System, kit and application for detecting miRNA and DNA mutation based on one-pot method of CRISPR-cas12a and split aptamer

Abstract

This invention relates to a system, kit, and application for the one-pot detection of miRNA and DNA mutations based on CRISPR-Cas12a and aptamers, belonging to the field of biomedical technology. The system for the one-pot detection of miRNA and DNA mutations based on CRISPR-Cas12a and aptamers includes: Cas12a protein, crRNA-R targeting miRNA, aptamer P1, aptamer P2, a linker, AO, and crRNA-D targeting DNA mutations. This invention combines CRISPR / Cas12a with a aptamer-based detection strategy to develop a label-free, direct detection system compatible with smartphones. This system can simultaneously detect EGFR mutations and miRNA-21 expression levels.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a system, kit, and application for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers. Background Technology

[0002] DNA mutations and abnormal microRNA (miRNA) expression are key drivers of lung cancer pathogenesis and progression. Among genetic alterations, mutations in EGFR, ALK, KRAS, and ROS1 genes are relatively common, with EGFR mutation being the most prevalent mutation type in non-small cell lung cancer (NSCLC). EGFR mutation detection serves not only as a crucial prognostic biomarker but also guides the selection of targeted therapy regimens. miRNAs such as miRNA-21, miRNA-155, miRNA-122, and miRNA-141 have been confirmed as important regulators of gene expression, participating in lung cancer development. Combining EGFR mutation analysis with miRNA-21 detection can overcome the limitations of single biomarker detection, thereby improving diagnostic accuracy and providing a more comprehensive molecular basis for prognostic assessment and drug resistance monitoring.

[0003] Traditional techniques face challenges in simultaneously detecting EGFR mutations and miRNA-21. The main obstacle is that miRNA must first be reverse transcribed into DNA before quantitative analysis, which not only increases operational complexity but can also lead to result variability. Furthermore, real-time PCR technology relies on specialized equipment and trained operators, limiting its application in point-of-care testing (POCT). The CRISPR / Cas12a system, with its high specificity, high sensitivity, programmability, and compatibility with mild reaction conditions, has become a promising next-generation nucleic acid detection platform, making it particularly suitable for POCT. Although the CRISPR / Cas12a system has been successfully applied to DNA mutation detection, it lacks RNA detection capabilities. Like traditional techniques, current CRISPR / Cas12a-based RNA detection technologies still rely on upstream reverse transcription or the use of probes to convert RNA into a crRNA-recognizable DNA form. In addition, the non-specific trans-cleavage activity of Cas12a limits its ability to simultaneously detect more than two targets without cross-interference. Therefore, there is an urgent need to construct a CRISPR / Cas12a system that can directly and simultaneously analyze EGFR mutations and miRNA-21. Summary of the Invention

[0004] The first objective of this invention is to provide a system for the one-pot detection of miRNA and DNA mutations based on CRISPR-Cas12a and aptamer. This system combines CRISPR / Cas12a with a aptamer-based detection strategy to achieve one-pot detection of miRNA-21 and EGFR L858R mutations.

[0005] The second objective of this invention is to provide a kit for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and apposition aptamers.

[0006] The third objective of this invention is to provide an application of a one-pot detection system for miRNA and DNA mutations based on CRISPR-Cas12a and aptamers.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers includes: Cas12a protein, crRNA-R targeting miRNA, apposition aptamer P1, apposition aptamer P2, linker, AO, and crRNA-D targeting DNA mutations.

[0008] Furthermore, the sequence of the crRNA-R targeting miRNA is shown in SEQ ID NO. 1; and the sequence of the crRNA-D targeting DNA mutation is shown in SEQ ID NO. 2.

[0009] Furthermore, the sequence of the split aptamer P1 is shown in SEQ ID NO. 3; the sequence of the split aptamer P2 is shown in SEQ ID NO. 4; and the sequence of the linker is shown in SEQ ID NO. 5.

[0010] Furthermore, the miRNA in the crRNA-R targeting miRNA is miRNA-21; the DNA mutation in the crRNA-D targeting DNA mutation is EGFR L858R DNA.

