Double-target miRNA single tube detection method based on combination of double recognition probe triggered RCA-CRISPR technologies

Abstract

The invention provides a double-recognition probe template for diagnosis or / and auxiliary diagnosis of acute myocardial infarction. The invention provides a double-recognition probe for diagnosis or / and auxiliary diagnosis of acute myocardial infarction. The invention also provides a kit for diagnosis or / and auxiliary diagnosis of acute myocardial infarction. The invention further provides a double-target miRNA single-tube detection method based on combination of a double-recognition probe triggered RCA-CRISPR technology, and the double-target miRNA single-tube detection method is used for detecting the target miRNA by adopting the kit and adopting single-tube method CRISPR-Cas12a fluorescence / lateral flow chromatography. Wherein the target miRNA is miRNA-1 (micro Ribonucleic Acid) and / or miRNA-208 (micro Ribonucleic Acid). According to the invention, high-sensitivity detection of double targets of miRNA-1 and miRNA-208 is realized, the sensitivity is improved by 2 orders of magnitude compared with that of a standard method, and the detection time is greatly shortened; the fluorescence spectrophotometer is used for carrying out accurate quantitative detection, the macroscopic instant detection can be realized through the lateral flow test strip, and the kit is suitable for different detection scenes, especially for basic laboratories with limited resources or on-site rapid detection.

Description

TECHNICAL FIELD

[0001] The application relates to a double-target miRNA single-tube detection method based on a double-identification probe triggered RCA-CRISPR technology combination, and belongs to the technical field of gene detection. BACKGROUND

[0002] microRNA (miRNA) is a class of endogenous non-coding single-stranded RNA with a length of about 21-25 nucleotides, and its significant feature is high homology between sequences, which puts a strict requirement on the development of detection technology capable of accurately distinguishing individual miRNA molecules. miRNA, like a "micro code" in the gene expression regulation network, realizes post-transcriptional gene expression regulation by combining the 3' untranslated region (3'UTR) of the target gene mRNA, and is deeply involved in core life processes such as cell proliferation, differentiation, apoptosis and tumor occurrence. Taking miRNA-1 as an example, as a key regulatory factor, its abnormal expression is closely related to the progression of various diseases such as skeletal muscle cell damage, myocardial lesions and malignant tumors. For example: Liu Xin, et al., Role of miRNA in the evolution of acute myocardial infarction, Chinese Journal of Arteriosclerosis, 2023, Vol. 31, No. 11, the article summarizes the feasibility of miRNA as an AMI diagnostic marker, its influence on AMI and related molecular mechanisms. Current research believes that miR-1 is a specific marker for the diagnosis of AMI, and its diagnostic significance for AMI within 3h of acute chest pain is great. Studies have found that the expression levels of miR-17-5p, miR-145-3p and miR-126-5p in AMI patients are significantly increased, and receiver operating characteristic curve analysis shows that miR-17-5p, miR-145-3p and miR-126-5p have good diagnostic efficiency for AMI. The levels of these three miRNAs were observed to increase in the early stage of AMI (within 4h after onset), and their diagnostic efficiency was higher than that of cardiac troponin. The time of increase of miR-208, miR-494, miR-499 and miR-1303 in AMI patients is later than the above three miRNAs, and the peak values of miR-208, miR-494 and miR-499 appear after 12h of onset, and the peak value of miR-1303 appears between 6h and 12h after onset or after 12h of onset, which can provide a longer time period for the diagnosis of AMI and reduce the probability of misdiagnosis and missed diagnosis. At present, the research on miRNA is mainly blood detection, as a blood marker.

[0003] There are multiple technical bottlenecks in using miRNA as RNA markers for diagnosing acute myocardial infarction. The first challenge comes from the short sequence and high homology of miRNA. There are only a few nucleotide differences between members of the miRNA family, which poses a great test for the specificity of the detection method and easily leads to "false positive" or "false negative" results. Secondly, miRNA-1 has extremely low abundance (0.01% of total RNA) and its expression level changes dramatically in pathological conditions (such as cancer and healthy samples, which can differ by thousands of times). Finally, the stability problem, miRNA is easily degraded by RNase in the sample, and the composition of clinical samples is complex, which increases the difficulty of accurate detection. At present, the existing miRNA detection technology also has obvious limitations. For example: Northern blot is considered as the "gold standard", but its operation is complicated, time-consuming, low sensitivity, and requires a large amount of sample, which is difficult to meet the needs of high-throughput and rapid clinical detection, and the efficiency is low. Real-time quantitative PCR (qRT-PCR) has high sensitivity and specificity and has become the mainstream in the laboratory, but it relies on the reverse transcription process and is easy to introduce bias; and it needs precise thermal cycler instruments and professional operators, the cost is relatively high, and the detection process is not "instantaneous", but more like "precision instruments in the laboratory", which is difficult to popularize to the bedside or resource-limited areas.

