An interferometric reflectance spectroscopy RNA detection chip

Abstract

The present invention belongs to the technical field of nucleic acid detection, and proposes an interference type reflective spectrum RNA detection sheet, including a glass slide, on which a first chromium layer, a first gold layer, a thiol-modified DNA layer, a DNA gel layer, a first chromium layer, and a second gold layer are sequentially arranged. The preparation method thereof comprises: washing the glass slide to obtain a decontaminated glass slide; plating a first gold layer on the surface of the decontaminated glass slide to obtain a glass slide I; washing the glass slide I, coating a layer of thiol-modified DNA oligonucleotide chain solution VI on the first gold layer after washing, and then soaking it in a DNA gel solution to obtain a glass slide II after washing; plating a second gold layer on the surface of the glass slide II again to obtain an interference type reflective spectrum RNA detection sheet, and the sequence of the DNA oligonucleotide chain VI in the DNA oligonucleotide chain solution VI is shown in SEQ ID NO: 10. Through the above technical scheme, the problems of complex operation, high cost, and long detection cycle of miRNA detection means in the prior art are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid detection and relates to an interference type reflection spectrum RNA detection sheet. Background Art

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding small single-stranded RNA molecules in cells, generally composed of 19-23 bases. Their biogenesis is a complex process. The alternating expression of miRNAs is associated with many diseases. A large number of observations have been made in linking abnormal expression levels of miRNAs to the occurrence and development of human diseases, genetic diseases, and altered immune system function. miRNAs can act as both oncogenes and tumor suppressors, emphasizing their importance in human cancer.

[0003] The analysis and detection of miRNA is a key technology for its biological function research, disease diagnosis and related gene drug development, but its detection is very difficult due to its short sequence, homology, low content and low abundance in cells. Traditional miRNA analysis and detection technologies mainly include: Northern hybridization analysis, real-time fluorescent polymerase chain reaction (RT-PCR), cloning technology and gene chip technology. These technologies have their own advantages and disadvantages in the application process, and the appropriate detection method can be selected according to the purpose of the experiment.

[0004] RT-PCR requires a small amount of sample and has high sensitivity, making it the most important method for detecting miRNA. However, due to the short fragments of miRNA, it is difficult to directly transcribe it into cDNA and use it as a template for PCR amplification, and it is also expensive.

[0005] Northern blotting technology is the main means of detecting miRNA expression. All miRNAs obtained through cloning and bioinformatics analysis need to be verified and confirmed by Northern blotting, which is the gold standard for miRNA detection. This method uses labeled DNA probes to hybridize with miRNA on nitrocellulose membranes and detects target bands through development. Northern blotting analysis often uses isotopes, fluorescence or nanogold to label DNA. This process is skillful and can detect the molecular size of the detected miRNA, highlight its abundance, and has simple instrument requirements. Therefore, it has been used for miRNA detection. However, this method has disadvantages such as time-consuming and labor-intensive operation, large sample consumption, and low accuracy of high-throughput detection.

[0006] Gene chip, also known as DNA chip, DNA microarray, oligonucleotide array, refers to the use of in situ synthesis (or microprinting) to solidify a large number of known nucleotides or cDNA on the surface of the support to form a DNA microarray, which is then hybridized with the amplified and labeled sample to detect the hybridization signal to achieve the detection or medical diagnosis of the sample. It is a faster, more extensive and more promising method to study miRNA expression. In chip analysis, reasonable probe design and effective miRNA labeling are particularly important and determine the performance of the chip. To fix thousands of miRNA probes on the chip at the same time, the first problem to be solved is how to ensure that all probes can hybridize at similar temperatures. Due to the limitation of the length of miRNA itself, the hybridization temperature cannot be unified by simply adjusting the length of the probe.

[0007] Therefore, it is of great significance to design a detection method with simple operation, low cost and short detection cycle. Summary of the invention

[0008] The present invention provides an interference reflection spectrum RNA detection sheet, which solves the problems of complex operation, high cost and long detection cycle of miRNA detection means in the prior art.

[0009] The technical solution of the present invention is achieved in this way:

[0010] An interference reflection spectrum RNA detection sheet comprises a glass slide, on which a first chromium layer, a first gold layer, a thiol-modified DNA layer, a DNA gel layer, a second chromium layer, and a second gold layer are sequentially arranged;

[0011] The sequence of the DNA oligonucleotide chain VI used in the thiol-modified DNA layer is shown in SEQ ID NO: 10;

[0012] The DNA gel is obtained by drying a DNA gel solution, and the DNA gel solution is a DNA gel-1 solution, a DNA gel-2 solution or a DNA gel-3 solution;

[0013] The preparation method of the DNA gel solution comprises the following steps:

[0014] A. Prepare DNA oligonucleotide chains Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, Ⅴ;

[0015] The sequence of the DNA oligonucleotide chain I is shown in SEQ ID NO: 1;

[0016] The sequence of the DNA oligonucleotide chain II is shown in SEQ ID NO: 2;

[0017] The sequence of the DNA oligonucleotide chain III-1 is shown in SEQ ID NO: 3;

[0018] The sequence of the DNA oligonucleotide chain III-2 is shown in SEQ ID NO: 4;

[0019] The sequence of the DNA oligonucleotide chain III-3 is shown in SEQ ID NO: 5;

[0020] The sequence of the DNA oligonucleotide chain IV-1 is shown in SEQ ID NO: 6;