[0011] Furthermore, the final concentration of the linker is 0.25-1.25 μM; the final concentration of the Cas12a protein is 5-75 nM; the final concentration of the crRNA-R targeting miRNA is 15-30 nM; the final concentration of the crRNA-D targeting DNA mutation is 15-30 nM; the final concentration of AO is 0.5-30 μM; the final concentration of the mitotic aptamer P1 is 0.25-1.25 μM; and the final concentration of the mitotic aptamer P2 is 0.25-1.25 μM.

[0012] Furthermore, the final concentration of the linker is 1 μM; the final concentration of the Cas12a protein is 25 nM; the final concentration of the crRNA-R targeting miRNA is 25 nM; the final concentration of the crRNA-D targeting DNA mutation is 25 nM; the final concentration of AO is 5 μM; the final concentration of the splitting aptamer P1 is 1 μM; and the final concentration of the splitting aptamer P2 is 1 μM.

[0013] A kit for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers, comprising the aforementioned system for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers.

[0014] The aforementioned system for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers, or the kit for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers, are used in the preparation of products for early diagnosis, molecular subtyping, and prognostic monitoring of lung cancer.

[0015] The detection method for miRNA and DNA mutations based on CRISPR-Cas12a and apposition aptamers in a one-pot assay includes the following steps: S1: Prepare a 10 μL Cas12a / crRNA-R solution by mixing 1 μL of 1 μM Cas12a, 2 μL of 500 nM crRNA-R, 4 μL of 10×NEB buffer r2.1, 2 μL of 20 μM linker and 1 μL of RNase inhibitor. S2: Mix 8 μL of nucleic acid sample with 10 μL of Cas12a / crRNA-R solution, incubate at 37 ℃ for 10 min, then continue incubation at 37 ℃ for 60 min. Subsequently, add 2 μL of 20 μM apposition aptamer P1, 2 μL of 20 μM apposition aptamer P2, 4 μL of 50 μM AO, and 12 μL of AO buffer. Incubate at 20-42 ℃ for 0-75 min. Perform the first fluorescence intensity measurement and analysis using the FluoroSignalAPP software on a smartphone. Then, mix 2 μL of 500 nM crRNA-D pre-added to the test tube cap by centrifugation, and incubate at 37 ℃ for 50 min. Perform the second fluorescence intensity measurement and analysis using the FluoroSignalAPP software on a smartphone.

[0016] The beneficial effects of this invention are: This invention combines CRISPR / Cas12a with a cleavage aptamer-based detection strategy to develop a label-free, direct detection system compatible with smartphones. This system can simultaneously detect EGFR mutations and miRNA-21 expression levels. This invention overcomes the key technical bottleneck of simultaneous nucleic acid analysis in lung cancer molecular profiling, particularly eliminating the need for reverse transcription and fluorescent labeling. By utilizing the unique trans-cleavage activity of RNA-activating and DNA-activating Cas12a, quantitative analysis of miRNA-21 and qualitative identification of EGFR L858R mutations can be achieved with only simple fluorescence readings.

[0017] This invention integrates smartphone-based RGB analysis technology via the FluoroSignalAPP, enabling point-of-care testing in resource-constrained environments and reducing reliance on specialized instruments. This integration strategy expands the application of CRISPR / Cas12a in multiplex nucleic acid detection, providing a rapid, economical, and easy-to-use tool for the simultaneous analysis of DNA and RNA biomarkers.

