Cyclic-rolling circle amplification method and kit for fluorescent quantitative detection of miRNA

Abstract

This invention discloses a method and kit for quantitative detection of circularized rolling circle amplified miRNAs. The method utilizes T4 RNA ligase to ligate the 3'-OH and 5'-phosphate ends of the target miRNA to form a circular miRNA. Using the circularized miRNA as a template, isothermal rolling circle amplification is performed using phi29 DNA polymerase to generate a large number of single-stranded DNA sequences with repetitive sequences. A universal molecular beacon is used to monitor the fluorescence signal of the amplification products in real time, achieving highly sensitive quantitative detection of miRNAs. This method has advantages such as high sensitivity, high specificity, and a wide linear detection range. It eliminates the need to design specific reverse transcription primers for each miRNA, can be extended to multiplex detection, and is suitable for clinical applications such as early tumor diagnosis, drug efficacy evaluation, and single-cell miRNA expression profiling.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid molecular detection and diagnostic technology, specifically to a method and kit for quantitative fluorescence detection of microRNA (miRNA) based on a combination of cyclization reaction and rolling circle amplification technology. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded small RNA molecules, approximately 18-25 nucleotides in length, that play a crucial role in gene expression regulation. Abnormal miRNA expression is closely associated with various human diseases, including tumors, cardiovascular diseases, and neurodegenerative diseases. Circulating miRNAs are stably present in bodily fluids such as blood and urine, exhibiting tissue-specific and disease-specific expression patterns, making them ideal non-invasive liquid biopsy biomarkers. Therefore, accurate, sensitive, and rapid detection of miRNA expression levels has significant clinical value for early disease diagnosis, personalized treatment, and prognostic assessment.

[0003] Currently, methods for miRNA detection mainly include Northern blotting, microarrays, and real-time quantitative PCR (RT-qPCR) based on reverse transcription. Northern blotting suffers from low sensitivity and cumbersome operation; microarrays have issues with insufficient specificity and poor quantitative accuracy; while RT-qPCR is the "gold standard" method, the short length and lack of poly(A) tails of miRNAs present challenges. Existing miRNA RT-qPCR methods mainly include stem-loop and tailing methods. The stem-loop method requires designing specific reverse transcription primers for each miRNA, resulting in high cost and low throughput; the tailing method requires two steps (tailing and reverse transcription), is time-consuming (4-6 hours), and the universal primers lack specificity.

[0004] Rolling circle amplification (RCA) technology uses phi29 DNA polymerase to continuously replicate circular DNA templates under isothermal conditions, resulting in significant signal amplification. However, miRNAs themselves are linear RNA molecules and cannot be directly used as RCA templates, limiting the application of this technology in miRNA detection. Therefore, there is an urgent need to develop a novel miRNA detection technology to overcome the limitations of existing methods. Summary of the Invention

[0005] In view of the problems existing in the current miRNA detection technology, the present invention aims to provide a novel method for quantitative detection of miRNA fluorescence and a dedicated kit to achieve highly sensitive, highly specific, simple and rapid quantitative analysis of miRNA. Specifically, the following technical problems need to be solved: (1) how to convert linear miRNA into a circular template that can be used for rolling circle amplification; (2) how to efficiently amplify miRNA signals under isothermal conditions; (3) how to design universal detection probes to avoid designing specific probes for each miRNA; (4) how to improve the sensitivity and specificity of detection and achieve the distinction of single base differences; (5) how to simplify the operation process, shorten the detection time and reduce the detection cost.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Scheme 1: This invention provides a circularization-rolling circle amplification method for quantitative detection of miRNA fluorescence, comprising the following steps: (1) Circulation reaction: using T4 RNA ligase to covalently link the 3'-OH end and the 5'-phosphate end of the miRNA to be tested to form a circular miRNA; (2) Rolling circle amplification reaction: using the circular miRNA as a template, adding DNA primers complementary to the miRNA sequence, and using phi29 DNA polymerase to perform isothermal rolling circle amplification reaction to generate a single-stranded DNA amplification product containing repetitive sequence units; adding a universal molecular beacon to the rolling circle amplification reaction system, wherein the molecular beacon hybridizes with the repetitive sequence units in the rolling circle amplification product and releases a fluorescence signal; (3) Fluorescence detection and quantitative analysis: monitoring the change in fluorescence intensity in real time, and calculating the concentration of miRNA in the sample to be tested based on the time when the fluorescence signal reaches the threshold or the comparison of the fluorescence intensity with the standard curve.

[0007] In the above detection method, the cyclization reaction system includes the RNA sample to be tested, T4 RNA ligase, and T4 RNA ligase buffer. The cyclization reaction conditions are: reaction temperature 16-20℃, reaction time 15-25 minutes; the ATP concentration in the reaction system is 0.5-2 mM, preferably 1 mM.

[0008] The cyclization reaction can be carried out in a conventional metal bath or water bath.

[0009] In the above detection method, a cyclization helper oligonucleotide is added to the cyclization reaction to improve cyclization efficiency. The cyclization helper oligonucleotide is complementary to a portion of the miRNA sequence to be tested, and has a length of 10-25 nucleotides, preferably 15-20 nucleotides. The cyclization helper oligonucleotide spatially brings the 5' and 3' ends of the miRNA closer together, which is beneficial for the ligation reaction.