[0021] The sequence of the DNA oligonucleotide chain IV-2 is shown in SEQ ID NO: 7;

[0022] The sequence of the DNA oligonucleotide strand IV-3 is shown in SEQ ID NO: 8;

[0023] The sequence of the DNA oligonucleotide chain V is shown in SEQ ID NO: 9;

[0024] B. Dissolve the DNA oligonucleotide chains Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, and Ⅴ in ultrapure water to obtain DNA oligonucleotide chain solutions Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, and Ⅴ, mix the DNA oligonucleotide chain solution Ⅰ and the DNA oligonucleotide chain solution Ⅴ, and react at 53.5-56.1°C for 1-2h. After the reaction, add the DNA oligonucleotide chain solution Ⅱ, and react at 67.5-70°C for 3.5-4h to obtain a DNA gel backbone solution;

[0025] C. When preparing DNA gel-1, the DNA oligonucleotide chain solution III-1 and the DNA oligonucleotide chain solution IV-1 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branch chain-1 solution; the DNA gel main chain solution obtained in step B and the DNA gel branch chain-1 solution obtained in step C are mixed to obtain a mixed solution, and the mixed solution is annealed. After the annealing is completed, the mixed solution is rotated and mixed at a speed of 60-75 rpm for 24 hours to obtain a DNA gel-1 solution;

[0026] When preparing DNA gel-2, the DNA oligonucleotide chain solution III-2 and the DNA oligonucleotide chain solution IV-2 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branch chain-2 solution; the DNA gel main chain solution obtained in step B and the DNA gel branch chain-2 solution obtained in step C are mixed to obtain a mixed solution, and the mixed solution is annealed. After the annealing is completed, the mixed solution is rotated and mixed at a speed of 60-75 rpm for 24 hours to obtain a DNA gel-2 solution;

[0027] When preparing DNA gel-3, the DNA oligonucleotide chain solution III-3 and the DNA oligonucleotide chain solution IV-3 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branch chain-3 solution; the DNA gel main chain solution obtained in step B and the DNA gel branch chain-3 solution obtained in step C are mixed to obtain a mixed solution, and the mixed solution is annealed. After annealing, the mixed solution is rotated and mixed at a speed of 60-75 rpm for 24 hours to obtain a DNA gel-3 solution.

[0028] Furthermore, the concentrations of the DNA oligonucleotide chain solutions I, II, III-1, III-2, III-3, IV-1, IV-2, IV-3, and V are all 100 μmol / L.

[0029] Furthermore, the mass ratio of the DNA oligonucleotide chain solution I, II, III-1, III-2, III-3, IV-1, IV-2, IV-3, and V is 100:100:(40-90):(40-90):(40-90):(40-90):(40-90):(40-90):1;

[0030] The mass ratio of the DNA oligonucleotide chain solution III-1 to the DNA oligonucleotide chain solution IV-1 is 1:1;

[0031] The mass ratio of the DNA oligonucleotide chain solution III-2 to the DNA oligonucleotide chain solution IV-2 is 1:1;

[0032] The mass ratio of the DNA oligonucleotide chain solution III-3 to the DNA oligonucleotide chain solution IV-3 is 1:1.

[0033] Furthermore, the annealing in step D is to react the mixed solution at 55-57° C. for 5 minutes, then react at 50-55° C. for 3 minutes, and finally cool it to 23-27° C. at a cooling rate of 1-2° C. / min.

[0034] Furthermore, the thickness of the first chromium layer and the second chromium layer are both 2 nm, and the thickness of the first gold layer and the second gold layer are both 15 nm.

[0035] A method for preparing an interferometric reflectance spectroscopy RNA detection sheet comprises the following steps:

[0036] S1, cleaning the glass slide to obtain a decontaminated glass slide, plating a first chromium layer on the surface of the decontaminated glass slide, and then plating a first gold layer on the surface of the first chromium layer to obtain glass slide I;

[0037] S2, washing the glass slide I to obtain a decontaminated glass slide I, dissolving the DNA oligonucleotide chain VI in ultrapure water to obtain a DNA oligonucleotide chain solution VI, coating the DNA oligonucleotide chain solution VI on the surface of the first gold layer of the decontaminated glass slide I, drying, and then washing the decontaminated glass slide I in deionized water, drying after washing, and then soaking the washed and dried decontaminated glass slide I in a DNA gel solution, and then taking the decontaminated glass slide I out of the DNA gel solution and drying it, and then immersing it in deionized water for washing and drying to obtain a glass slide II;

[0038] S3. Plate a second chromium layer on the surface of glass slide II, and then plate a second gold layer on the surface of the second chromium layer to obtain an interference reflection spectrum RNA detection sheet.

[0039] Furthermore, the concentration of the DNA oligonucleotide chain solution VI is 100 μmol / L.

[0040] Furthermore, the method for cleaning the glass slide or glass slide I includes: taking the glass slide or glass slide I, placing it in deionized water for ultrasonic cleaning, drying, then placing it in anhydrous ethanol for ultrasonic cleaning, drying, and placing it in deionized water again for ultrasonic cleaning and drying to obtain a decontaminated glass slide or decontaminated glass slide I.

