MiRNA (micro Ribonucleic Acid) detection method

Abstract

The invention belongs to the field of miRNA detection, and discloses a miRNA detection method, which can realize simple, rapid and specific detection of miRNA. The detection method comprises the following steps: step 1, preparing capture probe modified silicon dioxide microspheres; step 2, preparing reporter probe modified quantum dot fluorescent microspheres; 3, co-incubating target miRNA with the capture probe modified silicon dioxide microspheres prepared in the step 1 and the reporter probe modified quantum dot fluorescent microspheres prepared in the step 2 to form a compound; and 4, measuring the fluorescence intensity of the composite product in the step 3 by using a fluorospectro photometer, and detecting the target miRNA.

Description

Technical Field

[0001] The present invention belongs to the field of miRNA detection, and in particular, relates to a miRNA detection method. Background Art

[0002] miRNA is a non-coding small RNA composed of 19-22 nucleotides, which can regulate mRNA expression at the transcriptional or post-transcriptional level and play an important role in cell proliferation, differentiation, apoptosis and metabolism. Studies have found that abnormal expression of miRNA is closely related to the occurrence and development of tumors and is regarded as a potential marker for early diagnosis of tumors.

[0003] Traditional miRNA detection methods mainly include high-throughput sequencing, miRNA microarray and real-time fluorescence quantitative PCR (qPCR). Although these methods can achieve miRNA detection, they have limitations such as high cost, long time consumption and complex operation. Therefore, the development of simple, fast and reliable miRNA detection methods is of great significance in tumor diagnosis. Summary of the invention

[0004] In view of the existing problems, the present invention provides a method for detecting miRNA, which can realize simple, rapid and specific detection of miRNA.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: A miRNA detection method comprises the following steps: Step 1, preparing capture probe modified silica microspheres; Step 2, preparing quantum dot fluorescent microspheres modified with reporter probes; Step 3, the target miRNA is co-incubated with the capture probe modified silica microspheres prepared in step 1 and the reporter probe modified quantum dot fluorescent microspheres prepared in step 2 to form a complex; Step 4: Use a fluorescence spectrophotometer to measure the fluorescence intensity of the complex product of step 3 to detect the target miRNA.

[0006] As a preferred example, the particle size of the silica microspheres prepared in step 1 is 0.97 to 2.33 μm.

[0007] As a preferred example, the step 1 specifically includes: The first mixed solution and ammonia water are mixed evenly, and then the second mixed solution is added, stirred for reaction, centrifuged and the supernatant is discarded, and the precipitate is washed with ethanol to obtain silica microspheres; Dispersing the silica microspheres with ethanol, adding ammonia water and 3-aminopropyltriethoxysilane, and reacting at room temperature to obtain amino-containing silica microspheres; Dispersing the amino-modified silica microspheres with N,N-dimethylformamide, adding succinic anhydride, and reacting to obtain carboxylated silica microspheres; The carboxylated silica microspheres are dispersed with morpholineethanesulfonic acid solution, coupling reagents EDC and NHS are added to activate the carboxyl groups, and then the capture probes are added for nucleic acid coupling. After the reaction, the capture probes are centrifuged and washed with deionized water to obtain capture probe-modified silica microspheres.

[0008] As a preferred example, the first mixed solution comprises potassium chloride, deionized water, ethanol and tetraethyl silicate; the second mixed solution comprises ethanol and tetraethyl silicate.

[0009] As a preferred example, the particle size of the quantum dot fluorescent microspheres prepared in step 2 is 142 to 158 nm.

