MiRNA detection kit and application thereof

Abstract

The application discloses a miRNA detection kit and application thereof. The application triggers the connection of a padlock probe and rolling circle amplification through a target miRNA, forms a rolling circle amplification product containing a repeated Cas9 recognition unit, and further activates the trans-cleavage of a Cas9-crRNA / tracrRNA complex on a single-stranded fluorescent reporter probe, so that the detection of low-abundance miRNA is realized, and the information dimension and diagnostic accuracy are improved. The detection process is not dependent on a large sequencing platform, and the operation process is relatively simple, so that the application is suitable as a technical basis for non-invasive screening of colorectal cancer, risk assessment of adenoma, auxiliary typing diagnosis, and development of an in-vitro miRNA detection kit.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid molecular detection technology, specifically to a miRNA detection kit and its application. Background Technology

[0002] miRNAs are a class of endogenous non-coding RNAs with a length of approximately twenty nucleotides, and their expression levels are closely related to various physiological and pathological processes. Due to their short sequences, high homology, low abundance, and susceptibility to matrix interference in complex biological samples, sensitive, specific, and programmable in vitro detection of miRNAs is of great significance.

[0003] Existing CRISPR detection technologies mostly utilize the trans-cleavage activity of Cas12a or Cas13 to achieve signal output (Karlikow, M., Amalfitano, E., Yang, X. et al. CRISPR-induced DNA reorganization for multiplexed nucleic acid detection. Nat Commun 14, 1505(2023)). However, existing Cas12a / Cas13 systems have inherent limitations. For example, the Cas12a system primarily targets DNA, and detecting miRNA usually requires reverse transcription and isothermal amplification of the miRNA into DNA (e.g., RPA, RCA) before activating Cas12a, resulting in multiple steps. In recent years, the use of Cas12a to detect miRNA has increasingly employed split crRNA (CRISPR RNA), which splits the crRNA into two parts: scaffold RNA and miRNA acting as spacer RNA. While this method eliminates the need for reverse transcription and directly detects miRNA, it leads to increased nonspecific background in complex biological matrices. While the Cas13 system can directly recognize RNA, the trans-cleavage substrate of Cas13 is also RNA. The reporter probe is easily degraded by RNase in the environment, resulting in poor stability and requiring a strictly enzyme-free operating environment (Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, BhattacharyyaRP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Nucleic acid detection with CRISPR-Cas13a / C2c2. Science. 2017 Apr 28;356(6336):438-442.). In recent years, the trans-cleavage activity of Cas9 has been gradually confirmed for use in nucleic acid detection (Chen, J., Chen, Y., Huang, L. et al. Trans-nuclease activity of Cas9 activated by DNA or RNA target binding. Nat Biotechnol 43, 558–568 (2025)), but it still has significant limitations in miRNA detection scenarios.

[0004] Chinese patent application CN120330295A discloses a CRISPR-Cas9 nucleic acid detection method based on a single guide RNA splitting strategy. This method splits a single guide RNA into three parts: a tracrRNA (trans-activating CRISPR RNA) sequence, a DR sequence, and a spacer sequence. The tracrRNA and DR sequences are fixed sequences, while the spacer sequence is adjusted according to the target nucleic acid sequence. The target nucleic acid is detected through the trans-cleavage capability of the CRISPR-Cas9 system. This prior art demonstrates that Cas9 trans-cleavage activity can be used for miRNA detection, but its detection approach mainly relies on the direct activation of the Cas9 system by the target nucleic acid or a related recognition substrate. However, Cas9's trans-cleavage activity is very weak, making it difficult to directly detect miRNAs at levels as low as fM or even aM in clinical samples. Furthermore, it has a higher tolerance for single-base mismatches and poor specificity; changing the target requires changing the TS and TF chains, making the design cumbersome. In addition, fecal samples contain a large amount of PCR inhibitors, polysaccharides, bile salts, and nucleases, which can easily lead to high background noise in direct detection. Therefore, it is still necessary to develop a new miRNA detection method that organically combines the specific recognition, amplification, and Cas9 trans-cleavage signal output of the target miRNA. Summary of the Invention

[0005] Purpose of the invention: The first objective of this invention is to provide a miRNA detection kit.

[0006] The second objective of this invention is to provide an application of a miRNA detection kit in the preparation of colorectal cancer typing detection products.

[0007] The third objective of this invention is to provide a method for detecting miRNA based on rolling circle amplification combined with CRISPR-Cas9 trans-cleavage.