[0041] A method for using an interferometric reflectance spectroscopy RNA detection sheet comprises the following steps:

[0042] S1. Soak the RNA detection piece in deionized water and store it at 20°C for later use;

[0043] S2. Add the miRNA solution to be tested into the deionized water where the RNA detection piece is located, soak it for 4 to 6 hours, then take it out and soak it in new deionized water again, detect the reflection spectrum of the mirror surface, and record the data;

[0044] S3. Calculate the content of miRNA in the miRNA solution to be tested according to formulas (I) to (II);

[0045] d=(1+4x / m) 2 d1+17(Ⅰ)

[0046] λa=2ndcosθ(Ⅱ)

[0047] in:

[0048] d represents the distance from the upper surface of the first gold layer to the upper surface of the second gold layer, in nm;

[0049] x represents the amount of miRNA added to be tested, in nmol;

[0050] m represents the amount of DNA oligonucleotide chain III-1, DNA oligonucleotide chain III-2 or DNA oligonucleotide chain III-3 added to the DNA gel solution, in nmol;

[0051] d1 represents the particle size of the DNA gel prepared from the DNA oligonucleotide chain III-1, DNA oligonucleotide chain III-2 or DNA oligonucleotide chain III-3, in nm;

[0052] λ represents the maximum peak value of the ath characteristic peak, in nm;

[0053] a represents the peak order, which is a positive integer;

[0054] n represents the refractive index of the medium;

[0055] θ represents the incident angle.

[0056] The working principle and beneficial effects of the present invention are:

[0057] 1. In the present invention, a gold layer is used as a reflective layer, and a DNA gel synthesized from a specific DNA oligonucleotide chain is used as a thickness layer. The thin film interference principle is used to analyze miRNA. The operation is simple. It is only necessary to add the miRNA solution to the RNA detection sheet to detect the signal for qualitative detection. The wavelength can also be specifically analyzed by instruments such as a reflection spectrometer to calculate the thickness change between the two reflective layers, and the miRNA content in the solution can be calculated by a formula. In addition, the RNA detection sheet in the present invention has a short detection cycle, and the miRNA content in the solution can be calculated in only 5 hours, thereby reducing the RNA detection cost and solving the problems of complex operation, high cost and long detection cycle in the prior art.

[0058] 2. In the present invention, the thin film interference principle is used to analyze miRNA. Due to the volatility of light, the thin film interference effect will shield light of a certain wavelength due to the thickness of the RNA detection sheet, so that the RNA detection sheet itself displays a certain color. The change in the thickness of the RNA detection sheet will change the color displayed by the detection sheet, thereby detecting the output signal. At the same time, the signal is obvious and can be observed by colorimetry with the naked eye.

[0059] 3. In the present invention, the short DNA chain simulates the polymerization process of organic polymers and produces nanoscale DNA gel (200-600nm) by combining the main chain and the side chain. The present invention coats a layer of thiol-modified DNA oligonucleotide chain solution VI on the gold layer, so that the DNA gel is bound to the gold layer. The DNA gel undergoes a chain displacement reaction with the miRNA-21 in the miRNA-21 solution to be tested, resulting in a swelling effect, changing the distance between the two gold layers, thereby changing the color of the interference light, and calculating the content of miRNA in the solution through a formula.

[0060] 4. The DNA oligonucleotide chain III-1 of the present invention has a recognition interval consisting of a fragment completely complementary to miRNA-21, and is partially complementary to the DNA oligonucleotide chain IV-1, so the binding of the DNA oligonucleotide chain III-1 to the IV-1 chain can be competitively broken by miRNA-21; the recognition interval of the DNA oligonucleotide chain III-1 includes but is not limited to the miRNA-21 sequence. Therefore, the DNA oligonucleotide chain III-1 and the DNA oligonucleotide chain IV-1 can be sequenced according to the detection needs, such as the DNA oligonucleotide chain III-2 has a recognition interval consisting of a fragment completely complementary to miRNA-373, and is partially complementary to the DNA oligonucleotide chain IV-2, so the binding of the DNA oligonucleotide chain III-2 to the IV-2 chain can be competitively broken by miRNA-373; the DNA oligonucleotide chain III-3 has a recognition interval consisting of a fragment completely complementary to miRNA-155, and is partially complementary to the DNA oligonucleotide chain IV-3, so the binding of the DNA oligonucleotide chain III-3 to the IV-3 chain can be competitively broken by miRNA-155. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0062] Figure 1 This is a schematic diagram of the structure of the RNA detection chip;

[0063] In the picture: the upper and lower layers are the first gold layer and the second gold layer respectively, and the middle is DNA gel.

[0064] Figure 2 Transmission electron micrograph of a DNA gel. DETAILED DESCRIPTION

[0065] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] The preparation method of the DNA oligonucleotide chain solutions I, II, III-1, III-2, III-3, IV-1, IV-2, IV-3, V, and VI in the following embodiments comprises the following steps:

[0067] The purchased dry powdered DNA oligonucleotide chains Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, Ⅴ, Ⅵ were centrifuged at 12000 rpm for 90 s, and then ultrapure water was added to prepare 100 μmol / L DNA oligonucleotide chain solutions Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, Ⅴ, Ⅵ, respectively, and stored in a 4°C refrigerator for future use; the particle size of the DNA gel was observed using a transmission electron microscope.

[0068] The sequences of the DNA oligonucleotide chains used in the examples are shown in Table 1. The miRNAs to be tested are miRNA-21, miRNA-373, and miRNA-155, and their sequences are shown in Table 1.

