Rapid detection method of transgenic corn Reifeng 12-5

Abstract

The invention provides a rapid detection method of transgenic corn Reifeng 12-5. The rapid detection method comprises the following steps: (1) RPA reaction amplification, (2) test strip detection, and (3) PAH-Cu < 2 + > secondary amplification of a detection signal. According to the invention, AuNP-anti-Biotin is taken as a functional probe, an'antibody-hapten 'recognition system is adopted, an RPA technology is combined with a PAH-Cu < 2 + > deposition technology to doubly amplify a detection signal, trace detection of the transgenic maize Rusfeng 12-5 is realized, and the detection limit can reach 0.1 wt%.

Description

Technical Field

[0001] The invention belongs to the technical field of transgenic detection, and specifically relates to a rapid detection method for transgenic corn Ruifeng 12-5. Background Art

[0002] Corn is rich in nutrients and is a major food crop in my country. In 2020, the planting area has reached 4.1×10 7 hm 2 The insertion sequence of Ruifeng 12-5 is a glyphosate-resistant gene and insect-resistant gene with independent intellectual property rights in my country. The control effect of corn borer is 96%, which is a representative of my country breaking the monopoly of foreign patents. Once the genetically modified corn is approved for marketing, the supporting rapid detection means will be very necessary to maintain its long-term commercial development. At the same time, the convenient, rapid, sensitive and large-scale detection methods for genetically modified corn Ruifeng 12-5 and its deep-processed products will have urgent market demand and broad development prospects.

[0003] The present invention is based on the problem of low sensitivity in detecting low-content adulterated biotech foods, and prepares an "enhanced" colloidal gold test strip for the detection of Ruifeng 12-5. By establishing a dual signal amplification mechanism of RPA and PAH copper deposition to improve the sensitivity of detection, a low-cost colloidal gold test strip is selected to present the test results, thereby improving the portability of the detection. The Ruifeng 12-5 detection method of the present invention fully meets the requirements of rapid and trace detection. Summary of the invention

[0004] The purpose of the present invention is to develop a rapid detection method for transgenic corn Ruifeng 12-5. Specifically:

[0005] In a first aspect of the present invention, a rapid detection method for transgenic corn Ruifeng 12-5 is provided, the rapid detection method comprising:

[0006] (1) Using the specific primers of transgenic corn Ruifeng 12-5, RPA amplification was performed using the corn genome as a template.

[0007] (2) performing a colorimetric test strip test on the product obtained in step (1);

[0008] (3)PAH-Cu 2+ Amplify the detection signal twice.

[0009] Preferably, the specific primers include SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] Preferably, the specific primer is a modified specific primer.

[0011] Further preferably, the modification is selected from any one of FAM, FITC, biotin, dig or TAMRA.

[0012] In a specific embodiment of the present invention, the specific primers include:

[0013] RPA-F: 5'- / Dig / -SEQ ID NO: 1-3',

[0014] RPA-R: 5'- / Biotin / -SEQ ID NO: 2-3'.

[0015] Preferably, the color developing test strip comprises a colloidal gold test strip.

[0016] Preferably, the colloidal gold test strip contains AuNP-anti-Biotin as a functional probe.

[0017] Preferably, the (3) PAH-Cu 2+ The secondary amplification detection signal includes soaking the test strip after step (2) in PAH-Cu 2+ solution, take it out and soak it in ascorbic acid solution again, take it out and read the result.

[0018] Preferably, the Cu 2+ Derived from CuSO4 solution or CuCl2 solution;

[0019] In a specific embodiment of the present invention, the Cu 2+ Derived from CuSO4 solution.

[0020] Preferably, the PAH-Cu 2+ The solution is a mixture of PAH solution and CuSO4 solution.

[0021] Preferably, in PAH-Cu 2+ The cells are immersed in the solution for 5-10 min, preferably 6 min, and immersed in the ascorbic acid solution for 5-10 min, preferably 6 min.

[0022] Preferably, the PAH-Cu 2+ In solution, Cu 2+ The concentration is 1.25M and the PAH concentration is 10mg / mL;

[0023] Preferably, the ascorbic acid concentration is 100 mg / mL.

[0024] In a specific embodiment of the present invention, the rapid detection method comprises:

[0025] (1) Using SEQ ID NO: 1 and SEQ ID NO: 2 as specific primers, RPA amplification was performed using the corn genome as a template.

