A method for detecting miRNA155 based on CRISPR / Cas12a and exponential rolling circle amplification

Abstract

The application discloses a method for detecting miRNA155 based on CRISPR / Cas12a and exponential rolling circle amplification, and belongs to the medical detection field.The dumbbell probe carrying two enzyme recognition sites is introduced as a template, and the operation is simple, and an annealing program is not needed.The miRNA155 is subjected to exponential rolling circle amplification (T-ERCA), further, the amplification product ssDNA of the T-ERCA reaction is recognized by CRISPR / Cas12a, and the trans-cleavage activity thereof is activated, an exogenous single-stranded signal probe is cut, and a fluorescent signal is released.The double amplification effect of T-ERCA and CRISPR / Cas12a ensures that the T-ERCA / Cas12a detection system has high sensitivity, the detection range is from 1fM to 5nM, and the detection limit is 0.31fM.The method provided by the application is simple in operation, large instruments are not needed, a new idea and theoretical basis are provided for the CRISPR / Cas sensor platform in clinical diagnosis and detection.

Description

TECHNICAL FIELD

[0001] The application relates to a method for detecting miRNA155 based on CRISPR / Cas12a and exponential rolling circle amplification, and belongs to the medical detection field. BACKGROUND

[0002] MiRNA is a small non-coding RNA composed of about 20-25 nucleotides and having a regulatory function, which is involved in regulating biological processes such as cell differentiation, apoptosis, proliferation and signal transduction through post-transcriptional inhibition by binding to the 3'UTR of the target mRNA, and plays its biological regulation function. Studies have shown that the abnormal expression of miRNA is closely related to the occurrence and development of human cancer, and the high expression of miRNA155 is directly related to the occurrence of breast cancer, and miRNA has been recognized as an effective biomarker for cancer diagnosis and prognosis. Therefore, it is crucial to rapidly and accurately detect miRNAs in disease diagnosis and pathological analysis. Generally, miRNAs have inherent characteristics such as short length, easy degradation, sequence similarity and low abundance, and it is still a challenge to quantitatively detect them with high stability, high sensitivity and high selectivity. The most commonly used nucleic acid detection method at present is the signal amplification technology based on polymerase chain reaction (PCR), that is, adding molecular beacons to the PCR system to release fluorescence by the combination of molecular beacons and PCR products. However, the detection technology based on PCR is highly dependent on expensive and precise thermal cycling equipment, and requires special technical personnel to operate, which limits the application of PCR technology in low-resource areas, making it difficult to realize real-time detection and instant diagnosis on site.

[0003] The isothermal amplification technology developed in recent years is the most commonly used signal amplification method for miRNA detection, which is simpler and more convenient than PCR technology in terms of actual operation and instrument requirements, and it is free from the dependence on thermal cycling equipment, showing its good application prospect in clinical and on-site rapid diagnosis. It includes rolling circle amplification (RCA), catalytic hairpin assembly technology (CHA), hybridization chain reaction (HCR), strand displacement amplification (SDA) and exponential amplification. Among them, the SDA method involves strong non-specific amplification, and the CHA and HCR amplification driven by entropy change has low efficiency, and the high amplification efficiency of RCA and exponential amplification makes it an effective miRNA detection amplification strategy. The CRISPR / Cas biosensor is a new type of molecular diagnostic technology that has appeared in recent years, which is itself a signal amplification system, and can construct a cascade amplification super-sensitive miRNA diagnosis platform combined with RCA isothermal amplification, which is particularly suitable for the detection of low-abundance miRNA. In addition, the recognition of nucleic acid by CRISPR / Cas has sequence dependence, which can accurately distinguish single-base mutant targets, has high resolution, and ensures the high specificity of complex sample detection.

[0004] However, in the prior art, when using rolling circle amplification (RCA) reaction, a padlock probe and a target to be detected and T4 ligase are used for amplification process, resulting in non-specific amplification and background signal generation, reducing the sensitivity and accuracy of the detection method. SUMMARY

[0005] To solve the above technical problems, the present application develops a new isothermal amplification strategy T-ERCA / Cas12a system for miRNA155 detection. Figure 1 The detection process of the T-ERCA / Cas12a system is shown, including T-ERCA amplification reaction and CRISPR / Cas12a recognition two parts. First, in the presence of the target (miRNA-155), the T-ERCA reaction is triggered, and due to the fact that the amplification template of the dumbbell probe contains two nicking endonuclease Nt.BbvCI recognition sites, exponential cycle amplification can be achieved with the assistance of Phi29 DNA polymerase, generating a large amount of ssDNA product. Subsequently, the ssDNA product can be recognized by Cas12a / crRNA, activating the trans-cleavage activity of Cas12a / crRNA, and continuously shearing the exogenous fluorescent probe to generate a fluorescent signal, further amplifying the signal. Therefore, based on the dual amplification effect of T-ERCA and CRISPR / Cas12a, high sensitivity detection of miRNA-155 can be achieved.

