An isothermal amplification method for miR-146a and its application

Abstract

This invention discloses an isothermal amplification method for miR-146a and its application. The isothermal amplification method for miR-146a includes: mixing a cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTPs, levofloxacin enzyme, Nt.BbvCI nicking endonuclease, and water, and incubating at an isothermal temperature to amplify miR-146a; wherein the sequence of the cytosine-rich hairpin probe is shown in SEQ ID NO:1. The isothermal amplification method provided by this invention enables isothermal amplification of the cytosine-rich hairpin probe without relying on exogenous primers, reducing system complexity; simultaneously, it exhibits excellent detection sensitivity and specificity for miRNA-146a, showing promising application prospects in the pathophysiological development of type II diabetes.

Description

Technical Field

[0001] This invention belongs to the field of life and health and medical analysis, specifically relating to an isothermal amplification method for miR-146a and its application. Background Technology

[0002] Essentially, miRNAs belong to a class of single-stranded non-coding small RNAs capable of post-transcriptional regulation of gene expression. Since the discovery of the first miRNA more than two decades ago, the field of miRNA detection has experienced rapid development in bioanalytical and clinical diagnostic methods. Representative methods such as real-time polymerase chain reaction (RT-PCR), northern blotting (NB), microarrays, and fluorescence in situ hybridization (FISH) have made remarkable progress in miRNA detection. However, these methods also have obvious drawbacks. For example, for primer-guided RT-PCR, small errors introduced by exponential amplification can lead to false positives and negative results. NB is limited by sensitivity and universality, exhibiting weak specificity in distinguishing target miRNAs from their family members. FISH also has relatively low sensitivity for the quantitative analysis of miRNAs. Furthermore, the low copy number and high homology of miRNAs in bodily fluids pose significant challenges to the high sensitivity of these methods in detecting individual miRNAs. Therefore, the development of novel mi-reaction assays with robust detection performance remains imperative.

[0003] Diabetes mellitus (DM) is a global healthcare problem affecting more than 350 million people worldwide. Clinically, fasting blood glucose testing is a simple, common, and widely accepted screening method for diabetes. However, blood glucose levels are primarily used for the qualitative diagnosis of type 2 diabetes mellitus (T2DM) and cannot monitor the pathophysiological processes of disease development and progression. Furthermore, blood glucose levels are not meaningful for assessing the serious complications caused by T2DM. The severe global situation of T2DM necessitates the search for and detection of specific clinical biomarkers with significant potential to improve the diagnosis and treatment of T2DM. Extensive evidence suggests that aberrant expression of miR-146a is closely related to the pathophysiological development of type 2 diabetes and its complications; therefore, there is a promising prospect for developing an miRNA-146a as a biomarker for the diagnosis and prognosis of T2DM. Summary of the Invention

[0004] The main objective of this invention is to provide an isothermal amplification method for miRNA and its application in the detection of miR-146a, so as to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides an isothermal amplification method for miR-146a, comprising: mixing a cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTPs, clonosase, Nt.BbvCI nicking endonuclease with water and incubating at an isothermal temperature to achieve amplification of miR-146a; wherein the sequence of the cytosine-rich hairpin probe is shown in SEQ ID NO:1.

[0007] This invention also provides the application of the aforementioned isothermal amplification method for miR-146a in the detection of miR-146a.

[0008] This invention also provides a method for detecting miR-146a for non-diagnostic purposes, comprising:

[0009] A series of standard microRNA-21 solutions of different concentrations were mixed with cytosine-rich hairpin probes, NEBuffer, dNTPs, claenosase, Nt.BbvCI nicking endonuclease, and water, and incubated at an incubator. KCl and thioflavin T were then added for further incubation, followed by fluorescence detection to establish a standard curve of microRNA-21 concentration versus fluorescence intensity. The sequence of the cytosine-rich hairpin probe is shown in SEQ ID NO:1.

