Detection method of transgenic rice

Abstract

The invention discloses a transgenic rice detection method which comprises the following steps: respectively reacting gold nanoparticles with a DNA probe 1 or a DNA probe 2 in an aqueous solution to obtain the gold nanoparticle modified DNA probe 1 and the gold nanoparticle modified DNA probe 2; the DNA probe 1 modified by the gold nanoparticles and the DNA probe 2 modified by the gold nanoparticles are hybridized with sample DNA extracted from a rice sample, whether the rice sample is a transgenic rice sample or not is qualitatively judged through color change of the gold nanoparticles by adopting a dark-field microscope, or yellow and green gold nanoparticles are respectively counted, and whether the rice sample is a transgenic rice sample or not is judged. And quantitatively detecting the concentration of the target DNA molecules contained in the sample DNA. The invention provides a method for detecting transgenic rice by using a single-particle gold nanoparticle biosensing method, which realizes rapid quantitative detection of DNA molecules and qualitative detection of transgenic rice, and has the advantages of short time consumption and high detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a detection method for genetically modified rice. Background Art

[0003] The detection methods for genetically modified foods mainly include:

[0004] (1) Western blot detection technology

[0005] This detection uses polyacrylamide gel electrophoresis to separate the exogenous proteins in genetically modified foods, and makes them react and combine with chromogenic enzymes to obtain separated exogenous proteins, and then conducts separation tests on them. The main purpose of the test is to determine how much protein is contained in the genetically modified food and compare these proteins with the predetermined protein limits.

[0006] (2) Exogenous DNA detection technology

[0007] The main object of such detection is the characteristics of the gene sequences of the DNA fragments implanted into genetically modified crops. The transgenic DNA sequences are used for detection purposes and for detecting genetically modified foods as nucleic acids. The main transgenic detection technologies mainly detect promoters, genes, and terminators to facilitate the detection of genetically modified foods.

[0008] (3) Gene chip detection technology

[0009] The detection object of this technology is the genomic sequence of genetically modified crops. In actual application, the DNA of genetically modified crops needs to be arranged on a glass slide according to certain rules to obtain a microarray, and then specific software in the computer is used to calculate these gene sequences to obtain useful gene characteristics and information on biological impacts. The gene chip detection method has the advantages of reliability and accuracy.

[0010] (4) Biosensor technology

[0011] A biosensor is a device used for detection and analysis, usually completed by combining a biological recognition and a conversion element. The advantages of this type of detection technology are its simple operation and short time consumption, and it has extremely high selectivity. The disadvantages are its relatively poor stability and short service life, resulting in many limitations in actual applications.

[0012] However, the current detection technologies for genetically modified rice have problems such as complex operation, low sensitivity, and poor repeatability. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art, and provide a detection method for genetically modified rice with simple operation, good repeatability, and high sensitivity.

[0014] To solve the above technical problems, the technical solution proposed by the present invention is as follows:

[0015] A detection method for transgenic rice, comprising the following steps:

[0016] (1) React gold nanoparticles with DNA probe 1 or DNA probe 2 respectively in an aqueous solution to obtain DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles; the particle size of the gold nanoparticles is 35 - 45 nm (at this size, the nanoparticles are green, if the particle size is too small, the light is weak, and if the particle size is too large, the color will turn yellow); the nucleotide sequence of DNA probe 1 is as shown in SEQ ID NO:2, and the nucleotide sequence of DNA probe 2 is as shown in SEQ ID NO:3;

[0017] (2) Hybridize DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles with the sample DNA extracted from the rice sample, and use a dark - field microscope to qualitatively judge whether the rice sample is a transgenic rice sample through the color change of the gold nanoparticles, or quantitatively detect the concentration of the target DNA molecules contained in the sample DNA by counting the yellow and green gold nanoparticles respectively; the nucleotide sequence of the target DNA molecule is as shown in SEQ ID NO:1.

[0018] As a further improvement, the particle size of the gold nanoparticles is 40 nm.