[0010] As a preferred example, the step 2 specifically includes: Tetraethyl silicate, ethanol and deionized water are mixed evenly, then ammonia water is added, stirred for reaction, and then centrifuged and washed with ethanol to obtain silica particles; Disperse the silica particles with ethanol, add ammonia water and (3-mercaptopropyl)trimethoxysilane, stir at room temperature, and centrifuge and wash after reaction to obtain thiolated silica; disperse the thiolated silica with chloroform, add quantum dot chloroform solution dropwise under stirring, and react at room temperature to obtain quantum dot-modified silica; The quantum dot-modified silica is dispersed in ethanol, and ammonia water is added under vigorous stirring, and tetraethyl silicate is added dropwise, reacted at room temperature, and then washed with ethanol to obtain quantum dot fluorescent microspheres; Dispersing the quantum dot fluorescent microspheres with ethanol, adding ammonia water and 3-aminopropyltriethoxysilane, and reacting at room temperature to obtain amino quantum dot fluorescent microspheres; Dispersing the amino quantum dot fluorescent microspheres with N,N-dimethylformamide, adding succinic anhydride, and reacting to obtain carboxyl quantum dot fluorescent microspheres; The carboxylated quantum dot fluorescent microspheres were dispersed with morpholineethanesulfonic acid solution, EDC and NHS were added, activated at room temperature, and then the reporter probe was added. After the reaction, the reaction was performed and the reaction was centrifuged and washed with deionized water to obtain the quantum dot fluorescent microspheres modified with the reporter probe.

[0011] As a preferred example, the step 3 specifically includes: The quantum dot fluorescent microspheres modified with the reporter probes were added to the silica microsphere solution modified with the capture probes, and then the target miRNA was added, and the mixture was incubated in a constant temperature water bath. The final product was washed by centrifugation with PBS and finally dispersed in PBS.

[0012] As a preferred example, the temperature in the constant temperature water bath is 37.5°C, and the centrifugation is performed at 85g for 2 minutes.

[0013] As a preferred example, the incubation time in step 3 is 40 minutes.

[0014] As a preferred example, in step 3, the mass ratio of silica microspheres to quantum dot fluorescent microspheres is 1:8.

[0015] Beneficial effects: Compared with the prior art, the miRNA detection method of the present invention can achieve simple, rapid and specific detection of miRNA. The detection method of the present invention uses quantum dot fluorescent microspheres as fluorescent markers, silica microspheres as signal enrichment carriers, and utilizes a three-stage nucleic acid hybridization reaction to achieve simple and rapid detection of miRNA. The method does not require complex instruments and equipment and reaction systems, is simple and rapid, wherein step 3 only takes 40 minutes to complete the hybridization reaction, and the detection limit reaches 0.16 pM, has high specificity, and can achieve the distinction of single-base mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the principle of step 3 of the method of an embodiment of the present invention; Figure 2 This is a transmission electron microscope photo of the silica microspheres prepared in step 20 of the present example, wherein the scale of the transmission electron microscope photo is 1 μm; Figure 3 This is a particle size distribution diagram of silica microspheres prepared in step 20 of an example of the present invention; Figure 4 A transmission electron microscope photograph of the quantum dot fluorescent microspheres prepared in step 30 of the present example, wherein the scale of the transmission electron microscope photograph is 100 nm; Figure 5 This is a particle size distribution diagram of quantum dot fluorescent microspheres prepared in step 30 of an example of the present invention; Figure 6 The fluorescence response diagram of adding different mass ratios of silica microspheres and quantum dot fluorescent microspheres in the examples of the present invention; Figure 7 In the example of the present invention, step 40 is a fluorescence response diagram of hybridization at different times; Figure 8The figure is a graph showing the relationship between the fluorescence intensity of the product and the concentration of the target miR-21 in the example of the present invention; wherein, Figure A shows the fluorescence spectra of miR-21 at different concentrations, and Figure B shows the linear relationship between the fluorescence intensity of the product and the concentration of miR-21; Fig. 9 The fluorescence response diagram of the target miR-21 and other different sequences using the detection method of the example of the present invention; Fig.10 This is a correlation analysis of the actual sample detection results using the detection method of the example of the present invention and the qPCR detection method. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0018] A miRNA detection method according to an embodiment of the present invention comprises the following steps: Step 1: Prepare capture probe-modified silica microspheres.

