Method and kit for detecting miRNA-21 based on split-trigger recombination autocatalytic DNA cascade circuit

Abstract

The application belongs to the field of biosensing detection, and relates to a method and a kit for detecting miRNA-21 based on split-triggered recombination autocatalytic DNA cascade circuit. The detection method comprises the following steps: after being heated and denatured, hairpin probes H1, H2, H3 and H4 are slowly cooled to room temperature, then mixed with a to-be-detected solution, and then buffer solution and deionized water are added, and constant temperature incubation is carried out to obtain a reaction system I; hairpin probes Y1, Y2 and Y3 are added, constant temperature incubation is carried out to obtain a reaction system II; MgCl2 solution and a fluorescence reporter probe S are further added, and incubation is carried out in the dark, the obtained solution is diluted with deionized water, the fluorescence signal intensity is detected by a fluorescence spectrometer, and the concentration of miRNA-21 is obtained by substituting into a linear equation. The application does not need to participate in enzyme, and only needs a single constant temperature (37 DEG C) to complete the reaction, and has the advantages of low detection limit (as low as 33.4 fM), strong specificity, low background signal and good applicability of complex matrix.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and detection, and relates to a method for ultrasensitive detection of miRNA-21. Background Technology

[0002] Cancer, as a major global public health issue, seriously threatens human life and health. Abnormal expression of microRNAs (miRNAs) is closely related to the occurrence and development of various tumors, and their accurate detection is crucial for guiding clinical diagnosis and treatment of cancer. miRNA-21 is one of the most widely studied miRNAs in the fields of tumor molecular biology and clinical diagnostics. It exhibits abnormally high expression in various tumors, including breast cancer, lung cancer, cervical cancer, liver cancer, and colorectal cancer, and demonstrates good stability in bodily fluids such as serum, plasma, and urine, making it an ideal biomarker for clinical tumor diagnosis. However, miRNA-21 is characterized by its short sequence and extremely low abundance, posing a significant challenge to accurate quantification in low-concentration detection scenarios. Therefore, the development of highly sensitive and specific detection methods is still needed.

[0003] Currently, nucleic acid detection amplification technologies are mainly divided into two categories: enzyme-catalyzed amplification and enzyme-free amplification. Enzyme-catalyzed amplification, such as rolling circle amplification, ligase chain reaction, and loop-mediated isothermal amplification, has stringent requirements for the reaction environment and is prone to signal leakage and false positives. Enzyme-free amplification, such as catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), can avoid the defects of enzyme-catalyzed reactions, but single amplification strategies still have insufficient sensitivity and cannot meet the needs of accurate detection of low-abundance miRNAs. To improve detection performance, researchers have integrated various enzyme-free amplification strategies to construct cascaded DNA circuits. Among them, catalytic cascade circuits can drive the assembly of nanostructures through target-triggered cycles, but traditional cascade systems generally suffer from defects such as weak driving force, slow kinetics, and limited amplification efficiency.

[0004] DNAzymes, as catalytically active DNA oligonucleotides, possess advantages such as strong catalytic ability, structural stability, and ease of cascading with other signal transduction mechanisms, and are often used for biosensor signal output. Application CN119372320A discloses an APE1-mediated cascade signal amplification strategy for miRNA-21 detection. This amplification strategy requires the addition of the APE1 enzyme, and how to achieve self-catalytic cyclic signal amplification without enzyme addition remains a research challenge in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method and kit for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit, overcoming the shortcomings of existing miRNA detection technologies such as insufficient sensitivity, poor specificity, and limited amplification efficiency.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for detecting miRNA-21 based on a self-catalytic DNA cascade circuit triggered by cell division and recombination, comprising the following steps:

[0008] (1) Hairpin probes H1, H2, H3, and H4 were heat-denatured and then slowly cooled to room temperature. They were then mixed with the test solution, and buffer solution and deionized water were added. After constant temperature incubation, reaction system I was obtained. The nucleotide sequence of H1 in step (1) is shown in SEQ ID No. 1, the nucleotide sequence of H2 is shown in SEQ ID No. 2, the nucleotide sequence of H3 is shown in SEQ ID No. 3, and the nucleotide sequence of H4 is shown in SEQ ID No. 4. The concentration ratio of hairpin probes H1, H2, H3, and H4 in reaction system I is 1:1:1:1. The volume ratio of hairpin probes H1, H2, H3, H4, buffer solution, and deionized water is 1:1:1:1:8:2. The heat denaturation temperature in step (1) was 95℃ and the time was 5 min. The constant temperature incubation temperature was 37℃ and the time was 120 min.

[0009] (2) Hairpin probes Y1, Y2, and Y3 were added to reaction system I and incubated at a constant temperature to obtain reaction system II. The nucleotide sequence of Y1 in step (2) is shown in SEQ ID No. 5, the nucleotide sequence of Y2 is shown in SEQ ID No. 6, and the nucleotide sequence of Y3 is shown in SEQ ID No. 7. The concentration ratio of hairpin probe H1 in reaction system I to hairpin probes Y1, Y2, and Y3 in reaction system II is 1:1:1.5:1.5. The volume ratio of reaction system I to hairpin probes Y1, Y2, and Y3 is 15:1:1:1. The temperature for constant temperature incubation in step (2) is 37°C and the time is 180 min.

[0010] (3) Add MgCl2 solution and fluorescent reporter probe S to reaction system II, incubate in the dark, and dilute the resulting solution with deionized water to obtain the test solution; the concentration ratio of hairpin probe Y1 to MgCl2 and fluorescent reporter probe S in reaction system II in step (3) is 5:1:5; the nucleotide sequence of the fluorescent reporter probe S is shown in SEQ ID No.8, with -Cy3- linked between the 8th and 9th bases, -rA- linked between the 11th and 12th bases, and -BHQ2 linked at the 3' end. The incubation in step (3) is carried out at a temperature of 37℃ for 60 min.

[0011] (4) The fluorescence signal intensity of the test solution was detected by a fluorescence spectrometer, and the concentration of miRNA-21 was obtained by substituting it into the linear equation. In step (4), the excitation wavelength of the fluorescence spectrometer was 520 nm, the emission wavelength was 530 nm, and the acquisition range was 540~630 nm; the linear equation was y = 16.507lgC (miRNA-21) +107.38, R 2 =0.9916; where x is the concentration of the target miRNA-21 and y is the measured fluorescence signal value.

[0012] The specific operating steps are as follows:

[0013] (1) Hairpin probes H1, H2, H3 and H4 were denatured at 95 °C for 5 min and then slowly cooled to room temperature. They were then mixed with standard solutions of miRNA-21 at different concentrations in reaction tubes, and 10× CutSmart buffer system was added. The mixture was incubated at 37 °C for 120 min to complete the autocatalytic hybridization chain reaction (AHCR).

[0014] (2) Add the pretreated hairpin probes Y1, Y2 and Y3 to the reaction system of step (1), and continue to incubate at 37 °C for 180 min to complete the autocatalytic hairpin assembly (ACHA) and form a Y-type DNA structure.

[0015] (3) Keep the reaction system at a constant temperature of 37 °C, add MgCl2 solution and fluorescent reporter probe S to step (2), and incubate in the dark for 60 min so that the DNAzyme exposed in the Y-type DNA structure can catalyze the cleavage of the fluorescent reporter probe with the assistance of Mg²⁺.

[0016] (4) Add deionized water to the solution in step (3) to make up to 200 μL. Use an FL 7000 fluorescence spectrometer to detect the fluorescence signal intensity. Set the excitation wavelength to 520 nm, the emission wavelength to 530 nm, and the signal acquisition range to 540~630 nm.