[0008] Technical solution: To achieve the above objectives, the present invention provides a miRNA detection kit, the kit comprising a padlock probe, crRNA, and tracrRNA, wherein the nucleotide sequence of the padlock probe is shown in SEQ ID NO.3, the nucleotide sequence of the crRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.5.

[0009] The kit also includes a single-stranded fluorescence-quenching reporter probe with the nucleotide sequence TTTATTT.

[0010] The kit also includes the Cas9 protein.

[0011] The molar ratio of crRNA to tracrRNA is 1:1.

[0012] The molar ratio of the padlock probe to the miRNA is 10:1 to 10. 9 :1.

[0013] This invention also provides the application of a miRNA detection kit in the preparation of colorectal cancer typing detection products.

[0014] The detection target of the application includes miR-421, whose nucleotide sequence is shown in SEQ ID NO.1.

[0015] Preferably, the method for detecting rectal cancer typing involves detecting the concentration of target miRNA calculated from a standard curve under the same fecal RNA input conditions, and typing the test sample according to the target miRNA level.

[0016] The preferred method for colorectal cancer subtyping is to administer 200 ng RNA per reaction. When the target miRNA level is higher than 600 fM, it is identified as colorectal cancer (CRC); when the target miRNA level is between 300 fM and 600 fM, it is identified as adenomatous (CP); and when the target miRNA level is lower than 300 fM, it is identified as healthy (HC).

[0017] This invention also provides a method for detecting miRNA based on rolling circle amplification combined with CRISPR-Cas9 trans-cleavage, the method comprising the following steps:

[0018] (1) Mix the target miRNA in the sample to be tested with the padlock probe, and use ligase to make the padlock probe form a circular template;

[0019] (2) Perform rolling circle amplification on the circular template, and mix the obtained rolling circle amplification product with Cas9 protein, crRNA and tracrRNA to form a Cas9-crRNA / tracrRNA-RCA product complex system;

[0020] (3) Add a single-stranded fluorescent-quenched reporter probe to the composite system, detect the fluorescence signal, and calculate the content of the target miRNA based on the fluorescence intensity or fluorescence change rate;

[0021] The nucleotide sequence of the padlock probe is shown in SEQ ID NO.3, the nucleotide sequence of the crRNA is shown in SEQ ID NO.4, the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.5, and the miRNA includes miR-421, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0022] Preferably, the miRNA in step (1) needs to be complementary to both the 5' arm and the 3' arm of the padlock probe.

[0023] Preferably, the preferred step in forming the circular template is to prepare a 20 μL ligation reaction system, comprising: 13 μL DEPC water, 2 μL 10×SplintR ligation buffer, 2 μL 5 μM padlock probe, and 2 μL target miRNA standard. The mixture is heated at 90 °C for 5 min and then slowly cooled to room temperature to allow the target miRNA to fully hybridize with the 5' and 3' recognition arms of PP-421. Subsequently, 1 μL of SplintR ligase is added, and the mixture is incubated at 25 °C for 15 min to allow the padlock probe to ligate under the action of the target miRNA template, forming a circular DNA template.

[0024] Preferably, the rolling circle amplification in step (2) refers to expanding the circular template in step (1) in the presence of DNA polymerase and dNTPs to obtain a product containing multiple repeating Cas9 recognition units.

[0025] In step (2), the crRNA is characterized as a single-stranded CRISPR RNA in the type II CRISPR-Cas9 system. The crRNA includes a target recognition region at the 5' end and a tracrRNA binding region at the 3' end. The target recognition region is designed based on the repeat Cas9 recognition unit in the rolling circle amplification product and is complementary to the repeat Cas9 recognition unit sequence. The length of the target recognition region is 18 to 24 bases, preferably 20 bases. The tracrRNA binding region can complementarily pair with tracrRNA to form a Cas9-crRNA-tracrRNA complex.

[0026] Preferably, the tracrRNA in step (2) is a trans-activating CRISPRRNA in a type II CRISPR-Cas9 system. The tracrRNA includes a region that can complementarily pair with the tracrRNA binding region of crRNA, and a Cas9 binding region that can bind to Cas9 protein and maintain the activity of the Cas9-crRNA-tracrRNA complex. The tracrRNA does not contain a target recognition region that specifically complements the target miRNA or rolling circle amplification product, and the tracrRNA remains unchanged when the target miRNA is replaced. The molar ratio of crRNA to tracrRNA is 1:1.

[0027] The rolling circle amplification system in step (2) includes phi29 DNA polymerase, dNTPs, and phi29 DNA polymerase buffer.