[0019] As a further improvement, the gold nanoparticles are prepared by the following method: heat the chloroauric acid solution to boiling, and then add 1 wt% trisodium citrate solution and react until the color is stable.

[0020] As a further improvement, the reaction in step (1) includes: adding DNA probe 1 or DNA probe 2 to the aqueous solution of gold nanoparticles, waiting for 1 minute for the reaction, shaking well and then adding a citric acid buffer solution with pH 3.0, and reacting until the color does not change.

[0021] As a further improvement, the hybridization in step (2) includes: mixing DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles with the sample DNA extracted from the rice sample in water, adding a hybridization buffer solution, heating to 60 - 70 °C and maintaining for 5 - 10 min to make the DNA in a single - strand state, and then cooling to 20 - 35 °C for hybridization.

[0022] As a further improvement, the hybridization buffer solution is an aqueous solution of sodium chloride and sodium citrate, and the pH is adjusted to 7.0.

[0023] As a further improvement, after adding the hybridization buffer solution, heat to 65 °C and maintain for 5 min.

[0024] As a further improvement, quantitative detection is performed based on the fact that the ratio of the number of gold nanoparticle dimers to the total number of gold nanoparticles shows a linear relationship with the concentration of the target DNA molecule in the range of 0.001 μM to 1 μM.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention provides a single-particle gold nanoparticle biosensing method for detecting genetically modified rice. The principle of this method is based on DNA hybridization-induced formation of dimers or multimers between two nanoparticles, resulting in a color change from green to yellow under a dark-field microscope. Under a dark-field microscope, when gold nanoparticles of a specific size (about 40 nm) aggregate, the color changes from green to yellow, and the light intensity increases significantly. By separately counting the yellow and green gold nanoparticles, rapid quantitative detection of DNA molecules and qualitative detection of genetically modified rice are achieved. The time consumption is short (about 10 s), and the detection sensitivity can reach 0.001 μM.

[0027] This simple detection method greatly reduces the risk of sample contamination, avoids complex experimental operations, ensures good repeatability, and can automatically perform routine analysis. Compared with traditional fluorescent dyes and semiconductor nanocrystals, the nanoparticles used in the present invention are more biocompatible and optically more stable, without photobleaching and non-blinking. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of a single-molecule sandwich assay with plasmonic resonance nanoparticles;

[0030] Figure 2 It is a simulated color scattering image of two gold nanoparticles of different sizes and their dimers, where the upper left figure is an 18-nm monomer, the upper right figure is an 18-nm dimer, the lower left figure is a 40-nm monomer, and the lower right figure is a 40-nm dimer;

[0031] Figure 3 It is the ultraviolet-visible spectrum of gold nanoparticles and DNA probes modified with gold nanoparticles. From top to bottom, they are: 40-nm gold nanoparticles, DNA probe 1 modified with 40-nm gold nanoparticles, and DNA probe 2 modified with 40-nm gold nanoparticles;

[0032] Figure 4 These are dark-field images of gold nanoparticles and DNA probes modified with gold nanoparticles. From left to right, they are: 40-nm gold nanoparticles, DNA probe 1 modified with 40-nm gold nanoparticles, and DNA probe 2 modified with 40-nm gold nanoparticles.

[0033] Figure 5 These are representative images of real-time single-particle gold NP tracking during the hybridization of target DNA (at times 0 s, 2 s, 4 s, 5 s, and 7 s);

[0034] Figure 6 These are dark-field images of hybridization with different concentrations of target DNA (concentrations are 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM);

[0035] Figure 7 This is a linear relationship graph of the number of dimers accounting for the total number of nanoparticles under different concentrations of target DNA. Detailed implementation mode

[0036] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0038] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0039] The single-nanoparticle analysis of the present invention relies on a standard sandwich hybridization method to identify a single target molecule ( Figure 1 ). In the presence of a representative fragment of transgenic rice, namely the target DNA molecule (SEQ ID NO: 1), DNA probe 1 (SEQ ID NO: 2) and DNA probe 2 (SEQ ID NO: 3) are respectively complementary to half of the target DNA molecule sequence, resulting in the aggregation of two nanoparticles together, and the coupling effect of their plasmon resonance becomes significant, resulting in an obvious red shift of the plasmon resonance peak compared with that of a single gold nanoparticle. Therefore, the process of the gold nanoparticles changing from green to yellow can be photographed using a dark-field microscope and a color CCD camera.