[0010] In the above detection method, the rolling circle amplification reaction system includes: a circularization reaction product, a Ph29 DNA polymerase buffer, a Ph29 DNA polymerase, a dNTP mixture, DNA primers complementary to the miRNA sequence, and a universal molecular beacon. The rolling circle amplification reaction conditions are: reaction temperature 33-35℃; reaction time 40-50 minutes; dNTP concentration in the reaction system is 0.2-1 mM, preferably 0.5 mM; magnesium ion concentration is 5-15 mM, preferably 10 mM.

[0011] In the above detection method, the DNA primers complementary to the miRNA sequence added in the rolling circle amplification reaction can be random primers or specifically designed short primers.

[0012] In the above detection method, the universal molecular beacon added in the rolling circle amplification reaction has a stem-loop structure, including: a stem region consisting of 5-10 base pairs; a loop region of 15-25 nucleotides in length that is complementary to the repetitive sequence unit in the rolling circle amplification product; a 5' fluorescent group and a 3' quencher group.

[0013] A molecular beacon is a DNA probe with a hairpin structure. Its circular region sequence is complementary to the repeat unit in the rolling circle amplification product, while the stem region is formed by a self-complementary sequence. A fluorescent group is labeled at the 5' end, and a quencher group is labeled at the 3' end. When not hybridizing with the target, the molecular beacon maintains a hairpin structure, with the fluorescent group and quencher group close together, quenching the fluorescence. Upon hybridization with the amplification product, the hairpin structure opens, the fluorescent group and quencher group separate, and a fluorescent signal is emitted. Changes in fluorescence intensity are monitored using a real-time fluorescence detector; the rate and intensity of fluorescence signal enhancement are positively correlated with the concentration of the initial miRNA.

[0014] The phi29 DNA polymerase possesses high fidelity, strong strand displacement activity, and continuous synthesis capability. It can continuously replicate circular templates under isothermal conditions of 30-37°C to generate long single-stranded DNA products containing hundreds to thousands of repeating units complementary to the miRNA sequence. This process achieves exponential amplification of the original miRNA signal.

[0015] In the above detection method, the quantitative analysis is performed by measuring the time required for the fluorescence signal to reach a set threshold (similar to the Ct value in qPCR) or the endpoint value of the fluorescence intensity, and using a standard curve established with known concentrations of miRNA standards to calculate the absolute or relative concentration of miRNA in the unknown sample.

[0016] The above detection method has the following performance indicators: detection limit ≤ 0.1 fM; linear detection range from 0.1 fM to 100 pM; detection repeatability coefficient of variation CV < 5%; and the differential cycle threshold ΔCt ≥ 3 for miRNAs that can distinguish single-base differences.

[0017] The above detection method is applicable to the detection of miRNAs in various trace biological samples such as plasma, serum, urine, saliva, tissue homogenate, cell lysate, and exosome extract.

[0018] Option 2: The present invention also provides a miRNA fluorescence quantitative detection kit for implementing the above method, comprising the following components: (a) Reagents required for cyclization reaction: including T4 RNA ligase, T4 RNA ligase buffer, and cyclization helper oligonucleotides; (ii) Reagents required for rolling circle amplification reaction: including phi29 DNA polymerase, phi29 DNA polymerase buffer, dNTP mixture, DNA primers complementary to the miRNA sequence (random primers or specifically designed short primers), and universal molecular beacons; (iii) Positive control miRNA standard.

[0019] The kit described above also includes RNase inhibitors, nuclease-free water, miRNA standards at various concentrations, and / or negative control solutions.

[0020] The detection method and kit of this invention involve a universal molecular beacon with the nucleotide sequence: 5'-FAM-CGCGAGAACACAGCACGCG-DABCYL-3'. The universal molecular beacon of this invention produces good fluorescence signals for rolling circle amplification products of various miRNAs such as miR-21, miR-155, miR-210, and let-7a, and has universal detection capability.

[0021] Beneficial effects of the invention Compared with the prior art, the present invention has the following significant advantages: (1) High sensitivity: The invention adopts dual signal amplification of circularization-rolling circle amplification, and the detection limit is 0.1 fM (about 60 molecules), which is 1-2 orders of magnitude higher than conventional RT-qPCR. It can detect trace amounts of miRNA and meet the needs of single cell analysis, liquid biopsy and other applications.

[0022] (2) High specificity: Based on the substrate recognition specificity of T4 RNA ligase, the high fidelity of phi29 DNA polymerase and molecular beacon design, it can distinguish single-base differential miRNAs (such as let-7a and let-7b), with a specificity superior to traditional methods.

[0023] (3) Wide linear range: The detection range is from 0.1 fM to 100 pM, spanning 6 orders of magnitude, to meet the detection needs of miRNAs with different expression levels.

[0024] (4) Simple operation: Only two isothermal reactions, namely cyclization and rolling circle amplification, are required. No temperature cycling equipment is needed. Ordinary constant temperature device and fluorescence detector can be used to complete the process. The steps are simple and easy to standardize, making it suitable for clinical promotion.

[0025] (5) Fast and efficient: The whole process takes 2-3 hours (circularization 15-20 minutes, amplification 40-50 minutes), which is significantly shorter than the traditional RT-qPCR time of 4-6 hours.

[0026] (6) Low economic cost: The universal molecular beacon is compatible with the detection of multiple types of miRNAs, the amount of phi29 enzyme used is small, the isothermal reaction reduces the instrument requirements, and the overall cost is lower than that of commercial kits.