[0028] Preferably, the rolling circle amplification step involves using the circular DNA template as a template, adding phi29 DNA polymerase, dNTPs, phi29 DNA polymerase buffer, and DEPC water for rolling circle amplification. The rolling circle amplification reaction system includes: 20 μL of circular DNA template, 1 μL of 10×phi29 DNA polymerase buffer, 1.5 μL of dNTPs, 0.5 μL of phi29 DNA polymerase, and DEPC water to a final volume of 30 μL. The above reaction system is subjected to rolling circle amplification at 25–37 °C, preferably at 30 °C for 30 min. After the reaction, the product can be heat-inactivated at 65 °C–75 °C for approximately 10 min, preferably at 65 °C, to obtain the rolling circle amplification product.

[0029] Preferably, the step of forming the Cas9-crRNA-tracrRNA-RCA product complex system is as follows: crRNA and tracrRNA are mixed in an equimolar ratio in 1×NEBuffer r3.1, annealed at 95 °C for 5 min, and then naturally cooled to room temperature to form a crRNA-tracrRNA complex. SpyCas9 protein is then added, and the mixture is incubated at 25 °C for approximately 15 min to form a Cas9-crRNA-tracrRNA ternary complex. An appropriate amount of the rolling circle amplification product is added to a 200 μL PCR tube, using 1×NEBuffer r3.1 or an equivalent detection buffer as the reaction matrix. 12 μL of the pre-assembled Cas9-crRNA-tracrRNA complex is added, and the volume is brought to 200 μL with DEPC water to obtain the Cas9-crRNA-tracrRNA-RCA product complex system.

[0030] Preferably, the single-stranded fluorescent-quenched reporter probe in step (3) is characterized as a single-stranded DNA oligonucleotide labeled with a fluorescent group and a quenching group at both ends, wherein the single-stranded DNA oligonucleotide contains a T base or a C base, preferably a T-rich or C-rich sequence; the length of the single-stranded DNA oligonucleotide is 6 to 13 bases, preferably 7 bases. The single-stranded fluorescent-quenched reporter probe does not form a stable and specific complementary pair with the target miRNA, padlock probe, rolling circle amplification product, crRNA, or tracrRNA; when the Cas9-crRNA-tracrRNA complex is activated by the rolling circle amplification product, the single-stranded fluorescent-quenched reporter probe is cleaved by Cas9 as a trans-cleavage substrate, causing the fluorescent group and the quenching group to separate, thereby generating a fluorescent signal.

[0031] Preferably, the step of calculating the content of the target miRNA includes: placing the Cas9-crRNA-tracrRNA-RCA product complex system obtained in step (2) into a reaction vessel, adding a single-stranded fluorescent-quenched reporter probe to a final concentration of 10 nM to 1 μM, preferably 100 nM; using 1×NEBuffer r3.1 or an equivalent detection buffer as the reaction buffer system, and adding DEPC-treated water to a total reaction volume of 100 μL. After mixing, react at 25–42 °C for 15–60 min, preferably at 25–37 °C for 30 min, so that the Cas9-crRNA-tracrRNA complex activated by the RCA product trans-cleaves the single-stranded fluorescent-quenched reporter probe. Fluorescence detection can be performed by fluorescence spectroscopy scanning or point-to-point fluorescence reading. The fluorescence spectroscopy scanning conditions are an excitation wavelength of approximately 470–495 nm and a detection emission wavelength range of approximately 500–700 nm, with the FAM fluorescence signal at approximately 520 nm serving as the primary detection signal. The detection results can be expressed as endpoint fluorescence intensity and relative fluorescence enhancement value ΔFI. A standard curve is constructed with the logarithm of the target miRNA concentration on the x-axis and fluorescence intensity on the y-axis to calculate the content of the target miRNA.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) This invention triggers the circularization of a padlock probe using the target miRNA, followed by rolling circle amplification (RCA) to generate a long single-stranded DNA product containing multiple repeating Cas9 recognition units. This transforms low-abundance miRNA recognition events into repeating nucleic acid signals that can be recognized by the Cas9 complex. The signal is then amplified by Cas9 trans-cleavage of the fluorescent reporter probe. This system exhibits a concentration-dependent fluorescence response to miR-421 in the range of 1 fM to 10 pM, with a linear correlation coefficient R0. 2 With a limit of detection (LOD) as low as 0.81 fM, it can meet the high-sensitivity detection requirements for trace amounts of colorectal cancer-related miRNAs in fecal samples.

[0034] (2) The present invention adopts a separate crRNA-tracrRNA system. By changing the crRNA, different miRNA targets can be adapted. The tracrRNA, Cas9 protein and universal fluorescent reporter probe do not need to be redesigned for each target, which improves the programmability and scalability of the system.