[0040] Target DNA molecular sequence (SEQ ID NO: 1): GAATCC TGT TGC CGG TCT TGC GAT GATTAT DNA probe 1 sequence (SEQ ID NO: 2): ATAATCATC GCAAGA

[0041] DNA probe 2 sequence (SEQ ID NO: 3): CCG GCAACAGGATTC

[0042] Examples:

[0043] 1. Preparation of main solutions

[0044] (1) 1% chloroauric acid stock solution: Add ultrapure water to 1 g of chloroauric acid to obtain an aqueous solution, place it in a volumetric flask (100 mL), and make up the volume to the calibration line with ultrapure water.

[0045] (2) DNA modification buffer at pH 3.0: Add water to 0.2101 g of citric acid to obtain an aqueous solution, place it in a volumetric flask (100 ml), make up the volume to the calibration line with ultrapure water, and then add 1% hydrochloric acid solution and adjust the pH to 3.0 with a pH meter.

[0046] (3) 40 mM phosphate buffer: Add water to 0.12 g of sodium dihydrogen phosphate and 0.142 g of disodium hydrogen phosphate that have been weighed to obtain an aqueous solution, place it in a volumetric flask (50 mL), make up the volume to the calibration line with ultrapure water, and then add sodium hydroxide and adjust the pH to 7.4 with a pH meter.

[0047] (4) DNA hybridization buffer: Add water to 0.4383 g of sodium chloride and 0.2206 g of sodium citrate that have been weighed to obtain an aqueous solution, place it in a volumetric flask (50 mL), make up the volume to the calibration line with ultrapure water, and then add 1% hydrochloric acid solution and adjust the pH to 7.0 with a pH meter.

[0048] 2. Preparation of gold nanoparticles

[0049] (1) Preparation of containers

[0050] Clean the blue-capped reagent bottle, three-necked flask, cuvette and other required glassware with aqua regia in a fume hood, then rinse with ultrapure water 10 - 15 times, and then put them into a blast drying oven and dry for use, set the temperature at 105 °C.

[0051] (2) Preparation of gold nanoparticles

[0052] At room temperature, 100 mL of ultrapure water and 1 mL of chloroauric acid were placed in a three-necked flask, and then it was placed in a constant-temperature heating oil bath magnetic stirring pot. The condensation reflux device was turned on and heated to boiling. After boiling, 650 μL of trisodium citrate (1%) was quickly added, and after waiting for 15 minutes, it was observed that the solution gradually changed from colorless to purplish red, and the color was stable and would not change anymore.

[0053] (3) Detection by ultraviolet-visible spectrophotometer

[0054] The prepared gold nanoparticle solution was scanned with an ultraviolet-visible spectrophotometer, and the wavelength around 535 nm was the wavelength of the maximum absorption peak. The prepared gold nanoparticle solution was poured into a reagent bottle and stored refrigerated at 4 °C in the refrigerator.

[0055] Gold nanoparticles with an average particle size of about 40 nm were prepared by the above formula and method (there were slight differences in different batches, but within the range of 35 - 45 nm).

[0056] 3. Preparation of glass slides

[0057] Prepare aqua regia (30% hydrogen peroxide and 70% concentrated sulfuric acid by volume fraction). Place the glass slides and cover glasses in it for half an hour, wash them with ultrapure water, then ultrasonically clean them twice (10 minutes for each time), and finally dry them in a forced-air drying oven at 115 °C. After drying, store them in a petri dish to avoid contamination.