[0027] (7) No reverse transcription required: direct circularization amplification of miRNA avoids reverse transcription bias and improves detection accuracy and reproducibility.

[0028] (8) Small sample requirement: Only 1-10 μL of plasma or other trace samples are required, suitable for testing precious clinical samples. (9) Good reproducibility: The coefficient of variation within and between batches is <5%, and the results are reliable and comparable. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the technical process of the miRNA fluorescence quantitative detection method of the present invention; This demonstrates the complete process from the target miRNA through circularization, rolling circle amplification, and fluorescence signal detection. Figure 2 This is a schematic diagram of the molecular mechanism of the cyclization-rolling circle amplification principle of this invention; The molecular process of T4 RNA ligase catalyzing miRNA circularization, phi29 DNA polymerase performing rolling circle amplification, and molecular beacons hybridizing with amplification products to emit fluorescence is shown in detail. Figure 3 This is a schematic diagram of the structure of the universal molecular beacon of this invention; The stem-ring structure, positions of fluorescent and quenching groups of the molecular beacon, and conformational changes before and after binding to the target are shown. Figure 4 This is a standard curve diagram of the present invention; Standard curves were established using a series of concentrations of synthetic miR-21 standards (0.1 fM to 100 pM), with the x-axis representing the logarithm of miRNA concentration and the y-axis representing the fluorescence threshold time or fluorescence intensity, showing a good linear relationship (R² = 0.998). Figure 5 This is a diagram verifying the ability of the present invention to distinguish single base differences; The results of detection of three miRNAs, let-7a, let-7b and let-7c, which differ by only a single base, are shown. The fluorescence signal curves of the three miRNAs are clearly separated, and the threshold time difference ΔCt≥3, which proves that the method of the present invention can accurately distinguish miRNAs with highly similar sequences. Figure 6 This is an example diagram illustrating the clinical sample application of the present invention. The results of detecting miR-21 expression levels in the plasma of 10 liver cancer patients and 10 healthy controls were presented. The miR-21 expression in the liver cancer patient group was significantly higher than that in the healthy control group (P<0.001), which verifies the application value of the method of the present invention in clinical diagnosis. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0031] See Figures 1 to 3 This invention provides a method for quantitative detection of circularized-rolling-circular amplified miRNA using fluorescence, comprising the following steps: (1) Circulation reaction: using T4 RNA ligase to covalently link the 3'-OH end and the 5'-phosphate end of the miRNA to be tested to form a circular miRNA; (2) Rolling-circular amplification reaction: using the circular miRNA as a template, adding DNA primers complementary to the miRNA sequence, and using phi29 DNA polymerase to perform isothermal rolling-circular amplification reaction to generate a single-stranded DNA amplification product containing repetitive sequence units; adding a universal molecular beacon to the rolling-circular amplification reaction system, wherein the molecular beacon hybridizes with the repetitive sequence units in the rolling-circular amplification product and releases a fluorescence signal; (3) Fluorescence detection and quantitative analysis: monitoring the change in fluorescence intensity in real time, and calculating the concentration of miRNA in the sample to be tested based on the time when the fluorescence signal reaches the threshold or the comparison of the fluorescence intensity with the standard curve.

[0032] Figure 1 This is a schematic diagram of the technical process of the miRNA fluorescence quantitative detection method of the present invention, which shows the complete process from the target miRNA through the cyclization reaction, rolling circle amplification reaction to the detection of fluorescence signal. Figure 2 This is a schematic diagram of the molecular mechanism of the circularization-rolling circle amplification principle of the present invention, which shows in detail the molecular process of T4 RNA ligase catalyzing miRNA circularization, phi29 DNA polymerase performing rolling circle amplification, and molecular beacons hybridizing with amplification products to emit fluorescence. Figure 3 This is a schematic diagram of the structure of the universal molecular beacon of the present invention; it shows the stem-ring structure of the molecular beacon, the positions of the fluorescent group and the quenching group, and the conformational changes before and after binding with the target.

[0033] Example 1: Kit composition, detection method of the present invention, and standard operating procedure of the kit (I) Composition of the reagent kit of the present invention 1. First component: Reagents required for the cyclization reaction Includes: T4 RNA ligase (5 U / μL, New England Biolabs), 10×T4 RNA ligase buffer (500 mM Tris-HCl), and cyclized helper oligonucleotides (10 μM, designed according to the miRNA to be tested); mix the above reagents before use, aliquot into nuclease-free centrifuge tubes, and store at -20°C. 2. Second component: Reagents required for rolling circle amplification reaction Includes: phi29 DNA polymerase (10 U / μL, Thermo Fisher), 5×phi29 DNA polymerase reaction buffer (250 mM Tris-HCl), dNTP mixture (2.5 mM each, total 10 mM), DNA primers complementary to the miRNA sequence (10 μM, random / specific optional), universal molecular beacons. Store at -20°C. Mix all reagents before use. The sequence of the universal molecular beacon is 5'-FAM-CGCGAGAACACAGCACGCG-DABCYL-3', with a concentration of 10 μM; dissolved in TE buffer (pH 8.0) and stored at -20°C protected from light.

