Method for ultrasensitively detecting breast cancer miRNAs by constructing AND logic gate based on strand displacement driven RCA-Cas12a

Abstract

The invention belongs to the technical field of biological analysis, and particularly relates to application of strand displacement reaction, rolling circle amplification, a CRISPR-Cas12a system and in-vitro transcription in miRNAs detection. The invention provides a method for sensitively and specifically detecting breast cancer miR-21 and miR-155 by driving NRCA and LRCA amplification through strand displacement reaction, realizing efficient amplification of an activator, activating trans-cleavage of Cas12a in combination with transcriptional crRNA, and realizing fluorescence signal amplification. The method is convenient, sensitive, low in background interference and high in specificity, can be applied to simultaneous detection of miR-21 and miR-155 in breast cancer serum, can effectively distinguish healthy individuals from breast cancer patients, expands methodology of CRISPR-Cas12a in the field of in-vitro diagnosis, and shows a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis technology, and particularly relates to a method for ultrasensitive detection of breast cancer miRNAs (miR-21 and miR-155) by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a. Background Art

[0002] As the most common malignant tumor with the highest incidence and mortality rate among women globally, breast cancer's high metastatic characteristics significantly increase the clinical prognosis risk. Notably, approximately 90% of breast cancer-related deaths are not due to the primary lesions but secondary complications caused by the recurrence of metastatic lesions. Since these secondary tumors are mostly detected at an advanced stage, it poses a greater challenge to clinical diagnosis and treatment. Therefore, there is an urgent need to discover biomarkers to construct a new early diagnosis system for metastatic breast cancer, thereby improving early diagnosis and prognosis of patients.

[0003] In the field of oncology, specific biomarkers are crucial for accurate medical diagnosis and treatment. Recent research has provided evidence that microRNAs (miRNAs) act as key mediators in the complex dialogue between cancer cells and their host. However, during miRNA detection, the instability of RNA (such as contamination and degradation) is one of the major challenges. To address this issue, there is an urgent need to develop point-of-care testing (POCT) technologies and pre-treat RNA before detection. Currently, real-time quantitative polymerase chain reaction (RT-qPCR) and Northern blotting are conventional methods for miRNA detection. Although they perform well in terms of sensitivity and accuracy, these technologies rely on large equipment, which limits their application in portable detection and makes it difficult to popularize in resource-limited areas.

[0004] Recently, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR / Cas) systems have made significant progress in sensing applications. Among these CRISPR / Cas systems, CRISPR / Cas12a is a powerful detection tool because it has specific recognition capabilities for both single-stranded and double-stranded DNA. Cas12a has attracted much attention due to its cis- and trans-cleavage activities: the cis-cleavage activity can target and cleave the target DNA, while the trans-cleavage activity can non-specifically degrade single-stranded DNA (ssDNA). Notably, the trans-cleavage activity of Cas12a is only activated when the target DNA is fully paired with the complementary CRISPR RNA (crRNA). Although the nuclease activity of Cas proteins has expanded their applications in biosensing, their inherent sensitivity makes it difficult to directly detect trace targets. Therefore, to meet the high-sensitivity requirements of clinical detection, researchers have developed various methods of combining CRISPR / Cas12a systems with isothermal nucleic acid amplification techniques, such as opvCRISPR (RT-LAMP-CRISPR / Cas12a), HOLMESv2, and RCA-Cas12a. However, due to the compatibility of method components, sequences, and temperatures, simply combining the amplification method with CRISPR / Cas will result in reduced sensitivity. Summary of the Invention