[0058] 4. Modification of DNA probes

[0059] Centrifuge the 1 mL of gold nanomaterial prepared in step 2 (6000 rpm, 10 minutes). After the treatment, the supernatant needs to be removed, 100 μL of ultrapure water is added, and then 3 μL of DNA probe 1 or probe 2 (100 μM) diluted 1000 times is added. Wait for one minute for the reaction, shake well, and then add 200 μL of citric acid buffer solution with pH 3.0. Wait for 5 minutes - 2 hours and observe that the color does not change. Wash it three times with phosphate buffer solution (40 mM), and centrifuge each time (6000 rpm, 10 minutes) to remove the supernatant. Store the modified DNA material refrigerated at 4 °C in the refrigerator. The modified DNA probe 1 and the modified DNA probe 2 are obtained.

[0060] 5. Hybridization of target DNA

[0061] Mix 2 μL of the modified DNA probe 1 and 2 μL of the modified DNA probe 2 obtained in step 4 with 6 μL of the target DNA (100 μM) diluted 4000 times in 20 μL of deionized water. Then, add 30 μL of SSC hybridization buffer solution diluted eight times and heat at 65 °C for 5 minutes, and hybridize at room temperature for 1 hour.

[0062] 6. Extraction of Sample DNA

[0063] Take 4 - 5 g of the sample's stems and leaves, cut them into pieces, add liquid nitrogen at an extremely fast speed, then perform grinding treatment, and then put them into a centrifuge tube (50 ml). Then add about 25 mL of CTAB extraction solution that has been extracted and heated to 65°C, add an appropriate amount of β - mercaptoethanol to make the final concentration 1%, gently shake the centrifuge tube, wait until all substances are mixed evenly, and then soak it in water at 65°C for 60 - 90 minutes. During this process, gently invert the centrifuge tube every 5 minutes to prevent the substances inside from aggregating and caking. When the water temperature drops to room temperature, add an equal volume of chloroform (25 mL):isoamyl alcohol (24:1), shake it vigorously (60 r / min) until the organic phase turns yellow. Then perform centrifugation (4°C, 4000 r / min) for half an hour. Take another centrifuge tube (50 mL), add the supernatant, and then add the same volume of isopropanol at - 20°C to precipitate DNA until small cotton - like clusters float on the liquid surface. Use a glass rod to pick out the DNA into a 1.5 mL Eppendorf tube, add ethanol with a concentration of 70% and perform washing treatment (2 or 3 times). Soak it in 1 mL of TE (pH 8.0) at 65°C for 15 minutes to dissolve the DNA. After the temperature drops to room temperature, add 10 mg / mL RNaseA and soak it at 37°C for one hour to remove RNA. After purification, dissolve it in an appropriate amount (about 500 - 700 μL) of TE for standby.

[0064] 7. Hybridization of Sample DNA

[0065] Mix 2 μL of the modified DNA probe 1 obtained in step 4, 2 μL of the modified DNA probe 2, and 6 μL of the sample DNA obtained in step 6 in 20 μL of deionized water. Then, add 30 μL of SSC hybridization buffer diluted eight - fold, heat it at 65°C for 5 minutes, and hybridize at room temperature for 1 hour.

[0066] 8. Measurement with Dark - Field Microscope

[0067] Drop the DNA gold nanoparticle solution (obtained in steps 5 and 7) heated at 65°C for 5 minutes on a glass slide, quickly cover it with a coverslip, use DP73 CCD, immediately observe the process of the color change of gold particles from green to yellow during the hybridization process and record it on video, take a photo every 10 minutes, and end the observation and analysis after 1 hour of reaction.

[0068] 9. Results and Analysis

[0069] (1) Figure 2Shows simulated color scattering images of two gold nanoparticles of different sizes and their dimers. Figure 2 Based on the scattering spectra of individual gold nanoparticles, the dark-field scattering images of monomers (left figures) and dimers (right figures) of 18 nm (upper figures) and 40 nm (lower figures) gold NPs are calculated (i.e., the upper left figure is the 18 nm monomer, the upper right figure is the 18 nm dimer, the lower left figure is the 40 nm monomer, and the lower right figure is the 40 nm dimer). The distance between the two particles in the dimer is 10 nm. It can be seen that when the size of the gold nanoparticles is 18 nm, both the single nanoparticles and the dimers appear green, with only minor differences. When the size of the gold nanoparticles is 40 nm, there is an obvious green-to-yellow transition when forming dimers. Therefore, DNA hybridization analysis can be performed by simply counting the number of particles of different colors that appear in the CCD image.