[0034] 3. Third component: Positive control miRNA standard Synthetic standards containing miR-21 (or miR-16, etc., internal reference) are available in concentration series from 100 pM to 0.1 fM; dissolved in nuclease-free water, dispensed in small portions at -80℃, avoiding repeated freeze-thaw cycles. 4. Fourth component: Auxiliary reagents Includes RNase inhibitor (40 U / μL), nuclease-free water, and negative control (miRNA-free aqueous solution).

[0035] 5. Fifth Component: Supporting Consumables 0.2 mL optically clear 8-tube / 96-well PCR plate, RNase-free pipette tips, instruction manual and quality inspection report.

[0036] (II) Standard Operating Procedures for the Detection Method and Reagent Kit of the Invention The detailed standard operating procedure for miRNA detection using the kit of this invention is as follows: 1. Sample preparation Plasma / serum samples: Separate within 4 hours of blood collection, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, aliquot, and store at -80℃. Thaw at room temperature before use. Tissue samples: Immediately freeze fresh tissue in liquid nitrogen or immerse in RNA protection solution and store at -80℃. Extract total RNA according to standard methods before use. Cell samples: Collect cells, wash with PBS, lyse directly, or extract total RNA. Urine / saliva samples: Centrifuge to remove impurities, collect the supernatant for RNA extraction, or use directly.

[0037] 2. RNA extraction (if required) Use a commercially available RNA extraction kit (such as Qiagen, Thermo Fisher, etc.). Follow the instructions, and finally dissolve the RNA in 10-50 μL of nuclease-free water. Measure the RNA concentration and purity (OD260 / 280 should be between 1.8 and 2.0).

[0038] 3. Cycling reaction (1) Prepare the reaction system (20 μL) on ice: 2-5 μL of RNA sample to be tested (containing 1-100 ng total RNA or 1-10 μL plasma extract), 2 μL of 10×T4 RNA ligase buffer, 1 μL (5 U) of T4 RNA ligase, 1 μL (10 μM) of cyclized helper oligonucleotide, and nuclease-free water to a final volume of 20 μL; (2) Gently mix and then centrifuge briefly; (3) Incubate at 16℃ for 20 minutes (using a metal bath or PCR instrument); (4) Inactivate the enzyme by heating at 65℃ for 5 minutes; (5) Cool on ice or proceed directly to the next step.

[0039] 4. Rolling ring amplification reaction (1) Prepare the reaction system (25 μL) on ice: 5 μL of cyclization product (which can be diluted 1-10 times as needed), 5 μL of 5×phi29 DNA polymerase buffer, 1.25 μL of dNTP mixture (10 mM), 1 μL (10 U) of phi29 DNA polymerase, 1 μL (10 μM) of DNA primer complementary to the miRNA sequence, 0.5 μL (10 μM) of universal molecular beacon, and nuclease-free water to a final volume of 25 μL; (2) Gently mix and then centrifuge briefly; (3) Transfer to the optical tube or plate of the real-time PCR instrument; (4) Program settings: maintain a constant temperature of 35℃ for 45 minutes, and read the fluorescence value every 30 seconds; (5) After the reaction is complete, melting curve analysis can be performed: 65℃ for 30 seconds, then increase the temperature to 95℃ at a rate of 0.5℃ / 5 seconds, and continuously read the fluorescence value.

[0040] 5. Data Analysis (1) Derive the fluorescence amplification curve and melting curve; (2) Set the fluorescence threshold (usually 10 times the background fluorescence or set automatically by the software); (3) Read the Tt value of each sample; (4) Calculate the absolute or relative concentration of miRNA using a standard curve or the ΔΔCt method; perform standardization correction using an internal reference miRNA (such as miR-16).

[0041] 6. Quality Control (1) Each batch of tests should include: a positive control (a miRNA standard of known concentration), a negative control (a water or RNA sample without miRNA), and an internal control; (2) Check whether the Tt value of the positive control is within the expected range (deviation <10%). (3) Check whether the negative control has no obvious amplification signal (Tt>40 or no Tt value); (4) Check if the CV value of the technique repeatability is <5%; (5) Check whether the melting curve shows a single peak to confirm the product specificity.

[0042] Example 2: Optimization Experiment of Reaction Conditions for the Detection Method of the Present Invention In order to determine the optimal cyclization and rolling ring amplification reaction conditions, key parameters were optimized in this embodiment.

[0043] 1. Optimization of cyclization reaction (1) Standard cyclization reaction system (20 μL): containing 2 μL (10 pM) of the miRNA to be tested, 2 μL of 10×T4 RNA ligase buffer, 1 μL (5 U) of T4 RNA ligase, 1 μL (10 μM) of cyclization helper oligonucleotide, and the remainder is made up with nuclease-free water.

[0044] (2) Optimization direction and evaluation index: The fluorescence signal intensity and threshold time are used as evaluation indexes. The temperature (5 gradients such as 4℃, 16℃, etc.), time (5 gradients such as 10min, 15min, etc.), ATP concentration (5 gradients such as 0.1mM, 0.5mM, etc.) and whether or not cyclized auxiliary oligonucleotides are added are optimized. After the reaction, the enzyme is inactivated by heating at 65℃ for 5min before subsequent detection.

[0045] (3) Key optimization results: Temperature: 16-20℃ is the most efficient, with 16℃ being the best recommended. Time: 15-20 minutes yields the best results; extending the reaction time provides no gain and may even reduce it. ATP concentration: 0.5-2 mM is highly efficient, with 1 mM being optimal; Helper oligonucleotides: Addition increases efficiency by 3-5 times, enhances fluorescence signal, and shortens threshold time by 2-3 cycles.