[0035] (3) This invention employs a dual recognition and verification mechanism, which improves detection specificity. The first layer of recognition requires that the target miRNA must simultaneously pair complementaryly with both the 5' and 3' arms of the padlock probe in order to induce the padlock probe to ligate into a circular form under the action of SplintR ligase. The second layer of recognition requires that the repetitive sequence in the RCA product must pair complementaryly with the target recognition region of the crRNA in order to activate the trans-cleavage activity of Cas9. This system produces a significant fluorescent response to the target miRNA, while the signal of the non-target miRNA group is low, indicating that this dual recognition mechanism can reduce non-specific background signals and improve the selective recognition ability of the target miRNA.

[0036] (4) This invention is applicable to the in vitro detection of miRNAs in complex biological samples, especially to the detection of colorectal cancer-related miRNAs in fecal samples. Fecal samples from colorectal cancer patients (CRC, n=9), adenoma patients (CP, n=9), and healthy individuals (HC, n=15) were used as the detection targets. Under the same RNA input conditions, fecal qPCR and the fecal sCRAFT method of this invention were used for detection, respectively. The fecal sCRAFT detection results and the fecal qPCR detection results showed a consistent distribution trend, with higher miRNA levels in the CRC group, followed by the CP group, and lower levels in the HC group. This indicates that this invention can effectively detect colorectal cancer-related miRNAs in complex fecal matrices and can differentiate between colorectal cancer patients, adenoma patients, and healthy individuals based on differences in fecal miRNA expression. This detection process does not rely on large-scale sequencing platforms, and the operation procedure is relatively simple, making it suitable as a technical basis for non-invasive colorectal cancer screening, adenoma risk assessment, auxiliary subtyping diagnosis, and the development of in vitro miRNA detection kits. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the experimental principle.

[0038] Figure 2 The gel electrophoresis diagram confirms the padlock probe ligation triggered by the target miRNA and the formation of RCA products. Figure 2 In the diagram, A represents the principle of target miRNA triggering padlock probe ligation and rolling circle amplification. Figure 2B in the diagram represents gel electrophoresis verification under different reaction component conditions. M is the DNA Marker. Lane 1 is the padlock probe only, lane 2 is miRNA-421 only, lane 3 is the padlock probe and miRNA-421 only incubated, lane 4 is the padlock probe and miRNA-421 incubated with SplintR ligase, lane 5 is the padlock probe incubated with SplintR ligase and phi29 DNA polymerase and dNTPs added, and lanes 6, 7, and 8 are the padlock probe and miRNA-421 incubated with SplintR ligase and phi29 DNA polymerase and dNTPs added. The concentrations of miRNA-421 are 1 pM (lane 6), 1 nM (lane 7), and 1 μM (lane 8), respectively. Figure 2 C in the diagram represents the gel electrophoresis verification of RCA products formed when different miRNAs are used as templates.

[0039] Figure 3 This is a feasibility verification diagram for Cas9 trans-cleavage fluorescence detection. Lane 1 shows the cleavage results when SpyCas9, crRNA-tracrRNA, miRNA-421D, and ssDNA substrates are all present. Lane 2 shows the cleavage results when only SpyCas9 is absent. Lane 3 shows the cleavage results when only miRNA-421D is absent. Lane 4 shows the cleavage results when only crRNA-tracrRNA is absent.

[0040] Figure 4 This is a graph validating the specificity and anti-interference capability of the detection method, where... Figure 4 In the diagram, A represents the selectivity verification diagram for different miRNAs. Figure 4 B in the diagram represents the anti-interference capability verification.

[0041] Figure 5 The graph shows the relationship between fluorescence signal and miRNA concentration in the sample. Figure 5 In the graph, A represents the fluorescence response curves corresponding to different concentrations of miRNA-421. Figure 5 B in the graph represents the standard curve of the logarithm of miRNA-421 concentration versus fluorescence intensity.

[0042] Figure 6 This is a graph showing the test results of a clinical stool sample. Detailed Implementation

[0043] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0044] In the following examples, all nucleic acids used were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; DNA molecular weight standard markers were purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B600303-0020; SplintR ligase and its matching buffer were purchased from New England Biolabs (NEB), catalog number M0375S; phi29 DNA polymerase and its matching buffer were purchased from Beyotime Biotechnology Co., Ltd., catalog number D7053S; dNTPs were purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B500055-0250; SpyCas9 protein was purchased from New England Biolabs (NEB), catalog number M0386T; gel imaging was performed using a 3500 fully automated digital gel image analysis system (Shanghai Tianneng Life Science Co., Ltd.); fluorescence detection was performed using an F-320 fluorescence spectrophotometer (Tianjin Gangdong Technology Co., Ltd.). DEPC-treated water was used.