[0005] The present invention aims to overcome the problems of cumbersome operation steps, insufficient specificity, and sensitivity in the simultaneous detection of existing miRNA-155 and miRNA-21, and provides an AND logic gate platform for detecting dual miRNAs based on strand displacement-driven RCA-Cas12a. This platform combines RCA and Cas12a to achieve specific detection of miR-21 and miR-155 in breast cancer simultaneously. When miR-21 and miR-155 are present simultaneously, the hairpin probe unfolds under the action of strand displacement reaction, releasing the initiation sequences that can activate two RCA reactions. Two padlock probes specifically bind to the primers of Nicked RCA (NRCA) and Long RCA (LRCA) respectively, forming a dual-signal amplification pathway. Subsequently, the RCA amplification products, through base complementary pairing, serve as the primer strand and template strand of Branch RCA (BRCA) to complete the operation of the AND logic gate. Under the catalysis of phi29 DNA polymerase and exonuclease activity, the primers bind to the template and extend to generate a double-stranded DNA structure containing the PAM sequence, which can be precisely recognized by Cas12a and trigger its trans-cleavage activity, and finally realize the synchronous one-pot detection of dual miRNAs through fluorescence signal output.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] In the first aspect of the present invention, a method for detecting miR-21 and miR-155 using a strand-displacement-driven RCA-Cas12a AND logic gate platform is provided. The platform includes hairpin probes Hairpin-1 and Hairpin-2, padlock probes Padlock-1 and Padlock-2, T4 DNA ligase, phi29 DNA polymerase, nicking endonuclease Nt.BstNBI, crRNA template, Lba Cas12a, and FQ Reporter. Among them, the hairpin probes specifically recognize miR-21 and miR-155 (sequences are 5’-TCA ACA TCA GTC TGA TAA GCT ACG TAT AAA GCT TAT TCA GAC TGT TAT ATC-3’, SEQ ID NO: 1; 5’-ACC CCT ATC ACG ATT AGC ATT AAT TCA AAT GCT TAA TCG TGA TCTAG TG’, SEQ ID NO: 2); the padlock probes Padlock-1 and Padlock-2 are circularized under the action of T4 DNA ligase to form circular templates for NRCA and LRCA (sequences are 5’-CTG AAT AAG CTA AAA AAA AAT GGG TAA CCCGCC CTA CCC AGA AAA AAA CAG ATA TAA CAG T-3’, SEQ ID NO: 3; 5’-GAT TAA GCA TTAAAA AAA GAC TCG CGG GTT ACC CAT TAA AGA CTC AAA CAC TAG ATC AC-3’, SEQ ID NO: 4); the crRNA template (sequence is 5’-TGG GTA ACC CGC CCT ACC CAA TCT ACA CTT AGT AGA AATTAC CCT ATA GTG AGT CGT ATT A-3’, SEQ ID NO: 5).

[0008] In a preferred technical solution, the NRCA amplification product contains a nicking endonuclease Nt.BstNBI cleavage site 5’-GAGTCNNNN ▼ N-3’ (N is any base).

[0009] In a preferred technical solution, the sequence of the FQ Reporter is as follows: 5’-FAM-TTA TT-BHQ-3’.

[0010] The second aspect of the present invention provides a method for detecting miR-21 and miR-155 using the strand displacement-driven RCA-Cas12a-based AND logic gate platform described in the first aspect, comprising the following steps:

[0011] (1) Mix the crRNA template with the T7 promoter solution and incubate, followed by transcription to form crRNA;

[0012] (2) Mix the hairpin probe, miR-21 and miR-155, padlock probe, and T4 DNA ligase and incubate. Under the action of strand displacement reaction, the hairpin probe unfolds, and under the ligation of T4 DNA ligase, the two padlock probes are circularized to form circular templates of NRCA and LRCA respectively;

[0013] (3) Add phi29 buffer, phi29 DNA polymerase, and nicking endonuclease Nt.BstNBI to the reaction system of step (2). Under the catalysis of phi29 DNA polymerase, a large amount of NRCA and LRCA amplification products are obtained. Under the action of nicking endonuclease Nt.BstNBI, the NRCA amplification product is fragmented. The monomerized NRCA amplicon serves as a secondary primer. After hybridization with the LRCA amplicon, under the action of the 3'-to-5' exonuclease activity and amplification effect of phi29 DNA polymerase, BRCA is triggered to generate long dsDNA or ssDNA of different lengths containing PAM;

[0014] (4) Mix Lba Cas12a with the transcription product crRNA of step (1), and then add the reaction mixture of step (3) and continue to incubate for CRISPR / Cas12a cleavage reaction;

[0015] (5) Detect the fluorescence signal of the reaction mixture of step (4) by an F-7000 fluorescence spectrophotometer.