[0046] (4) Standard cyclization reaction conditions: 16℃ reaction for 20 minutes, ATP concentration of 1mM, and addition of cyclization auxiliary oligonucleotides.

[0047] 2. Optimization of Rolling Ring Amplification Reaction (1) Experimental design: Using the cyclized miR-21 product of Example 2 as a template, a 25 μL reaction system was prepared: 5 μL of cyclized product, 5 μL of 5×phi29 DNA polymerase buffer, optimized concentration of dNTP mixture, 1 μL (10 U) of phi29 DNA polymerase, 1 μL (10 μM, containing both random and specific) DNA primers complementary to the miRNA sequence, 0.5 μL (10 μM) of universal molecular beacon, and nuclease-free water to make up to 25 μL.

[0048] (2) Parameter optimization results: Temperature: Amplification efficiency is high at 30-37℃, and fluorescence signal is strongest at 33-35℃; both excessively high and low temperatures will inhibit enzyme activity, with the optimal temperature being 33-35℃. Time: Fluorescence signals reach a high level after 40-50 minutes; extending to 60 minutes does not result in significant increases. Considering overall detection efficiency, the optimal time is determined to be 45 minutes. dNTP concentration: 0.2-1 mM yields good results, with 0.5 mM offering the best cost-effectiveness; both excessively high and low concentrations have drawbacks, and the optimal concentration is 0.5 mM (for each type). Mg²⁺ concentration: 5-15 mM results in high amplification efficiency, with 10 mM yielding the best results; abnormal concentrations can lead to limited enzyme activity or non-specific amplification. The optimal concentration is 10 mM. Primer type: Random primers have strong universality but slightly higher background, while specific primers have high amplification efficiency and low background; random primers are selected for multiplex detection, while specific primers are selected for high-sensitivity detection of single miRNA.

[0049] (3) Standard reaction conditions: 35℃ for 45 minutes, dNTP concentration 0.5 mM, Mg²⁺ concentration 10 mM, primer type selected as needed.

[0050] Example 3: Design and Validation of Universal Molecular Beacons

[0051] 1. Design principles of universal molecular beacons Stem region: A 5-8 bp self-complementary sequence is used, preferably with high GC content to ensure stability while avoiding interference with target hybridization due to excessive structural stability. The stem region sequence of this invention is 5'-CGCG...CGCG-3', 6-7 bp in length, with a melting temperature (Tm) of 55-65℃; The loop region, 18-22 nt in length, is complementary to the repeat unit of the rolling circle amplification product. The design must meet three requirements: high sequence specificity and no cross-hybridization; GC content of 40%-60% to match the hybridization temperature; and no self-secondary structure or dimer formation. Fluorescent / Quenching Groups: The 5' end is labeled with a fluorescent group (such as FAM, ROX, Cy3, Cy5), and the 3' end is labeled with a quenching group (such as DABCYL, BHQ1, BHQ2). The core requirement is that the spectra of the two groups fully overlap to achieve efficient quenching. Versatility: By analyzing the sequence characteristics of multiple miRNAs through bioinformatics, and identifying the conserved common sequences of the rolling circle amplification products of circularly targeted miRNAs, a single molecular beacon can be used to detect multiple miRNAs.

[0052] 2. Specific sequence Universal molecular beacon sequence: 5'-FAM-CGCGAGAACACAGCACGCG-DABCYL-3' The bolded portion is the stem region sequence, and the middle fragment is the loop recognition sequence complementary to the amplified product.

[0053] 3. Performance Verification (1) Specificity: The test detected fully complementary, single-base mismatch, triple-base mismatch, and non-complementary target DNA. The fluorescence signal of fully complementary targets was 100%, the signal of single-base mismatches decreased to 30%-40%, the signal of triple-base mismatches was less than 10%, and the signal of non-complementary sequences was close to the background level (<5%), showing good specificity. (2) Sensitivity: Detection of target DNA at concentrations ranging from 10 pM to 1 fM showed a significantly higher fluorescence signal than the background at 1 fM concentration, with a good linear relationship between signal intensity and target concentration (R² = 0.995); (3) Stability: The molecular beacon was stored at -20℃, 4℃, and room temperature. At -20℃, there was no significant performance change after 3 months; at 4℃, performance decreased by less than 10% after 1 month; and at room temperature, performance decreased by approximately 20% after 1 week. Storage at -20℃, protected from light, is recommended. (4) Universality: It can detect miRNA rolling circle amplification products such as miR-21, miR-155, miR-210, and let-7a, and all produce good fluorescence signals, thus having universal detection capability.

[0054] Example 4: Establishment of the Standard Curve This embodiment uses a series of synthetic miR-21 standards at various concentrations to establish a standard curve for quantitative detection, such as... Figure 4 As shown.

[0055] 1. Experimental steps: S1. Prepare a series of miR-21 standards at concentrations of 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, and 0.1 fM, with three replicates for each concentration; S2. Perform cyclization and rolling circle amplification reactions according to the standard operating procedures of the detection method and kit of the present invention in Example 1; S3. Monitor fluorescence signals in real time and record the fluorescence intensity change curve of each sample over time; S4. Determine the fluorescence threshold (usually set to 10 times the mean background fluorescence), and calculate the time required for the fluorescence signal of each sample to reach the threshold (Threshold Time, Tt), similar to the Ct value in qPCR; S5. The logarithm of miR-21 concentration (log 10 Plot a standard curve with [concentration] as the x-axis and Tt as the y-axis.