[0069] In the embodiment, formulas (I) and (II) are as follows:

[0070] d=(1+4x / m) 2 d1+17 (Ⅰ)

[0071] λa=2ndcosθ (Ⅱ)

[0072] in:

[0073] d represents the distance from the upper surface of the first gold layer to the upper surface of the second gold layer, in nm;

[0074] x represents the amount of miRNA added to be tested, in nmol;

[0075] m represents the amount of DNA oligonucleotide chain III-1, DNA oligonucleotide chain III-2 or DNA oligonucleotide chain III-3 added to the DNA gel solution, in nmol;

[0076] d1 represents the particle size of the DNA gel prepared from DNA oligonucleotide chain III-1, DNA oligonucleotide chain III-2 or DNA oligonucleotide chain III-3, in nm;

[0077] λ represents the maximum peak value of the ath characteristic peak, in nm;

[0078] a represents the peak order, which is a positive integer;

[0079] n represents the refractive index of the medium;

[0080] θ represents the angle of incidence;

[0081] All the light rays detected by the spectrum are incident perpendicular to the mirror surface, so cosθ=1, and no refraction occurs, n is 1, and it has nothing to do with the refractive index of the medium. Therefore, λ is proportional to the distance d between the two reflectors. When a is a certain peak order, the increase of d will lead to the increase of λ.

[0082] Table 1 Sequences of DNA and miRNA oligonucleotide chains used in the experiment

[0083]

[0084] (I) Detection of miRNA-21

[0085] Example 1

[0086] S1. Take a glass slide, put it in a beaker with deionized water and ultrasonically clean it for 1 minute, then put it in an electric blast drying oven to dry it, then put the dried glass slide in a beaker with anhydrous ethanol and ultrasonically clean it for 1 minute, put it in an electric blast drying oven to dry it after cleaning, take out the glass slide after drying, put it in a new beaker with deionized water and ultrasonically clean it for 1 minute, and get a decontaminated glass slide after drying;

[0087] S2. Using a magnetron sputtering coating device, a 2 nm first chromium layer is plated on the surface of the decontaminated glass slide, and then a 15 nm first gold layer is plated on the surface of the first chromium layer to obtain a glass slide I;

[0088] S3. Wash the glass slide I according to the method in step S1 to obtain a decontaminated glass slide I, take 100 μL of the DNA oligonucleotide chain solution VI and apply it on the first gold layer surface of the decontaminated glass slide I, put the decontaminated glass slide I coated with the DNA oligonucleotide chain solution VI into an electric hot air drying oven for drying, take out the decontaminated glass slide I and wash it in deionized water for 5 hours, wash and remove the soluble impurities on the surface of the medium layer and then dry it, then put the dried decontaminated glass slide I into a DNA gel solution and soak it for 12 hours, then take out the decontaminated glass slide I from the DNA gel solution and dry it, then immerse it in deionized water for washing and drying to obtain a glass slide II;

[0089] S4. Using magnetron sputtering coating equipment, a 2 nm second chromium layer is plated on the surface of the glass slide II, and then a 15 nm second gold layer is plated on the surface of the second chromium layer to obtain an interference reflection spectrum RNA detection sheet;

[0090] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0091] S6. After adding the miRNA-21 solution containing 3 μmol miRNA-21 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in a static state for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0092] S7. The content of miRNA-21 in the miRNA-21 solution to be tested was calculated to be 3.12 μmol according to formulas (I) to (II). The results are shown in Table 2;

[0093] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0094] A. Prepare oligonucleotide chain solution I containing 100 μmol of DNA oligonucleotide chain I, DNA oligonucleotide chain solution II containing 100 μmol of DNA oligonucleotide chain II, DNA oligonucleotide chain solution III containing 40 μmol of DNA oligonucleotide chain III, DNA oligonucleotide chain solution IV containing 40 μmol of DNA oligonucleotide chain IV, and DNA oligonucleotide chain solution V containing 1 μmol of DNA oligonucleotide chain V;

[0095] B. Add the DNA oligonucleotide chain solution I and DNA oligonucleotide chain solution V in step A to centrifuge tube a and mix them. Place centrifuge tube a in a PCR instrument, set the temperature to 56.1°C, and react for 1 hour. After the reaction is completed, add DNA oligonucleotide chain solution II to centrifuge tube a, set the temperature to 68.8°C, and react for 4 hours to obtain a DNA gel backbone solution.

[0096] C. Add DNA oligonucleotide chain solution III-1 and DNA oligonucleotide chain solution IV-1 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 52.7°C, and the reaction time to 1 hour to obtain DNA gel branched chain-1 solution;

[0097] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-1 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place the mixed solution into a PCR instrument for annealing. The annealing procedure is to react at 55°C for 5 minutes, then at 50°C for 3 minutes, and finally cool from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 hours to obtain a DNA gel solution.

[0098] Table 2

[0099]

[0100] Example 2

[0101] S1. Take a glass slide, put it in a beaker with deionized water and ultrasonically clean it for 1 minute, then put it in an electric blast drying oven to dry it, then put the dried glass slide in a beaker with anhydrous ethanol and ultrasonically clean it for 1 minute, put it in an electric blast drying oven to dry it after cleaning, take out the glass slide after drying, put it in a new beaker with deionized water and ultrasonically clean it for 1 minute, and get a decontaminated glass slide after drying;

[0102] S2. Using a magnetron sputtering coating device, a 2 nm first chromium layer is plated on the surface of the decontaminated glass slide, and then a 15 nm first gold layer is plated on the surface of the first chromium layer to obtain a glass slide I;