[0016] Preferably, it is characterized in that the hairpin probe binds to the padlock probe through toehold-assisted strand displacement reaction.

[0017] Preferably, it is characterized in that nicking endonuclease Nt.BstNBI cleaves the NRCA amplification product, and its product can not only serve as a primer for BRCA but also activate the NRCA reaction again to achieve cyclic amplification.

[0018] Preferably, it is characterized in that the use of phi29 DNA polymerase not only provides power for NRCA and LRCA to constitute a dual-signal amplification path but also utilizes its exonuclease activity to generate primers required to activate BRCA and activate the trans-cleavage of Cas12a.

[0019] A third aspect of the present invention provides an application of a method for detecting miR-21 and miR-155 using a strand displacement-driven RCA-Cas12a-based AND logic gate platform as described in the first aspect.

[0020] In a preferred technical solution, the samples to be detected include sera of breast cancer patients and normal sera.

[0021] Compared with the prior art, a method for ultrasensitively detecting breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a has the following beneficial effects:

[0022] (1) The method for ultrasensitively detecting breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a of the present invention has high sensitivity, and the double signal amplification of rolling circle amplification NRCA and LRCA can effectively improve the detection sensitivity.

[0023] (2) The method for ultrasensitively detecting breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a of the present invention has extremely high specificity. The strand displacement reaction-driven RCA-Cas12a endows this method with extremely high specificity and the ability to recognize single-base mismatches.

[0024] (3) The method for ultrasensitively detecting breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a of the present invention has low background interference. The optimized design of the padlock probe effectively optimizes the background signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To enhance the further understanding of the present invention, the following will briefly introduce the drawings required for the specific implementation methods. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 It is a schematic diagram of the principle of the method for ultrasensitively detecting breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a of the present invention.

[0027] Figure 2 It is a non-denaturing polyacrylamide gel electrophoresis diagram for verifying the feasibility of the method for detecting miR-21 in the present invention.

[0028] Figure 3 It is a non-denaturing polyacrylamide gel electrophoresis diagram for verifying the feasibility of the method for detecting miR-155 in the present invention.

[0029] Figure 4 It is a fluorescence spectrogram for verifying the feasibility of the method in the present invention.

[0030] Figure 5 Schematic diagram of AND logic gate relationship for verifying the feasibility of the method in the present invention.

[0031] Figure 6 Graph showing the experimental results of optimizing the dosage of T4 DNA ligase and the ligation reaction time.

[0032] Figure 7 Graph showing the experimental results of optimizing the RCA reaction time and the reaction time of Cas12a cleavage.

[0033] Figure 8 Graph for sensitivity analysis of different concentrations of miRNAs in the present invention.

[0034] Figure 9 Graph showing the non-linear and linear relationships of fluorescence signal fitting for different concentrations of miRNAs in the present invention.

[0035] Figure 10 Fluorescence spectrogram and bar chart for verifying the specificity of the method in the present invention.

[0036] Figure 11 Graph showing the real-time fluorescence response and linear relationship of RT-qPCR analysis of different concentrations of miRNA standards.

[0037] Figure 12 In this figure, A is a bar chart of the detection of miRNAs in breast cancer patients and normal sera by the method of the present invention and RT-qPCR; B is a box plot of the detection of miRNAs in breast cancer patients and normal sera by the method of the present invention and RT-qPCR. Detailed implementation manners

[0038] Sequence listing:

[0039]

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The materials cited herein and their citations will be incorporated by reference.

[0042] Equivalent technologies of the specific embodiments described that can be understood by those skilled in the art through conventional experiments will be included in this application.

[0043] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. In the quantitative experiments of the following examples, three repeated experiments are set, and the results are averaged. The instrument and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instrument and equipment; the experimental materials used in the following examples, unless otherwise specified, are all obtained by purchasing from a conventional biochemical reagent store.