[0056] 2. Experimental Results: (1) The fluorescence amplification curves of miR-21 at different concentrations showed typical S-shaped curves. The higher the concentration, the earlier the curve appeared and the smaller the Tt value. (2) The standard curve shows the Tt value versus log 10 There is a good linear relationship between [concentration] and [concentration], and the linear regression equation is: Tt = -3.42 × log 10 [concentration] + 25.6; the correlation coefficient R² = 0.998, indicating a very good linear relationship. Amplification efficiency calculation: based on the slope of the standard curve, the amplification efficiency E = 10^(-1 / slope) - 1 = 10^(-1 / -3.42) - 1 = 96.5%, close to the ideal 100%, indicating that the amplification reaction is highly efficient and stable; (3) Detection limit: At a concentration of 0.1 fM, all three replicate samples showed obvious fluorescence signals and the coefficient of variation (CV) of the Tt value was <5%, thus the detection limit of this method was determined to be 0.1 fM (equivalent to approximately 60 molecules / reaction system). (4) Linear range: from 0.1 fM to 100 pM, spanning 6 orders of magnitude, the standard curves all maintain a good linear relationship, proving that this method has a wide linear detection range; (5) Reproducibility: The standard deviation of Tt values ​​of 3 replicates at each concentration point is <0.3 and CV is <3%, indicating that the method has good reproducibility.

[0057] Table 1. Detection results of miR-21 standards at different concentrations. miR-21 concentration Tt value (mean ± SD) CV(%) 100 pM 12.3±0.2 1.6 10 pM 15.7±0.3 1.9 1 pM 19.1±0.2 1.0 100 fM 22.5±0.4 1.8 10 fM 25.9±0.5 1.9 1 fM 29.3±0.6 2.0 0.1 fM 32.8±1.0 3.0

[0058] This embodiment verifies the ability of the method of the present invention to distinguish miRNAs with highly similar sequences, especially miRNA family members that differ by only a single base.

[0059] 1. Experimental Design: (1) Three members of the let-7 family were selected: let-7a, let-7b, and let-7c. Their sequences are highly similar, differing by only 1-2 bases. let-7a: 5'-UGAGGUAGUAGGUUGUAUAGUU-3'; let-7b: 5'-UGAGGUAGUAGGUUGUGUGGUU-3'; let-7c: 5'-UGAGGUAGUAGGUUGUAUGGUU-3' (2) Prepare three synthetic miRNA standards at equal concentrations (10 pM). Detection was performed according to the detection method and kit standard operating procedure of the present invention in Example 1, with three replicates for each miRNA. (3) Compare the fluorescence amplification curves and Tt values ​​of the three miRNAs.

[0060] 2. Experimental Results: (1) All three miRNAs were successfully detected and produced obvious fluorescent signals; (2) The fluorescence amplification curves were clearly separated, and the Tt values ​​of the three miRNAs were significantly different: let-7a: Tt = 15.8±0.3; let-7b: Tt = 19.2±0.4; let-7c: Tt = 16.5±0.3; ΔTt between let-7a and let-7b = 3.4, ΔTt between let-7a and let-7c = 0.7, and ΔTt between let-7b and let-7c = 2.7; (3) Based on the amplification efficiency, the concentration difference corresponding to ΔTt = 3.4 is about 10-fold, which proves that this method can clearly distinguish these highly similar miRNAs. (4) Further analysis revealed that the difference in Tt value was related to the subtle differences in miRNA circularization and amplification efficiency, which may be related to the secondary structure of the sequence and the sequence preference of ligase and polymerase.

[0061] Figure 5 This is a verification diagram of the single-base difference discrimination capability of the present invention, showing the detection results of three miRNAs, let-7a, let-7b and let-7c, which differ only in a single base. The fluorescence signal curves of the three miRNAs are clearly separated, and the threshold time difference ΔCt≥3, proving that the method of the present invention can accurately distinguish miRNAs with highly similar sequences.

[0062] This experiment demonstrates that the method of the present invention has extremely high sequence specificity and can accurately distinguish miRNAs with only a single base difference, which is difficult to achieve with many traditional methods (such as Northern blot and microarray hybridization).

[0063] Example 6: Detection of miRNAs in clinical plasma samples This embodiment demonstrates the application of the method of the present invention in actual clinical samples, detecting the expression level of miR-21 in the plasma of liver cancer patients and healthy controls. The detection method is the same as the standard operation used in the kit in Example 1.

[0064] 1. Sample Source: Peripheral blood samples were collected from 10 patients with hepatocellular carcinoma (pathologically confirmed) and 10 age-matched healthy volunteers. Plasma was separated within 4 hours after blood collection, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at -80℃ for later use.

[0065] 2. RNA extraction: Total RNA was extracted from 200 μL of plasma using a commercial RNA extraction kit (such as Qiagen miRNeasySerum / Plasma Kit); the extracted RNA was dissolved in 20 μL of nuclease-free water.