[0006] The present application provides a composition containing CRISPR / Cas12a protein, dumbbell probe carrying two nicking endonuclease recognition sites, crRNA, fluorescent signal probe, DNA polymerase, and nicking endonuclease.

[0007] In one embodiment, the nicking endonuclease recognizes and cuts the dumbbell probe.

[0008] In one embodiment, the nicking endonuclease is selected from one or more of Nt.BbvCI and / or Nb.BtsI.

[0009] In one embodiment, the nucleotide sequence of the dumbbell probe is shown in SEQ ID NO. 2.

[0010] In one embodiment, the 5' end of the dumbbell probe is modified by a phosphate group.

[0011] In one embodiment, the nucleotide sequence of the crRNA is shown in SEQ ID NO. 6.

[0012] In one embodiment, the nucleotide sequence of the fluorescent signal probe is shown in SEQ ID NO. 3, and the 5' end of the fluorescent signal probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0013] In an embodiment, the fluorescent group comprises HEX, FAM, ROX, FITC, Cy3 or Cy5, and the quenching group comprises BHQ1, BHQ2, BHQ3, TRMRA, MGBNFQ or DABCYL.

[0014] In an embodiment, the DNA polymerase comprises phi29 polymerase.

[0015] In an embodiment, the Cas12a protein is selected from one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.

[0016] The present application provides a detection kit for detecting miRNA155, which contains the above-mentioned composition.

[0017] The present application provides the use of the above-mentioned composition or the above-mentioned kit in detecting miRNA155, which is not for the purpose of diagnosis and treatment of diseases.

[0018] In an embodiment, the kit contains dNTP.

[0019] In an embodiment, the use comprises the following steps:

[0020] (1) Synthesis of dumbbell probe: using DNA ligase to close the dumbbell probe carrying two nicking endonuclease recognition sites.

[0021] (2) T-ERCA reaction: mixing the dumbbell probe synthesized in step (1), DNA polymerase, restriction endonuclease and dNTP for amplification reaction to obtain amplification product ssDNA.

[0022] (3) CRISPR / Cas12a recognition and fluorescence signal collection: mixing the ssDNA obtained in step (2) with nucleotide sequence crRNA shown in SEQ ID NO. 6, Cas12a protein and fluorescence signal probe for reaction.

[0023] In an embodiment, in step (1), the DNA ligase is T4 DNA ligase.

[0024] In an embodiment, in step (1), after the dumbbell probe solution and T4 DNA ligase buffer are treated at 95°C for 3 min, they are slowly cooled to room temperature for 20 min, T4 DNA ligase is added, and incubation is performed at 37°C for 1 h; exonuclease is added and incubated for 2 h; and finally, inactivation is performed at 80°C for 20 min.

[0025] In an embodiment, the exonuclease includes Exol and Exoll.

[0026] In an embodiment, in step (2), 20 μL of 0.4 μM closed-loop dumbbell probe, 0.4 U / μL phi29 polymerase, 0.3 U / μL restriction endonuclease, and 250 mM dNTP are subjected to an amplification reaction.

[0027] In an embodiment, in step (2), the amplification reaction is performed at 37°C for 100 min.

[0028] In an embodiment, in step (3), 4 μL of the amplification product ssDNA, 2 μL of 1 μM crRNA, 2 μL of 1 μM Cas12a, and 2 μL of a fluorescent signal probe are thoroughly mixed.

[0029] In an embodiment, in step (3), after the mixing reaction is performed at 37°C for 20 min, ddH2O is added, and the fluorescence spectrum and fluorescence signal intensity are collected by a fluorescence spectrophotometer (PerkinElmer),

[0030] In an embodiment, the excitation wavelength of the fluorescence spectrophotometer is 490 nm, and the emission wavelength is 560 nm.