[0045] The nucleotide sequences of the miRNA, probe, and reporter system involved in the following examples are shown in Table 1.

[0046] Table 1

[0047]

[0048] Example 1: Detection of target miRNA using rolling circle amplification combined with CRISPR-Cas9 trans-cleavage detection method.

[0049] 1. Principle of Rolling Circle Amplification Combined with CRISPR-Cas9 Trans-Cut Detection Method

[0050] like Figure 1 As shown, the detection method of this invention includes three steps: target miRNA recognition ligation, rolling circle amplification, and CRISPR-Cas9 trans-cleavage fluorescence readout. First, the target miRNA hybridizes complementaryly with the recognition arms at both ends of the padlock probe, bringing the 5' and 3' ends of the padlock probe closer together, and then ligating them under the action of a ligase to form a circular DNA template. Subsequently, rolling circle amplification is performed using the circular DNA template to generate a long single-stranded DNA product containing repeating Cas9 recognition units. Further, the rolling circle amplification product binds to the Cas9-crRNA-tracrRNA complex, activating the trans-cleavage activity of Cas9 on the single-stranded fluorescence-quenching reporter probe, causing the fluorescent group to separate from the quenching group and generate a fluorescence signal. By constructing a fluorescence signal standard curve, quantitative detection of the target miRNA in the sample can be achieved.

[0051] 2. Using miR-421 as the target miRNA to trigger padlock probe ligation

[0052] The steps for triggering the ligation of the padlock probe into a circular DNA template using the target miRNA are as follows: Prepare a 20 μL ligation reaction mixture, comprising: 13 μL DEPC water; 2 μL 10×SplintR ligation buffer; 2 μL PP-421 (sequence shown in SEQ ID NO.3), 5 μM; and 2 μL miR-421 (sequence shown in SEQ ID NO.1). Heat the mixture at 90 °C for 5 min, then slowly cool to room temperature to allow the target miRNA to fully hybridize with the 5' and 3' recognition arms of the padlock probe PP-421. Then add 1 μL of SplintR ligase and incubate at 25 °C for 15 min to allow PP-421 to ligate under the action of the target miRNA template, forming a circular DNA template. After the reaction, a portion of the ligation product was directly analyzed by gel electrophoresis.

[0053] Figure 2 A in the diagram illustrates the principle of target miRNA triggering padlock probe ligation and rolling circle amplification in this invention. The target miRNA first hybridizes with complementary regions at both ends of the padlock probe, bringing the 5' and 3' ends of the probe closer together. Under the action of SplintR ligase, the two ends of the padlock probe are ligated to form a circular template. Subsequently, this circular template undergoes rolling circle amplification in the presence of phi29 DNA polymerase and dNTPs, generating a long single-stranded DNA product containing repetitive sequences.

[0054] Figure 2 B in the diagram represents the gel electrophoresis verification results under different reaction component conditions. Lane 1 is the control group containing only the padlock probe PP-421, showing the migration band of the padlock probe itself; Lane 2 is the control group containing only the target miR-421, showing the migration signal of the short miRNA; Lane 3 is the control group where PP-421 and miR-421 are co-incubated without the addition of SplintR ligase, mainly showing the signal of the unligated probe or hybridization complex, without forming obvious ligation products; Lane 4 is the ligation reaction group where PP-421, miR-421, and SplintR ligase are added simultaneously, and ligation-related bands with different migration positions compared to the unligated probe can be observed, indicating that the target miR-421 can serve as a template to promote the ligation of the padlock probe into a circular form.

[0055] 3. Rolling circle amplification (RCA) reaction

[0056] The rolling circle amplification (RCA) reaction steps are as follows: The product obtained from the ligation reaction described above is used as the RCA template. Phy29 DNA polymerase, dNTPs, Phy29 DNA polymerase buffer, and DEPC water are added for rolling circle amplification. The RCA reaction system includes: 20 μL of the ligation product from the previous step, 1 μL of 10× Phy29 DNA polymerase buffer, 1.5 μL of dNTPs, 0.5 μL of Phy29 DNA polymerase, and DEPC water to a final volume of 30 μL. The reaction system is incubated at 30 °C for 30 min. After the reaction, the product can be heat-inactivated at 65 °C for approximately 10 min. The resulting RCA product is used for subsequent Cas9 trans-cleavage detection. The RCA product is verified by gel electrophoresis. The results are as follows: Figure 2 As shown in Figure B, lanes 6, 7, and 8 reveal that a significant RCA product signal was generated only in the system where the target miRNA was present and successfully induced PP-421 circularization. In contrast, no RCA product signal appeared in the control system (lanes 1, 2, 3, 4, and 5) lacking the target miRNA, ligase, or key RCA components. This result indicates that the RCA reaction depends on the circular template induced by the target miRNA, which can convert the target miRNA recognition event into a long single-stranded DNA product containing repeating Cas9 recognition units.