[0017] (5) Plot a standard curve with the logarithm of the concentration of miRNA-21 standard solution as the abscissa and the corresponding fluorescence signal intensity as the ordinate, and establish a linear regression equation.

[0018] (6) Using miRNA-155, miRNA-200c, miRNA-141, miRNA-let 7a, and Pax-5a as interfering sequences, parallel experiments were conducted according to steps (1) to (4), and the selectivity of the method was examined by comparing the fluorescence signal response values.

[0019] (7) Prepare simulated actual samples by mixing 1% human serum with different concentrations of miRNA-21, and verify the detection performance of the method in complex matrices by following the above steps.

[0020] Preferably, the concentrations of hairpin probes H1, H2, H3, and H4 are all 10 μM, with a molar ratio of 1:1:1:1; the concentrations of hairpin probes Y1, Y2, and Y3 are 10 μM, with a molar ratio of 1:1.5:1.5; the concentration gradient of the miRNA-21 standard solution is 0 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM; the 10× CutSmart buffer consists of 200 mM Tris-Ac, 500 mM Kac, 100 mM MgAc2, and 1 g / mL BSA, with a pH of 7.9.

[0021] Preferably, the concentration of the MgCl2 solution is 2 mM, and the fluorescent reporter probe S is a Cy3 / BHQ2 double-labeled oligonucleotide chain with the sequence 5'-CCCCGGAG / Cy3 / CGG / rA / TGGCCGAGGCTGG / BHQ2 / -3', at a concentration of 10 μM. The probe cleavage site contains RNA bases (Ra).

[0022] Preferably, fluorescence detection is performed using an FL 7000 fluorescence spectrometer with an excitation wavelength of 520 nm, an emission wavelength of 530 nm, and a collection range of 540~630 nm.

[0023] Preferably, the concentrations of the interfering sequences miRNA-155, miRNA-200c, miRNA-141, let-7a, and Pax-5a are all 10 μM.

[0024] Preferably, the incubation time for the AHCR reaction is 120 min; the incubation time for ACHA assembly is 180 min; and the incubation time for DNAzyme-catalyzed cleavage of the fluorescent reporter probe in the dark is 60 min.

[0025] The detection principle in this invention is as follows: The target miRNA-21 acts as a promoter, opening the H1 hairpin and sequentially initiating the hybridization chain reaction of H2, H3, and H4. A splitting CHA trigger chain is generated at the 5′ end of H2 and the 3′ end of H4. This trigger chain activates the Y1 hairpin, which assembles with Y2 and Y3 to form a Y-shaped DNA structure. The target-mimicking sequences on Y2 and Y3 are spatially close, reconstructing a complete HCR initiation sequence, which in turn activates a new round of AHCR, forming an AHCR-ACHA autocatalytic feedback loop, achieving exponential signal amplification. Simultaneously, the exposed sequences of Y1 and Y3 form an active DNA enzyme under the action of Mg²⁺, cyclically cleaving the fluorescent reporter probe and releasing a strong fluorescent signal. Without a target, the hairpin probes coexist stably, without triggering, assembly, or signal output. This invention achieves ultrasensitive, highly specific, and highly interference-resistant detection of miRNA-21 through an integrated design of enzyme-free isothermal function, self-catalytic feedback, and Y-type DNAzyme signal output. It maintains excellent performance in complex biological samples and is suitable for the accurate detection of low-abundance nucleic acid biomarkers in clinical settings.

[0026] The present invention has the following beneficial effects:

[0027] 1. This invention constructs an enzyme-free autocatalytic feedback cascade circuit. Through the synergistic cycle of autocatalytic hybridization chain reaction (AHCR) and autocatalytic hairpin assembly (ACHA), it achieves exponential amplification of target signals, effectively overcoming the sensitivity bottleneck of traditional amplification techniques. This type of autocatalytic DNA circuit uses separation-triggered recombination as its core, forming a self-feedback cycle through the reverse catalysis of the initial reaction by the product, converting trace targets into significantly enhanced fluorescence readings. It maintains high specificity and stability even in complex biological samples, providing a novel technical pathway for the ultrasensitive detection of low-abundance miRNAs and early disease diagnosis.