[0066] 3. miRNA detection: 5 μL of extracted RNA was used for circularization and rolling circle amplification (using the standard operating procedure of the detection method and kit of the present invention in Example 1); three technical replicates were set for each sample; the internal control miRNA (miR-16) was detected at the same time for data standardization; the absolute concentration was calculated using the miR-21 standard curve.

[0067] 4. Experimental Results: miR-21 and miR-16 signals were successfully detected in all samples, with no detection failures. The plasma miR-21 concentration range was 5-20 fM in the healthy control group and 50-500 fM in the hepatocellular carcinoma (HCC) patient group. Statistical analysis showed that the miR-21 expression level in the HCC patient group was significantly higher than that in the healthy control group (P < 0.001, Mann-Whitney U test). After standardization using miR-16 as an internal reference, the miR-21 / miR-16 ratio in the HCC patient group was 8-15 times that in the healthy control group. ROC curve analysis showed that the AUC (area under the curve) for using miR-21 as a diagnostic marker to distinguish between HCC patients and healthy individuals was 0.92. When the cutoff value was set at 30 fM, the sensitivity was 85% and the specificity was 90%.

[0068] Table 2. Detection results of some samples Sample number Group miR-21 concentration (fM) miR-16 concentration (fM) miR-21 / miR-16 ratio HC-1 healthy 8.5 120 0.071 HC-2 healthy 12.3 135 0.091 HC-3 healthy 6.8 98 0.069 BC-1 patient 156 142 1.099 BC-2 patient 234 128 1.828 BC-3 patient 89 115 0.774 BC-4 patient 412 156 2.641 Figure 6 The following is an example of the clinical application of the present invention, showing the detection results of miR-21 expression levels in the plasma of 10 liver cancer patients and 10 healthy controls. The miR-21 expression in the liver cancer patient group was significantly higher than that in the healthy control group (P<0.001), which verifies the application value of the method of the present invention in clinical diagnosis.

[0069] This experiment demonstrates that the detection method of the present invention can be successfully applied to the detection of clinical plasma samples and has good clinical diagnostic value.

[0070] Example 7: Method Performance Evaluation This embodiment systematically evaluated various performance indicators of the method of the present invention, as detailed below: 1. Sensitivity (Limit of Detection): Using serially diluted miR-21 standards, the lowest concentration that could produce a significantly higher signal than the background (signal-to-noise ratio > 3) was determined. Results: The limit of detection was 0.1 fM, equivalent to approximately 60 molecules / 25 μL reaction system.

[0071] 2. Linearity Range: A standard curve was established in the range of 0.1 fM to 100 pM to evaluate the linear relationship. Results: The linear range spanned 6 orders of magnitude, with R² = 0.998, demonstrating excellent linearity.

[0072] 3. Intra-batch precision: Samples of the same concentration were tested 10 times within the same batch, and the CV value was calculated. Results: The intra-batch CV for 10 pM miR-21 was 2.8%, and the intra-batch CV for 1 pM was 3.5%.

[0073] 4. Inter-batch precision: The CV value was calculated for testing the same sample from different batches (3 different dates). Results: The inter-batch CV for 10 pM miR-21 was 4.2%, and the inter-batch CV for 1 pM was 5.1%.

[0074] 5. Accuracy (Recovery): Different amounts of miR-21 standard were added to plasma samples of known concentrations, and the recovery rate was calculated after detection. Results: The spiked recovery rate was 95-108%, with an average of 102%, demonstrating the high accuracy of the method.

[0075] 6. Specificity: When detecting let-7a standard, 10-fold concentrations of let-7b, let-7c, or other miRNAs were added to observe whether cross-reactivity occurred. Results: The presence of other miRNAs did not affect the detection of let-7a (bias <5%), demonstrating the good specificity of the method.

[0076] 7. Comparison with the gold standard method: miR-21 in 30 plasma samples were detected simultaneously using the method of this invention and a commercial RT-qPCR kit (TaqMan method). Results: The detection results of the two methods were highly correlated (Pearson correlation coefficient r = 0.94, P < 0.001), but the method of this invention had a lower limit of detection (0.1 fM vs 1 fM), shorter detection time (2.5 hours vs 4.5 hours), and lower cost (approximately 50%).

[0077] 8. Stability: The reaction system was tested after being placed at room temperature for different times to evaluate reagent stability. Results: The cyclization reaction mixture showed no significant performance change after 2 hours at room temperature, and the rolling circle amplification mixture remained stable after 1 hour at room temperature. It is recommended to store reagents on ice or at 4°C and prepare them fresh for optimal results.

[0078] Example 8: Clinical application case of the method of the present invention 1. Early diagnosis of tumors: Application scenario: The method of this invention can be used to detect the expression level of tumor-associated miRNAs (such as miR-21, miR-155, miR-210, etc.) in plasma to assist in the early diagnosis of tumors.

[0079] Case Study: In a study involving 50 patients with early-stage liver cancer and 50 healthy controls, plasma miR-21 expression was detected using the method of this invention. The results showed that plasma miR-21 levels were significantly elevated in patients with early-stage liver cancer. Using 30 fM as the diagnostic threshold, the sensitivity reached 82%, the specificity reached 88%, and the AUC was 0.90, demonstrating good diagnostic value.

[0080] 2. Monitoring of drug efficacy: Application scenario: Monitoring changes in circulating miRNA levels before and after treatment in cancer patients to assess treatment efficacy.