[0031] Beneficial effects:

[0032] 1) T-ERCA introduces a dumbbell structure to amplify the template, and when the closed loop is connected, no additional padlock probe is needed. For amplification of the DNA template, self-pairing can be used to pull the connection ends together for T4 ligase closed loop connection. This reduces the amplification procedure and also avoids the non-specific amplification and background signal generation caused by the introduction of the padlock probe. In addition, the dumbbell structure amplification template closed loop process does not require a target miRNA as a connection probe. Compared with traditional RCA, T-ERCA avoids the problem of inaccurate detection and low sensitivity caused by miRNA degradation due to a long connection process.

[0033] 2) The dumbbell structure of T-ERCA amplification template carries two nicking endonuclease sites. The rolling circle amplification product will be cut at the nicking endonuclease site, and the product can be used as a rolling circle amplification primer for the next step of amplification, realizing exponential signal amplification. Compared with traditional RCA and other isothermal amplification, it has higher amplification efficiency and detection sensitivity.

[0034] 3) CRISPR / Cas12a itself is a signal amplification system, combined with T-ERCA, a cascade signal amplification system can be established to further improve the detection sensitivity. The amplification product ssDNA of T-ERCA reaction is recognized by CRISPR / Cas12a and activates its transcleavage activity to cut the exogenous single-stranded signal probe and release the fluorescence signal. The dual amplification effect of T-ERCA and CRISPR / Cas12a ensures that the T-ERCA / Cas12a detection system has high sensitivity, with a detection range from 1 fM to 5 nM and a detection limit of 0.31 fM.

[0035] 4) CRISPR / Cas12a transcleavage makes it have signal readout function, without the need for additional signal readout system, which simplifies the T-ERCA / Cas12a system and shortens the detection time.

[0036] 5) The experimental execution temperature of the CRISPR / Cas system combined with isothermal amplification technology is well matched, T-ERCA is easy to integrate, and single-tube non-opening detection can be realized, avoiding pollution and reducing the detection procedure, avoiding the use of thermal cycler instruments, and being suitable for deployment as a clinical POCT detection.

[0037] 6) Compared with traditional polymerase chain reaction, T-ERCA has the characteristics of mild reaction conditions and high amplification efficiency.

[0038] 7) T-ERCA amplification and CRISPR / Cas12a recognition have sequence dependence, and the T-ERCA / Cas12a detection system enhances the specificity of miRNA155 detection, which can accurately distinguish miRNA155 from its family homologous miRNA.

[0039] 8) Given the programmability of CRISPR / Cas12a, T-ERCA / Cas12a can be modified into a universal platform for detecting other miRNAs, providing new ideas and theoretical basis for CRISPR / Cas sensor platform in clinical diagnosis and detection. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1Schematic diagram of T-ERCA / Cas12a system detection. (A) The synthesis process of dumbbell probe (DP); (B) Schematic diagram of T-ERCA combined with CRISPR / Cas12 (T-ERCA / Cas12a) for detection of miRNA-155.

[0041] Figure 2 Verification of the feasibility of T-ERCA / Cas12a system. (A) Polyacrylamide gel electrophoresis (PAGE) analysis of crRNA; (B) Fluorescence response spectrum of T-ERCA / Cas12a system based on different target samples; (C) Polyacrylamide gel electrophoresis analysis of dumbbell probe (DP) and T-ERCA / Cas12a system.

[0042] Figure 3 Optimization of detection conditions. (A) Optimization of dumbbell probe (DP) concentration; (B) Optimization of amplification time; (C) Optimization of CRISPR / Cas12a cleavage time, in columnar chart A, left for control group, right for experimental group.

[0043] Figure 4 Detection performance of T-ERCA / Cas12a system. (A) Fluorescence spectrum of T-ERCA / Cas12a for different concentrations of miRNA-155 (1 fM to 5 nM); (B) Calibration curve of miRNA-155 concentration according to fluorescence change, linear relationship between fluorescence change and concentration logarithm; (C) Corresponding logarithmic calibration curve in the linear range of 1 fM to 50 pM; (D) Corresponding logarithmic calibration curve in the linear range of 0 pM to 5 nM.

[0044] Figure 5 Specificity, repeatability and stability of T-ERCA / Cas12a sensing system. (A, B) Fluorescence spectrum of different interfering substances, and corresponding fluorescence intensity; (C) Repeatability; (D) Stability.