[0044] Example 1

[0045] A method for constructing an AND logic gate ultrasensitive detection of breast cancer miRNAs based on strand displacement-driven RCA-Cas12a, and the specific synthesis steps are as follows:

[0046] (1) Preparation of crRNA: First, anneal a mixture containing 1 μL of T7 promoter (100 μM), 1 μL of crRNA template (100 μM), 2 μL of 10× AmpliScribe T7 reaction buffer, and 10 μL of RNase-free water at 95 °C for 5 min and slowly cool to room temperature. Then, add 1.5 μL of NTP (100 mM for each NTP), 2 μL of 100 mM DTT, 0.5 μL of RNase inhibitor, and 2 μL of T7 RNA polymerase to mix and prepare a 20 μL transcription reaction, and react at 37 °C for 2 h. Then, add 2 μL of DNaseI to the reaction solution and store at 37 °C for 1 h to degrade the DNA template. Quantify with Nanodrop2000 and store at -80 °C.

[0047] (2) Preparation of RCA-Cas12a reaction: Add 1 μL of hairpin probe (100 nM), 1 μL of padlock (100 nM), and 1 μL of target (100 nM) to 1 μL of 10× T4 DNA ligase buffer, and then add DEPC-treated water to 10 μL. The hairpin probe, padlock, and target are annealed at 95 °C for 5 min and then gradually cooled to room temperature. After adding 5 U of T4 DNA ligase, incubate at 37 °C for 30 min and stop by heating at 80 °C for 10 min. Subsequently, add 2 μL of 10× phi29 buffer, 1 μL of 20 mM dNTPs, 5 U of Nt.BbvCI nicking endonuclease, and 5 U of phi29 DNA polymerase to the above reaction system. React at 37 °C for 40 min and inactivate by heating at 80 °C for 10 min. Add 1 μL of Cas12a (2 μM), 1 μL of crRNA (2 μM), and 2 μL of F-Q reporter (1 μM) to the reaction solution. After incubating at 37 °C for 60 min, add 70 μL of a solution of DEPC-treated water to measure fluorescence, and record with an F-7000 fluorescence spectrophotometer under 480 nm excitation light.

[0048] Example 2

[0049] Feasibility analysis of constructing an AND logic gate for ultrasensitive detection of breast cancer miRNAs markers based on strand displacement-driven RCA-Cas12a

[0050] To investigate the feasibility of the method of the present invention for the detection of miR-21 and miR-155, non-denaturing polyacrylamide gel electrophoresis analysis was used, and the results are shown in Figure 2 、 3 . Then fluorescence spectroscopy was used for analysis, and the results are shown in Figure 4 、 5 .

[0051] As can be seen from Figure 2 , when the padlock probe Padlock-1, the hairpin probe Hairpin-1 and miR-21 were incubated together, a clear band with a slow migration rate was observed, indicating that Padlock-1 successfully hybridized with the primer (lane 2) exposed after the binding of Hairpin-1 and miR-21 (lane 4), and after adding T4 DNA polymerase, ExonucleaseⅠ and phi29 DNA polymerase for amplification, a very bright band was observed (lane 8), which proved that the product of LRCA was obtained.

[0052] As can be seen from Figure 3 , when the padlock probe Padlock-2, the hairpin probe Hairpin-2 and miR-155 were incubated together, a clear band with a slow migration rate was observed, indicating that Padlock-2 successfully hybridized with the primer (lane 2) exposed after the binding of Hairpin-2 and miR-155 (lane 4), and after adding T4 DNA polymerase, ExonucleaseⅠ, phi29 DNA polymerase and Nt.BstNBI nicking endonuclease for amplification, many bands of different lengths were produced (lane 8), which indicated that the NRCA reaction produced a large amount of amplified ssDNA.