[0010] Furthermore, the test sample containing microRNA-21 was mixed with cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTP, clanoside, Nt.BbvCI nicking endonuclease, and water and incubated at a constant temperature. Then, KCl and thioflavin T were added and incubation continued. The fluorescence intensity of the test sample was measured and compared with the standard curve to obtain the concentration of miR-146a in the test sample.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] (1) The isothermal amplification method provided by the present invention is very useful for a multi-functionalized cytosine-rich hairpin probe. Unlike traditional isothermal amplification technology, which requires directional chain polymerization of exogenous primers, it only uses isothermal amplification and continues through a single-temperature incubation period, which reduces the complexity of the system.

[0013] (2) This invention provides a unique time-resolved amplification mode responsible for the controllable configuration conversion of C-HP with cascaded signal amplification capability. Without the need for any auxiliary probes or complex sequence arrangements, C-HP can rapidly and efficiently accumulate the number of G-quadruplexes at isothermal conditions.

[0014] (3) The sequence analysis of C-HP in this invention shows that the probe has a simple structure, complete function, low cost and flexible design. C-HP-based TR-SDA can be easily applied to the field of molecular medicine that requires molecular diagnosis of gene-related diseases. The TR-SDA strategy has considerable practical application potential in the fields of biomedical research and disease diagnosis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the miR-146a sensing and detection principle based on a time-resolved sensor in a typical embodiment of the present invention.

[0017] Figure 2 This is a fluorescence signal diagram illustrating the feasibility of miR-146a detection in a typical embodiment of the present invention;

[0018] Figures 3a-3b This is a standard curve of miR-146a within a linear concentration range in a typical embodiment of the present invention. Detailed Implementation

[0019] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The technical solution of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0020] Specifically, as one aspect of the technical solution of this invention, an isothermal amplification method for miR-146a includes: mixing a cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTPs, Klenow enzyme, Nt.BbvCl nicking endonuclease, and water and incubating at an isothermal temperature to achieve amplification of miR-146a; wherein, the sequence of the cytosine-rich hairpin probe (C-HP) is shown in SEQ ID NO:1.

[0021] In some preferred embodiments, the sequence of the cytosine-rich hairpin probe (denoted as C-HP) is as follows: AAACGTAACCCTAACCCTAAccctaaccctgcTGAGG AACCCATGGAATTCAGTTC AAACGTAgcagggttaggg.

[0022] In some preferred embodiments, the cytosine-rich hairpin probe is capable of binding to the miR-146a complementary sequence and the nick sequence.

[0023] In some preferred embodiments, the volume ratio of the cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTP, clanoside, and Nt.BbvCI nicking endonuclease is 2.5–5.0: 5.0–10.0: 2.5–5.0: 2.0–5.0: 0.5–2.0: 1.0–5.0.

[0024] Specifically: 2.5–5.0 μL C-HP (1 μM / μL), 5.0–10.0 μL miR-146a at a specific concentration, 2.5–5.0 μL 10×NEBuffer, 2.0–5.0 μL dNTP, 0.5–2.0 μL Klenow, and 1.0–5.0 μL Nt (10 U / μL).

[0025] In some preferred embodiments, the concentration of the cytosine-rich hairpin probe is 4*10⁻⁶. -1 μM / μL.

[0026] In some preferred embodiments, the isothermal amplification method includes: mixing a cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTP, clanoside, Nt.BbvCI nicking endonuclease with water and incubating at 37°C for 1–3 h to allow G-quadruplexes to accumulate, thereby amplifying miR-146a.

[0027] Specifically, the isothermal amplification method for miR-146a in this invention is a time-resolved strand displacement amplification (TR-SDA) technique.

[0028] Another aspect of this invention provides the application of the aforementioned isothermal amplification method for miR-146a in the detection of miR-146a.