[0045] Figure 6 Fluorescence response of T-ERCA / Cas12a detection to total RNA extracted from MCF-7 cells and L02 cells. (A, B) Fluorescence response of total RNA of two cell lines. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0047] The reagents involved in the following examples are as follows:

[0048] HiScribe T7 High Yield RNA Synthesis Kit, Exonuclease I (Exo I), Exonuclease III (Exo III), T4 DNA Ligase, phi29 Polymerase, NEBuffer 2.0 and Nicking Endonuclease Nt.BbvCI were purchased from New England BioLabs (Beijing, China). RNase-free H2O, miRcute miRNA Isolation and Purification Kit and RNAprep Kit were purchased from Tiangen (Beijing, China). Cas12a (Cpf1) was purchased from Guangzhou Meigao Biotechnology. TE buffer, DEPC-treated water, dNTP mixture and PAGE related reagents (Acryl / Bis 30% solution (29:1), nucleic acid dye, TBE buffer, DNA loading buffer, loading buffer and DNA marker) were purchased from Sangon Biotechnology (Shanghai, China). All DNA sequences were synthesized by Shanghai Shengong Bioengineering Co., Ltd. and purified by HPLC.

[0049] The relevant probes and oligonucleotide sequences involved in the following examples are shown in Table 1.

[0050] Table 1 Probes and oligonucleotide sequences used in the present application

[0051]

[0052] “p” represents a phosphate group modification.

[0053] Example 1 A detection method for detecting miRNA155 with high sensitivity and low background

[0054] Specifically, the following steps are included:

[0055] (1) crRNA synthesis and purification: 1 μL T7 promoter (100 μM), 1 μL crRNA template (100 μM) and 16 μL RNase-free H2O were mixed well and linearly annealed at 95 °C to room temperature for 20 min. Then, 10 μL NTP and 2 μL T7 RNA polymerase were added, and the mixture was incubated at 37 °C for 12 hours to transcribe a large amount of crRNA. Then, 2 μL DNase I was added and reacted at 37 °C for 2 hours to digest the DNA template. Finally, the crRNA was purified using the miRcute miRNA Isolation and Purification Kit and quantified using the NanoDrop 2000C (Thermo Fisher), and then stored at -20 °C for subsequent use.

[0056] (2) Synthesis of dumbbell probe: First, linear template dumbbell probe solution (TP, 40 mM, SEQ ID NO. 2) and T4 DNA ligase buffer were placed in a PCR machine, treated at 95 °C for 3 min, and then slowly cooled to room temperature for 20 min. Then 2 pL of T4 DNA ligase (400 U / pL) was added and incubated at 37 °C for 1 h in an oven; 2 pL of Exol (20 U / pL) and 2 pL of ExoIII (20 U / pL) were added and incubated for 2 h; placed in a PCR machine, inactivated at 80 °C for 20 min, to form a closed-loop dumbbell probe (DP).

[0057] (3) T-ERCA reaction: 20 pL of mixed solution containing closed-loop dumbbell probe (DP, 0.4 pM) in step (2), different concentrations of miRNA155, phi29 polymerase (0.4 U / pL), Nt.BbvCI (0.3 U / pL), 1 x phi29 polymerase buffer and dNTP (250 mM) were placed in an oven and reacted at 37 °C for 100 min to obtain the amplification product (ssDNA).

[0058] (4) CRISPR / Cas12a recognition and fluorescence signal collection: 4 pL of amplification product in step (3), 2 pL of NEBuffer 2.0, 2 pL of crRNA (1 pM) in step (1), 2 pL of Cas12a (1 pM), 2 pL of FQ-reporter signal probe and 9 pL of RNase-free H2O were mixed well using a vortex. The mixture was placed in an oven and reacted at 37 °C for 20 min. After that, the FQ-reporter signal probe was cleaved by CRISPR / Cas12a, producing a yellow fluorescence signal. 80 pL of ddH2O was added and the fluorescence spectrum and fluorescence signal intensity were collected by a fluorescence spectrophotometer (PerkinElmer) with an excitation wavelength of 490 nm and an emission wavelength of 560 nm.

[0059] Example 2 Verification of the feasibility of T-ERCA / Cas12a system for detecting miRNA-155

[0060] Specifically, the following steps were taken:

[0061] The detection of miRNA-155 was carried out according to the method in Example 1, and the reaction products in each step of Example 1 were analyzed by electrophoresis using 10% non-denaturing polyacrylamide gel. The reaction products were mixed with DNA loading buffer at a volume ratio of 5:1 and transferred to the gel well. The gel was placed in TBE buffer (1 x) at a voltage of 100 V for 100 min. Finally, the electrophoresis gel was placed in nucleic acid dye for 12 min and the image was collected by JENA UV solo Imager.