[0053] As can be seen from Figure 4 , when the target miR-21 or the target miR-155 was absent, the detected fluorescence signal was extremely weak as the background signal. However, when both the target miR-21 and the target miR-155 were present, through strand displacement reaction-driven RCA-Cas12a, compared with the control group, the fluorescence signal was significantly enhanced. It is shown that the synergistic effect of miR-21 and miR-155 can trigger a large increase in the fluorescence signal, which indicates that this logic gate platform is feasible. Therefore, the schematic diagram of the AND logic gate relationship can also be obtained, as shown in Figure 5 .

[0054] Example 3

[0055] To test the effects of different reaction conditions on the ultrasensitive detection of breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a.

[0056] (1) Optimization of the amount of T4 DNA ligase

[0057] The preparation method of the sensor was the same as that in Example 1, except that the amount of T4 DNA ligase was changed to 0.5, 2, 5, 10, 15 U. The results are shown in Figure 6 A.

[0058] As can be seen from Figure 6 A, when the amount of T4 DNA ligase reached 5 U, the signal-to-noise ratio was the highest and the fluorescence signal was the best.

[0059] (2) Optimization of the ligation reaction time

[0060] The preparation method of the sensor was the same as that in Example 1, except that the ligation reaction time was changed to 5, 10, 20, 30, 50 min. The results are shown in Figure 6 B.

[0061] As can be seen from Figure 6 B, when the ligation reaction time was 30 min, the signal-to-noise ratio was the highest.

[0062] (3) Optimization of the RCA reaction time

[0063] The preparation method of the sensor was the same as that in Example 1, except that the RCA reaction time was changed to 20, 30, 40, 50, 60 min. The results are shown in Figure 7 A.

[0064] As can be seen from Figure 7 A, when the RCA reaction time was 40 min, the signal-to-noise ratio was the highest and the fluorescence signal was the best.

[0065] (4) Optimization of the Cas12 cleavage reaction time

[0066] The preparation method of the sensor was the same as that in Example 1, except that the Cas12 cleavage reaction time was changed to 20, 30, 40, 50, 60 min. The results are shown in Figure 7 B.

[0067] As can be seen from Figure 7 B, when the Cas12 cleavage reaction time was 50 min, the signal-to-noise ratio was the highest and the fluorescence signal was the best.

[0068] Example 4

[0069] Performance analysis experiment of ultrasensitive detection of breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a.

[0070] (1) Sensitivity detection

[0071] The preparation method of the AND logic gate platform based on strand displacement-driven RCA-Cas12a was the same as that in Example 1, except that the concentrations of target molecules miR-21 and miR-155 were changed to 0 fM, 0.5 fM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 5 nM. The results are shown in Figure 8 .

[0072] From Figure 8 it can be seen that the fluorescence intensity is positively correlated with the concentration of target molecules.

[0073] (2) Standard curve establishment

[0074] Using Figure 8 the fluorescence intensities corresponding to different miR-21 and miR-155 concentrations in Figure 9 .

[0075] From Figure 9 it can be seen that there is a good linear relationship between the logarithm of the double target concentration (lgC miRNAs ) and the fluorescence intensity, and the regression equation is F = 351.25 + 344.83lgC miRNAs (R 2 = 0.9958). Calculated based on the 3σ / k rule, the detection sensitivity of this sensor for miR-21 and miR-155 can reach 156.3 aM. The results show that the method of the present invention has high detection sensitivity.

[0076] (3) Specificity detection

[0077] The preparation method of the AND logic gate platform based on strand displacement-driven RCA-Cas12a was the same as that in Example 1, except that the target molecules were changed to a mixture of several other miRNAs (including miR-18a, miR-200b, miR-141, and let-7a) as interferents. The results are shown in Figure 10 .

[0078] From Figure 10 it can be seen that it is found that when Mixed miRNAs exist or any one of miR-21 and miR-155 exists alone, it can be ignored just like the background signal, indicating that the method of the present invention has excellent specificity.

[0079] Example 5

[0080] Practical sample application test for ultrasensitive detection of breast cancer miRNAs by constructing an AND logic gate based on strand displacement-driven RCA-Cas12a.