[0004] Therefore, in order to break through the above limitations, the present application focuses on solving the detection problem of trace miRNA-1 in biological samples. Nucleic acid amplification is the core strategy to enhance the target signal. Among many amplification technologies, isothermal amplification has obvious advantages because it can efficiently amplify the target signal at a constant temperature, avoiding the thermal cycling process required by traditional PCR. Among them, the rolling circle amplification (RCA) technology is often used for miRNA analysis due to its simple design, outstanding specificity, etc. However, although traditional RCA can amplify the signal, it usually requires long primers and complex probe design, and the detection sensitivity is sometimes limited by the amplification efficiency.

[0005] In order to further improve the accuracy and sensitivity of detection, the CRISPR / Cas system is introduced. As a "revolutionary tool" in the field of gene editing, it is known for its precise target recognition and signal self-amplification effect mediated by trans-cleavage activity. Coupling nucleic acid amplification (such as RCA) with the CRISPR / Cas system is expected to synergistically improve the detection performance. Given that the RCA product is a long single-stranded DNA (ssDNA) repeat sequence, the CRISPR / Cas12a (Cpf1) is selected in the V-A type CRISPR system. The unique advantage of Cas12a is that it can not only specifically recognize double-stranded DNA (dsDNA), but more importantly, it can directly recognize single-stranded DNA (ssDNA) without relying on specific TTTV protospacer adjacent motif (PAM) sequences, thereby triggering strong trans-cleavage activity to amplify the detection signal.

[0006] Currently, the conventional rolling circle amplification combined with the CRISPR / Cas12a system method still faces some limitations:

[0007] ① These methods usually use miRNA as the connecting template of the padlock probe, and the connecting process needs to rely on high-temperature annealing program, which increases the reaction steps, and high temperature also leads to the degradation of miRNA. The reported patents, such as application number 202211460172.8, invention name: a method for detecting miRNA155 based on CRISPR / Cas12a and exponential rolling circle amplification, although the reaction conditions are optimized, it does not need to rely on high-temperature annealing program, but this method still has obvious limitations: this method is used for the diagnosis of breast cancer, if used for the diagnosis of emergency, such as myocardial infarction, the golden rescue window is 120 minutes after the onset, the detection timeliness requirement is extremely high, and the method needs to be carried out in two independent reaction steps in turn, which leads to a long detection time (2 hours), which is difficult to meet the core needs of "fast results" in clinical first aid, thereby delaying the judgment and subsequent intervention of the disease; on the other hand, there is an opening cover link in the operation process, which significantly increases the risk of sample cross contamination or external environmental interference, affecting the accuracy and reliability of the detection results;

[0008] ② Due to the specificity of the connecting reaction process, only a single target can be detected;

[0009] ③ Although the rolling circle amplification technology has high sensitivity, the concentration in different substrates is different, which requires accurate detection of miRNA from highly complex media. Therefore, the current detection method based on this combination still has obvious shortcomings in the degree of operation process simplification, anti-pollution ability, detection time and overall performance optimization. SUMMARY

[0010] In view of the above shortcomings of the prior art, the present application aims to provide a double recognition probe template based on the combination of rolling circle amplification (RCA) and CRISPR / Cas12a technology for rapid detection of miRNA-1 and miRNA-208, in order to solve the problems of complex operation, limited detection target, insufficient sensitivity and specificity of the existing miRNA detection method.

[0011] The present application provides a double recognition probe template for diagnosing or / and assisting in diagnosing acute myocardial infarction, the DNA sequence of which is shown in SEQ ID NO: 3.

[0012] The present application provides the use of the double recognition probe template in the preparation of a kit for diagnosing or / and assisting in diagnosing acute myocardial infarction.