[0103] S3. Wash the glass slide I according to the method in step S1 to obtain a decontaminated glass slide I, take 100 μL of the DNA oligonucleotide chain solution VI and apply it on the first gold layer surface of the decontaminated glass slide I, put the decontaminated glass slide I coated with the DNA oligonucleotide chain solution VI into an electric hot air drying oven for drying, take out the decontaminated glass slide I and wash it in deionized water for 5 hours, wash and remove the soluble impurities on the surface of the medium layer and then dry it, then put the dried decontaminated glass slide I into a DNA gel solution and soak it for 12 hours, then take out the decontaminated glass slide I from the DNA gel solution and dry it, then immerse it in deionized water for washing and drying to obtain a glass slide II;

[0104] S4. Using magnetron sputtering coating equipment, a 2 nm second chromium layer is plated on the surface of the glass slide II, and then a 15 nm second gold layer is plated on the surface of the second chromium layer to obtain an interference reflection spectrum RNA detection sheet;

[0105] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0106] S6. After adding the miRNA-21 solution containing 1 μmol miRNA-21 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in a static state for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0107] S7. The content of miRNA-21 in the miRNA-21 solution to be tested was calculated according to formula (I) to (II) to be 1.07 μmol. The results are shown in Table 3;

[0108] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0109] A. Prepare oligonucleotide chain solution I containing 100 μmol of DNA oligonucleotide chain I, DNA oligonucleotide chain solution II containing 100 μmol of DNA oligonucleotide chain II, DNA oligonucleotide chain solution III containing 62.5 μmol of DNA oligonucleotide chain III, DNA oligonucleotide chain solution IV containing 62.5 μmol of DNA oligonucleotide chain IV, and DNA oligonucleotide chain solution V containing 1 μmol of DNA oligonucleotide chain V;

[0110] B. Add the DNA oligonucleotide chain solution I and DNA oligonucleotide chain solution V in step A to centrifuge tube a and mix them. Place centrifuge tube a in a PCR instrument, set the temperature to 53.5°C, and react for 1.5 hours. After the reaction is completed, add DNA oligonucleotide chain solution II to centrifuge tube a, set the temperature to 70°C, and react for 3.5 hours to obtain a DNA gel backbone solution.

[0111] C. Add DNA oligonucleotide chain solution III-1 and DNA oligonucleotide chain solution IV-1 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 55.8°C, and the reaction time to 1.5h to obtain DNA gel branched chain-1 solution;

[0112] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-1 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 55°C for 5 minutes, then at 50°C for 3 minutes, and finally cool from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 hours to obtain a DNA gel solution.

[0113] Table 3

[0114]

[0115] Example 3

[0116] S1. Take a glass slide, put it in a beaker with deionized water and ultrasonically clean it for 1 minute, then put it in an electric blast drying oven to dry it, then put the dried glass slide in a beaker with anhydrous ethanol and ultrasonically clean it for 1 minute, put it in an electric blast drying oven to dry it after cleaning, take out the glass slide after drying, put it in a new beaker with deionized water and ultrasonically clean it for 1 minute, and get a decontaminated glass slide after drying;

[0117] S2. Using a magnetron sputtering coating device, a 2 nm first chromium layer is plated on the surface of the decontaminated glass slide, and then a 15 nm first gold layer is plated on the surface of the first chromium layer to obtain a glass slide I;

[0118] S3. Wash the glass slide I according to the method in step S1 to obtain a decontaminated glass slide I, take 100 μL of the DNA oligonucleotide chain solution VI and apply it on the first gold layer surface of the decontaminated glass slide I, put the decontaminated glass slide I coated with the DNA oligonucleotide chain solution VI into an electric hot air drying oven for drying, take out the decontaminated glass slide I and wash it in deionized water for 5 hours, wash and remove the soluble impurities on the surface of the medium layer and then dry it, then put the dried decontaminated glass slide I into a DNA gel solution and soak it for 12 hours, then take out the decontaminated glass slide I from the DNA gel solution and dry it, then immerse it in deionized water for washing and drying to obtain a glass slide II;

[0119] S4. Using magnetron sputtering coating equipment, a 2 nm second chromium layer is plated on the surface of the glass slide II, and then a 15 nm second gold layer is plated on the surface of the second chromium layer to obtain an interference reflection spectrum RNA detection sheet;

[0120] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0121] S6. After adding the miRNA-21 solution containing 3 μmol miRNA-21 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in a static state for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0122] S7. The content of miRNA-21 in the miRNA-21 solution was calculated according to formula (I) to (II) and was 3.09 nmol. The results are shown in Table 4;

[0123] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0124] A. Prepare oligonucleotide chain solution I containing 100 μmol of DNA oligonucleotide chain I, DNA oligonucleotide chain solution II containing 100 μmol of DNA oligonucleotide chain II, DNA oligonucleotide chain solution III containing 90 μmol of DNA oligonucleotide chain III, DNA oligonucleotide chain solution IV containing 90 μmol of DNA oligonucleotide chain IV, and DNA oligonucleotide chain solution V containing 1 μmol of DNA oligonucleotide chain V;

[0125] B. Add the DNA oligonucleotide chain solution I and DNA oligonucleotide chain solution V in step A to centrifuge tube a and mix them. Place centrifuge tube a in a PCR instrument, set the temperature to 55.9°C, and react for 2 hours. After the reaction is completed, add DNA oligonucleotide chain solution II to centrifuge tube a, set the temperature to 67.5°C, and react for 4 hours to obtain a DNA gel backbone solution.