[0019] Preferably, the step 1 specifically includes: Step 101, uniformly mix the first mixed solution and ammonia water, then add the second mixed solution, stir the reaction, centrifuge and discard the supernatant, wash the precipitate with ethanol, and prepare silica microspheres. The first mixed solution includes potassium chloride, deionized water, ethanol and tetraethyl silicate. The second mixed solution includes ethanol and tetraethyl silicate.

[0020] Step 102: Disperse the silica microspheres with ethanol, add ammonia water and 3-aminopropyltriethoxysilane, and react at room temperature to obtain amino-modified silica microspheres. Here, the room temperature is 18-22°C.

[0021] Step 103: Disperse the amino-modified silica microspheres with N,N-dimethylformamide, add succinic anhydride, and obtain carboxylated silica microspheres after reaction.

[0022] Step 104, dispersing the carboxylated silica microspheres with morpholineethanesulfonic acid solution, adding coupling reagents EDC (English full name: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, Chinese full name: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (English full name: N-Hydroxy succinimide, Chinese full name: N-hydroxysuccinimide) to activate the carboxyl group, and then adding the capture probe for nucleic acid coupling. After the reaction, centrifugation and washing with deionized water are performed to obtain capture probe-modified silica microspheres.

[0023] Step 2: Prepare quantum dot fluorescent microspheres modified with reporter probes.

[0024] Preferably, step 2 specifically includes: Step 201, tetraethyl silicate, ethanol and deionized water are mixed evenly, then ammonia water is added, stirred for reaction, and then washed with ethanol and centrifuged to obtain silicon dioxide particles; Step 202, dispersing the silica particles with ethanol, adding ammonia water and (3-mercaptopropyl)trimethoxysilane, stirring at room temperature for reaction and centrifuging to obtain thiolated silica; dispersing the thiolated silica with chloroform, adding quantum dot chloroform solution dropwise under stirring, reacting at room temperature to obtain quantum dot-modified silica; Step 203, dispersing the quantum dot-modified silica in ethanol, adding ammonia water and dropping tetraethyl silicate under vigorous stirring, reacting at room temperature, and washing with ethanol to obtain quantum dot fluorescent microspheres; Step 204, dispersing the quantum dot fluorescent microspheres with ethanol, adding ammonia water and 3-aminopropyltriethoxysilane, and reacting at room temperature to obtain amino quantum dot fluorescent microspheres; Step 205, dispersing the amino quantum dot fluorescent microspheres with N,N-dimethylformamide, adding succinic anhydride, and obtaining carboxylated quantum dot fluorescent microspheres after reaction; Step 206, dispersing the carboxylated quantum dot fluorescent microspheres with morpholineethanesulfonic acid solution, adding coupling reagents EDC and NHS to activate the carboxyl group, and then adding the reporter probe, and washing by centrifugation with deionized water after the reaction to obtain the quantum dot fluorescent microspheres modified with the reporter probe.

[0025] Step 3: The target miRNA is co-incubated with the capture probe-modified silica microspheres prepared in step 1 and the reporter probe-modified quantum dot fluorescent microspheres prepared in step 2 to form a silica microsphere / target miRNA / quantum dot fluorescent microsphere complex.

[0026] Preferably, step 3 specifically comprises: adding the quantum dot fluorescent microspheres modified with the reporter probe to the silica microspheres modified with the capture probe, then adding the target miRNA, incubating in a constant temperature water bath to form a complex, washing by centrifugation, and finally dispersing in PBS (full name in English: phosphate buffered saline, full name in Chinese: phosphate buffered saline). Preferably, the temperature in the constant temperature water bath is 37.5°C, the centrifugation process is a centrifugal force of 85g, and centrifugation for 2 minutes. Preferably, the mass ratio of silica microspheres to quantum dot fluorescent microspheres is 1:8. Preferably, the incubation time of step 3 is 40 minutes.

[0027] Step 4: Use a fluorescence spectrophotometer to measure the fluorescence intensity of the complex product of step 3 to detect the target miRNA.