[0057] 4. Assembly and trans-cleavage of the Cas9-crRNA / tracrRNA complex

[0058] This embodiment is an experiment to verify whether the complex assembly and trans cleavage can be successful. The DNA sequence corresponding to miR-421, namely miR-421D (sequence shown in SEQ ID NO.2), is used to simulate its RCA product. The actual detection is performed using the RCA product of miR-421.

[0059] The steps for Cas9-crRNA / tracrRNA complex assembly and trans-cleavage detection are as follows:

[0060] S1: Mix crRNA (sequence shown in SEQ ID NO.4) and tracrRNA (sequence shown in SEQ ID NO.5) in an equimolar ratio, anneal at 95 °C for 5 min, and then allow to cool naturally to room temperature to form a crRNA-tracrRNA complex. Then add SpyCas9 protein and incubate at 25 °C for about 15 min to form a Cas9-crRNA-tracrRNA ternary complex.

[0061] S2: Add an appropriate amount of miR-421D to a 200 μL PCR tube. Use 1×NEBuffer r3.1 (New England Biolabs (NEB) Co., Ltd., catalog number B6003V) as the reaction matrix, add 12 μL of pre-assembled Cas9-crRNA-tracrRNA complex, then add 20 μL of 2.5 μM ssDNA substrate (FAM-TTTTTTTTTT (SEQ ID NO.11)), and bring the volume to 200 μL with DEPC water.

[0062] S3: After incubating the reaction system at room temperature for 30 min, run a gel electrophoresis to verify the reaction.

[0063] The results are as follows Figure 3 As shown, in the complete system containing SpyCas9, crRNA-tracrRNA, miR-421D, and ssDNA substrate, cleavage bands of the ssDNA substrate were observed in the gel; however, no cleavage products were observed in the control group lacking Cas9, crRNA-tracrRNA, miR-421D, or the ssDNA substrate. This result indicates that miR-421D can activate the trans-cleavage activity of the Cas9-crRNA-tracrRNA complex.

[0064] Example 2: Verification of the specificity and anti-interference capability of the rolling circle amplification combined with CRISPR-Cas9 trans-cutting detection method.

[0065] 1. Perform dual-target validation to ensure the specificity of the detection method.

[0066] The dual-target validation includes: a first layer validating the complementary pairing and circularization of the target miRNA with the recognition arms at both ends of the padlock probe; and a second layer validating the complementary pairing of the RCA product with crRNA and Cas9 trans-cleavage activation. To verify the target recognition specificity of the detection system of this invention, this embodiment uses PP-421 as the padlock probe. Under the same reaction conditions, target miR-421 and different non-target miRNAs are added to examine whether different miRNAs can trigger PP-421 to form a circular structure and further undergo RCA amplification. miR-421, miR-21 (sequence shown in SEQ ID NO. 6), miR-155 (sequence shown in SEQ ID NO. 7), miR-10b (sequence shown in SEQ ID NO. 10), miR-221 (sequence shown in SEQ ID NO. 8), and miR-561 (sequence shown in SEQ ID NO. 9) are used as template nucleic acids. Each group was treated with the same concentrations of PP-421, SplintR ligase, phi29 DNA polymerase, and dNTPs, and the padlock probe ligation reaction and RCA amplification reaction were performed sequentially according to the conditions described in Example 1. After the reaction, a portion of the amplification products was taken for gel electrophoresis detection.