[0026] (2) Detect the amplified product using colloidal gold test strips.

[0027] (3) Mix the PAH solution and CuSO4 solution to obtain PAH-Cu 2+ Solution, in which Cu 2+ The concentration is 1.25M and the PAH concentration is 10mg / mL;

[0028] (4) Place the test strip after step (2) on the PAH-Cu 2+ The samples were immersed in the solution and incubated for 6 min, and the samples were immersed in the ascorbic acid solution and incubated for 6 min.

[0029] The second aspect of the present invention provides a detection kit for transgenic corn Ruifeng 12-5, the detection kit comprising reagents, primers, color test strips, PAH solution, Cu 2+ solution and / or ascorbic acid solution.

[0030] Preferably, the reagents and primers are those required for RPA reaction.

[0031] Preferably, the reagents include enzyme, Primer Free Rehydration buffer, and magnesium acetate;

[0032] Preferably, the magnesium acetate solution is 280 mM and is used for RPA amplification catalysis reaction;

[0033] Preferably, the concentration of the primer is 10 μM.

[0034] Further preferably, the primers include SEQ ID NO: 1 and SEQ ID NO: 2;

[0035] Preferably, the specific primer is a modified specific primer.

[0036] Further preferably, the modification is selected from any one of FAM, FITC, biotin, dig or TAMRA.

[0037] In a specific embodiment of the present invention, the specific primers include:

[0038] RPA-F: 5'- / Dig / -SEQ ID NO: 1-3',;

[0039] RPA-R: 5'- / Biotin / -SEQ ID NO: 2-3'.

[0040] Preferably, the color developing test strip comprises a colloidal gold test strip.

[0041] Preferably, the Cu 2+ The solution includes a CuSO4 solution or a CuCl2 solution.

[0042] Further preferably, the Cu 2+ It is a CuSO4 solution.

[0043] The third aspect of the present invention provides a rapid detection method as described above, and / or the use of the detection kit as described above.

[0044] Preferably, the application includes detecting the transgenic corn Ruifeng 12-5 in transgenic products.

[0045] In order to achieve the purpose of the present invention, an "enhanced" colloidal gold test strip was constructed using AuNP-anti-Biotin as a functional probe, an "antibody-hapten" recognition system, and RPA technology combined with polyallylamine hydrochloride (PAH) copper deposition technology to double amplify the detection signal, thereby realizing trace detection of genetically modified corn Ruifeng 12-5.

[0046] The detection and analysis principle of this method: When the genetically modified corn Ruifeng 12-5 component is present, a product with digoxigenin modification on one end and biotin modification on the other end is amplified through the RPA reaction. The running buffer mixed with the RPA product migrates on the colloidal gold test paper through capillary force. First, at the binding pad, the biotin in the product will bind to the AuNP-anti-Biotin to form a product-AuNP-anti-Biotin complex. Continue chromatography, the digoxigenin antibody at the T line captures the product labeled with digoxigenin, thereby forming a sandwich structure of digoxigenin antibody-product-AuNP-anti-Biotin, and a red line appears. Continue chromatography, and the excess AuNP-anti-Biotin is captured by the biotin secondary antibody at the C line, so that the C line turns red. Soak the paper strips in PAH-Cu in turn. 2+ First, PAH has a certain viscosity and adheres to the AuNPs on the test strip. Then, the rich amine groups of PAH further bind to Cu 2+ A low reduction potential complex is formed, and finally, in the presence of ascorbic acid reducing agent, a Cu shell is formed on the surface of AuNPs, which appears as a black line.

[0047] The present invention has at least the following advantages and beneficial effects:

[0048] The present invention establishes a rapid detection method for transgenic corn Ruifeng 12-5. First, the genomic DNA of transgenic corn Ruifeng 12-5, i.e., RPA reaction template DNA, is extracted by a kit. Then, the extracted target is amplified for the first time by RPA amplification reaction. Finally, the signal on the colloidal gold test strip is amplified for the second time by PAH copper deposition technology, and the test result is read.

[0049] (I) This method uses AuNPs to modify and prepare AuNP-anti-Biotin, adopts the "antibody-hapten" recognition system, uses RPA technology for the first signal amplification, combines PAH copper deposition technology for the second signal amplification, and presents the test results with colloidal gold test strips.