[0028] 2. This invention achieves exponential signal amplification through a bidirectional feedback loop of autocatalytic hybridization chain reaction and autocatalytic hairpin assembly, significantly improving detection sensitivity. It requires no enzyme participation and only a single isothermal temperature (37 ℃) to complete the reaction. It boasts advantages such as a low detection limit (as low as 33.4 fM), high specificity, low background signal, and excellent applicability to complex matrices. It enables precise quantitative detection of trace nucleic acid biomarkers and is widely used in early tumor diagnosis, clinical testing, and biosensor technology. Employing an enzyme-free isothermal detection system, requiring only a 37 ℃ reaction throughout, it eliminates the need for complex instruments and expensive reagents. It offers advantages such as simple operation, low background signal, good stability, and high specificity, effectively distinguishing the target from various interfering sequences.

[0029] 3. This invention achieves exponential signal amplification and highly specific fluorescence detection of the target microRNA-21 (miRNA-21) by constructing a dual-module autocatalytic feedback system integrating autocatalytic hybridization chain reaction (AHCR) and autocatalytic hairpin assembly (ACHA). It maintains excellent detection performance in complex biological matrices such as 1% human serum, possesses good anti-interference capabilities and practical application potential, and has strong circuit programmability, allowing for expansion to the detection of various nucleic acid biomarkers, thus having a wide range of applications. Attached Figure Description

[0030] 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 of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of a self-catalytic DNA cascade circuit based on cell division-triggered recombination and its application in programmable nucleic acid biosensing.

[0032] Figure 2 The fluorescence spectrum is the result of measuring the experimental scheme to verify its feasibility, as determined in Example 1.

[0033] Figure 3 The results are the detection results of the standard sample according to Example 1; A: Fluorescence spectrum corresponding to the fluorescence intensity and the concentration of the target substance in Example 1; B: Linear graph of fluorescence intensity and the concentration of the target substance, with the inset being a broken line graph of fluorescence intensity and the logarithm of the concentration of the target substance.

[0034] Figure 4 This is the result of selective investigation in Example 1.

[0035] Figure 5 The results are for the actual sample; A: Fluorescence spectrum of the actual sample measured with respect to the concentration of the target analyte; B: Linear relationship between the peak value of the fluorescence spectrum and the logarithm of the target gene concentration, with the inset showing a broken line graph of fluorescence intensity versus the logarithm of the target analyte concentration. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0038] The nucleotide sequence involved in this invention is as follows:

[0039] Example

[0040] The detection method for miRNA-21 based on the autocatalytic DNA cascade circuit triggered by cell division and recombination includes the following steps:

[0041] (1) Hairpin probes H1, H2, H3, and H4 were heated at 95 °C for 5 min and then slowly cooled to room temperature to form a stable hairpin structure. 1 μL of 10 μM H1, 1 μL of 10 μM H2, 1 μL of 10 μM H3, 1 μL of 10 μM H4, 1 μL of miRNA-21 standard solution of different concentrations, 8 μL of ultrapure water, and 2 μL of 10× CutSmart buffer were added to a centrifuge tube in sequence, mixed well, and incubated at 37 °C for 120 min to complete the autocatalytic hybridization chain reaction (AHCR).

[0042] (2) Add 1 μL of 10 μM Y1, 1 μL of 15 μM Y2 and 1 μL of 15 μMY3 to the reaction system of step (1), mix well, and continue to incubate at 37 °C for 180 min to complete the autocatalytic hairpin assembly (ACHA) and form a Y-type DNA structure.