[0126] C. Add DNA oligonucleotide chain solution III-1 and DNA oligonucleotide chain solution IV-1 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 57.5°C, and the reaction time to 2h to obtain DNA gel branched chain-1 solution;

[0127] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-1 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 57°C for 5 minutes, then at 55°C for 3 minutes, and finally cool from 55°C to 23°C with a cooling amplitude of 2°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 75 rpm for 12 hours to obtain a DNA gel solution.

[0128] Table 4

[0129]

[0130] Accuracy experiment

[0131] According to the RNA detection sheet samples prepared in Example 1, miRNA-21 solutions containing 0.5 nmol, 1.0 nmol, and 1.5 nmol miRNA-21 were detected respectively, and the results obtained are shown in Table 5 below.

[0132] Table 5

[0133] Addition amount μmol 0.5 1.0 1.5 Peak value of the first characteristic peak in the visible light range nm 449.6 485.9 535.8 miRNA content nmol 0.52 0.97 1.56

[0134] It can be seen that the RNA detection sheet provided by the present invention has a simple detection operation. It only needs to add the miRNA solution into the RNA detection sheet to detect the signal for qualitative detection, with high accuracy. At the same time, it reduces the cost of RNA detection and solves the problems of complex operation, high cost and long detection cycle in the prior art.

[0135] (II) Detection of miRNA-373

[0136] Example 4

[0137] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 1;

[0138] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0139] S6. After adding the miRNA-373 solution containing 3 μmol miRNA-373 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in the deionized water for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0140] S7. The content of miRNA-373 in the miRNA-373 solution to be tested was calculated according to formula (I) to (II) to be 3.13 μmol. The results are shown in Table 6;

[0141] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0142] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 1;

[0143] C. Add DNA oligonucleotide chain solution III-2 and DNA oligonucleotide chain solution IV-2 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 52.7°C, and the reaction time to 1 hour to obtain DNA gel branched chain-2 solution;

[0144] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-2 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place the mixed solution into a PCR instrument for annealing. The annealing procedure is to react at 55°C for 5 minutes, then at 50°C for 3 minutes, and finally cool from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 hours to obtain a DNA gel solution.

[0145] Table 6

[0146]

[0147] Example 5

[0148] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 2;

[0149] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0150] S6. After adding the miRNA-373 solution containing 1 μmol miRNA-373 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in the deionized water for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0151] S7. The content of miRNA-373 in the miRNA-373 solution to be tested was calculated according to formulas (I) to (II) to be 1.15 μmol. The results are shown in Table 7;

[0152] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0153] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 2;

[0154] C. Add DNA oligonucleotide chain solution III-2 and DNA oligonucleotide chain solution IV-2 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 55.8°C, and the reaction time to 1.5h to obtain DNA gel branched chain-2 solution;

[0155] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-2 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 55°C for 5 minutes, then at 50°C for 3 minutes, and finally cool from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 hours to obtain a DNA gel solution.

[0156] Table 7

[0157]

[0158] Example 6

[0159] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 3;

[0160] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0161] S6. After adding the miRNA-373 solution containing 3 μmol miRNA-373 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in the deionized water for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0162] S7. The content of miRNA-373 in the miRNA-373 solution was calculated according to formula (I) to (II) to be 3.29 nmol. The results are shown in Table 8;

[0163] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0164] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 3;

[0165] C. Add DNA oligonucleotide chain solution III-2 and DNA oligonucleotide chain solution IV-2 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 57.5°C, and the reaction time to 2h to obtain DNA gel branched chain-2 solution.

[0166] D. Place the DNA gel main chain solution obtained in step B and the DNA gel side chain-2 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 57°C for 5 minutes, then react at 55°C for 3 minutes, and finally cool from 55°C to 23°C with a cooling amplitude of 2°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 75 rpm for 12 hours to obtain a DNA gel solution.

[0167] Table 8

[0168]

[0169] (III) Detection of miRNA-155

[0170] Example 7

[0171] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 1;

[0172] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0173] S6. After adding the miRNA-155 solution containing 3 μmol miRNA-155 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in a static state for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0174] S7. The content of miRNA-155 in the miRNA-155 solution to be tested was calculated according to formula (I) to (II) to be 2.99 μmol. The results are shown in Table 9;

[0175] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0176] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 1;

[0177] C. Add DNA oligonucleotide chain solution III-3 and DNA oligonucleotide chain solution IV-3 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 52.7°C, and the reaction time to 1 hour to obtain DNA gel branched chain-3 solution;

[0178] D. Place the DNA gel main chain solution obtained in step B and the DNA gel branch chain-3 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place the mixed solution into a PCR instrument for annealing. The annealing program is 55°C for 5 min, then 50°C for 3 min, and finally cooling from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 h to obtain a DNA gel solution.

[0179] Table 9

[0180]

[0181] Example 8

[0182] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 2;

[0183] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0184] S6. After adding the miRNA-155 solution containing 1 μmol miRNA-155 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in the deionized water for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0185] S7. The content of miRNA-155 in the miRNA-155 solution to be tested was calculated to be 1.11 μmol according to formulas (I) to (II). The results are shown in Table 10;

[0186] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0187] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 2;

[0188] C. Add DNA oligonucleotide chain solution III-3 and DNA oligonucleotide chain solution IV-3 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 55.8°C, and the reaction time to 1.5h to obtain DNA gel branched chain-3 solution;

[0189] D. Place the DNA gel main chain solution obtained in step B and the DNA gel branch chain-3 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 55°C for 5 minutes, then at 50°C for 3 minutes, and finally cool from 50°C to 27°C with a cooling amplitude of 1°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 60 rpm for 12 hours to obtain a DNA gel solution.