[0050] (ii) By optimizing the experimental conditions of the LFAs reaction system, the optimal addition amount of RPA product, AuNP-anti-Biotin, running buffer and the optimal running buffer type were obtained; 2+ The system experimental conditions were optimized to obtain the optimal Cu 2+ The source is related to the concentration of PAH, CuSO4, AA solution and immersion time. Compared with the traditional detection method, the qualitative result determination by naked eyes has a faster detection time and higher detection sensitivity, which meets the needs of rapid detection.

[0051] (III) The test strip-based sensing platform established by this method achieved highly specific recognition and highly sensitive detection of the transgenic corn Ruifeng 12-5, avoiding false positives, and the qualitative detection limit of the transgenic corn Ruifeng 12-5 could reach 0.1wt%. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the detection principle of genetically modified corn Ruifeng 12-5.

[0053] Figure 2 Characterization of AuNPs, Au@PAH-Cu NPs and AuNP-anti-Biotin, where A is the TEM image of AuNPs, B is the TEM image of Au@PAH-Cu NPs, and C is the UV-visible absorption spectrum of AuNPs, Au@PAH-Cu NPs and AuNP-anti-Biotin.

[0054] Figure 3 The figure is a verification diagram of RPA reaction and PAH copper deposition enhancement, wherein A is an agarose gel diagram of RPA reaction, B is a colloidal gold test strip diagram, 1 in A is a positive amplification product, and 2 is a negative amplification product.

[0055] Figure 4Optimization of the LFAs system, where A is RPA product with different volumes, B is AuNP-anti-Biotin with different volumes, and C is different types of running buffer (a: 0.002% Triton X-100 + 2% BSA + 1x PBS + 0.05% Tween 20; b: 10mM Tris-HAc + 0.002% Triton X-100 + 2% BSA + 1x PBS + 0.05% Tween 20; c: 2% BSA + 4x SSC + 0.05% Tween 20; d: 2% BSA + 4xSSC + 0.002% Triton X-100 + 0.05% Tween 20; e: 10mM Tris-HAc + 2% BSA + 4xSSC + 0.002% Triton X-100 + 0.05% Tween 20; f: 0.01M PBS+0.05% Tween 20), D is running buffer with different volumes.

[0056] Figure 5 PAH-Cu 2+ System optimization, where A is different Cu 2+ Source, B is different Cu 2+ concentration, C is different PAH concentrations, D is different ascorbic acid concentrations, and E is PAH-Cu 2+ The solution immersion incubation time is optimized, and F is the ascorbic acid solution immersion incubation time optimization, wherein the ordinate is the peak area, representing the depth of the T line color.

[0057] Figure 6 The sensitivity of the detection of genetically modified corn Ruifeng 12-5, where A is the detection using only RPA amplification, and B is the detection using RPA amplification followed by PAH-Cu 2+ The deposition means amplifies the signal.

[0058] Figure 7 This is the specificity of the detection of genetically modified corn Ruifeng 12-5, where the vertical axis is the peak area, representing the depth of the T line color. DETAILED DESCRIPTION

[0059] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0060] In the present invention, each optimization experiment uses the best value obtained from the previous optimization experiment as a parameter for subsequent experiments. For example, the present invention first optimizes the amount of RPA product added with a colloidal gold addition amount of 2 μL, a buffer addition amount of 100 μL, and a buffer type of e to obtain the optimal addition amount of the optimal RPA product. Subsequent experiments all use this value, and so on.

[0061] Subsequently, the scheme optimized by the above experiment was used as the basic scheme to 2+ Source: Cu 2+ concentration, PAH concentration, ascorbic acid concentration, PAH-Cu 2+ The solution immersion incubation time and the ascorbic acid solution immersion incubation time were optimized according to the above principles.

[0062] Doing Cu 2+ When the source was, the PAH concentration was 13.32 mg / ml, Cu 2+ The concentration is 1.334M, the ascorbic acid concentration is 100mg / mL, and the PAH-Cu 2+ The incubation time for the solution immersion and the ascorbic acid solution immersion were both 10 min.

[0063] Example 1 Establishment of a rapid detection method for transgenic corn Ruifeng 12-5

[0064] 1. Experimental Materials

[0065] Biotin antibody, digoxigenin antibody, biotin secondary antibody, sodium dihydrogen phosphate (NaH2PO4), sodium hydrogen phosphate (Na2HPO4), copper sulfate, polyallylamine hydrochloride, etc. were purchased from commercial products.