[0018] Experimental results show that the CRISPR-Cas12a and aptamer-based one-pot method for detecting miRNA and DNA mutations in this invention exhibits a linear range of 5-500 nM for miRNA-21 detection. 2 The detection limit is 1.26 nM (0.99825), and the detection limit is 1.26 nM. It has high specificity for interfering miRNAs and single nucleotide variants and excellent detection accuracy. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the detection principle of a one-pot method for detecting miRNA and DNA mutations based on CRISPR-Cas12a and apposition aptamers. Figure 2 The following diagrams illustrate the feasibility results in Experiment Example 1: A shows the fluorescence signal detection results of trans cleavage of 10 nt and 20 nt single-stranded DNA substrates; B shows the AO fluorescence signal generated by aptamer assembly after trans cleavage of the linker mediated by miRNA-21 or DNA-21 activation of Cas12a; C shows a comparison of the standardized trans cleavage efficiency of 5, 10, 20, and 29 nt single-stranded DNA substrates; and D shows the trans cleavage efficiency analysis of DAP-10 and 29 nt single-stranded DNA substrates by miRNA-21-activated Cas12a. Figure 3 This is a diagram showing the trans cleavage results of Cas12a activated by miRNA-21 and DNA-21 on a 29 nt single-stranded DNA substrate in Experiment Example 1; Figure 4This diagram illustrates the feasibility of a CRISPR-Cas12a and aptamer-based one-pot method for detecting miRNA and DNA mutations with smartphone-assisted detection. A shows a comparison of AO fluorescence intensity in the presence and absence of aptamers P1 and P2; B shows a comparison of AO fluorescence intensity in the presence and absence of the EGFR L858R DNA target; C shows the PAGE analysis results of the G-quadruplex reverse cleavage product generated by EGFR L858R activating Cas12a; D shows a comparison of fluorescence intensity between the first and second readings under four experimental conditions; E shows the color display effect of smartphone imaging under different linker concentrations; and F shows the correlation analysis between AO fluorescence intensity and its green channel value. Figure 5 The diagram shows the optimization results of the conditions in Experiment Example 2. In the diagram, A is the optimized assembly time of AO with apposition aptamers P1 and P2, B is the optimized AO concentration, C is the optimized linker concentration, D is the optimized Cas12a concentration, E is the optimized crRNA-R concentration, F is the optimized crRNA-D concentration, G is the optimized reaction temperature for miRNA-21 to activate Cas12a, and H is the optimized time for miRNA-21 to activate Cas12a, resulting in trans-cleavage of the linker. Figure 6 This is the free energy diagram of the secondary structure of the G-quadriplex complex at 37 °C in Experimental Example 2; Figure 7 The stability graph of the AO fluorescence signal over time in Experiment Example 2 is shown. Figure 8 Figures show the performance analysis and real sample detection results of the CRISPR-Cas12a and aptamer one-pot method for detecting miRNA and DNA mutations in Experiments 3 and 4. In Experiment 4, A is the change of AO green channel signal intensity under different concentrations of miRNA-21 treatment, B is the standard curve of miRNA-21 detection in the concentration range of 5-500 nM, C is the green channel signal intensity at three plasma spiking concentrations, D is the miRNA-21 specific detection result, E is the EGFR L858R mutation specific detection result, and F is the real sample detection result. Figure 9 The images show the interface of the FluoroSignalAPP, where A is the software startup screen, B is the image acquisition and storage screen, C is the ISO and exposure time selection screen, D is the image import screen, E is the image analysis screen, F is the analysis report generation screen, G is the analysis result export screen, and H is the historical data management screen. Detailed Implementation

[0020] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0021] LbCas12a protein (Cpf1) and NEB buffer r2.1 were purchased from Editgene Ltd. (Guangzhou, China); the AGRNAex Pro RNA extraction kit was from AGAccurate Biology. The column-based animal genomic DNA purification kit was from Sangon Biotech (Shanghai, China), and all oligonucleotides were synthesized and purified by HPLC by Sangon Biotech. All nucleic acid solutions were prepared using DEPC-treated water (for RNA) or TE buffer (for DNA). Auramine O (AO) fluorescent dye was purchased from Aladdin Biochemical Technology Ltd. (Shanghai, China). AO buffer contained 20 mM Tris-HCl, 20 mM potassium chloride, and 25 mM magnesium chloride, at pH 7.4.

[0022] The sequences involved in this invention are shown in Table 1.

[0023] Table 1. Sequences involved in this invention

[0024] Example 1 The system for detecting miRNA and DNA mutations based on the CRISPR-Cas12a and aptamer one-pot method in Example 1 includes the following components: 1 μL of Cas12a at a concentration of 1 μM, 2 μL of crRNA-R at a concentration of 500 nM, 4 μL of 10×NEB buffer r2.1, 2 μL of linker at a concentration of 20 μM, 1 μL of RNase inhibitor, 2 μL of aptamer P1 at a concentration of 20 μM, 2 μL of aptamer P2 at a concentration of 20 μM, 4 μL of AO at a concentration of 50 μM, 12 μL of AO buffer, and 2 μL of crRNA-D at a concentration of 500 nM.