[0028] Preferably, the silica microspheres prepared in step 1 have a particle size of 0.97 to 2.33 μm. The quantum dot fluorescent microspheres prepared in step 2 have a particle size of 142 to 158 nm. Micrometer-sized silica microspheres have a large surface area and can bind more signal molecules; while the quantum dot fluorescent microspheres are nanometer-sized, which is conducive to the hybridization reaction. At the same time, a large number of quantum dots are encapsulated inside the microspheres, which have higher fluorescence intensity and better stability. The combination of two microspheres of different sizes can improve the detection sensitivity.

[0029] In step 1 of the above method, the capture probe is modified with silica microspheres by covalent bonding, and the capture probe is coupled to the surface of the silica microspheres. In step 2, the reporter probe is modified with quantum dot fluorescent microspheres by covalent bonding, and the reporter probe is coupled to the surface of the quantum dot fluorescent microspheres. For the target miRNA, a capture probe and a reporter probe are designed for specific hybridization. The capture probe is complementary to a portion of the sequence of the target miRNA, and the reporter probe is complementary to another portion of the sequence of the target miRNA. In step 3, the target miRNA, the silica microspheres modified with the capture probe, and the quantum dot fluorescent microspheres modified with the reporter probe are co-incubated, and the target miRNA undergoes a three-segment hybridization reaction with the capture probe and the reporter probe on the surface of the microspheres (corresponding to English: Three-segment hybridization, referred to as TSH in this article). In step 3, as Figure 1 As shown, part of the target miRNA hybridizes with the capture probe, and the other part hybridizes with the reporter probe to form a completely complementary stable double-stranded chain, and the quantum dot fluorescent microspheres are bound to the surface of the silica microspheres. This three-stage hybridization reaction utilizes the coaxial stacking effect to improve the specificity of target miRNA detection. The stability of the DNA double-strand is mainly affected by two factors: complementary pairing of bases and the stacking effect of adjacent bases. Base stacking refers to the dipole-dipole interaction between planar aromatic groups in two adjacent nucleotides. Since the long target miRNA is completely complementary to the short capture probe and reporter probe, the adjacent bases at the gap of the two short chains produce a coaxial stacking effect, which increases the stability of the hybridization and the melting temperature of the double-stranded chain. When a base mutation occurs, the coaxial stacking effect between the bases is interrupted, reducing the efficiency and thermal stability of the hybrid double-stranded chain formation. Therefore, as the number of base mutations increases, the fluorescence intensity of the three-stage hybridization product gradually decreases, which is conducive to the distinction between the target sequence and the mutant sequence.

[0030] In the detection method of the above embodiment, when the target miRNA is present, the capture probe and the reporter probe hybridize with the miRNA to form a three-stage hybridization structure, and the quantum dot fluorescent microspheres are bound to the surface of the silica microspheres to generate a detection signal. The present invention realizes non-amplification, simple and rapid detection of miRNA through a three-stage nucleic acid hybridization reaction, combined with the signal enrichment effect of the microspheres and the high fluorescence of the quantum dot fluorescent microspheres.

[0031] Taking miR-21 as an example, an example is provided below according to the detection method of the present invention.

[0032] A method for detecting miR-21, comprising: Step 10: Design of capture probe H1, reporter probe H2, target miR-21 and other nucleic acid sequences.

[0033] For miR-21, a capture probe H1 and a reporter probe H2 were designed that can specifically hybridize. All nucleic acid sequences were synthesized by GenScript. The 5' end of the capture probe H1 and the 3' end of the reporter probe H2 were amino-modified. The sequences are shown in Table 1. The mutated bases are in bold italics in Table 1.