[0067] The results are as follows Figure 2 As shown in Figure C, M represents the DNA marker; lane 1 is the target miR-421 group, and lanes 2-6 are the non-target miRNA groups: miR-21, miR-155, miR-10b, miR-221, and miR-561, respectively. A clear high-molecular-weight RCA product band was observed in lane 1, indicating that miR-421 can complementaryly pair with the recognition arms at both ends of PP-421, inducing PP-421 to circulate and subsequently undergo RCA amplification. In contrast, no clear high-molecular-weight RCA product was observed in lanes 2-6, with only weak background bands or no amplified signal, indicating that the aforementioned non-target miRNAs cannot effectively induce PP-421 circulation or produce effective RCA amplification products. These results demonstrate that PP-421 has good sequence recognition specificity for target miR-421; only target miRNAs matching the recognition arms at both ends of the padlock probe can trigger circulation and subsequent RCA reactions. This result demonstrates the first-layer target verification mechanism in the detection system of this invention, namely, the target miRNA must be correctly paired with the 5' and 3' recognition arms of the padlock probe in order to initiate the subsequent signal amplification process.

[0068] Furthermore, to verify the specificity of the second-layer recognition, the RCA amplification products of miR-421, miR-21, miR-155, miR-10b, miR-221 and miR-561 were subjected to Cas9-crRNA-tracrRNA complex trans-cleavage fluorescence detection.

[0069] The assembly of the Cas9-crRNA / tracrRNA complex and the trans-cleavage fluorescence detection steps are as follows:

[0070] S1: Same as S1 in Example 1.

[0071] S2: Add an appropriate amount of miR-421D to a 200 μL PCR tube, use 1×NEBuffer r3.1 as the reaction matrix, add 12 μL of pre-assembled Cas9-crRNA-tracrRNA complex, then add 20 μL of 1 μM single-stranded fluorescent-quenched reporter probe FQ (sequence TTTATTT), and make up to 200 μL with DEPC water.

[0072] S3: Fluorescence detection was performed using an F-320 fluorescence spectrophotometer. The excitation wavelength was set to 470 nm, and the emission spectrum was scanned in the range of 500–700 nm. The fluorescence intensity at 520 nm was taken as the detection signal. The final detection signal was evaluated using the relative fluorescence enhancement value ΔFI.

[0073] The results are as follows Figure 4 As shown, compared with the blank control group (reaction system without target miRNA) and the non-target miRNA groups (miR-21, miR-155, miR-10b, miR-221, miR-561), the target miR-421 group produced a significantly increased fluorescence signal, while the relative fluorescence enhancement values ​​of the other non-target miRNA groups remained at a level comparable to the blank control. This result indicates that after the pre-ligation and amplification steps, only the RCA product triggered by the target miR-421 can be strictly complementary to the pre-defined crRNA and effectively activate the trans-cleavage activity of Cas9, thereby producing a significant fluorescence output; non-target miRNAs cannot form an effective final signal.

[0074] 2. Verify the anti-interference capability of the detection method.

[0075] Using miR-421 as the target miRNA, the fluorescence response of the system containing only miR-421 and the system containing a mixture of miR-421 and non-target miRNA (Mix) were detected under different concentration conditions.

[0076] The results are as follows Figure 4As shown, under different concentrations of 100 fM, 100 pM, and 1 nM, the system containing the target miR-421 all produced significant fluorescence signals. When non-target miRNAs were added to the system simultaneously to form a mixed sample, the fluorescence intensity was close to that of the miR-421-only group, with no significant signal inhibition or abnormal increase observed. These results indicate that the detection system of this invention can stably recognize the target miR-421 even in the context of complex miRNA coexistence, demonstrating good anti-interference ability and detection reliability.

[0077] Example 3 Sensitivity testing of rolling circle amplification combined with CRISPR-Cas9 trans-cutting detection method

[0078] Different concentrations of miR-421 standards were prepared, including 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM. Equal volumes of each standard were added to the detection system, and padlock probe ligation, RCA amplification, and Cas9 trans-cleavage fluorescence detection were performed according to the methods described in Examples 1 and 2. A blank control group without target miRNA was included. Three parallel wells were used for each experiment. Fluorescence detection was performed using fluorescence spectroscopy scanning or point-to-point fluorescence reading. The fluorescence spectroscopy scanning conditions were: excitation wavelength of 470 nm, scanning range of 500–700 nm, with the FAM fluorescence signal at 520 nm used as the primary detection signal. Detection results can be expressed as endpoint fluorescence intensity, relative fluorescence enhancement value ΔFI, or real-time fluorescence growth rate.

[0079] The results are as follows Figure 5 As shown in Figure A, the FAM fluorescence signal gradually increases with increasing miR-421 concentration, indicating that the system of this invention has a concentration-dependent response to miR-421. Furthermore, a standard curve was established with the logarithm of miR-421 concentration C (x) as the abscissa and fluorescence intensity (y) as the ordinate. The results are shown in Figure A. Figure 5 As shown in Figure B, within the range of 1 fM to 10 pM, the fluorescence intensity exhibits a good linear relationship with the miR-421 concentration, with the linear equation being: y = 266.6x + 753.7, and the linear correlation coefficient R0. 2 =0.9670. The limit of detection (LOD) is calculated as LOD = 3σ / k, where σ is the standard deviation of the fluorescence signal in the blank control group, and k is the slope of the linear regression of the standard curve. The calculated LOD for miR-421 in this system is 0.81 fM.