[0025] Example 2 The one-pot method for detecting miRNA and DNA includes the following steps: S1: Prepare a Cas12a / crRNA-R solution by mixing 1 μL of 1 μM Cas12a, 2 μL of 500 nM crRNA-R, 4 μL of 10×NEB buffer r2.1, 2 μL of 20 μM linker, and 1 μL of RNase inhibitor.

[0026] S2: 8 μL of nucleic acid sample was mixed with 10 μL of Cas12a / crRNA-R solution and incubated at 37 °C for 10 min. The mixture was then transferred to 37 °C and incubated for another 60 min to ensure complete cleavage of the linker by RNA-activated Cas12a. Next, 2 μL of 20 μM P1, 2 μL of 20 μM P2, 4 μL of 50 μM AO, and 12 μL of AO buffer were added to initiate AO fluorescence detection. After incubation at 37 °C for 15 min, the first fluorescence intensity measurement and analysis were performed using the FluoroSignal app on a smartphone. Finally, 2 μL of 500 nM crRNA-D, pre-added to the tube cap, was mixed by centrifugation and incubated at 37 °C for another 50 min to complete the second fluorescence intensity measurement and analysis.

[0027] Detection Principle: Aptamers P1 and P2 self-assemble into a G-quadruplex structure via the linker, thereby activating the fluorescence signal of AO. When miRNA-21 is absent, the fluorescence intensity of AO reaches its peak. In the presence of miRNA-21, miRNA-21 activates Cas12a to trans-cleave the linker, resulting in a decrease in the fluorescence intensity of AO. This decrease in fluorescence intensity allows for the quantitative analysis of miRNA-21. Furthermore, the presence of the EGFR L858R mutant can activate Cas12a. The self-assembled G-quadruplex then acts as a substrate for CRISPR / Cas12a, further reducing the fluorescence intensity of AO. This decrease in AO fluorescence intensity helps identify the occurrence of the EGFR L858R mutation.

[0028] from Figure 4 As can be seen from D, when only miRNA-21 is present, the initial fluorescence intensity is lower than that of the blank control, while the second fluorescence intensity remains stable. When miRNA-21 and the EGFR L858R mutant are present simultaneously, the initial fluorescence reading decreases due to the influence of miRNA-21, and the second reading further decreases after the addition of crRNA-D, indicating that EGFR L858R-activated Cas12a undergoes trans-cleavage.

[0029] Experimental Example 1 Feasibility analysis 1. 25 nM Cas12a and 25 nM crRNA-R were reacted in 10×NEB buffer r2.1 at 37 °C for 30 min to form a Cas12a / crRNA-R complex. The Cas12a / crRNA-R complex was then mixed with 500 nM of single-stranded DNA substrates of different lengths (5 nt, 10 nt, 20 nt, and 29 nt), and 100 nM miRNA-21 was added simultaneously to initiate the Cas12a trans-cleavage reaction. The reaction was terminated by heating at 95 °C for 5 min after 80 min to inactivate Cas12a. Finally, polyacrylamide gel electrophoresis (PAGE) and fluorescence detection were used to characterize and analyze the trans-cleavage effect on substrates of different lengths. The results showed that the miRNA-21-activated Cas12a protein had almost no trans-cleavage activity against the fluorescence-quenched reporter sequence of the 5 nt single-stranded DNA substrate. Trans-cleavage products of 10 nt and 20 nt single-stranded DNA substrates were detected using a secondary Cas12a reporter system, while the 29 nt single-stranded DNA substrate was evaluated by its ability to excite AO fluorescence upon assembly with apposition aptamers P1 and P2. Control reactions were performed in parallel for each substrate length, using an equal amount of uncut single-stranded DNA substrate instead of the trans-cleavage product. Figure 2 As can be seen from A in the graph, the decrease in fluorescence signal gradually intensifies as the length of the single-stranded DNA substrate increases from 10 nt to 20 nt. Figure 3 As can be seen, the band corresponding to the 29 nt single-stranded DNA substrate showed significant attenuation, confirming that the 29 nt single-stranded DNA substrate was cleaved by Cas12a activated by miRNA-21. From Figure 2 As shown in Figure C, trans-cleavage efficiency increases with increasing single-stranded DNA substrate length, with 29 nt single-stranded DNA substrates exhibiting the highest activity. By comparing the trans-cleavage effects of linear 29 nt single-stranded DNA substrates with their fully folded aptamers (DAP-10) forming G-quadruplex structures, [the following analysis is needed to determine the optimal trans-cleavage efficiency]. Figure 2 As shown in Figure D, the decrease in AO fluorescence intensity induced by the linear 29 nt single-stranded DNA substrate during trans cleavage was significantly greater than that induced by the DAP-10 aptamer. This indicates that, under trans cleavage mode, miRNA-21-activated Cas12a has a higher cleavage efficiency for linear single-stranded DNA than for structured DNA, validating the rationale for using the 29 nt single-stranded DNA substrate as the primary trans cleavage substrate. The 29 nt single-stranded DNA serves as the linker.