[0034] Table 1 Capture probe, reporter probe, target miR-21 and other nucleic acid sequences

[0035] Step 20, preparing capture probe-modified silica microspheres, comprising: Step 201, 11.1 mL of the first mixed solution and 0.5 mL of ammonia water were mixed evenly, 3.9 mL of the second mixed solution was added at a rate of 0.1 mL / min, stirred at room temperature for 8 hours, centrifuged at 85g for 10 minutes, the supernatant was discarded, and the precipitate was washed with ethanol 3 times to obtain silica microspheres (referred to as Beads). The first mixed solution contained 0.00138 g of potassium chloride, 0.945 g of deionized water, 7.539 g of ethanol, and 0.1648 g of tetraethyl silicate. The second mixed solution contained 2.612 g of ethanol and 0.412 g of tetraethyl silicate.

[0036] Step 202, dispersing silica microspheres with 20 mL of ethanol, adding 200 mL of ammonia water and 100 mL of 3-aminopropyltriethoxysilane, and reacting at room temperature for 12 hours to obtain amino-containing silica microspheres; Step 203, dispersing the amino-modified silica microspheres with 10 mL of N,N-dimethylformamide, adding 10 mL of succinic anhydride (10 mg / mL), and reacting for 24 hours to obtain carboxylated silica microspheres.

[0037] Step 204: Disperse the carboxylated silica microspheres with 10 mL of 0.1 M morpholineethanesulfonic acid solution at pH 6.0, add 100 mL of 50 mg / mL EDC and 100 mL of 50 mg / mL NHS, and react at room temperature for 30 minutes; then add 20 mL of 10 mM capture probe H1, and continue to react for 4 hours. Wash by centrifugation with deionized water three times to obtain capture probe-modified silica microspheres (referred to as Bead-H1).

[0038] The morphology of the prepared Bead-H1 was characterized by transmission electron microscopy (TEM). Figure 2 As shown. Figure 2 It can be seen that the Bead particle size is uniform and has good monodispersity. The particle size statistics were performed using Nano Measurer 1.2 software. The results are as follows: Figure 3 shown. Figure 3 In the figure, the horizontal axis represents the particle size of silica microspheres, unit: mm; the vertical axis represents the proportion of silica microspheres with different particle sizes. Figure 3 It can be seen that the Bead particle size is 1.65±0.68μm, showing a normal distribution trend.

[0039] Step 30, preparing quantum dot fluorescent microspheres modified with reporter probes, specifically comprising: Step 301, 100 mL of tetraethyl silicate, 5 mL of ethanol and 100 mL of deionized water were mixed evenly; 300 mL of ammonia water was added, stirred at room temperature for 6 hours, centrifuged at 8600 g for 10 minutes, and washed with ethanol three times to obtain silica particles; Step 302, dispersing the silica particles with 12 mL of ethanol, adding 120 mL of ammonia water and 30 mL of (3-mercaptopropyl)trimethoxysilane, stirring at room temperature for 12 hours, and centrifuging to obtain thiolated silica; dispersing the thiolated silica with 10 mL of chloroform, and adding 2.5 mL of quantum dot chloroform solution dropwise under stirring, wherein the quantum dot concentration is 4 mg / mL, and reacting at room temperature for 30 minutes to obtain quantum dot-modified silica; Step 303, further coating the outer layer of silicon shell: disperse the obtained quantum dot-modified silica in 5 mL of ethanol, add 0.8 mL of 2.0 wt% ammonia water under vigorous stirring (stirring speed is 1200 rpm), and add 30 mL of tetraethyl silicate every 20 minutes, a total of 90 mL of tetraethyl silicate. React at room temperature for 6 hours, wash with ethanol 3 times, and obtain quantum dot fluorescent microspheres (referred to as Qbead); Step 304, using 20 mL of ethanol to disperse the quantum dot fluorescent microspheres, adding 200 mL of ammonia water and 100 mL of 3-aminopropyltriethoxysilane, reacting at room temperature for 12 hours to obtain amino quantum dot fluorescent microspheres; Step 305: Use 10 mL of N,N-dimethylformamide to disperse the amino quantum dot fluorescent microspheres, add 10 mL of succinic anhydride (10 mg / mL), and react for 24 hours to obtain carboxylated quantum dot fluorescent microspheres.