[0081] (1) Establishing a standard curve by RT-qPCR method

[0082] After extracting total miRNAs from different cell lines and clinical serum samples, according to the instructions of miRNA 1st Strand cDNA Synthesis Kit (bystem-loop), the corresponding cDNA templates were prepared. Ensure strict compliance with the operating steps of the kit to guarantee the quality and efficiency of cDNA synthesis. Add 10 μL of ChamQ Universal SYBR Qpcr MasterMix, 0.4 μL of forward primer with a concentration of 10 μM, 0.4 μL of reverse primer with a concentration of 10 μM, 1 μL of reverse transcription reaction product, and 8.2 μL of DEPC-treated water into a solution with a total reaction volume of 20 μL. Use the QuantStudio 3 RealTime system to measure miRNAs. The reaction program is as follows: First, perform pre-denaturation at 95 °C for 5 min; then perform initial denaturation at 95 °C for 15 s; then perform 40 cycles, each cycle including denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s; finally, perform final denaturation at 95 °C for 15 s, fluorescence signal acquisition at 60 °C for 60 s, and melting curve analysis at 95 °C for 15 s. The results are shown in Figure 11 A. According to Figure 11 the CT value corresponding to the intersection point of the A threshold line and the fluorescence spectrum for linear fitting, and the results are shown in Figure 11 B.

[0083] From Figure 11 B, it can be seen that from 10 -13 M to 10 -9 M, there is a good linear relationship between the Ct value and the logarithm of the miRNAs concentration, and the regression equation is Ct = 29.5502 - 3.4718lgC (R 2 = 0.9994).

[0084] (2) Analyzing clinical serum samples by constructing an AND logic gate ultrasensitive detection of breast cancer miRNAs based on strand displacement-driven RCA-Cas12a and the gold standard RT-qPCR

[0085] After obtaining the serum component by centrifuging the fresh whole blood sample at 10,000 rpm for 8 min, 1 mL of serum was mixed with 15 mL of perchloric acid solution in proportion, and protein macromolecule precipitation was achieved by vortex oscillation at room temperature for 5 min. Subsequently, the mixed solution was centrifuged twice (10,000 rpm, 10 min), and the separated supernatant was transferred to a cryotube and stored in an ultra-low temperature refrigerator at -80 °C for later use. Take 0.5 mL of the serum sample and place it in a centrifuge tube, add 1 mL of miRNA extraction solution, and lyse for 5 - 10 min to fully release miRNA. Add 0.2 mL of CHCl3 to the centrifuge tube, shake vigorously for 30 s, and let it stand for 3 min to stratify the mixture. Centrifuge at 12,000 rpm and 4 °C for 10 min to separate the upper aqueous phase and transfer it to a new centrifuge tube. Add an equal volume of isopropanol to the above centrifuge tube, mix well and let it stand overnight at room temperature to fully precipitate miRNA. Centrifuge at 12,000 rpm and 4 °C for 10 min, discard the supernatant, and retain the miRNA precipitate. Add 1 mL of 75% ethanol for washing to remove residual impurities. Centrifuge at 12,000 rpm and 4 °C for 3 min, discard the supernatant, dry at room temperature for 10 min, and finally add 30 μL of DEPC-treated water to completely dissolve miRNA for subsequent experiments. Use a Nanodrop2000C spectrophotometer to measure the concentration of the total miRNA extracted. Detect miR-21 and miR-155 in 6 normal serum samples and 6 breast cancer patient sera respectively using the strand displacement-driven RCA-Cas12a-based AND logic gate platform and the gold standard RT-qPCR, and perform quantification using the standard curve constructed in Example 5. The results are shown in Figure 12 A and 12B.

[0086] From Figure 12 A and Figure 12 B, it can be seen that the average levels of miR-21 and miR-155 in the sera of breast cancer patients are significantly higher than the corresponding levels in the sera of healthy people (results of the proposed biosensor, magenta columns), which are consistent with the results of RT-qPCR detection (light purple columns). In addition, the differences in the expressions of miR-21 and miR-155 between the cancer patient group and the healthy people group were analyzed by t-test, and it was found that the levels of miR-21 and miR-155 in breast cancer patients were significantly higher than those in healthy people (t-test, p < 0.001). These results indicate that this method can accurately identify the differences in the levels of miR-21 and miR-155 between breast cancer patients and healthy controls, and has great potential for clinical diagnostic applications.