[0062] like Figure 2 As shown in Figure A, the crRNA, after being digested with DNase I, still showed a clear single electrophoretic band, indicating that the crRNA was successfully synthesized. Figure 2 As shown in Figure C, the band in lane 1 has a low electrophoretic migration rate and is a product band of hybridization between the linear template probe (TP) and itself. In the presence of T4 DNA ligase, a band with a high electrophoretic migration rate can be observed in lane 2, which is attributed to the formation of a circular dumbbell probe (DP). These results indicate that the circular dumbbell probe (DP) was successfully synthesized. In the absence of target miRNA-155, only the DP band is observed in lane 3 after the T-ERCA reaction. However, in the presence of miRNA-155, lane 4 shows bands with different electrophoretic migration rates, corresponding to the amplified single strand, the dumbbell probe, and the hybridized strand formed by the complementary single strand of the amplified product and the dumbbell probe, respectively. This indicates that miRNA-155 triggers the T-ERCA reaction to generate a large amount of amplified ssDNA.

[0063] like Figure 2 As shown in Figure B, compared to the control group, in the presence of miRNA-155, the T-ERCA reaction was triggered by the miRNA-155 target, generating a large amount of ssDNA, which in turn activated CRISPR / Cas12a to cleave the fluorescent signal probe, producing a large amount of fluorescence. However, when the target miRNA-155 was absent, no ssDNA was generated, and therefore the trans-cleavage activity of Cas12a / crRNA was not activated, resulting in a negligible fluorescence response. Based on these results, this protocol can be used for the sensitive detection of miRNA-155.

[0064] Example 3: Optimization of Experimental Parameters for the Detection Method

[0065] The specific steps are as follows:

[0066] (1) Optimization of DP concentration in the T-ERCA reaction step

[0067] The only difference from step (3) of Example 1 was the DP concentration. T-ERCA reactions were performed with DP concentrations of 0.1 μM, 0.4 μM, and 1 μM. Fluorescence values ​​were detected in the experimental groups with different DP concentrations. A control group without the target miRNA-155 was also included to detect background fluorescence values. Figure 3 As shown in Figure A, the fluorescence response was strongest when the DP template concentration was 0.4 μM. Increasing the DP concentration led to nonspecific amplification, resulting in a stronger background signal; therefore, 0.4 μM DP was used as the optimal reaction condition.

[0068] (2) Optimization of reaction time in the T-ERCA reaction step

[0069] The difference between Example 1 step (3) is only the reaction time, set the reaction time for 40, 60, 80, 100, 120 and 140 min for T-ERCA reaction, detect the fluorescence value of the experimental group at different reaction time, and set the control group without target miRNA-155 to detect the background fluorescence value. As shown in Figure 3 B, with the extension of reaction time, the fluorescence net response value gradually increases, and reaches the peak at 100 min, and then decreases, it can be seen that the extension of reaction time will lead to high background signal.

[0070] Fluorescence net response value (ΔF) = fluorescence value of experimental group - fluorescence value of control group

[0071] (3) Optimization of reaction time in CRISPR / Cas12a recognition and fluorescence signal collection reaction step

[0072] The difference between Example 1 step (4) is only the reaction time of cleavage, set the cleavage reaction time for 5, 10, 15, 20, 25 and 30 min for T-ERCA reaction, detect the fluorescence value of the experimental group at different cleavage reaction time, and set the control group without target miRNA-155 to detect the background fluorescence value.

[0073] As shown in Figure 3 C, after CRISPR / Cas12a cleavage for 20 min, the fluorescence net response value reaches the peak. The extension of CRISPR / Cas12a cleavage time does not increase the fluorescence net response value, but reduces it, which is caused by the enhancement of background signal.

[0074] Example 4 Detection performance of T-ERCA / Cas12a system

[0075] (1) Standard curve and detection limit

[0076] In order to evaluate the detection performance of T-ERCA / Cas12a system, a series of concentrations of miRNA-155 were quantitatively analyzed under the experimental conditions optimized in Example 3. As shown in Figure 4 A, the intensity of fluorescence signal gradually increases with the increase of miRNA-155 concentration (1fM-5nM). Figure 4 B shows the calibration curve of fluorescence net response value with the change of miRNA-155 concentration, the fluorescence net response value (ΔF) is linearly related to the logarithm of miRNA-155 concentration. When miRNA-155 is 1fM to 50pM ( Figure 4 C), the fitting equation is ΔF1=33.73*logC+29.26 (R 2 =0.996); when miRNA-155 is 50pM to 5nM,Figure 4 D), the fitting equation is AF2=301.99*logC-1211.98 (R 2 =0.994). The limit of detection (LOD) is 0.31 fM.