[0013] The application provides a double recognition probe for diagnosing or / and assisting in diagnosing acute myocardial infarction, which is prepared by the following steps: firstly, heat annealing is performed to allow the double recognition probe template to self-assemble into a partially complementary double-stranded skeleton; secondly, gap closure is completed at 37 DEG C in the presence of T4 DNA ligase and its special buffer; and finally, a complete double recognition probe (D-probe) is obtained.

[0014] It comprises the following steps:

[0015] a. The double recognition probe template, ddH2O and T4 DNA ligase buffer are prepared into a mixture, which is annealed at 95 DEG C for 5 minutes and slowly cooled to room temperature;

[0016] b. T4 DNA ligase is added, and the obtained double recognition probe is incubated at 37 DEG C.

[0017] The application provides a kit for diagnosing or / and assisting in diagnosing acute myocardial infarction, which comprises the double recognition probe, CRISPR / Cas12a protein, dNTP mixture, crRNA, phi29 DNA polymerase reaction buffer, phi29 DNA polymerase, Cas12a enzyme reaction buffer and reporter probe.

[0018] The final concentrations of the raw materials in the kit are as follows:

[0019] The double recognition probe template is 0.05-0.2 μM, the CRISPR / Cas12a protein is 0.5 mM, the dNTP mixture is 100 nM, the phi29 DNA polymerase reaction buffer is 1x, the phi29 DNA polymerase is 0.067 U / μL, the Cas12a enzyme reaction buffer is 1x, and the fluorescent reporter probe or lateral flow reporter probe is 500 nM.

[0020] The application further provides a double-target miRNA single-tube detection method based on the double recognition probe triggered RCA-CRISPR technology, which adopts the kit and a single-tube method.

[0021] The CRISPR-Cas12a fluorescence / lateral flow chromatography is used for detecting the target miRNA, and the target miRNA is miRNA-1 and / or miRNA-208.

[0022] Specifically, it comprises the following steps:

[0023] a. The sample to be detected is taken, and the target miRNA is pretreated by diluting the miRNA target with DEPC water;

[0024] b, tube bottom RCA: phi29 DNA polymerase isothermal rolling circle amplification for 20 min at 37 DEG C with double recognition probe as template to generate long ssDNA specific to target; after centrifugal mixing, the Cas12a / crRNA complex pre-set on the tube cover recognizes the amplification product, activates the reverse cleavage activity for 40 min, continuously cuts the reporter probe, and the released fluorescence or gold label strip signal is read by a spectrometer or lateral flow test strip to realize quantitative / qualitative detection of miRNA.

[0025] Further, the specific method of the b step is:

[0026] The double recognition probe is mixed with 2 muL target miRNA, 1 muL dNTP mixture (10 mM), 2 muL phi29 DNA polymerase reaction buffer, 2 muL phi29 DNA polymerase (6.7*10 2 units / ml) and 3 muL ddH2O, and is added to the bottom of the reaction tube; 3 muL ddH2O, 2 muL crRNA (1 muM), 2 muL Cas12a (1 muM) protein, 2 muL Cas12a enzyme reaction buffer (buffer 1) and 1 muL of the mixture of fluorescent reporter probe or lateral flow reporter probe (10 muM) are added to the tube cover; then, amplification is carried out at 37 DEG C for 20 min to obtain a large amount of long repeated single-stranded DNA, and then the CRISPR / Cas system in the tube cover is mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and reaction is carried out at 37 DEG C for 40 min; the fluorescence signal released by the reporter probe is detected by a fluorescence spectrometer, or the test line signal is observed by a lateral flow test strip, and the detection result of miRNA is obtained. The concentration of the double recognition probe is 200 nM.

[0027] The sample is derived from urine or serum; the fluorescent reporter probe is 5'-FAM-TTATTATT-BHQ1-3' or lateral flow reporter probe 5'-FAM-TTATTATT-Biotin-3';

[0028] The fluorescence signal is collected by a fluorescence spectrometer at an excitation wavelength of 490 nm and an emission wavelength of 500 nm-700 nm.

[0029] The application also provides a method for diagnosing or / and assisting in diagnosing acute myocardial infarction for non-therapeutic purposes, which is to detect the content of miRNA-1 and / or miRNA-208 in urine or blood by using the double-target miRNA single-tube detection method based on the double-recognition probe triggered RCA-CRISPR technology.