[0070] (2) Figure 3 Shows the UV-Vis spectra of gold nanoparticles and DNA probes modified with gold nanoparticles. From top to bottom, they are: 40 nm gold nanoparticles, DNA probe 1 modified with 40 nm gold nanoparticles, and DNA probe 2 modified with 40 nm gold nanoparticles. Figure 4 Shows the dark-field images of gold nanoparticles and DNA probes modified with gold nanoparticles. From left to right, they are: 40 nm gold nanoparticles, DNA probe 1 modified with 40 nm gold nanoparticles, and DNA probe 2 modified with 40 nm gold nanoparticles. In the dark images, the gold nanoparticles exhibit almost the same scattered light color, indicating that the colloidal nanoparticles are well monodispersed and have a good spherical shape. The maximum absorption peaks of the 40 nm gold nanoparticles, DNA probe 1 modified with 40 nm gold nanoparticles, and DNA probe 2 modified with 40 nm gold nanoparticles are all at a wavelength of 535. It can be seen from the dark-field images that the gold nanoparticles appear green and are circular in shape.

[0071] (3) Real-time observation of DNA hybridization-induced gold nanoparticle aggregation:

[0072] To demonstrate the utility of single-particle analysis of plasmonic nanoparticles, a real-time NP tracking experiment was conducted to directly detect the color changes induced by hybridization. Figure 5It is a representative image of real-time single-particle gold NP tracking during the hybridization of target DNA. At the beginning, the probe gold nanoparticles without target DNA modification perform Brownian motion and gradually diffuse during the motion. Their collisions are determined by thermal motion, and the adhesion time between them can be ignored. However, after the complementary target DNA molecules are added to the hybridization solution, since the two probe gold particles anneal with the complementary target DNA molecules to form stable dimers. From the typical time-series images shown in the figure, it can be seen that at 0 seconds, the two gold nanoparticles gradually approach, separate at 4 seconds, collide again at 5 seconds, and bind at 7 seconds, resulting in an observable obvious green-to-yellow transition in intensity (more than 2 times).

[0073] (4) Hybridization effects of different concentrations of target DNA:

[0074] To quantify the sensitivity of this method, 6 μL of target DNA with concentrations ranging from 0.001 to 10 μM were respectively taken, and then 2 μL of each of the modified DNA probe 1 and DNA probe 2 were added to a centrifuge tube containing 30 μL of SSC hybridization buffer, heated in a constant temperature water bath at 65 °C for 5 min, then hybridized at room temperature for 1 h, and then 5 μL was evenly spread on a glass slide.

[0075] Figure 6 It shows the dark-field images of hybridization of different concentrations of target DNA (concentrations are 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM respectively), and a graph of the number of yellow spots counted as a function of the target DNA concentration. Figure 7 It is the linear relationship between the number of dimers and the total number of nanoparticles at different concentrations of target DNA.

[0076] It can be seen that the number of dimers decreases continuously with the decrease of concentration at different concentrations, and the yellow particles are significantly more than the green particles at high concentrations. There is a good linear relationship from 0.001 μM to 1 μM. When the concentration of target DNA further increases, the count reaches a plateau. This is because, at high concentrations of the target, DNA hybridization occurs not only between two single gold nanoparticles. In other words, oligomers can also form in the presence of a large number of targets, and as the concentration continuously decreases, when the concentration reaches 0.001 μM, the proportion of yellow particles in the image is already less than 20%.

[0077] Through the above experiments, the following conclusions can be drawn:

[0078] 1) The optimal size of the gold nanoparticles used in this experiment is about 40 nm in size and above 532 in wavelength. Because when the size of the gold nanoparticles is 18 nm, both single nanoparticles and dimers appear green, and there is no obvious green-to-yellow transition when forming dimers.

