Probe system and method for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex

Abstract

The invention provides a probe system and method for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex, and belongs to the technical field of molecular probe fluorescence sensing. Target miRNA-driven PER amplification, PER product-induced G tetramer conformational change and G-Quadruplex-ThT signal'lightening 'mechanism are organically combined to detect the AKI-related miRNA. A low-background label-free signal amplification system is constructed for accurate detection of target miRNA, has the characteristics of high sensitivity, strong specificity, simplicity in operation and the like, and still keeps good performance in an actual sample. Besides, by designing the dumbbell-shaped hairpin probe HP, specific detection of different targets can be realized, and the establishment of the technical platform provides a new method for research and development of a kit for simultaneously realizing rapid detection of disease biomarkers such as small molecules, nucleic acid, protein and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular probe fluorescence sensing, and particularly relates to a probe system and method for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex. Background Art

[0002] Acute kidney injury (AKI), a common clinical complication characterized by a rapid and rapid decline in renal function, is associated with a variety of other high-mortality conditions and is a significant contributor to increased mortality and healthcare costs. AKI can be caused by a variety of factors, including sepsis, severe dehydration, surgical bleeding, cirrhosis, ischemia-reperfusion injury, nephrotoxic medications, and urinary tract obstruction. AKI is common among hospitalized patients worldwide. According to the International Society of Nephrology (ISN), the incidence of AKI in hospitalized patients ranges from 14.7% to 31.5%, with specific figures varying depending on population, region, research methodology, and healthcare setting. The World Health Organization (WHO) 2019 global statistics indicate that over 13 million people develop acute kidney injury annually, with the incidence of AKI in intensive care units reaching 30%-60%. According to ISN research, the mortality rate within one week of AKI in low- and middle-income countries is as high as 12%. Another ISN study explored the identification and management of AKI in resource-poor settings. The results showed a 15.8% mortality rate following AKI, with most AKI patients being young, without prior medical history, or major comorbidities. ISN-related studies indicate that even milder AKI symptoms can lead to adverse long-term outcomes, such as proteinuria, hypertension, or chronic kidney disease (CKD). For example, up to a quarter of patients with community-acquired AKI may not fully recover and eventually develop CKD. The high morbidity and mortality rates of acute kidney injury make it a major global public health challenge, posing a significant threat to patients' lives and health, incurring a significant economic burden, and placing a severe test on the healthcare system.

[0003] Traditional diagnostic criteria for AKI include: an absolute increase in serum creatinine >26 μmol / L (0.3 mg / dL) within 48 hours; a 50-99% increase in serum creatinine (1.50-1.99 × baseline) over 7 days; or a decrease in urine output to less than 0.5 mL / kg per hour for 6 hours or longer. However, due to the mechanism of Scr production, the Scr increase after AKI is delayed, making it difficult to diagnose and intervene in the early stages of kidney damage. Furthermore, Scr levels may be affected by drugs that compete with the renal tubular transport mechanism (organic cation transporters), such as trimethoprim, dronedarone, pyrimethamine, salicylates, and cimetidine; chemotherapy drugs such as olaparib, imatinib, and bosutinib; and antiretroviral drugs such as dolutegravir and cobicistat. This can lead to an overly high Scr level and an inaccurate overdiagnosis of AKI. In addition, the Scr content in different individuals is also affected by gender, age, dietary habits, body muscle content, etc. Therefore, the development of a timely and specific detection method is important for the diagnosis and prognosis of AKI.

[0004] In recent years, a number of potential early markers of AKI have garnered significant attention, including kidney injury molecule-1 (KIM-1), angiotensin-converting enzyme 2 (ANG-2), N-acetyl-β-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), troponin, and serum cystatin C. As essential regulators of diverse pathophysiological processes, miRNAs have also been implicated in the development and treatment of AKI. Dysregulated miRNA expression plays a crucial role in the progression of AKI and is a key regulator of a range of signaling cascades involved in the pathogenesis of renal disease both in vivo and in vitro. When AKI develops, elevated miRNA levels store mRNA transcripts in processing bodies, inhibiting mRNA translation and leading to downstream protein translation blockade. Therefore, abnormal miRNA levels specifically expressed in renal tissue and the periphery could serve as novel biomarkers for the diagnosis of renal disease, potentially enabling non-invasive diagnosis of AKI through liquid biopsy techniques. These results offer greater clinical relevance than traditional diagnostic targets.