[0030] The double recognition probe comprises a complementary region of miRNA-1 and miRNA-208, and a complementary region of crRNA, and the prefabricated double recognition probe can be directly used in subsequent detection reaction without a high-temperature annealing step. The double recognition probe can form a dumbbell structure through an annealing program, and form a circular DNA under the action of T4 DNA ligase without a ligation probe. The target miRNA is miRNA-1 or miRNA-208, and the final concentration is 2fM-200pM, and the actual detection limit (2fM) is two orders of magnitude higher than that of the standard method RT-qPCR (200fM). The CRISPR / Cas reaction system in the tube cover is mixed with the rolling circle amplification product through transient centrifugation, which effectively reduces the cross contamination caused by the opening operation while shortening the operation time, and the total detection time is 1 hour. The present application can use lateral flow analysis method to detect miRNA-1 in real time, the fluorescence reporter probe is replaced by lateral flow reporter probe 5'-FAM-TTATTATT-Biotin-3', after the end, 70 muL ddH2O is added, the lateral flow test strip is inserted into the reaction tube for 2 minutes, and the result can be directly judged by naked eye. The detection method of the present application has a signal deviation of ≤±15% in a sample matrix containing 5% urine or serum.

[0031] The present inventors' team first found that the urine non-coding nucleic acid miRNA-1 can be used as a myocardial infarction diagnostic marker (Cheng Y, Tan N, Yang J, et al. A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond).

[0032] 2010; 119(2): 87-95. https: / / doi.org / 10.1042 / CS20090645). This finding is of great significance, which breaks the limitations of traditional blood markers, realizes non-invasive sampling, and greatly improves the convenience of clinical detection. However, there are still two key shortcomings when miRNA-1 is used alone as a diagnostic marker for myocardial infarction: first, the specificity is insufficient. miRNA-1 is not a specific marker for myocardial cells, and there is a certain level of expression in tissues such as skeletal muscle and smooth muscle. Second, the early signal coverage is not complete. Although miRNA-1 can be released into urine in the early stage of myocardial ischemia, its concentration increases less in non-ST segment elevation myocardial infarction (NSTEMI) with less myocardial cell necrosis, which may be missed due to "insufficient signal strength", resulting in missed treatment window and affecting prognosis. Another non-coding nucleic acid marker miRNA-208 has also been clinically confirmed to be associated with myocardial infarction. miRNA-208 has strong specificity and is a specific marker for myocardial cells. It is mainly stored in the sarcomere of myocardial cells. Only when the myocardial cells undergo irreversible necrosis and the sarcomere structure is broken, can it be released into the body fluid in large quantities, effectively avoiding interference from other tissue damage. Therefore, the cooperative detection of miRNA-208 can effectively make up for the lack of specificity of miRNA-1 and greatly exclude the interference of other organ damage such as skeletal muscle damage and renal dysfunction. The essence of this scheme is to solve the defects of a single marker by "complementary properties" to achieve "1+1>2" in diagnostic ability. On the other hand, relying on the release characteristics of miRNA-1 in the early stage of ischemia, it can fill the blind area of miRNA-208 in early diagnosis, covering the whole course from myocardial ischemic stress to cell necrosis. The synergistic effect of the two ultimately realizes the highly accurate diagnosis of acute myocardial infarction (AMI), providing a more reliable detection basis for early clinical intervention and improving patient prognosis.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] (1) The method of the present application realizes high sensitivity detection of miRNA-1 and miRNA-208 double targets, and the sensitivity is improved by 2 orders of magnitude compared with the standard method. This is mainly due to the synergistic effect of rolling circle amplification and

[0035] CRISPR / Cas12a. Rolling circle amplification technology can efficiently amplify target sequences under constant temperature conditions, producing a large amount of long repeated single-stranded DNA, significantly amplifying the detection signal. Secondly, the CRISPR / Cas12a system can activate the cis and trans cleavage activity of Cas12a after recognizing the amplification product, making full use of the two kinds of cleavage activity to realize cascade amplification while outputting signals.