[0029] Another aspect of the present invention provides a method for detecting miR-146a for non-diagnostic purposes, comprising:

[0030] A series of standard microRNA-21 solutions of different concentrations were mixed with cytosine-rich hairpin probes, NEBuffer, dNTPs, clanosides, Nt.BbvCI nicking endonuclease, and water, and incubated at an incubator. KCl and thioflavin T (denoted as ThT) were then added for further incubation, followed by fluorescence detection to establish a standard curve of microRNA-21 concentration versus fluorescence intensity. The sequence of the cytosine-rich hairpin probe is shown in SEQ ID NO:1.

[0031] Furthermore, the test sample containing microRNA-21 was mixed with cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTP, clanoside, Nt.BbvCI nicking endonuclease, and water and incubated at a constant temperature. Then, KCl and thioflavin T were added and incubation continued. The fluorescence intensity of the test sample was measured and compared with the standard curve to obtain the concentration of miR-146a in the test sample.

[0032] In some preferred embodiments, the cytosine-rich hairpin probe is capable of binding to the miR-146a complementary sequence and the nick sequence.

[0033] In some preferred embodiments, the volume ratio of the cytosine-rich hairpin probe, NEBuffer, dNTP, clanoside, and Nt.BbvCI nicking endonuclease is 2.5–5.0: 2.5–5.0: 2.0–5.0: 0.5–2.0: 1.0–5.0.

[0034] In some preferred embodiments, the concentration of the cytosine-rich hairpin probe is 4*10⁻⁶. -1 μM / μL.

[0035] In some preferred embodiments, the detection method includes: mixing a series of standard microRNA-21 solutions of different concentrations with cytosine-rich hairpin probes, NEBuffer, dNTPs, clanosides, Nt.BbvCI nicking endonuclease, and water, and incubating at 37°C for 1–3 h; then adding KCl and thioflavin T and continuing incubation for 5–15 min to obtain the G-quadruplex / thioflavin T complex;

[0036] Furthermore, fluorescence detection was performed on the G-quadruplex / thioflavin T complex to establish a standard curve of microRNA-21 concentration versus fluorescence intensity.

[0037] Specifically, a series of standard microRNA-21 solutions of different concentrations were mixed with cytosine-rich hairpin probes, NEBuffer, dNTPs, clanokinase, Nt.BbvCI nicking endonuclease, and water, and incubated at 37°C for 1–2 h. Then, 26–35 μL of KCl (50 mM) and 1–5 μL of ThT (2 mM) were added and incubated for approximately 5–15 min. Finally, 70–100 μL of sterile deionized water was added to ensure sufficient sample volume.

[0038] In some preferred embodiments, the detection method includes: mixing a test sample containing microRNA-21 with a cytosine-rich hairpin probe, miR-146a, NEBuffer, dNTPs, clanoside, Nt.BbvCI nicking endonuclease, and water, and incubating at 37°C for 1–3 h; then adding KCl and thioflavin T and continuing incubation for 5–15 min; and obtaining the fluorescence intensity of the test sample by testing.

[0039] In some preferred embodiments, the cytosine-rich hairpin probe (denoted as C-HP) is designed to determine whether such isothermal amplification is time-resolved. A C-HP with five T bases at its 3' end is designed, and similar oligonucleotides are renamed C-HP-T. The main difference between C-HP-based time-resolved isothermal amplification and C-HP-T-based amplification is that the G-quadruplex cannot be reused. Therefore, if the signal amplification rate of C-HP is higher than that of C-HP-T, there is undoubtedly time-resolved polymerization on C-HP, which helps to improve signal gain.

[0040] The 5' end of the C-HP in this invention is designed with 7 bases, which is identical to a portion of the loop (the structure of C-HP belongs to a "stem-loop" hairpin structure, where the loop refers to the ring-shaped portion of the hairpin structure). This design facilitates the reuse of the peeled G-quadruplex, which can then be used as a primer to guide the hybridization and replication of new C-HPs. Over time, the initially added target miR-146 will create more and more G-quadruplex units.