[0030] 2. The splitting aptamer P1, splitting aptamer P2, linker, and AO were mixed, and AO buffer was added to adjust the volume to 20 μL. After vortexing, the mixture was incubated at 37 ℃ for 15 min to ensure complete assembly of the complex. The entire reaction solution was then transferred to a quartz cuvette for fluorescence detection. The emission intensity at 546 nm (excitation wavelength 480 nm) was measured using an FS5 fluorescence spectrometer (Livingston, Edinburgh Instruments, UK). The final concentrations of splitting aptamer P1, splitting aptamer P2, and linker were all 1 μM, and the final concentration of AO was 20 μM. Figure 4 As can be seen from Figure A, a strong fluorescence signal was observed only when the splitting aptamer P1, splitting aptamer P2, and linker were present simultaneously. This confirms that fluorescence activation strictly depends on the three components co-assembling into a G-quadruplex structure. The absence of any component will hinder the formation of the complex and maintain the fluorescence-off state.

[0031] 3. Mix 0.5 μL of 1 μM Cas12a and 1 μL of 0.5 μM crRNA-D and incubate at 37 °C for 15 minutes to obtain the Cas12a / crRNA-D RNP complex. Mix 2 μL of 5 μM EGFR L858R with the above Cas12a / crRNA-D RNP complex. Then add the G-quadruplex complex composed of aptamer P1, aptamer P2, linker, and AO as the trans-cleavage substrate and adjust the final reaction volume to 20 μL with AO buffer, so that the final concentrations of aptamer P1, aptamer P2, and linker are all 1 μM, and the final concentration of AO is 20 μM. After incubation at 37 °C for 1 hour, the fluorescence intensity of AO is measured using an FS5 fluorescence spectrophotometer. Figure 4 As shown in Figure B, AO fluorescence remained at its highest level in the absence of EGFR L858R; however, the fluorescence intensity decreased significantly after the addition of EGFR L858R, confirming that EGFR L858R-activated Cas12a achieved efficient trans-cleavage of the G-quadriplex. Figure 4 As shown in Figure C, compared to the intact band in lane 6, lane 7, containing EGFR L858R, exhibits significant substrate trans-cleavage, while lane 8, without EGFR L858R, remains intact. This confirms that the assembled G-quadruplex structure is an effective trans-cleavage substrate for DNA activation of Cas12a.

[0032] 4. Smartphone-Assisted Detection System: 1 μL of linker at different concentrations, 1 μL of 20 μM apposition aptamer P1, 1 μL of 20 μM apposition aptamer P2, and 2 μL of 50 μM AO were mixed and added to AO buffer, bringing the final volume to 20 μL. The mixture was then incubated at 37 ℃ for 25 min. The signals were read using the FluoroSignalAPP application on a HUAWEI Pura 70 smartphone and an FS5 fluorescence spectrophotometer. A handheld UV lamp was used as the excitation source. The FluoroSignalAPP was used to capture images, and the green channel of the images was used as the analysis signal. The reliability of the smartphone-assisted detection system was verified by comparing the detection results of the smartphone and the FS5 fluorescence spectrophotometer. Figure 4 As can be seen from E, the color of the acquired image varies with different linker concentrations. Quantitative extraction of the green channel intensity from the image revealed an excellent linear correlation between it and the corresponding fluorescence intensity measured by the FS5 fluorescence spectrophotometer: Y = 0.00145X - 2.70327, R... 2 =0.98388. The FluoroSignalAPP includes three core functional modules: image acquisition, region of interest (ROI) selection, and ROI RGB value extraction.