[0066] The nucleic acid sequences used in the experiment are as follows (from 5' to 3'):

[0067] RPA-F: 5'- / Dig / -GTCGTTTTCCCGCCTTCAGTT-3'(5'- / Dig / -SEQ ID NO: 1-3'),

[0068] RPA-R: 5'- / Biotin / -GGTGCCTGGAAGACAAGTTCTA-3' (5'- / Dig / -SEQ ID NO: 2-3').

[0069] 2. Design principle of rapid detection method for genetically modified corn Ruifeng 12-5

[0070] When the genetically modified corn Ruifeng 12-5 component is present, a product with digoxigenin modification on one end and biotin modification on the other end is amplified through RPA reaction. The running buffer mixed with the RPA product migrates on the LFAs through capillary force. First, at the binding pad, the biotin in the product will bind to AuNP-anti-Biotin to form a product-AuNP-anti-Biotin complex. Continue chromatography, the digoxigenin antibody at the T line captures the digoxigenin-labeled product, thereby forming a sandwich structure of digoxigenin antibody-product-AuNP-anti-Biotin, and a red line appears. Continue chromatography, and the excess AuNP-anti-Biotin is captured by the biotin secondary antibody at the C line, so that the C line turns red. Soak the paper strips in PAH-Cu in turn. 2+ First, PAH has a certain viscosity and will adhere to the AuNPs on the test strip. Then, the rich amine groups of PAH will further bind to Cu 2+ Finally, in the presence of AA reducing agent, a Cu shell is formed on the surface of AuNPs, showing black lines (see Figure 1 ).

[0071] 3. Preparation of AuNPs, Au@PAH-Cu NPs and AuNP-anti-Biotin

[0072] Synthesis of AuNPs: AuNPs were prepared by reduction method. Washing utensils. Soak the round-bottom flask, magnetic stirring bar and measuring cylinder in aqua regia overnight, pour out the acid, wash the residual acid with deionized water, add ultrapure water to the round-bottom flask and boil, pour out the water, wipe off the residual water and set aside. Preparation of AuNPs. Add 100mL of 1mM HAuCl4 solution to the round-bottom flask and heat to boiling; add 10mL of 38.8mM trisodium citrate solution and accelerate stirring; keep stirring and boiling the above solution until the color of the solution turns wine red or cherry red, and stop heating. Recovery of AuNPs. After the above solution is cooled to room temperature under stirring, it is placed in a clean 50mL centrifuge tube and stored in a 4℃ refrigerator. Characterization of AuNPs. The prepared AuNPs solution was characterized by UV-visible spectroscopy and transmission electron microscopy to determine its particle size, shape and uniformity.

[0073] Preparation of Au@PAH-Cu NPs: 500 μL of synthesized AuNPs were placed in a 1.5 mL centrifuge tube, centrifuged at 6000×g for 20 min, the supernatant was discarded, 1 mL of 1 mg / mL PAH and 300 μL of 100 μg / mL CuSO4 were added, the mixture was mixed up and down in a mixer for 30 min, 400 μL of 100 mg / mL AA was added, and the mixture was mixed for 30 min. Then, the mixture was centrifuged at 6000×g for 10 min, the supernatant was discarded, and 100 μL of water was added to resuspend the mixture. The result was the Au@PAH-Cu NP solution. Subsequently, an electron microscope image was taken of the prepared Au@PAH-Cu NP to prove the growth of colloidal gold, which can be used for signal amplification.

[0074] Preparation of AuNP-anti-Biotin: Antibody modification of synthesized AuNPs was performed as follows: 1 mL of previously prepared AuNPs was placed in a 1.5 mL centrifuge tube, 2 μg of biotin antibody was added, and the mixture was placed on a mixer for 1 h, 100 μL of 10 wt% BSA (prepared and used on the same day) was added, and the mixture was mixed up and down for 5 min, and then 100 μL of 10 wt% NaCl solution was added, and the mixture was mixed up and down for 2 h, and finally centrifuged at 9000 rpm for 20 min, and the supernatant was discarded. The resulting precipitate was AuNPs modified with biotin antibody (AuNP-anti-Biotin), and it was placed in a 4°C refrigerator for later use. ( Figure 2 )