[0041] This invention utilizes the 3' end of C-HP as a primer to directly polymerize along the downstream stem in the absence of a target miR. Although this extension can form a complete G-rich sequence, the fragment is tightly locked by the long stem, completely suppressing the conformational change of the G-rich sequence to a G-quadruplex. After introducing the target miR-146a and hybridizing with region III on the C-HP ring, miR-146a guides C-HP to undergo intermolecular replication along the downstream ring and stem. Simultaneously, the 3' end of C-HP also achieves directional intramolecular replication along its downstream stem. Without the need for any auxiliary probes or complex sequence arrangements, C-HP can rapidly and efficiently accumulate G-quadruplex ligands at isothermal conditions.

[0042] This invention allows for a large collection of G-quadruplexes, a special type of DNA structure formed from non-canonical hoogsteen-type base pairing. Once bound to ThT, the resulting G-quadruplex / ThT complex rapidly outputs a detectable fluorescent signal proportional to the miR-146a concentration, thereby avoiding covalent modification of the fluorophore on the probe and achieving label-free sensing of miR-146a.

[0043] The detection method provided in this invention can detect miR-146a with extremely high sensitivity. miR-146a is a potential biomarker for type 2 diabetes, and the expression levels of miR-146a in healthy individuals and patients with T2DM were measured. As a proof-of-concept study, TR-SDA is a powerful molecular diagnostic method that can greatly assist future clinical research.

[0044] Specifically, the isothermal amplification method for miR-146a in this invention, when used for miR-146a detection, includes:

[0045] After preparing the relevant chemicals and reagents, deionized water with a resistivity of 18.2 MΩ / CM obtained from the Milli-A10 system (Millipore) was used to prepare the buffer solution and for the determination.

[0046] After the test samples were analyzed by gel electrophoresis, the final visualization of the gel was performed on a gel imaging system (Hangzhou Longgene Scientific Instruments Co., Ltd.).

[0047] For the analysis of miR-146a, fluorescence analysis was performed based on TR-SDA.

[0048] Specific testing methods include:

[0049] 1. Chemical substances and reagents

[0050] All oligonucleotides used in the experiment were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and dissolved in 1×TE buffer. 10×NEB sustained-release solution (500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 10mM DTT, pH 7.9), 1×TE (10mM Tris-HCl, 1mM EDTA, 1mM EDTA, pH 7.8-8.2), dNTPS (dATP, dGTP, dCTP, dTTP), 5×TBE (445mM Tris(hydroxymethyl)aminomethane, 445mM Boric Acid, 10mM EDTA, pH 8.0-8.6), 25-500bp DNA labeling, 6×DNA loading buffer, sterile water, thiamine T (THT), KF-Poly (5U / μL) and Nt.BbvCl (10U / μL) were ordered from Sangon Biotech (Shanghai) Co., Ltd. (China). KCL was ordered from China National Pharmaceutical Group Corporation. Deionized water with a resistivity of 18.2 MΩ / CM obtained from the Milli-A10 system (Millipore) was used to prepare buffer solutions and for measurements.

[0051] 2. Gel electrophoresis analysis

[0052] For gel electrophoresis analysis, 2 μL of 6× loading buffer, 0.5 μL of nucleic acid acid dye (100×SYBR Green I), and 8 μL of the test sample were premixed and then loaded into a freshly prepared 12% non-denaturing polyacrylamide gel. Subsequently, gel electrophoresis was performed at 80V for 90 minutes in 1×TBE buffer. Final visualization of the gel was performed on a gel imaging system (Hangzhou Longgene Scientific Instruments Co., Ltd.).