[0033] Experimental Example 2 Conditional optimization 1. Splitting aptamer assembly time: From Figure 5 As can be seen from A, at 37 ℃, the fluorescence intensity of AO gradually increases with the increase of the assembly time of splitting aptamers P1 and P2 with the linker, and reaches a plateau after 15 min, indicating that the G-quadruplex structure has been fully formed.

[0034] 2. Optimization of AO Concentration: Maintaining the concentrations of apse aptamer P1, apse aptamer P2, and linker at 1 µM, the AO concentration was optimized. Figure 5 As shown in B, the fluorescence intensity of AO increases with increasing AO concentration, reaching its highest value at 5 µM. The conformational stability of the assembled G-quadruplex complex is confirmed by the predicted secondary structure obtained through minimum free energy analysis. Figure 6 It can be seen that the free energy of the secondary structure is -52.01 kcal / mol, indicating that the secondary structure of the G-tetrachain complex has good stability. From... Figure 7 It can be seen that the AO fluorescence signal remained stable within 72 hours, demonstrating the excellent temporal stability of the reporting system under the detection conditions.

[0035] 3. CRISPR / Cas12a component concentration optimization: From Figure 5 As shown in Figure C, the difference in AO fluorescence between the reaction system containing miRNA-21 and the reaction system without miRNA-21 increases with increasing linker concentration, reaching a peak at a linker concentration of 1 µM. Figure 5 As can be seen from D, when the Cas12a concentration is 25 nM, the difference in AO fluorescence intensity between the reaction system containing miRNA-21 and the reaction system without miRNA-21 reaches its peak. From Figure 5 As can be seen from E and F, the difference in AO fluorescence between the two reaction systems reaches its peak when the concentrations of crRNA-R and crRNA-D are 25 nM.

[0036] 4. Optimization of reaction temperature and time for miRNA-21 activation of Cas12a: By activating the Cas12a / crRNA-R complex with 2 μL of 5 μM miRNA-21, from... Figure 5 As can be seen from G, when the incubation time was controlled at 75 min and the reaction temperature at 37 ℃, the AO fluorescence difference between the reaction system containing miRNA-21 and the reaction system without miRNA-21 reached its peak. From Figure 5 As can be seen from H, when the reaction temperature is controlled at 37 °C, the fluorescence signal rapidly decreases before 60 min, and then gradually enters the kinetic plateau phase. Furthermore, at 60 min, there are sufficient linkers for the subsequent assembly of the G-quadruplex and the generation of fluorescence signals in the second detection step. Therefore, 60 min was chosen as the optimal cutting time to ensure stable and reliable signal readings without compromising the integrity of the two-stage detection strategy.

[0037] Experimental Example 3 Performance Analysis 1. Standard Curve and Linear Range: The optimal conditions obtained in Experiment 2 were used to detect different concentrations of miRNA. Figure 8 As can be seen from A, the intensity of the green channel in AO continuously decreases with increasing miRNA-21 concentration. From... Figure 8 As can be seen from B, within the concentration range of 5 nM-500 nM, the green channel intensity (Y) correlates with the logarithm of the target concentration (X=lgC). miRNA-21 The correlation coefficient (Rm) shows a linear relationship with nM. The standard curve equation is Y = -41.748221X + 203.41176, and the correlation coefficient (Rm) is [missing value]. 2 The value is 0.99825.

[0038] 2. Limit of Detection (LOD): The LOD was determined by analyzing blank samples (n=6). The threshold signal was defined as the average intensity of the green channel of the sample plus three standard deviations (mean + 3SD). Substituting this threshold into the standard curve equation Y=-41.748221X+203.41176, the LOD of miRNA-21 was calculated to be 1.26 nM.

[0039] 3. Recovery and Precision Assessment: Low, medium, and high concentrations (10, 100, and 500 nM) of miRNA-21 were added to the plasma of healthy individuals, respectively. Figure 8 As can be seen from C, the signal intensity of the green channel decreases as the target concentration increases. Table 2 shows that the detection method of this invention exhibits high accuracy and good repeatability.