[0075] 4. Preparation of LFAs

[0076] Soak the conjugate pad in boric acid at pH 8 for 30 minutes, take it out and place it in an oven at 37°C for 2 hours, and store it in a dry environment for later use; spray the prepared 1 mg / mL digoxin antibody on a 25 mm × 30 cm nitrocellulose membrane at a uniform speed (1 μL / cm) using a scriber, and the formed line is the detection line T line. Spray 1 mg / mL biotin secondary antibody on the appropriate position of the nitrocellulose membrane at the same speed to obtain the quality control line, i.e., C line; place the above nitrocellulose membrane in a 37°C oven for 3 hours, and store it at 4°C for later use; assemble the absorbent pad, conjugate pad, sample pad, and nitrocellulose membrane on a 60 mm × 30 cm PVC backing in a certain manner (each part has an overlapping thickness of 2 mm); use a programmable slitting machine to cut the above assembled LFAs, each finished product has a width of 3.5 mm, and store it at dry room temperature.

[0077] 5. PAH-Cu 2+ System preparation

[0078] PAH solutions, CuSO4 solutions and AA solutions of different concentrations were prepared and placed in a 4°C refrigerator for later use. Then, PAH solutions of different concentrations were mixed with CuSO4 solutions to prepare PAH-Cu2+ Solution

[0079] 6. Detection of genetically modified corn Ruifeng 12-5

[0080] First, the genomic DNA of transgenic Ruifeng 12-5 was extracted using Tiangen's plant genome extraction kit. Second, RPA amplification was performed using the extracted genomic DNA as a template. Then, 6 μL of the RPA amplification product was added to 100 μL of running buffer and mixed well. The LFAs pre-dropped with 2.5 μL of AuNP-anti-Biotin on the conjugate pad were inserted into the mixture. Under capillary force, the mixture migrated on the LFAs. Finally, the test strip was soaked in PAH-Cu 2+ Solution for 6 minutes, take out, soak in AA solution for 6 minutes, take out, and read the results. If there is genetically modified corn Ruifeng 12-5 component, two black bands visible to the naked eye are T and C lines. Use ImageJ software to process the T line. Each sample needs to be tested three times in parallel, and the average of the three times is calculated.

[0081] Example 2 Detection Condition Optimization Results

[0082] 1. RPA reaction and PAH copper deposition enhancement verification

[0083] Primers were verified by agarose gel electrophoresis. Figure 3 As shown in A, the amplified product fragment is 94bp. The positive sample has a clear band on the gel image here, while the negative control group (genomic DNA replaced with ddH2O) has no band. This proves that RPA has amplified the product and the primer can be used for subsequent experiments. 2+ solution, take it out and soak it in AA solution again. Figure 3 B), the color of the T line is significantly more distinct, indicating that PAH copper deposition enhances the signal.

[0084] 2. Optimization of LFAs reaction system

[0085] The relevant factors of the LFAs system affect its analytical performance. Therefore, these factors (RPA product, AuNP-anti-Biotin, amount of running buffer added, and type of running buffer) were optimized to obtain high analytical performance. The color depth of the T line was analyzed by image J software, and the quantitative indicator was the peak area. ( Figure 4 )

[0086] 3. PAH-Cu 2+ System Optimization

[0087] In order to make the PAH copper deposition effect better, PAH-Cu 2+ Cu in the system 2+ The source and PAH, CuSO4, AA solution concentration and immersion time were optimized to obtain high analytical performance. The color depth of the T line before and after enhancement was analyzed by Image J software, the quantitative indicator was the peak area, and the data was processed to make a difference map. ( Figure 5 )

[0088] Example 3 Sensitivity determination of the detection method

[0089] According to the above optimization system, the transgenic corn Ruifeng 12-5 was mixed with ordinary corn in a certain mass ratio to prepare a mixture containing different percentages (a: 100wt%, b: 50wt%, c: 25wt%, d: 10wt%, e: 1wt% and f: 0.1wt%, g: 0wt%) of transgenic corn Ruifeng 12-5, and the genome was extracted for detection. Figure 6 It can be seen that 1-100wt% samples can be detected, that is, TL has color. As the genetically modified component increases, the color of T also deepens. When the genetically modified component is 0.1wt%, the T line of the test strip is almost invisible. 2+ The quantitative index is the peak area, that is, after the PAH copper deposition signal is enhanced, the color of the T line is significantly deepened, and the detection limit can reach 0.1wt%.