[0053] 3. TR-SDA-based fluorescence analysis of miR-146a

[0054] Before detecting miR-146a, 1 μM C-HP was heated to 95 °C for 5 minutes and then slowly cooled to room temperature to stabilize its hairpin structure. For miR-146a detection, a 25 μL reaction mixture containing 2.5 μL C-HP (1 μM), 5.0 μL miR-146a at a specific concentration, 2.5 μL 10×NEBuffer, 2.0 μL dNTP, 0.5 μL Klenow, and 1.0 μL Nt.BbvCl was incubated at 37 °C for 1 h. Then, 26 μL KCl (50 mM) and 1 μL ThT (2 mM) were added and mixed with the reaction mixture and incubated for approximately 5 minutes. Finally, 70 μL of sterile deionized water was added to provide sufficient sample volume for direct detection via fluorescence recording on an F97 Pro fluorescence spectrophotometer (Shanghai Spectrophotometer Technology Co., Ltd.). The excitation wavelength was chosen to be 425 nm, and the fluorescence emission wavelength was set to 430-620 nm. The slit width for both excitation and emission was set to 10 nm, and the PMT detector voltage was set to 650 V.

[0055] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0056] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0057] Example 1

[0058] (1) Preparation of time-resolved sensor: Add 1 μM C-HP to PCR tube, heat to 95℃ for 5 min for denaturation, then cool to 25℃ at a rate of 1℃ per min, and keep at 25℃ for 30 min to make the formed time-resolved sensor structure more stable, and then store at 4℃ for later use.

[0059] (2) Feasibility steps for time-resolved sensor detection of miR-146a: 25 μL of miR-146a reaction system contains 2.5 μL of C-HP (1 μM), 2.5 μL of miR-146a at a specific concentration, 2.5 μL of 10×NEBuffer, 2.0 μL of dNTP, 0.5 μL of Klenow, 1.0 μL of Nt.BbvCl and 14 μL of deionized water, and then incubates at 37 °C for 1 h.

[0060] (3) Detection of fluorescence signal: The fluorescence signal in the system was detected using a fluorescence spectrophotometer. Before fluorescence detection, the reaction product needed to be processed. 26 μL of KCl (50 mM) and 1 μL of ThT (2 mM) were added to the reaction product in (2), mixed thoroughly, and incubated for about 5 min. Finally, 70 μL of sterile deionized water was added to ensure sufficient sample volume for detection. The parameters for fluorescence sensing detection were: excitation and emission slit width of 10 nm, PMT detector voltage of 650 V; excitation wavelength of the fluorescent group (FAM) of 425 nm, and collection range of emission wavelength of 430 nm to 620 nm.

[0061] (4) Establishment of the standard curve: Different concentrations of miR-146a (0-10 nmol / L) were added to (2), and fluorescence was detected after the reaction. The fluorescence detection steps were the same as in (3). The fluorescence produced by different concentrations of miR-146a was recorded in detail. Based on this, a standard curve for the detection of samples of different concentrations of miR-146a was established with different concentrations of miR-146a as the abscissa and the fluorescence output signal value as the ordinate. The standard curve established through experiments is: F 490 =29.71C miR-146a +304.9, correlation coefficient R 2 =0.9878; Standard curve as follows Figures 3a-3b As shown;

[0062] (5) Quantitative detection of actual samples: Using actual samples of normal human serum as the detection target, fluorescence detection was performed according to the same processing methods in (1), (2), (3) and (4). The obtained fluorescence detection values ​​were substituted into the standard curve to calculate the concentration of miR-146a in normal human serum.

[0063] Example 2

[0064] (1) Preparation of time-resolved sensor: Add 1 μM C-HP to PCR tube, heat to 95℃ for 5 min for denaturation, then cool to 25℃ at a rate of 1℃ per min, and keep at 25℃ for 30 min to make the formed time-resolved sensor structure more stable, and then store at 4℃ for later use.

[0065] (2) Feasibility steps for time-resolved sensor detection of miR-146a: 25 μL of miR-146a reaction system contains 2.5 μL of C-HP (1 μM), 2.5 μL of miR-146a at a specific concentration, 2.5 μL of 10×NEBuffer, 2.0 μL of dNTP, 0.5 μL of Klenow, 1.0 μL of Nt.BbvCl and 14 μL of deionized water, and then incubates at 37 °C for 1 h.