[0005] The primer exchange reaction (PER) is a signal amplification technology that can autonomously synthesize and assemble single-stranded DNA molecules according to a pre-set reaction pathway and sequence. Signal amplification or controlled synthesis of single-stranded DNA products is achieved through strand displacement between a designed DNA hairpin primer (typically 7-9 bases) and an auxiliary DNA strand. The long single-stranded DNA products generated by PER can contain multiple repeating sequences, which can be designed to bind dyes to produce fluorescent signals or serve as efficient reporter molecules or pre-amplifiers for subsequent detection. Thioflavin T (ThT) is a commonly used water-soluble fluorescent dye whose fluorescence signal changes closely with binding to specific structures. When free in solution, ThT exhibits very weak fluorescence (almost no signal) because its excited-state energy is dissipated non-radiatively due to intramolecular rotation. ThT is embedded in the terminal tetrad plane or groove region of the G-quadruplex through electrostatic interaction and π-π stacking. Its molecular rotation is restricted, non-radiative decay is suppressed, and fluorescence is significantly enhanced. By utilizing the "turn-on" or "light-up" property of ThT molecules, G-Quadruplex-ThT can be used as an activation signal molecule to construct a label-free fluorescent biosensing strategy for target miRNA detection.

[0006] Currently, proteins such as neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), matrix metalloproteinase-9 (MMP-9), cystatin C (CysC), sodium-hydrogen exchanger 3 (NHE3), and kidney injury molecule-1 (KIM-1) in the blood are often considered biomarkers for the early diagnosis of AKI. However, their timeliness is poor, and their content and distribution are easily affected by the internal environment. Compared with protein biomarkers, RNA biomarkers have high specificity for predicting AKI and can show characteristic abnormalities in the early stages of AKI. However, the low abundance of miRNAs in body fluids makes them difficult to detect with high sensitivity. Early identification and intervention are not only key to kidney protection but also an important strategy for improving overall prognosis. Therefore, the development of new, highly specific and sensitive miRNA detection methods is of great significance for the early prevention and treatment of AKI. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a probe system and method for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex, aiming to use target-induced primer exchange reaction to obtain DNA repeat sequences with downstream signal activation, provide more initiators for the "lighting up" of ThT signal molecules, and establish a rapid, label-free miRNA detection technology platform; this technology can realize primer exchange-repeat sequence generation-signal activation reaction, which can effectively simplify the experimental operation process, reduce background signals and improve detection sensitivity. The nucleic acid detection platform technology is used to develop a constant temperature detection technology for AKI-specific miRNA.

[0008] To achieve the above objectives, this solution first provides a probe system for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex, wherein the probe system includes a hairpin probe HP, a hairpin probe Primer, Bst DNA polymerase, a hairpin probe M1 and K + In the presence of target miRNA, the target miRNA specifically binds to the hairpin probe HP to detect the target miRNA; The sequence of the hairpin probe HP is shown in SEQ ID NO. 1; The sequence of the hairpin probe Primer is shown in SEQ ID NO. 2; The sequence of the hairpin probe M1 is shown in SEQ ID NO. 3; The sequence of the target miRNA is shown in SEQ ID NO. 4.