[0043] (3) Add 1 μL of 2 mM MgCl2 solution and 1 μL of 10 μM fluorescent reporter probe S to the system in step (2), and incubate at 37 °C for 60 min under light-protected conditions to allow the DNAzyme in the Y-shaped structure to catalyze the cleavage of the fluorescent reporter probe; (4) Add 180 μL of deionized water to the reaction solution, mix thoroughly, and then use an FL 7000 fluorescence spectrometer to detect the fluorescence signal. The excitation wavelength is 520 nm, the emission wavelength is 530 nm, and the acquisition range is 540~630 nm.

[0044] Figure 1This is a schematic diagram of the detection principle in this embodiment. Using the target miRNA-21 as a promoter, the H1 hairpin is opened, sequentially initiating the hybridization chain reaction of H2, H3, and H4. A splitting CHA trigger chain is generated at the 5′ end of H2 and the 3′ end of H4. The trigger chain activates the Y1 hairpin, which assembles with Y2 and Y3 to form a Y-shaped DNA structure. The target-mimicking sequences on Y2 and Y3 are spatially close, reconstructing a complete HCR initiation sequence, which in turn activates a new round of AHCR, forming an AHCR-ACHA autocatalytic feedback loop, achieving exponential signal amplification. Simultaneously, the exposed sequences of Y1 and Y3 form an active DNAzyme under the action of Mg²⁺, cyclically cleaving the fluorescent reporter probe and releasing a strong fluorescent signal. Without a target, the hairpin probes coexist stably, without triggering, assembly, or signal output. This invention achieves ultrasensitive, highly specific, and highly interference-resistant detection of miRNA-21 through an integrated design of enzyme-free isothermal function, self-catalytic feedback, and Y-type DNAzyme signal output. It maintains excellent performance in complex biological samples and is suitable for the accurate detection of low-abundance nucleic acid biomarkers in clinical settings.

[0045] Depend on Figure 2 As can be seen in the figure, curve a is the fluorescence spectrum measured by the reaction solution containing the target gene, curve b is the fluorescence spectrum measured by the reaction solution without the target gene, and curve c is the fluorescence spectrum measured by the reaction solution without Mg. 2+ The fluorescence spectra of the detection solution are shown in Figure d. Curve d represents the fluorescence spectrum of the detection solution that does not undergo the CHA reaction. Curve e contains only the S chain. Figure 2 It can be seen that the difference in fluorescence signal peak values ​​between the presence and absence of the target gene is significant, and the signal-to-noise ratio reaches nearly 3 times, verifying the feasibility of this experimental scheme.

[0046] Figure 3 A shows the fluorescence spectra of the standard sample as measured according to Example 1, corresponding to the concentration of the target analyte. Within the target gene concentration range of 100 fM to 100 nM, there is a good linear relationship between the peak value of the fluorescence spectrum and the logarithm of the target gene concentration, with the linear regression equation being y = 16.507lgC. (miRNA-21) +107.38, detection limit as low as 33.4 fM ( Figure 3 B).

[0047] Example 2

[0048] The detection method for miRNA-21 based on the autocatalytic DNA cascade circuit triggered by cell division, and the exploration of its specificity and selectivity, are as follows:

[0049] (1) Hairpin probes H1, H2, H3, and H4 were heated at 95 °C for 5 min and then slowly cooled to room temperature to form a stable hairpin structure. 1 μL of 10 μM H1, 1 μL of 10 μM H2, 1 μL of 10 μM H3, and 1 μL of 10 μM H4 were added to centrifuge tubes, along with 1 μL of 10 μM miRNA-155, miRNA-200c, miRNA-141, let-7a, and Pax-5a standard solutions, 8 μL of ultrapure water, and 2 μL of 10× CutSmart buffer. The mixtures were then incubated at 37 °C for 120 min to complete the autocatalytic hybridization chain reaction (AHCR).