[0040] Step 306: Disperse the carboxylated quantum dot fluorescent microspheres with 10 mL of 0.1 M morpholineethanesulfonic acid solution at pH 6.0, add 100 mL of 50 mg / mL EDC and 100 mL of 50 mg / mL NHS, and react at room temperature for 30 minutes; then add 20 mL of 10 mM reporter probe H2, and continue to react for 4 hours. Wash by centrifugation with deionized water for 3 times to obtain reporter probe-modified quantum dot fluorescent microspheres (referred to as Qbead-H2).

[0041] The morphology of the prepared Qbead-H2 was characterized by TEM. Figure 4 As shown. Figure 4 It can be seen that the Qbead particle size is uniform and has good monodispersity. The Qbead particle size is counted using Nano Measurer 1.2 software, as shown in the figure. Figure 5 The horizontal axis represents the particle size of quantum dot fluorescent microspheres, unit: nm; the vertical axis represents the proportion of quantum dot fluorescent microspheres with different particle sizes. Figure 5 It can be seen that the particle size of Qbead is 150±8nm, showing a normal distribution trend.

[0042] Step 40, co-incubating the target miR-21 with the Bead-H1 prepared in step 20 and the Qbead-H2 prepared in step 30 to form a complex, specifically comprising: 50 mL of 0.16 mg / mL Qbead-H2 was added to 50 mL of 0.02 mg / mL Bead-H1 solution, and then 2 mL of 10 nM miR-21 was added. The mixture was incubated in a constant temperature water bath at 37.5°C. The formed complex was centrifuged at 85g for 2 minutes, washed and dispersed in 200 mL PBS.

[0043] In step 40, the 5' end of H1 and the 3' end of H2 are amino modified. The 3' end and 5' end of the target miR-21 are complementary to H1 and H2 respectively, and hybridization forms a completely complementary stable double chain, and finally forms a silica microsphere / miR-21 / quantum dot fluorescent microsphere complex.

[0044] The mass concentration of Qbead-H2 in step 40 was changed to 0.02 mg / mL, 0.04 mg / mL, 0.08 mg / mL, 0.12 mg / mL, and 0.2 mg / mL respectively, and other conditions remained unchanged to carry out hybridization reaction. The fluorescence intensity of the product was analyzed by fluorescence spectrophotometer. The results are as follows: Figure 6 shown. Figure 6 In the figure, the horizontal axis represents the mass ratio of the two microspheres added, and the vertical axis represents the fluorescence intensity of the product. Figure 6It can be seen that when the mass ratio of Bead-H1 to Qbead-H2 is 1:8, the reporter signal reaches a higher level, and there is no statistical difference with the signal level generated by the mass ratio of 1:10. Therefore, 1:8 is the optimal reaction mass ratio of Bead-H1 to Qbead-H2.

[0045] In step 40, the reaction was carried out in a constant temperature water bath at 37.5°C for 10, 20, 30, 40, 50 and 60 minutes respectively, and other conditions remained unchanged. The fluorescence intensity of the product was analyzed by a fluorescence spectrophotometer, and the results were as follows: Figure 7 shown. Figure 7 In the figure, the horizontal axis represents different hybridization times, and the vertical axis represents the fluorescence intensity of the product. Figure 7 It can be seen that the reporter signal reaches the highest after 40 minutes of hybridization. Therefore, 40 minutes is the optimal time for hybridization reaction.

[0046] Step 50: Use a fluorescence spectrophotometer to measure the fluorescence intensity of the complex product of step 40 to detect the target miRNA.