[0009] Based on a general inventive concept, this solution also provides a method for detecting acute kidney injury miRNA using a probe system for non-diagnostic purposes, comprising the following steps: S1. Probe pretreatment: Prepare the synthesized hairpin probe HP, Primer, and M1 lyophilized powder into 100 μM stock solutions with DEPC water and store at 4°C until use. S2. Annealing of hairpin probes: Anneal the hairpin probes HP and M1 at 95°C for 5 min and then slowly cool them to room temperature to allow them to self-assemble into hairpin structures through complementary base pairing. Store them in a refrigerator at 4°C until use. S3. Target-activated primer exchange reaction: First, incubate the miRNA to be tested with 1 µM hairpin probe HP. Once the miRNA to be tested is fully bound to the hairpin probe HP, add the hairpin probe Primer and react at 37°C for 30 min. Then, add Bst DNA polymerase, reaction materials dATP, dTTP, dCTP, and 1× Bst Reaction Buffer required for the primer exchange reaction. React at 65°C for 1 h, then incubate at 80°C for 2 min to inactivate the enzyme. S4. G-tetramer structure formation: After the primer exchange reaction in step S2 is completed, hairpin probe M1 and K are added to the system. + , forming a G-tetramer structure; S5. Signal activation and fluorescence detection of G-Quadruplex-ThT: Add 40 µM Thioflavin T to the system prepared in step S4, react at 37°C in PBS buffer for 30 min, and collect the fluorescence spectrum using an RF-6000 fluorescence spectrophotometer.

[0010] Preferably, the incubation temperature in step S3 is 37° C., and the incubation time is 30 min.

[0011] Preferably, the pH of the 1× Bst Reaction Buffer in step S3 is 8.8, containing 20 mM Tris-HCl, 10 mM (NH4 )2 SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100.

[0012] Preferably, the PBS buffer in step S5 has a pH of 7.4 and contains 50 mM KCl.

[0013] Preferably, in step S5, the fluorescence spectrophotometer is set to an excitation wavelength Ex=450 nm, an emission wavelength Em=465-600 nm, the excitation and emission slit widths are set to 5 nm and 5 nm respectively, and the voltage is set to 700 V.

[0014] Preferably, the sample to be tested for miRNA in step S3 is urine.

[0015] The mechanism of this probe in detecting AKI: This protocol designed a dumbbell-shaped hairpin structure, HP. Upon encountering its target, miR-21, miR-21 specifically binds to the hairpin at one end of HP, opening the loop and exposing the stem sequence a' (AATA AGA GAT CATC) complementary to the primer (a). Primer (a) binds to the HP (a') sequence through complementary base pairing, providing a site for the PER reaction. Subsequently, under the action of the strand-displacing DNA polymerase Bst, the primer is extended by replicating the b domain (ACT AAA TTCA). This extended primer uses the HP 5' stem region sequence b' (TGAA TTT AGT) as a template to produce the PER product, PER-P, with the ab sequence (GATG ATC TCT TATT ACT AAA TTCA). This process is terminated by three consecutive G bases introduced adjacent to the HP 5' b domain. The replicated b domain competes with the b domain on the hairpin probe HP through a random walk process of three-way branch migration. Finally, the replicated domain b is replaced, and the extended primer can spontaneously separate from the hairpin and release into the system. The "dumbbell-shaped" hairpin probe HP acts as a template to participate in the next round of PER cycle, generating a large number of ab sequences. The large number of products (ab) produced by the PER process can specifically open the G-rich sequence hairpin probe M1, exposing the originally closed G-rich sequence, and then in K + With the help of ThT, a highly folded G-tetramer structure is formed. ThT is embedded in its spatial structure to release a high-intensity fluorescent signal, thereby achieving accurate quantitative detection of AKI-specific miRNA in patient urine. This invention can achieve specific activation and label-free amplification detection of different target miRNAs by designing different "dumbbell-shaped" hairpin probes HP and primers.

[0016] The sensitivity and specificity of this detection system are reflected in: (1) High sensitivity: The “dumbbell-shaped” probe design and target-driven specific PER amplification provide more initiation sequences for downstream signal transduction, thereby improving the detection sensitivity. Using G-Quadruplex as a specific chimeric receptor for the ThT signal molecule, a G-Quadruplex-ThT signal “lighting up” model is constructed, which can significantly reduce the background signal and improve the detection signal-to-noise ratio.

[0017] (2) High specificity: The current AKI diagnosis relies on serum creatinine (Scr) and urine volume assessment, which have fundamental flaws, leading to delayed clinical diagnosis and increased risk of misdiagnosis. Serum creatinine has multiple limitations as a marker of renal function. PER has programmability, autonomous catalysis, and signal amplification capabilities. Using miRNA as a marker, it utilizes the principle of DNA base complementary pairing to achieve precise design and target-specific activation, and has single-base discrimination capabilities.