[0090] Example 4 Specificity verification of the detection method

[0091] According to the above optimized system, the genetically modified corn Ruifeng 12-5 was specifically detected. Five genetically modified crops (a: 10% genetically modified corn Ruifeng 12-5 (the genetically modified corn Ruifeng 12-5 was mixed with ordinary corn at a mass ratio of 1:9), b: genetically modified corn MON810, c: genetically modified soybean MON87769, d: genetically modified corn GA21, e: genetically modified rice TT51-1) were selected for detection. The results are as follows Figure 7 As shown, except for 10% of the transgenic corn Ruifeng 12-5 showing two lines, the rest of the samples only showed line C. The quantitative index is the peak area. The results show that the sensor designed by the present invention has good specificity. ( Figure 7 )

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A rapid detection method for transgenic corn Ruifeng 12-5, characterized in that: The rapid detection method comprises the following steps: (1) Using the specific primers of transgenic maize Ruifeng 12-5, RPA amplification was performed using the maize genome as a template; (2) performing a colorimetric test strip test on the product obtained in step (1); (3)PAH-Cu 2+ Amplify the detection signal twice.

2. The rapid detection method according to claim 1, characterized in that: The specific primers include SEQ ID NO: 1 and SEQ ID NO: 2; Preferably, the specific primer is a modified specific primer; Further preferably, the modification is selected from any one of FAM, FITC, biotin, dig or TAMRA; Preferably, the specific primers include: RPA-F: 5'- / Dig / -SEQ ID NO: 1-3', and RPA-R: 5'- / Biotin / -SEQ ID NO: 2-3'.

3. The rapid detection method according to claim 1 or 2, characterized in that: The color developing test strip comprises a colloidal gold test strip.

4. The rapid detection method according to any one of claims 1 to 3, characterized in that: The step (3) PAH-Cu 2+ The secondary amplification detection signal includes soaking the test strip after step (2) in PAH-Cu 2+ solution, take it out and soak it in ascorbic acid solution again, take it out and read the result.

5. The rapid detection method according to any one of claims 1 to 4, characterized in that: The Cu 2+ Derived from CuSO4 solution or CuCl2 solution; Preferably, the Cu 2+ Derived from CuSO4 solution; Preferably, the PAH-Cu 2+ The solution is a mixture of PAH solution and CuSO4 solution.

6. The rapid detection method according to any one of claims 4-5, characterized in that: In PAH-Cu 2+ Soak and incubate in the solution for 5-10 min, and soak and incubate in the ascorbic acid solution for 5-10 min; Preferably, in PAH-Cu 2+ The samples were immersed in the solution for 6 min and then immersed in the ascorbic acid solution for 6 min.

7. The rapid detection method according to any one of claims 4 to 6, characterized in that: The PAH-Cu 2+ In solution, Cu 2+ The concentration is 1.25M and the PAH concentration is 10mg / mL; and / or, The ascorbic acid concentration is 100 mg / mL.

8. A detection kit for transgenic corn Ruifeng 12-5, characterized in that: The detection kit includes reagents, specific primers, color test strips, PAH solution, Cu 2+ solution and / or ascorbic acid solution; Preferably, the reagents and specific primers are the reagents and primers required for the RPA reaction; Further preferably, the specific primers include SEQ ID NO: 1 and SEQ ID NO: 2; Preferably, the specific primer is a modified specific primer; Further preferably, the modification is selected from any one of FAM, FITC, biotin, dig or TAMRA; Preferably, the specific primers include: RPA-F: 5'- / Dig / -SEQ ID NO: 1-3', and RPA-R: 5'- / Biotin / -SEQ ID NO: 2-3'; Preferably, the color developing test strip comprises a colloidal gold test strip; Preferably, the Cu 2+ The solution includes a CuSO4 solution or a CuCl2 solution; Further preferably, the Cu 2+ It is a CuSO4 solution.

9. A rapid detection method according to any one of claims 1 to 7, and / or use of the detection kit according to claim 8 in gene detection.

10. The use according to claim 9, characterized in that The application includes detecting the genetically modified corn Ruifeng 12-5 in genetically modified products.

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