[0066] (3) Detection of fluorescence signal: The fluorescence signal in the system was detected using a fluorescence spectrophotometer. Before fluorescence detection, the reaction product needed to be processed. 26 μL of KCl (50 mM) and 1 μL of ThT (2 mM) were added to the reaction product in (2), mixed thoroughly, and incubated for about 5 min. Finally, 70 μL of sterile deionized water was added to ensure sufficient sample volume for detection. The parameters for fluorescence sensing detection were: excitation and emission slit width of 10 nm, PMT detector voltage of 650 V; excitation wavelength of the fluorescent group (FAM) of 425 nm, and collection range of emission wavelength of 430 nm to 620 nm.

[0067] (4) Establishment of the standard curve: Different concentrations of miR-146a (0-10 nmol / L) were added to (2), and fluorescence was detected after the reaction. The fluorescence detection steps were the same as in (3). The fluorescence produced by different concentrations of miR-146a was recorded in detail. Based on this, a standard curve for the detection of samples of different concentrations of miR-146a was established with different concentrations of miR-146a as the abscissa and the fluorescence output signal value as the ordinate. The standard curve established through experiments is: F 490 =29.71C miR-146a +304.9, correlation coefficient R 2 =0.9878; Standard curve as follows Figures 3a-3b As shown;

[0068] (5) Quantitative detection of actual samples: The actual sample of type II diabetes was used as the detection target. Fluorescence detection was performed according to the same processing methods in (1), (2), (3) and (4). The obtained fluorescence detection values ​​were substituted into the standard curve to calculate the concentration of miR-146a in the serum of type II diabetes.

[0069] Example 3

[0070] (1) Preparation of time-resolved sensor: Add 1 μM C-HP to PCR tube, heat to 95℃ for 5 min for denaturation, then cool to 25℃ at a rate of 1℃ per min, and keep at 25℃ for 30 min to make the formed time-resolved sensor structure more stable, and then store at 4℃ for later use.

[0071] (2) Feasibility steps for time-resolved sensor detection of miR-146a: 25 μL of miR-146a reaction system contains 5.0 μL of C-HP (1 μM), 2.5 μL of miR-146a at a specific concentration, 2.5 μL of 10×NEBuffer, 3.0 μL of dNTP, 1.0 μL of Klenow, 2.0 μL of Nt.BbvCl and 9.0 μL of deionized water, and then incubates at 37 °C for 1 h.

[0072] (3) Detection of fluorescence signal: The fluorescence signal in the system was detected using a fluorescence spectrophotometer. Before fluorescence detection, the reaction product needed to be processed. 26 μL of KCl (50 mM) and 1 μL of ThT (2 mM) were added to the reaction product in (2), mixed thoroughly, and incubated for about 5 min. Finally, 70 μL of sterile deionized water was added to ensure sufficient sample volume for detection. The parameters for fluorescence sensing detection were: excitation and emission slit width of 10 nm, PMT detector voltage of 650 V; excitation wavelength of the fluorescent group (FAM) of 425 nm, and collection range of emission wavelength of 430 nm to 620 nm.

[0073] (4) Establishment of standard curve: Add different concentrations of miR-146a (0-10 nmol / L) to (2), and detect fluorescence after the reaction. The fluorescence detection steps are the same as in (3). Record the fluorescence generated by different concentrations of miR-146a in detail. Based on this, establish a standard curve for the detection of samples with different concentrations of miR-146a with different concentrations of miR-146a as the x-axis and the fluorescence output signal value as the y-axis.

[0074] (5) Quantitative detection of actual samples: Using actual samples of normal human serum as the detection target, fluorescence detection was performed according to the same processing methods in (1), (2), (3) and (4). The obtained fluorescence detection values ​​were substituted into the standard curve to calculate the concentration of miR-146a in normal human serum.