[0018] (3) Low cost: ThT is a water-soluble fluorescent dye. After interacting with DNA G-Quadruplex, the fluorescent signal is significantly enhanced. A label-free signal output model (G-Quadruplex-ThT) based on G-Quadruplex "lighting up" ThT can be constructed, which can effectively reduce experimental costs.

[0019] (4) Strong versatility: Specific "dumbbell-shaped" hairpin probes can be designed for different targets. Combined with G-Quadruplex-ThT activated by circulating PER products, this technology can achieve highly sensitive and label-free detection of different disease marker targets (such as proteins, nucleic acids, small molecules, ions, etc.). This technology system has strong versatility and a wide range of applications, and can effectively solve the problem of complex design of multiple detection reaction systems in conventional technologies.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This protocol innovatively uses miRNA in the urine of AKI patients as a promoter, and utilizes the primer exchange reaction PER to generate DNA products with multiple repeat sequences, providing multiple G tetramer binding sites for the activation of ThT signaling molecules. A PER-based G-Quadruplex-ThT activation signal amplification system is constructed, and a precise quantitative detection technology for AKI-specific miRNA based on patient urine is developed, which can achieve highly sensitive, label-free and rapid detection of AKI marker miRNA.

[0021] (2) This scheme innovatively combines the target miRNA-driven PER amplification, the PER product-induced G tetramer conformational change, and the G-Quadruplex-ThT signal "lighting up" mechanism to construct a low-background, label-free signal amplification system for the precise detection of target miRNA. It has the characteristics of high sensitivity, strong specificity, and simple operation, and still maintains good performance in actual samples. In addition, through the design of the "dumbbell-shaped" hairpin probe HP, specific detection of different targets (proteins, nucleic acids, small molecules, ions, etc.) can be achieved. The establishment of this technology platform provides a new method for the development of rapid detection kits for disease biomarkers such as small molecules, nucleic acids, and proteins.

[0022] (3) Compared with existing detection technologies, it has the advantages of being fast, accurate, economical, and independent of expensive equipment. It provides a promising solution for the field of AKI detection and is of great significance for improving the accuracy of AKI diagnosis and the precision of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a schematic diagram of the experimental principle of Example 1; Figure 2 The electrophoresis verification of probe assembly and feasibility analysis of PER reaction in Example 2; Figure 3 This is a feasibility analysis of the PER system and target response in Example 2; Figure 4 This is the target miRNA detection performance analysis in Example 3; Figure 5 This is a linear graph of the fluorescence response of target miRNA at different concentrations in Example 3; Figure 6 This is the specificity analysis of the PER system and different miRNA detection in Example 4; Figure 7 Comparison of fluorescence intensity at 520 nm after incubation of the PER system with different miRNAs in Example 4. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0026] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0027] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0028] The probes involved in the present invention were purchased from Shanghai Sangon Biotechnology Co., Ltd., as shown in Table 1:

[0029] Example 1 A probe system for detecting AKI-related miRNAs based on primer exchange reaction and G-quadruplex.

[0030] (1) Probe pretreatment: Prepare the lyophilized powders of the synthesized hairpin probes HP, Primer, M1, and miR-21 into 100 μM stock solutions using DEPC water and store them at 4 °C for later use.

[0031] (2) Annealing of hairpin probes: Anneal the hairpin probes HP and M1 at 95 °C for 5 min and then slowly cool them to room temperature to allow them to self-assemble into a hairpin structure through base complementary pairing. Store them in a refrigerator at 4 °C for later use.