[0050] (2) Add 1 μL of 10 μM Y1, 1 μL of 15 μM Y2 and 1 μL of 15 μMY3 to the reaction system of step (1), mix well, and continue to incubate at 37 °C for 180 min to complete the autocatalytic hairpin assembly (ACHA) and form a Y-type DNA structure.

[0051] (3) Add 1 μL of 2 mM MgCl2 solution and 1 μL of 10 μM fluorescent reporter probe S to the system in step (2), and incubate at 37 °C for 60 min under light-protected conditions to allow the DNAzyme in the Y-shaped structure to catalyze the cleavage of the fluorescent reporter probe; (4) Add 180 μL of deionized water to the reaction solution, mix thoroughly, and then use an FL 7000 fluorescence spectrometer to detect the fluorescence signal. The excitation wavelength is 520 nm, the emission wavelength is 530 nm, and the acquisition range is 540~630 nm.

[0052] Considering practical requirements, the specificity and selectivity of this method for specific targets were examined. The effects of miRNA-155, miRNA-200c, miRNA-141, let-7a, and Pax-5a as interfering agents on the sensing system were investigated. Experimental results confirmed that the sensing system showed low or almost no response to different interfering components, but a significant response to specific targets, indicating good selectivity and specificity. Figure 4 ).

[0053] Application examples

[0054] Spiked samples were prepared using 1% human serum (volume fraction), and the above steps were followed to verify the detection performance of the method in actual samples.

[0055] The testing steps are as follows:

[0056] (1) Hairpin probes H1, H2, H3, and H4 were heated at 95 °C for 5 min and then slowly cooled to room temperature to form stable hairpin structures. 1 μL of 10 μM miRNA-21 was added to 1% human serum and reacted at 32 °C for 1 h. Then, 1 μL of 10 μM H1, 1 μL of 10 μM H2, 1 μL of 10 μM H3, 1 μL of 10 μM H4, 8 μL of ultrapure water, and 2 μL of 10× CutSmart buffer were added to centrifuge tubes, mixed, and incubated at 37 °C for 120 min to complete the autocatalytic hybridization chain reaction (AHCR).

[0057] (2) Add 1 μL of 10 μM Y1, 1 μL of 15 μM Y2 and 1 μL of 15 μMY3 to the reaction system of step (1), mix well, and continue to incubate at 37 °C for 180 min to complete the autocatalytic hairpin assembly (ACHA) and form a Y-type DNA structure.

[0058] (3) Add 1 μL of 2 mM MgCl2 solution and 1 μL of 10 μM fluorescent reporter probe S to the system in step (2), and incubate at 37 °C for 60 min under light-protected conditions to allow the DNAzyme in the Y-shaped structure to catalyze the cleavage of the fluorescent reporter probe; (4) Add 180 μL of deionized water to the reaction solution, mix thoroughly, and then use an FL 7000 fluorescence spectrometer to detect the fluorescence signal. The excitation wavelength is 520 nm, the emission wavelength is 530 nm, and the acquisition range is 540~630 nm.

[0059] Figure 5 A shows the fluorescence spectra of actual samples as measured in Example 1, corresponding to the fluorescence intensity and the concentration of the target analyte. Within the target gene concentration range of 500 fM - 100 nM, there is a good linear relationship between the peak value of the fluorescence spectrum and the logarithm of the target gene concentration, with a linear regression equation of y = 30.29lgC(miRNA-21) + 123.29, and a detection limit as low as 166.7 fM. Figure 5 (B) These findings make autocatalytic cascade circuits a highly sensitive and specific miRNA detection tool, paving the way for their application in complex biological environments. This method shows promising potential for identifying real samples.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit, characterized in that, The steps are as follows: (1) Hairpin probes H1, H2, H3 and H4 were thermally denatured and then slowly cooled to room temperature. They were then mixed with the solution to be tested, and then buffer solution and deionized water were added. After incubation at a constant temperature, reaction system I was obtained. (2) Add hairpin probes Y1, Y2 and Y3 to reaction system I, and incubate at a constant temperature to obtain reaction system II; (3) Add MgCl2 solution and fluorescent reporter probe S to reaction system II, incubate in the dark, and dilute the resulting solution with deionized water to obtain the test solution; (4) The fluorescence signal intensity of the test solution was detected by a fluorescence spectrometer, and the concentration of miRNA-21 was obtained by substituting it into the linear equation.

2. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 1, characterized in that: In step (1), the nucleotide sequence of H1 is shown in SEQ ID No. 1, the nucleotide sequence of H2 is shown in SEQ ID No. 2, the nucleotide sequence of H3 is shown in SEQ ID No. 3, and the nucleotide sequence of H4 is shown in SEQ ID No. 4; the concentration ratio of hairpin probes H1, H2, H3 and H4 in reaction system I is 1:1:1:1; the volume ratio of hairpin probes H1, H2, H3, H4, buffer solution and deionized water is 1:1:1:1:8:

2.

3. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 2, characterized in that: In step (1), the temperature for thermal denaturation is 95°C and the time is 5 min; the temperature for constant temperature incubation is 37°C and the time is 120 min.

4. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 2 or 3, characterized in that: In step (2), the nucleotide sequence of Y1 is shown in SEQ ID No. 5, the nucleotide sequence of Y2 is shown in SEQ ID No. 6, and the nucleotide sequence of Y3 is shown in SEQ ID No. 7; the concentration ratio of hairpin probe H1 in reaction system I to hairpin probes Y1, Y2, and Y3 in reaction system II is 1:1:1.5:1.5; the volume ratio of reaction system I to hairpin probes Y1, Y2, and Y3 is 15:1:1:

1.

5. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 4, characterized in that: In step (2), the constant temperature incubation is 37°C and the time is 180 min.

6. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 5, characterized in that: In step (3), the concentration ratio of hairpin probe Y1 to MgCl2 and fluorescent reporter probe S in reaction system II is 5:1:5; the nucleotide sequence of the fluorescent reporter probe S is shown in SEQ ID No.8, with -Cy3- linked between the 8th and 9th bases, -rA- linked between the 11th and 12th bases, and -BHQ2 linked at the 3' end.

7. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to any one of claims 1-5, characterized in that: In step (3), the incubation in the dark is carried out at a temperature of 37°C for 60 minutes.

8. The method for detecting miRNA-21 based on a cell division-triggered recombination autocatalytic DNA cascade circuit according to claim 7, characterized in that: In step (4), the excitation wavelength of the fluorescence spectrometer is 520 nm, the emission wavelength is 530 nm, and the acquisition range is 540~630 nm; the linear equation is y = 16.507lgC (miRNA-21) +107.38, R 2 =0.9916; where x is the concentration of the target miRNA-21 and y is the measured fluorescence signal value.

9. A detection kit based on the method described in any one of claims 1-8.

10. The reagent kit according to claim 9, characterized in that: The kit contains hairpin probes H1, H2, H3, H4, Y1, Y2, Y3 and fluorescent reporter probe S, as well as a 10×CutSmart buffer system and MgCl2 solution; The nucleotide sequence of H1 is shown in SEQ ID No. 1, the nucleotide sequence of H2 is shown in SEQ ID No. 2, the nucleotide sequence of H3 is shown in SEQ ID No. 3, and the nucleotide sequence of H4 is shown in SEQ ID No.

4. The nucleotide sequence of Y1 is shown in SEQ ID No. 5, the nucleotide sequence of Y2 is shown in SEQ ID No. 6, the nucleotide sequence of Y3 is shown in SEQ ID No. 7, and the nucleotide sequence of the fluorescent reporter probe S is shown in SEQ ID No.

8. The 8th and 9th bases are directly linked to -Cy3-, the 11th and 12th bases are directly linked to -rA-, and the 3' end is linked to -BHQ2.

Images