[0047] A. About the sensitivity of the detection method: Use PBS to prepare miR-21 standard solutions with concentrations of 0, 1, 5, 10, 50, 100 and 500 pM. Mix 25 mL of 0.02 mg / mL Bead-H1, 5 mL of different concentrations of miR-21 and 25 mL of 0.16 mg / mL Qbead-H2, hybridize at 37.5°C for 40 minutes, centrifuge at 85g for 2 minutes, resuspend the precipitate in 200 mL PBS, and analyze the fluorescence intensity of the final product using a fluorescence spectrophotometer. Figure 8 In (A), the horizontal axis represents the emission wavelength of the quantum dots, and the vertical axis represents the fluorescence intensity of the product. Figure 8 In (A), within the range of 1 pM to 500 pM, as the concentration of miR-21 increased, the fluorescence intensity of the product gradually increased.

[0048] The logarithm of miR-21 concentration (log C miR-21 ) as the horizontal axis and the product fluorescence intensity as the vertical axis, and a standard curve is drawn. The results are shown in Figure 8 (B) shown. Figure 8 In (B), within the range of miR-21 concentration of 1 pM-500 pM, the fluorescence intensity of the product showed a good linear relationship with the logarithm of the miR-21 concentration. Through linear fitting, the linear equation Y=31.2-9.2lgX was obtained, and the correlation coefficient R 2 =0.990. Where Y represents the fluorescence intensity of the product and X represents the concentration of miR-21. The minimum detection limit of this method for miR-21 was calculated to be 0.16 pM.

[0049] B. Specificity of the detection method: 5 mL of 100 pM target miR-21, single-base mismatch sequence (1mut), three-base mismatch sequence (3mut), five-base mismatch sequence (5mut) and other miRNA sequences (miR-24, miR-31, miR-143 and miR-22) were mixed with 25 mL, 0.02 mg / mL Bead-H1 and 25 mL, 0.16 mg / mL Qbead-H2, respectively, and hybridized at 37.5°C for 40 minutes. The mixture was centrifuged at 85g for 2 minutes, and the precipitate was resuspended in 200 mL PBS. The fluorescence intensity of the final product was analyzed using a fluorescence spectrophotometer. The results are shown in Figure 2. Fig. 9 shown. Fig. 9 In the figure, the horizontal axis represents different nucleic acid sequences, and the vertical axis represents the ratio of the fluorescence intensity generated by different sequences to the fluorescence intensity of the blank control (I / I0). Fig. 9 In the experiment, the fluorescence signal generated by miR-21 can be significantly distinguished from the signal response generated by 1 mut, 3 mut, 5mut and other sequences. This shows that this detection method can well distinguish base mismatch sequences and other miRNAs and has high specificity.

[0050] C. Quantitative detection of miR-21 in actual samples: Total RNA was extracted from cancerous tissue specimens and adjacent tissue specimens of 7 prostate cancer patients. 5 mL of total RNA solution was mixed with 25 mL of 0.02 mg / mL Bead-H1 and 25 mL of 0.16 mg / mL Qbead-H2, hybridized at 37.5°C for 40 minutes, centrifuged at 85g for 2 minutes, and the precipitate was resuspended in 200 mL PBS. The fluorescence intensity of the final product was analyzed using a fluorescence spectrophotometer. Figure 8 (B) The standard curve was drawn to calculate the concentration of miR-21 and compared with the qPCR detection results. Fig.10 shown. Fig.10 In the figure, the ordinate represents the molar concentration of miR-21 in different samples calculated by the method of the present invention, and the abscissa represents the molar concentration of miR-21 in different samples calculated by qPCR. The detection result (Y) of the detection method and the qPCR detection result (X) were subjected to correlation regression analysis, and the linear regression equation was Y=1.034X+6.728, the correlation coefficient r=0.9826, and P<0.0001. This shows that the two methods have a good correlation.

[0051] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above specific embodiments. The above specific embodiments and the description in the specification are only for further illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the claims and their equivalents.

Claims

1. A miRNA detection method, characterized in that: The method comprises the following steps: Step 1, preparing capture probe modified silica microspheres; Step 2, preparing quantum dot fluorescent microspheres modified with reporter probes; Step 3, the target miRNA is co-incubated with the capture probe modified silica microspheres prepared in step 1 and the reporter probe modified quantum dot fluorescent microspheres prepared in step 2 to form a complex; Step 4: Use a fluorescence spectrophotometer to measure the fluorescence intensity of the complex product of step 3 to detect the target miRNA.