[0032] (3) Target activation PER reaction: First, the miRNA to be tested was incubated with 1 μM hairpin probe HP at 37 °C for 30 min. If the miRNA to be tested was the target miR-21, the miRNA would fully bind to the hairpin probe HP. Then, the hairpin probe Primer (a) was added and reacted at 37 °C for 30 min to allow it to bind to the a' domain on HP. Then, the Bst enzyme required for the PER reaction, the reaction raw materials dNTPs (dATP, dTTP, dCTP) and the buffer required for the enzyme (1× Bst Reaction Buffer, pH 8.8, containing 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100) were added. The reaction was carried out at 65 °C for 1 h. The enzyme was then inactivated by keeping it at 80 °C for 2 min. Finally, the feasibility of target activation was verified by polyacrylamide gel electrophoresis.

[0033] (4) Formation of G-tetramer structure: After the PER reaction is completed, a large amount of PER product (ab) reacts with the G-rich hairpin probe M1. Ab can specifically bind to the hairpin probe M1 and open the hairpin structure that was originally combined with its own reverse complement, exposing the G-rich sequence that was originally masked by the hairpin structure. + In the presence of ions, a G-tetramer structure can be formed at one end of M1, and the binding of PER product to M1 can be verified by polyacrylamide gel electrophoresis.

[0034] (5) Signal activation and fluorescence detection of G-Quadruplex-ThT: 40 μM ThT was added to the above system and reacted at 37 °C for 30 min in PBS buffer (pH 7.4, 50 mM KCl). The fluorescence spectrum of the target miRNA-activated PER reaction was collected using an RF-6000 fluorescence spectrophotometer. The excitation wavelength Ex = 450 nm, the emission wavelength Em = 465-600 nm, the excitation and emission slit widths were set to 5 nm and 5 nm, respectively, and the voltage was set to 700 V.

[0035] Detection mechanism Figure 1 shown.

[0036] Example 2 DNA probe assembly and PER feasibility analysis were investigated.

[0037] 12% polyacrylamide gel electrophoresis (PAGE) was used to investigate the DNA probe assembly and PER process. Figure 2 As shown, lane 1 is a DNA marker, lane 2 is miR-21, lane 3 is the PER-P chain, lane 4 is the M1 probe, and lane 5 is PER-P + M1. As the molecular weight increases, it can be seen that the PER product ab sequence can bind to the hairpin probe M1. Lane 6 is the HP probe, lane 8 is a 1:1 mixture of HP and target miR-21 assembled at 37°C for 30 minutes, and lane 7 is a 1:1 mixture of HP, target, and primer assembled at 37°C for 30 minutes. The results show that miR-21 and HP can assemble to form a stable double-stranded probe structure. Furthermore, with the addition of the primer sequence, the molecular weight gradually increases, indicating that the HP probe assembles well with the target and primer, forming a sandwich structure that triggers the PER reaction.

[0038] Subsequently, a fluorescence spectrophotometer was used to investigate the PER reaction initiated by the target miR-21. Utilizing the characteristic that the fluorescence of the PER product activated DNAG tetramer (split-G-Quadruplex) and interacted with ThT, a label-free fluorescence signal output mode (G-Quadruplex-ThT) was constructed in which G-Quadruplex "lit up" ThT. The results were as follows: Figure 3As shown in the figure, ThT molecules have almost no fluorescence signal in the solution. However, when the target miR-21 is present, the primer-mediated PER reaction can be initiated, thereby activating the G tetramer structure. The fluorescence signal is significantly enhanced after co-incubation with ThT molecules. When the target miR-21 is not present, the PER reaction cannot proceed, but M1, as a hairpin structure rich in G sequences, can bind to ThT and show a certain background signal, but the overall fluorescence intensity is quite different from that of the experimental group, indicating that the system has the feasibility of responding to the target miR-21 and is expected to be used for label-free and specific detection of related target miRNAs.

[0039] Example 3 PER and target response performance analysis were investigated.

[0040] In order to evaluate the responsiveness of the PER system to the target miR-21 molecule in detail, we investigated the fluorescence response behavior of the PER system with different concentrations of miR-21 (10 nM, 50 nM, 100 nM, 200 nM, 250 nM, 300 nM, 500 nM). Figure 4 As shown in Figure 5, the fluorescence signal gradually increased with increasing miR-21 concentration, showing a linear relationship within the range of 10-500 nM. The linear regression equation was y = 3.77x + 3602, with a linear correlation coefficient R² = 0.98. The results indicate that the method can achieve sensitive detection of the target miR-21.