[0040] Table 2 Recovery and Reproducibility

[0041] 4. Specificity analysis: miRNA-155, miRNA-122, and miRNA-141 were analyzed individually, and also co-detected with miRNA-21. Figure 8 As can be seen from D, the green channel signal was significantly reduced in samples containing only miRNA-21. Figure 8 As can be seen from the E in the figure, this invention clearly distinguishes between the EGFR L858R mutant and wild-type sequences, demonstrating high single-base resolution.

[0042] Test Example 4 Real sample analysis Total RNA and DNA were extracted from NCI-H1975 (human lung adenocarcinoma cells) and BEAS-2B (human normal bronchial epithelial cells). Total RNA was isolated using the AG RNAex Pro RNA Extraction Kit according to the manufacturer's instructions, and genomic DNA was purified using the Ezup Column-Based Animal Genomic DNA Extraction Kit. Equal volumes of RNA and DNA extracted from the same cell line were mixed to form nucleic acid samples for analysis, and detection was performed according to the procedure in Example 2. Figure 8 As can be seen from F in the results, the initial detection showed that the signal intensity of miRNA-21 in NCI-H1975 cells was significantly lower than that in BEAS-2B cells. P <0.001). In the second detection, the NCI-H1975 sample showed a further decrease in signal ( PThe result (<0.01) confirmed the presence of the EGFR L858R mutation. These results indicate that this method can serve as an integrated platform for simultaneously analyzing miRNA expression and DNA mutation status, providing supporting evidence for its potential application in the molecular diagnosis of lung cancer. Significant difference analysis showed that the fluorescence intensity of BEAS-2B cells also decreased slightly in the second detection, but this change was within the error range and not statistically significant (P=0.6058).

[0043] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A system for detecting miRNA and DNA mutations based on CRISPR-Cas12a and split aptamer one-pot method, characterized in that, include: Cas12a protein, crRNA-R targeting miRNA, mitochondrial aptamer P1, mitochondrial aptamer P2, linker, AO, and crRNA-D targeting DNA mutations.

2. The system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers according to claim 1, characterized in that, The sequence of the crRNA-R targeting miRNA is shown in SEQ ID NO. 1; the sequence of the crRNA-D targeting DNA mutation is shown in SEQ ID NO.

2.

3. The system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers according to claim 1, characterized in that, The sequence of the split aptamer P1 is shown in SEQ ID NO. 3; the sequence of the split aptamer P2 is shown in SEQ ID NO. 4; and the sequence of the linker is shown in SEQ ID NO.

5.

4. The system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers according to claim 1, characterized in that, The miRNA in the crRNA-R targeting miRNA is miRNA-21; the DNA mutation in the crRNA-D targeting DNA mutation is EGFR L858R DNA.

5. The system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers according to claim 1, characterized in that, The final concentration of the linker is 0.25-1.25 μM; the final concentration of the Cas12a protein is 5-75 nM; the final concentration of the crRNA-R targeting miRNA is 15-30 nM; the final concentration of the crRNA-D targeting DNA mutation is 15-30 nM; the final concentration of AO is 0.5-30 μM; the final concentration of the splitting aptamer P1 is 0.25-1.25 μM; and the final concentration of the splitting aptamer P2 is 0.25-1.25 μM.

6. The system for detecting miRNA and DNA mutations based on a one-pot method using CRISPR-Cas12a and apposition aptamers according to claim 5, characterized in that, The final concentration of the linker is 1 μM; the final concentration of the Cas12a protein is 25 nM; the final concentration of the crRNA-R targeting miRNA is 25 nM; the final concentration of the crRNA-D targeting DNA mutation is 25 nM; the final concentration of AO is 5 μM; the final concentration of the splitting aptamer P1 is 1 μM; and the final concentration of the splitting aptamer P2 is 1 μM.

7. A kit for detecting miRNA and DNA mutations using a one-pot method based on CRISPR-Cas12a and aptamers, characterized in that... The system comprising the one-pot method for detecting miRNA and DNA mutations based on CRISPR-Cas12a and apposition aptamers as described in claim 1.

8. The system for detecting miRNA and DNA mutations based on CRISPR-Cas12a and aptamer in a one-pot method as described in any one of claims 1-6, or the kit for detecting miRNA and DNA mutations based on CRISPR-Cas12a and aptamer in a one-pot method as described in claim 7, in the preparation of products for early diagnosis, molecular subtyping, and prognostic monitoring of lung cancer.

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