[0077] (2) Specificity

[0078] Select miRNA-21, let-7a and random RNA as interference substances to evaluate the specificity of T-ERCA / Cas12a system for miRNA-155 detection, and add 1 nM concentration. Set blank control. T-ERCA / Cas12a detects the fluorescence spectrum changes caused by different interference substances, only the target miRNA-155 causes significant fluorescence signal, and the fluorescence signal intensity is significantly enhanced Figure 5 A and Figure 5 B), the fluorescence intensity changes caused by other substances are negligible, indicating that T-ERCA / Cas12a has excellent selectivity for target miRNA-155.

[0079] (3) Reproducibility

[0080] In order to further study the sensing characteristics of the method, its reproducibility and stability were further verified. The concentration of the target miRNA-15 was set to 1 nM, and 3 consecutive tests were performed in five experimental groups to explore the reproducibility of the method. As shown in Figure 5 C, the relative standard deviation is 1.01%, indicating that the T-ERCA / Cas12a system has good reproducibility.

[0081] Example 5 Application of T-ERCA / Cas12a in actual samples

[0082] Select human breast cancer cells (MCF-7) as positive cells, and human normal liver cells (LO2) as negative control, extract total RNA from the two kinds of cells. Under the same concentration (500 ng / μL), take 2 μL total RNA as test sample, and use T-ERCA / Cas12a for detection. The results are shown in Figure 6 As shown in Figure 6 A), the fluorescence signals caused by the two cell lines are significantly different, and the fluorescence intensity caused by MCF-7 cells is significantly higher than that of LO2 cells *** <0.001) Figure 6B). These results show that the level of miRNA-155 in MCF-7 cells is much higher than that in normal L02 cells, which is due to the high expression of miRNA-155 in breast cancer. All these results show that the proposed T-ERCA / Cas12a sensing system has potential application value in the detection of important disease markers miRNA.

[0083] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A composition characterized in that, The composition contains a CRISPR / Cas12a protein, a dumbbell probe carrying two nicking endonuclease recognition sites, a crRNA, a fluorescent signal probe, a DNA polymerase, and a nicking endonuclease; the nicking endonuclease recognizes and cuts the amplification product containing the dumbbell probe, and the nicking endonuclease includes Nt.BbvCI and / or Nb.BtsI; The nucleotide sequence of the dumbbell probe is shown in SEQ ID NO.

2. The nucleotide sequence of the crRNA is shown in SEQ ID NO. 6; the nucleotide sequence of the fluorescent signal probe is shown in SEQ ID NO. 3, and the 5' end of the fluorescent signal probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

2. The composition of claim 1, wherein, The DNA polymerase includes phi29 polymerase, and the Cas12a protein is selected from one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a.

3. A detection kit for detecting miRNA 155, characterized by, The detection kit contains the composition of claim 1 or 2.

4. Use of the composition of claim 1 or 2 or the kit of claim 3 in detecting miRNA155, which is not for the purpose of diagnosis and treatment of diseases.

5. Use according to claim 4, characterized in that, The use includes the following steps: (1) Synthesis of dumbbell probe: using a DNA ligase to circularize the dumbbell probe carrying two nicking endonuclease recognition sites; (2) T-ERCA reaction: mixing the dumbbell probe synthesized in step (1), DNA polymerase, restriction endonuclease, and dNTP for amplification reaction to obtain ssDNA amplification product; (3) CRISPR / Cas12a recognition and fluorescent signal collection: mixing the ssDNA obtained in step (2) with crRNA with a nucleotide sequence shown in SEQ ID NO. 6, Cas12a protein, and fluorescent signal probe for reaction.

6. Use according to claim 5, characterized in that, In step (2), the amplification reaction conditions are 37℃ for 100 min.

7. Use according to claim 5, characterized in that, In step (3), the mixing reaction conditions are 37℃ for 20 min, then adding ddH2O, and collecting the fluorescence spectrum and fluorescence signal intensity by a fluorescence spectrophotometer.

Images