[0190] Table 10

[0191]

[0192]

[0193] Example 9

[0194] The method for preparing the interferometric reflectance spectroscopy RNA detection sheet (i.e., steps S1-S4) is the same as in Example 3;

[0195] S5. Place the RNA detection sheet flat and soak it in deionized water, detect the reflection spectrum of the mirror surface, and record the data as data I;

[0196] S6. After adding the miRNA-155 solution containing 3 μmol miRNA-155 to be tested into the deionized water where the RNA detection piece is located, the RNA detection piece is placed in a static state for 5 hours, and then the RNA detection piece is taken out and immersed in new deionized water, and the reflection spectrum of the mirror surface is detected and the data is recorded as data II;

[0197] S7. The content of miRNA-155 in the miRNA-155 solution was calculated according to formula (I) to (II) to be 3.22 nmol. The results are shown in Table 11.

[0198] Wherein, the preparation method of DNA gel solution comprises the following steps:

[0199] The method for preparing the DNA gel backbone solution (i.e., steps A and B) is the same as in Example 3;

[0200] C. Add DNA oligonucleotide chain solution III-3 and DNA oligonucleotide chain solution IV-3 into centrifuge tube b and mix them. Place centrifuge tube b in a PCR instrument, set the temperature to 57.5°C, and the reaction time to 2h to obtain DNA gel branched chain-3 solution;

[0201] D. Place the DNA gel main chain solution obtained in step B and the DNA gel branch chain-3 solution obtained in step C into centrifuge tube c and mix them to obtain a mixed solution. Place centrifuge tube c into a PCR instrument for annealing. The annealing procedure is to react at 57°C for 5 minutes, then react at 55°C for 3 minutes, and finally cool from 55°C to 23°C with a cooling amplitude of 2°C / min. Place the annealed mixed solution on a rotating mixer for rotation mixing at a rotation speed of 75 rpm for 12 hours to obtain a DNA gel solution.

[0202] Table 11

[0203]

[0204] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Sun Congxin <120> Interference reflection spectrum RNA detection sheet and preparation method thereof <160> 13 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 40 <212> DNA <213> Artificial sequence <220> <223> <400> 1 agacgtcata gttaacgact tctgatcata agtttcagtt 40 <210> 2 <211> 35 <212> DNA <213> Artificial sequence <220> <223> <400> 2 agtcgttaac tatgacgtct aaacttatga tcaga 35 <210> 3 <211> 27 <212> DNA <213> Artificial sequence <220> <223> <400> 3 tcaacatcag tctgataagc taaactg 27 <210> 4 <211> 28 <212> DNA <213> Artificial sequence <220> <223> <400> 4 acacccccaaa atcgaagcac ttcaactg 28 <210> 5 <211> 27 <212> DNA <213> Artificial sequence <220> <223> <400> 5 cccctatcac gattagcatt aaaactg 27 <210> 6 <211> 16 <212> DNA <213> Artificial sequence <220> <223> <400> 6 actgatgttg aaactg 16 <210> 7 <211> 15 <212> DNA <213> Artificial sequence <220> <223> <400> 7 tttggggtgt aactg 15 <210> 8 <211> 15 <212> DNA <213> Artificial sequence <220> <223> <400> 8 gtgatagggg aactg 15 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> <400> 9 aactgaaact tatgatcaga 20 <210> 10 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Nucleotide chain with thiol group at the 3' end <400> 10 agtcgttaac tatgacgtct aaaaaaaaaash 30 <210> 11 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> <400> 11 uagcuuauca gacugauguu ga 22 <210> 12 <211> twenty three <212> RNA <213> Artificial sequence <220> <223> <400> 12 gaagugcuuc gauuuugggg ugu 23 <210> 13 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> <400> 13 uuaaugcuaa ucgugauagg gg 22