[0080] The results demonstrate that the present invention, by combining RCA with Cas9 trans-cleavage signal output, can achieve highly sensitive detection of fM-level low-abundance miRNAs, making it suitable for the detection of trace colorectal cancer-related miRNAs in complex samples such as feces.

[0081] Example 4: Application of Rolling Circle Amplification Combined with CRISPR-Cas9 Transcutting Detection Method in the Subtyping and Diagnosis of Colorectal Cancer

[0082] Fecal samples were collected from colorectal cancer patients, adenoma patients, and healthy individuals at Nanjing Pukou People's Hospital (ethics approval numbers: 2026-SR-001, 2026-SR-002). The colorectal cancer patient group was designated as the CRC group (n = 9 samples); the adenoma patient group as the CP group (n = 9 samples); and the healthy individual group as the HC group (n = 15 samples). After collection, miRNA was extracted from all samples according to the instructions of the fecal RNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B518811-0050). The extracted RNA was tested for concentration and purity, and then the same mass of RNA (e.g., 200 ng RNA per reaction) was used for subsequent detection.

[0083] The method of this invention (hereinafter referred to as fecal sCRAFT) was used to detect the level of target miRNA in each fecal sample. The specific steps included: mixing the fecal miRNA sample with the corresponding padlock probe according to the steps of Example 1, so that the target miRNA triggered the padlock probe to form a loop, followed by RCA amplification to generate an RCA product containing a repeating Cas9 recognition unit; then adding the Cas9-crRNA-tracrRNA complex and FQ (sequence TTTATTT) according to the steps of Example 2, detecting the fluorescence signal, and calculating the relative content or concentration of the target miRNA in the sample according to the standard curve established in Example 4.

[0084] In this embodiment, qPCR was used to perform parallel detection on the same batch of fecal RNA samples as a control method (fecal qPCR). The qPCR detection was performed according to the instructions of the corresponding miRNA reverse transcription and real-time PCR kits (Nanjing Novizan Biotechnology Co., Ltd., catalog numbers MR101-01 and Q312-02).

[0085] The results are as follows Figure 6 As shown. Figure 6In the table, the top row shows the Stool-qPCR detection results, and the bottom row shows the Stool-sCRAFT detection results of this invention. The CRC, CP, and HC groups are labeled with different colors, with sample numbers of CRC (n = 9), CP (n = 9), and HC (n = 15), respectively. The results show that the fecal sCRAFT and fecal qPCR detection results exhibit a consistent distribution trend. Specifically, the CRC group averaged greater than 600 fM, the CP group averaged between 300 and 600 fM, and the HC group averaged less than 300 fM. These results demonstrate that the detection method of this invention can effectively detect colorectal cancer-related miRNAs in the complex matrix of feces and can differentiate between colorectal cancer patients, adenoma patients, and healthy individuals based on differences in fecal miRNA expression.

[0086] Therefore, the method of the present invention can be used for in vitro detection of colorectal cancer-related miRNAs in fecal samples, and can serve as a technical basis for non-invasive screening of colorectal cancer, adenoma risk assessment, auxiliary subtyping diagnosis, and the development of miRNA detection kits.

Claims

1. A miRNA detection kit, characterized in that, The detection kit contains a padlock probe, crRNA, tracrRNA, and Cas9 protein. The nucleotide sequence of the padlock probe is shown in SEQ ID NO.3, the nucleotide sequence of the crRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the tracrRNA is shown in SEQ ID NO.

5.

2. The miRNA detection kit according to claim 1, characterized in that, The kit also includes a single-stranded fluorescence-quenched reporter probe with the nucleotide sequence TTTATTT.

3. The miRNA detection kit according to claim 1, characterized in that, The molar ratio of the crRNA to the tracrRNA is 1:

1.

4. The miRNA detection kit according to claim 1, characterized in that, The molar ratio of the padlock probe to the miRNA is 10:1 to 10. 9 :

1.

5. The use of the miRNA detection kit according to claims 1-4 in the preparation of a colorectal cancer typing detection product.

6. The application according to claim 5, characterized in that, The target of the application includes miR-421, whose nucleotide sequence is shown in SEQ ID NO.1.

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