[0041] Example 4 Investigate the target specificity analysis of the PER system.

[0042] To investigate the specificity of the PER system for detecting miR-21 among AKI-related miRNAs, we selected different miRNAs highly expressed in the urine of AKI patients: miR-24 (SEQ ID NO. 8), miR-155 (SEQ ID NO. 9), and miR-200b (SEQ ID NO. 10), as well as target miR-21 with different base mismatches: single-base mismatch SmiR-21 (SEQ ID NO. 5), double-base mismatch DmiR-21 (SEQ ID NO. 6), and triple-base mismatch TmiR-21 (SEQ ID NO. 7), and examined the detection specificity of the PER system in detail. The results are shown in Figure 2. Figure 6 As shown in Figure 7, when the target miR-21 was present, the fluorescence intensity of the detection system was significantly higher than that of the control group, and it had the ability to distinguish single-base mismatches, indicating that the PER system had good selectivity and specificity for the target miR-21.

[0043] The above is only a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiment. For those skilled in the art, improvements and modifications obtained without departing from the technical concept of the present invention should also be considered as the scope of protection of the present invention.

Claims

1. A probe system for detecting AKI-related miRNA based on primer exchange reaction and G-quadruplex, characterized in that: The probe system includes a hairpin probe HP, a hairpin probe Primer, Bst DNA polymerase, a hairpin probe M1 and K + In the presence of target miRNA, the target miRNA specifically binds to the hairpin probe HP to detect the target miRNA; The sequence of the hairpin probe HP is shown in SEQ ID NO. 1; The sequence of the hairpin probe Primer is shown in SEQ ID NO. 2; The sequence of the hairpin probe M1 is shown in SEQ ID NO. 3; The sequence of the target miRNA is shown in SEQ ID NO.

4.

2. A method for detecting acute kidney injury miRNA using the probe system according to claim 1 for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Probe pretreatment: Prepare the synthesized hairpin probe HP, Primer, and M1 lyophilized powder into 100 μM stock solutions with DEPC water and store at 4°C until use. S2. Annealing of hairpin probes: Anneal the hairpin probes HP and M1 at 95°C for 5 min and then slowly cool them to room temperature to allow them to self-assemble into hairpin structures through complementary base pairing. Store them in a refrigerator at 4°C until use. S3. Target-activated primer exchange reaction: First, incubate the miRNA to be tested with 1 µM hairpin probe HP. Once the miRNA to be tested is fully bound to the hairpin probe HP, add the hairpin probe Primer and react at 37°C for 30 min. Then, add Bst DNA polymerase, reaction materials dATP, dTTP, dCTP, and 1× Bst Reaction Buffer required for the primer exchange reaction. React at 65°C for 1 h, and then incubate at 80°C for 2 min to inactivate the enzyme. S4. G-tetramer structure formation: After the primer exchange reaction in step S2 is completed, hairpin probe M1 and K are added to the system. + , forming a G-tetramer structure; S5. Signal activation and fluorescence detection of G-Quadruplex-ThT: Add 40 µM Thioflavin T to the system prepared in step S4. The mixture was reacted at 37°C in PBS buffer for 30 min. The fluorescence spectrum was collected using an RF-6000 fluorescence spectrophotometer.

3. The method according to claim 2, characterized in that The incubation temperature in step S3 is 37° C., and the incubation time is 30 min.

4. The method according to claim 2, characterized in that The pH of the 1× Bst Reaction Buffer in step S3 is 8.8, and it contains 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , and 0.1% Triton X-100.

5. The method according to claim 2, characterized in that The PBS buffer in step S5 has a pH of 7.4 and contains 50 mM KCl.

6. The method according to claim 2, characterized in that In step S5, the fluorescence spectrophotometer is set to an excitation wavelength Ex=450 nm, an emission wavelength Em=465-600 nm, an excitation slit width and an emission slit width of 5 nm and 5 nm respectively, and a voltage of 700 V.

7. The method according to claim 2, characterized in that The sample to be tested for miRNA in step S3 is urine.

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