[0087] Note: The serum specimens of breast cancer patients and normal individuals were obtained from the Hunan Provincial Key Laboratory of Regional Genetic Birth Defect Prevention and Control, Changsha Maternal and Child Health Care Hospital Affiliated to Hunan Normal University (Ethical Approval Number: EC-20240828-09).

[0088] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in this technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A method for detecting miR-21 and miR-155 based on a strand displacement-driven RCA-Cas12a AND logic gate platform, characterized in that The platform includes hairpin probes Hairpin-1 and Hairpin-2, padlock probes Padlock-1 and Padlock-2, T4 DNA ligase, phi29 DNA polymerase, nicking endonuclease Nt.BstNBI, crRNA template, LbaCas12a, and FQ Reporter; wherein, the hairpin probes specifically recognize miR-21 and miR-155, and the sequences are as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the padlock probes Padlock-1 and Padlock-2 are circularized under the action of T4 DNA ligase to form circular templates of NRCA and LRCA, and the sequences are as shown in SEQ ID NO: 3 and SEQ ID NO: 4; the crRNA template has a sequence as shown in SEQ ID NO:

5.

2. The logic gate platform according to claim 1, wherein The NRCA amplification product contains a nick endonuclease Nt.BstNBI cleavage site 5'-GAGTCNNNN ▼ N-3' (where N is any base).

3. The logic gate platform according to claim 1, characterized in that The sequence of the FQ Reporter is as follows: 5’-FAM-TTATT-BHQ-3’.

4. A method for detecting miR-21 and miR-155 based on a strand displacement-driven RCA-Cas12a AND logic gate platform, characterized in that, It includes the following steps: (1) Mix the crRNA template with the T7 promoter solution and incubate it to transcribe crRNA. (2) Mix the hairpin probes, miR-21 and miR-155, padlock probes, and T4 DNA ligase and incubate them. The hairpin probes are unfolded under the action of strand displacement reaction, and the two padlock probes are circularized under the ligation action of T4 DNA ligase to form circular templates of NRCA and LRCA respectively. (3) Add phi29 buffer, phi29 DNA polymerase, and nicking endonuclease Nt.BstNBI to the reaction system of step S2. Under the catalysis of phi29 DNA polymerase, a large number of NRCA and LRCA amplification products are obtained. Under the action of nicking endonuclease Nt.BstNBI, the NRCA amplification products are fragmented. The monomerized NRCA amplicons are used as secondary primers. After hybridizing with the LRCA amplicons, under the action of the 3’-to-5’ exonuclease activity and amplification effect of phi29 DNA polymerase, BRCA is triggered to generate long dsDNA or ssDNA of different lengths containing PAM. (4) Mix Lba Cas12a with the transcription product crRNA of step (1), and then add the reaction mixture of step (3) and continue to incubate for CRISPR / Cas12a cleavage reaction. (5) Detect the fluorescence signal of the reaction mixture of step (4) by an F-7000 fluorescence spectrophotometer.

5. The method according to claim 4, characterized in that, The hairpin probe binds to the padlock probe through toehold-assisted strand displacement reaction.

6. The method according to claim 4, wherein The nicking endonuclease Nt.BstNBI cleaves the NRCA amplification product, and its product can not only serve as a primer for BRCA but also activate the NRCA reaction again to achieve circular amplification.

7. The method according to claim 4, wherein The use of phi29 DNA polymerase not only provides power for NRCA and LRCA to constitute a dual-signal amplification path but also utilizes its exonuclease activity to generate primers required to activate BRCA and activate the trans-cleavage of Cas12a.

8. Use of the method for detecting miR-21 and miR-155 based on the strand displacement-driven RCA-Cas12a AND logic gate platform according to any one of claims 1-7.

9. The application according to claim 8, wherein, The samples to be detected include the sera of breast cancer patients and normal sera.

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