[0079] 2) The color change during hybridization is relatively fast. Generally, it can be stabilized as a yellow dimer in about 10 seconds.

[0080] 3) When the target DNA concentration is between 0.001 μM and 1 μM, there is a linear relationship, and its correlation coefficient R 2 is 0.9805, and the trend line is Y = 0.1471X + 0.0507.

[0081] (5) Analysis of actual samples: The samples used in the experiment were rice near the Agricultural University. After extracting DNA in step 6, DNA probe 1 and DNA probe 2 were added for hybridization analysis. The hybridization situation was observed under a dark-field microscope. During the waiting process, no dimers or multimers were formed between the gold nanoparticles, and the color did not change from green to yellow and there was no obvious change in light intensity. Therefore, it can be proved that the purchased samples are not genetically modified crops.

[0082] In summary, in the present invention, gold nanoparticles are covalently modified with two oligonucleotide sequences through gold-thiol chemistry. Under favorable annealing conditions, the target DNA molecules in the sample solution complementary to the two probe sequences can absorb gold nanoparticles together in a sandwich form, generating dimers or oligomers, and the color changes from green to yellow under a dark-field microscope, realizing the qualitative detection of genetically modified foods and the quantitative detection of DNA in genetically modified foods, and successfully realizing the detection of DNA by the sandwich method at the single-molecule level.

[0083] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Therefore, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A detection method for genetically modified rice, characterized in that, It includes the following steps: (1) React gold nanoparticles with DNA probe 1 or DNA probe 2 respectively in an aqueous solution to obtain DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles; the particle size of the gold nanoparticles is 35 - 45 nm; the nucleotide sequence of the DNA probe 1 is as shown in SEQ ID NO:2, and the nucleotide sequence of the DNA probe 2 is as shown in SEQ ID NO:3; (2) Hybridize DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles with the sample DNA extracted from the rice sample, and qualitatively determine whether the rice sample is a genetically modified rice sample by the color change of the gold nanoparticles using a dark - field microscope, or quantitatively detect the concentration of the target DNA molecules contained in the sample DNA by counting the yellow and green gold nanoparticles respectively; the nucleotide sequence of the target DNA molecule is as shown in SEQ ID NO:

1.

2. The detection method of the genetically modified rice according to claim 1, characterized in that The particle size of the gold nanoparticles is 40 nm.

3. The detection method of the genetically modified rice according to claim 2, wherein The gold nanoparticles are prepared by the following method: heat the chloroauric acid solution to boiling, and then add 1 wt% trisodium citrate solution and react until the color is stable.

4. The detection method of the genetically modified rice according to any one of claims 1 to 3, characterized in that, The reaction in step (1) includes: add DNA probe 1 or DNA probe 2 to the aqueous solution of gold nanoparticles, wait for 1 minute for the reaction, shake well and then add a citric acid buffer solution with pH 3.0, and react until the color does not change.

5. The detection method of the genetically modified rice according to any one of claims 1 to 3, characterized in that, The hybridization in step (2) includes: mix DNA probe 1 modified with gold nanoparticles and DNA probe 2 modified with gold nanoparticles with the sample DNA extracted from the rice sample in water, add a hybridization buffer solution, heat to 60 - 70 °C and maintain for 5 - 10 min to make the DNA in a single - strand state, and then cool to 20 - 35 °C for hybridization.

6. The detection method of the transgenic rice according to claim 5, characterized in that, The hybridization buffer solution is an aqueous solution of sodium chloride and sodium citrate, and the pH is adjusted to 7.

0.

7. The detection method of the transgenic rice according to claim 5, wherein After adding the hybridization buffer solution, heat to 65 °C and maintain for 5 min.

8. The detection method of the transgenic rice according to any one of claims 1 to 3, characterized in that Quantitative detection is carried out according to the ratio of the number of gold nanoparticle dimers to the total number of gold nanoparticles, which has a linear relationship with the concentration of the target DNA molecule in the range of 0.001 μM to 1 μM.

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