[0075] Example 4

[0076] (1) Preparation of time-resolved sensor: Add 1 μM C-HP to PCR tube, heat to 95℃ for 5 min for denaturation, then cool to 25℃ at a rate of 1℃ per min, and keep at 25℃ for 30 min to make the formed time-resolved sensor structure more stable, and then store at 4℃ for later use.

[0077] (2) Feasibility steps for time-resolved sensor detection of miR-146a: 25 μL of miR-146a reaction system contains 4.0 μL of C-HP (1 μM), 2.5 μL of miR-146a at a specific concentration, 2.5 μL of 10×NEBuffer, 4.0 μL of dNTP, 1.5 μL of Klenow, 3.0 μL of Nt.BbvCl and 7.5 μL of deionized water, and then incubates at 37 °C for 1 h.

[0078] (3) Detection of fluorescence signal: The fluorescence signal in the system was detected using a fluorescence spectrophotometer. Before fluorescence detection, the reaction product needed to be processed. 26 μL of KCl (50 mM) and 1 μL of ThT (2 mM) were added to the reaction product in (2), mixed thoroughly, and incubated for about 5 min. Finally, 70 μL of sterile deionized water was added to ensure sufficient sample volume for detection. The parameters for fluorescence sensing detection were: excitation and emission slit width of 10 nm, PMT detector voltage of 650 V; excitation wavelength of the fluorescent group (FAM) of 425 nm, and collection range of emission wavelength of 430 nm to 620 nm.

[0079] (4) Establishment of standard curve: Add different concentrations of miR-146a (0-10 nmol / L) to (2), and detect fluorescence after the reaction. The fluorescence detection steps are the same as in (3). Record the fluorescence generated by different concentrations of miR-146a in detail. Based on this, establish a standard curve for the detection of samples with different concentrations of miR-146a with different concentrations of miR-146a as the x-axis and the fluorescence output signal value as the y-axis.

[0080] (5) Quantitative detection of actual samples: The actual sample of type II diabetes was used as the detection target. Fluorescence detection was performed according to the same processing methods in (1), (2), (3) and (4). The obtained fluorescence detection values ​​were substituted into the standard curve to calculate the concentration of miR-146a in the serum of type II diabetes.

[0081] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0082] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method for detecting miR-146a for non-diagnostic purposes, characterized in that include: A series of standard miR-146a solutions of different concentrations were mixed with a cytosine-rich hairpin probe, NEBuffer, dNTPs, clanosides, Nt.BbvCI nicking endonuclease, and water, and incubated at 37°C for 1–3 h. KCl and thioflavin T were then added, and incubation continued for 5–15 min. Fluorescence detection was then performed to establish a standard curve of miR-146a concentration versus fluorescence intensity. The sequence of the cytosine-rich hairpin probe is shown in SEQ ID NO:

1. The cytosine-rich hairpin probe can bind to both the complementary and nicked sequences of miR-146a. Furthermore, the test sample containing miR-146a was mixed with a cytosine-rich hairpin probe, NEBuffer, dNTPs, clanosides, Nt.BbvCI nicking endonuclease, and water, and incubated at 37°C for 1–3 h. Then, KCl and thioflavin T were added and incubation continued for 5–15 min. The fluorescence intensity of the test sample was measured and compared with the standard curve to obtain the concentration of miR-146a in the test sample.

2. The detection method according to claim 1, characterized in that: The volume ratio of the cytosine-rich hairpin probe, NEBuffer, dNTP, clenosase, and Nt.BbvCI nicking endonuclease is 2.5~5.0:2.5~5.0:2.0~5.0:0.5~2.0:1.0~5.

0.

3. The detection method according to claim 2, characterized in that: The concentration of the cytosine-rich hairpin probe is 4*10 -1 μM / μL.

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