[0081] Case Study: Blood samples were collected from 20 patients with colorectal cancer who received chemotherapy to measure miR-21 levels before treatment, during treatment (after the second cycle), and after treatment. Results showed that patients who responded to treatment experienced a significant decrease in miR-21 levels (average decrease of 65%), while patients who did not respond to treatment or whose disease progressed showed no significant change or even an increase in miR-21 levels, suggesting that miR-21 can serve as a dynamic biomarker for monitoring treatment efficacy.

[0082] 3. Exosomal miRNA analysis: Application scenario: Isolating exosomes from plasma and analyzing the miRNAs they carry for liquid biopsy.

[0083] Case Study: Exosomes were isolated from the plasma of cancer patients (ultracentrifugation), and exosomal RNA was extracted and detected using the method of this invention. Due to the extremely low levels of miRNA in exosomes, the ultra-high sensitivity (0.1 fM) of this method ensures accurate detection. Studies have found that exosomal miR-21 levels are closely related to tumor metastasis and can serve as a prognostic indicator.

[0084] In summary, this invention provides an innovative miRNA detection technology that achieves highly sensitive (0.1 fM), highly specific (single-base differentiation), rapid (2-3 hours), and economical miRNA quantitative analysis by organically combining circularization-rolling circle amplification and universal molecular beacon detection. This method has demonstrated excellent performance in clinical sample testing and shows broad promise in clinical applications such as early tumor diagnosis and treatment monitoring, and is expected to become an important technical means for liquid biopsy and precision medicine.

Claims

1. A method for quantitative detection of circularized-rolling-circular amplified miRNA using fluorescence, characterized in that, Includes the following steps: (1) Circulation reaction: The 3'-OH end and 5'-phosphate end of the target miRNA are covalently linked by T4 RNA ligase to form a circular miRNA; (2) Rolling circle amplification reaction: Using the circular miRNA as a template, DNA primers complementary to the miRNA sequence are added, and isothermal rolling circle amplification reaction is performed using phi29 DNA polymerase to generate a single-stranded DNA amplification product containing repetitive sequence units; a universal molecular beacon is added to the rolling circle amplification reaction system, and the molecular beacon releases a fluorescent signal after hybridization with the repetitive sequence units in the rolling circle amplification product; (3) Fluorescence detection and quantitative analysis: The fluorescence intensity change is monitored in real time, and the concentration of miRNA in the target sample is calculated based on the time when the fluorescence signal reaches the threshold or the comparison of the fluorescence intensity with the standard curve.

2. The detection method according to claim 1, characterized in that, The cyclization reaction system includes the RNA sample to be tested, T4 RNA ligase, and T4 RNA ligase buffer. The cyclization reaction conditions are: reaction temperature 16-20℃, reaction time 15-25 minutes; and ATP concentration in the reaction system is 0.5-2 mM.

3. The detection method according to claim 1, characterized in that, The cyclization reaction is enhanced by adding a cyclization auxiliary oligonucleotide, which is complementary to the partial sequence of the miRNA to be tested and has a length of 10-25 nucleotides.

4. The detection method according to claim 1, characterized in that, The rolling circle amplification reaction system includes: cyclization reaction product, phi29 DNA polymerase buffer, phi29 DNA polymerase, dNTP mixture, DNA primers complementary to the miRNA sequence, and universal molecular beacons; the rolling circle amplification reaction conditions are: reaction temperature 33-35℃; reaction time 40-50 minutes; dNTP concentration in the reaction system is 0.2-1 mM; magnesium ion concentration is 5-15 mM.

5. The detection method according to claim 1 or 4, characterized in that, The DNA primers complementary to the miRNA sequence can be random primers or specifically designed short primers.

6. The detection method according to claim 1 or 4, characterized in that, The universal molecular beacon has a stem-loop structure, comprising: a stem region consisting of 5-10 base pairs; a loop region of 15-25 nucleotides in length that is complementary to the repetitive sequence unit in the rolling circle amplification product; a 5' fluorescent group and a 3' quencher group.

7. The detection method according to claim 1, characterized in that, The detection performance indicators are as follows: detection limit ≤ 0.1 fM; linear detection range from 0.1 fM to 100 pM; detection repeatability coefficient of variation CV < 5%; able to distinguish miRNAs with single-base differences, differential cycle threshold ΔCt ≥ 3.

8. The detection method according to claim 1, characterized in that, It is suitable for the detection of miRNAs in various biological samples, including plasma, serum, urine, saliva, tissue homogenate, cell lysate, and exosome extract.

9. A miRNA fluorescence quantitative detection kit, characterized in that, It includes the following components: (a) Reagents required for cyclization reaction: including T4 RNA ligase, T4 RNA ligase buffer, and cyclization helper oligonucleotides; (ii) Reagents required for rolling circle amplification reaction: including phi29 DNA polymerase, phi29 DNA polymerase buffer, dNTP mixture, DNA primers complementary to miRNA sequence, and universal molecular beacons; (iii) Positive control miRNA standard; The sequence of the universal molecular beacon is 5'-FAM-CGCGAGAACACAGCACGCG-DABCYL-3', which produces good fluorescence signals for rolling circle amplification products of multiple miRNAs such as miR-21, miR-155, miR-210, and let-7a, and has universal detection capability.

10. The detection kit according to claim 9, characterized in that, It also includes RNase inhibitors, nuclease-free water, miRNA standards at various concentrations, and / or negative control solutions.

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