Claims

1. An interferometric reflectance spectroscopy RNA detection sheet, characterized in that: The method comprises a glass slide, on which a first chromium layer, a first gold layer, a thiol-modified DNA layer, a DNA gel layer, a second chromium layer, and a second gold layer are sequentially arranged; The sequence of the DNA oligonucleotide chain VI used in the thiol-modified DNA layer is shown in SEQ ID NO: 10; The DNA gel is obtained by drying a DNA gel solution, and the DNA gel solution is a DNA gel-1 solution, a DNA gel-2 solution or a DNA gel-3 solution; The preparation method of the DNA gel solution comprises the following steps: A. Prepare DNA oligonucleotide chains Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, Ⅴ; The sequence of the DNA oligonucleotide chain I is shown in SEQ ID NO: 1; The sequence of the DNA oligonucleotide chain II is shown in SEQ ID NO: 2; The sequence of the DNA oligonucleotide chain III-1 is shown in SEQ ID NO: 3; The sequence of the DNA oligonucleotide chain III-2 is shown in SEQ ID NO: 4; The sequence of the DNA oligonucleotide chain III-3 is shown in SEQ ID NO: 5; The sequence of the DNA oligonucleotide chain IV-1 is shown in SEQ ID NO: 6; The sequence of the DNA oligonucleotide chain IV-2 is shown in SEQ ID NO: 7; The sequence of the DNA oligonucleotide strand IV-3 is shown in SEQ ID NO: 8; The sequence of the DNA oligonucleotide chain V is shown in SEQ ID NO: 9; B. Dissolve the DNA oligonucleotide chains Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, and Ⅴ in ultrapure water to obtain DNA oligonucleotide chain solutions Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, and Ⅴ, mix the DNA oligonucleotide chain solution Ⅰ and the DNA oligonucleotide chain solution Ⅴ, and react at 53.5-56.1°C for 1-2h. After the reaction, add the DNA oligonucleotide chain solution Ⅱ, and react at 67.5-70°C for 3.5-4h to obtain a DNA gel backbone solution; C. When preparing DNA gel-1, the DNA oligonucleotide chain solution III-1 and the DNA oligonucleotide chain solution IV-1 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branched chain-1 solution; D. Mixing the DNA gel main chain solution obtained in step B and the DNA gel side chain-1 solution obtained in step C to obtain a mixed solution, annealing the mixed solution, and rotating the annealed mixed solution at a speed of 60 to 75 rpm for 24 hours to obtain a DNA gel-1 solution; When preparing DNA gel-2, the DNA oligonucleotide chain solution III-2 and the DNA oligonucleotide chain solution IV-2 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branch chain-2 solution; the DNA gel main chain solution obtained in step B and the DNA gel branch chain-2 solution obtained in step C are mixed to obtain a mixed solution, and the mixed solution is annealed. After the annealing is completed, the mixed solution is rotated and mixed at a speed of 60-75 rpm for 24 hours to obtain a DNA gel-2 solution; When preparing DNA gel-3, the DNA oligonucleotide chain solution III-3 and the DNA oligonucleotide chain solution IV-3 of step B are mixed, and reacted at 52.7-57.5° C. for 1-2 hours to obtain a DNA gel branch chain-3 solution; the DNA gel main chain solution obtained in step B and the DNA gel branch chain-3 solution obtained in step C are mixed to obtain a mixed solution, and the mixed solution is annealed. After annealing, the mixed solution is rotated and mixed at a speed of 60-75 rpm for 24 hours to obtain a DNA gel-3 solution.

2. The interferometric reflectance spectroscopy RNA detection sheet according to claim 1, characterized in that: The concentrations of the DNA oligonucleotide chain solutions Ⅰ, Ⅱ, Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅳ-1, Ⅳ-2, Ⅳ-3, and Ⅴ are all 100 μmol / L.

3. The interferometric reflectance spectroscopy RNA detection sheet according to claim 2, characterized in that: The mass ratio of the DNA oligonucleotide chain solution I, II, III-1, III-2, III-3, IV-1, IV-2, IV-3, and V is 100:100:(40-90):(40-90):(40-90):(40-90):(40-90):(40-90):1; The mass ratio of the DNA oligonucleotide chain solution III-1 to the DNA oligonucleotide chain solution IV-1 is 1:1; The mass ratio of the DNA oligonucleotide chain solution III-2 to the DNA oligonucleotide chain solution IV-2 is 1:1; The mass ratio of the DNA oligonucleotide chain solution III-3 to the DNA oligonucleotide chain solution IV-3 is 1:

1.

4. The interferometric reflectance spectroscopy RNA detection sheet according to claim 1, characterized in that: The annealing in step D is to react the mixed solution at 55-57° C. for 5 minutes, then react at 50-55° C. for 3 minutes, and finally cool it to 23-27° C. at a cooling rate of 1-2° C. / min.

5. The interferometric reflectance spectroscopy RNA detection sheet according to claim 1, characterized in that: The thickness of the first chromium layer and the second chromium layer are both 2 nm, and the thickness of the first gold layer and the second gold layer are both 15 nm.

6. The method for preparing an interferometric reflectance spectroscopy RNA detection sheet according to claim 1, characterized in that: The following steps are involved: S1, cleaning the glass slide to obtain a decontaminated glass slide, plating a first chromium layer on the surface of the decontaminated glass slide, and then plating a first gold layer on the surface of the first chromium layer to obtain glass slide I; S2, washing the glass slide I to obtain a decontaminated glass slide I, dissolving the DNA oligonucleotide chain VI in ultrapure water to obtain a DNA oligonucleotide chain solution VI, coating the DNA oligonucleotide chain solution VI on the surface of the first gold layer of the decontaminated glass slide I, drying, then washing the decontaminated glass slide I in deionized water, drying after washing, and then soaking the washed and dried decontaminated glass slide I in a DNA gel solution, then taking the decontaminated glass slide I out of the DNA gel solution and drying, and then immersing it in deionized water for washing and drying to obtain a glass slide II; the sequence of the DNA oligonucleotide chain VI is shown in SEQ ID NO: 10; S3. Plate a second chromium layer on the surface of glass slide II, and then plate a second gold layer on the surface of the second chromium layer to obtain an interference reflection spectrum RNA detection sheet.

7. The method for preparing an interferometric reflectance spectroscopy RNA detection sheet according to claim 6, characterized in that: The concentration of the DNA oligonucleotide chain solution VI is 100 μmol / L.

8. The method for preparing an interferometric reflectance spectroscopy RNA detection sheet according to claim 6, characterized in that: The method for cleaning the glass slide or glass slide I comprises: taking the glass slide or glass slide I, placing it in deionized water for ultrasonic cleaning, drying, then placing it in anhydrous ethanol for ultrasonic cleaning, drying, placing it in deionized water again for ultrasonic cleaning and drying, to obtain a decontaminated glass slide or decontaminated glass slide I.

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