2. The method according to claim 1, characterized in that The particle size of the silica microspheres prepared in step 1 is 0.97 to 2.33 μm.

3. The method according to claim 1, characterized in that: The step 1 specifically includes: The first mixed solution and ammonia water are mixed evenly, and then the second mixed solution is added to carry out stirring reaction, after the reaction is completed, the supernatant is discarded by centrifugation, and the precipitate is washed with ethanol to obtain silica microspheres; Dispersing the silica microspheres with ethanol, adding ammonia water and 3-aminopropyltriethoxysilane, and reacting at room temperature to obtain amino-containing silica microspheres; Dispersing the amino-modified silica microspheres with N,N-dimethylformamide, adding succinic anhydride, and reacting to obtain carboxylated silica microspheres; The carboxylated silica microspheres are dispersed with morpholineethanesulfonic acid solution, coupling reagents EDC and NHS are added to activate the carboxyl groups, and then the capture probes are added for nucleic acid coupling. After the reaction, the capture probes are centrifuged and washed with deionized water to obtain capture probe-modified silica microspheres.

4. The method according to claim 3, characterized in that The first mixed solution comprises potassium chloride, deionized water, ethanol and tetraethyl silicate; the second mixed solution comprises ethanol and tetraethyl silicate.

5. The method according to claim 1, characterized in that The particle size of the quantum dot fluorescent microspheres prepared in step 2 is 142 to 158 nm.

6. The method according to claim 1, characterized in that The step 2 specifically includes: Tetraethyl silicate, ethanol and deionized water are mixed evenly, ammonia water is added and stirred for reaction, and then centrifugation and washing are performed with ethanol to obtain silica particles; Dispersing the silica particles with ethanol, adding ammonia water and (3-mercaptopropyl)trimethoxysilane, reacting at room temperature, and centrifuging to obtain thiolated silica; dispersing the thiolated silica with chloroform, adding quantum dot chloroform solution dropwise under stirring, and reacting at room temperature to obtain quantum dot-modified silica; The quantum dot-modified silica is dispersed in ethanol, and ammonia water is added under vigorous stirring, and tetraethyl silicate is added dropwise, reacted at room temperature, and then washed with ethanol to obtain quantum dot fluorescent microspheres; Dispersing the quantum dot fluorescent microspheres with ethanol, adding ammonia water and 3-aminopropyltriethoxysilane, and reacting at room temperature to obtain amino quantum dot fluorescent microspheres; Dispersing the amino quantum dot fluorescent microspheres with N,N-dimethylformamide, adding succinic anhydride, and reacting to obtain carboxyl quantum dot fluorescent microspheres; The carboxylation quantum dot fluorescent microspheres were dispersed with morpholineethanesulfonic acid solution, coupling reagents EDC and NHS were added for carboxyl activation, and then the reporter probe was added. After the reaction, the deionized water was used for centrifugal washing to obtain the quantum dot fluorescent microspheres modified with the reporter probe.

7. The method according to claim 1, characterized in that The step 3 specifically includes: The quantum dot fluorescent microspheres modified with the reporter probes are added to the silica microsphere solution modified with the capture probes, and then the target miRNA is added, and the mixture is incubated in a constant temperature water bath. The formed complex is washed by centrifugation with PBS and finally dispersed in PBS.

8. The method according to claim 7, characterized in that The temperature in the thermostatic water bath is 37.5° C., the incubation time is 30 to 60 minutes, and the centrifugation is performed at 85 g for 2 minutes.

9. The method according to claim 8, characterized in that The incubation time in step 3 is 40 minutes.

10. The method according to claim 1, characterized in that In step 3, the mass ratio of the silica microspheres to the quantum dot fluorescent microspheres is 1:8.

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