic soybean according to claim 3, wherein the RPA amplification system comprises 24pmol each of the forward primer and the reverse primer, 5pmol of probe, and 50ng of DNA template in 50 μ l reaction system.
5. The method for detecting the RPA of yellow 6106 in transgenic soybean according to claim 4, wherein the RPA amplification system is 50 μ l of total system, 29.5 μ l of rehydration buffer, 2.5 μ l of 280 μm M magnesium acetate solution, and the balance is water.
6. The method for detecting RPA of yellow 6106 in transgenic soybean according to any one of claims 3 to 5, wherein the RPA amplification procedure is a 39 ℃ reaction for 20 minutes.
7. Use of the detection primer and probe combination of claim 1, the detection kit of claim 2, and/or the detection method of any one of claims 3-6 for detecting seed resources of yellow 6106 in transgenic soybean.
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