[0036] (2) The pre-prepared specific dual recognition probe can be directly used in the detection process, which eliminates the high-temperature annealing step, realizes constant temperature single tube detection, and greatly shortens the detection time (1 hour);

[0037] (3) The dual recognition probe contains a recognition sequence complementary to the miRNA and a target sequence recognized by the CRISPR / Cas12a, and can trigger the amplification process only in the presence of the target, and the targeting of the CRISPR / Cas12a further ensures the specificity of the detection;

[0038] (4) The CRISPR / Cas system in the present application is placed inside the reaction tube cover, and mixing with the rolling circle amplification product is realized by transient centrifugation, which avoids the opening operation in the middle of the way, effectively reduces the risk of cross contamination, and improves the detection efficiency.

[0039] (5) The present application can not only carry out accurate quantitative detection through the fluorescence spectrometer, but also can realize instant detection visible to the naked eye through the lateral flow test strip, which is suitable for different detection scenes, especially for the resource-limited primary laboratory or on-site rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure A is a schematic diagram of the dual-target miRNA rapid detection method of the present application.

[0041] Figure 2 Figure A is a schematic diagram of the dual recognition probe, wherein the complementary region of crRNA is indicated by the arrow, and the miRNA recognition region is on both sides; Figure 2 Figure B is a PAGE image of the synthesis reaction of the dual recognition probe and the rolling circle amplification reaction product, lane 1: dual recognition probe template; lane 2: product after synthesis of the dual recognition probe; lane 3: rolling circle amplification product in the absence of target; lane 4: rolling circle amplification product in the presence of miRNA-1 target; Figure 2 Figure C is a PAGE image of the rolling circle amplification and CRISPR / Cas12a combined reaction, lane 1: rolling circle amplification and CRISPR / Cas12a combined reaction product in the absence of target; lane 2: rolling circle amplification and CRISPR / Cas12a combined reaction product in the presence of target; lane 3: crRNA.

[0042] Figure 3 Figure A is a schematic diagram of the "one-pot method" reaction of the rolling circle amplification and the CRISPR / Cas12a process directly mixed; Figure 3 Figure B is a schematic diagram of the "single tube method" reaction of the rolling circle amplification and the CRISPR / Cas12a process separated; Figure 3 Figure C is the fluorescence spectrum obtained by the detection method in the presence or absence of target; Figure 3 Figure A is a fluorescence spectrum obtained by the detection method; Figure 3 Figure D is a fluorescence spectrum obtained by the detection method in the presence or absence of target;Figure 3 B fluorescence spectrum obtained by the detection method.

[0043] Figure 4 The feasibility of detecting miRNA-208 according to the present application, wherein Figure 4 A is an OR logic gate constructed by taking miRNA-1 and miRNA-208 as input signals; Figure 4 B is a fluorescence spectrum obtained by the detection method in the presence or absence of a miRNA-208 target.

[0044] Figure 5 The result graph after optimizing the method according to the present application: Figure 5 A is the fluorescence value after the reaction of different concentrations of double recognition probes (D-probes); Figure 5 B is the time ratio of the reaction before and after centrifugation, and the fluorescence value corresponding to the reaction under different time ratios.

[0045] Figure 6 A is the fluorescence spectrum of target miRNA-1 at different concentrations of 2fM-200pM; Figure 6 B is the fluorescence response of miRNA-1 at different concentrations of 0-200pM, wherein the insert is the linear relationship between the fluorescence intensity (ΔF) and the logarithm of the concentration of miRNA-1 in the range of 2fM-200pM; Figure 6 C is Figure 6 B is an enlarged view; the error bar represents the standard deviation of three independent measurements.

[0046] Figure 7 The fluorescence spectrum of detecting different concentrations of miRNA-1 (0-200pM) by reverse transcription quantitative PCR method.

[0047] Figure 8 A is the fluorescence value after the reaction of different miRNAs at a concentration of 200pM; Figure 8 A is the fluorescence response of 5 different experimental groups at a concentration of 2pM, and the error bar represents the standard deviation of three independent measurements.

[0048] Figure 9 A is the detection principle diagram of the lateral flow test strip based on the method according to the present application; Figure 9 B is the test strip detection result diagram after the reaction with or without a target, and the target concentration is 2nM.

[0049] Figure 10 A is the principle diagram of the negative and positive detection results of the lateral flow test strip; Figure 10 B is the test strip detection result diagram of target miRNA-1 at different concentrations of 0-2nM; Figure 10C is the concentration of 200 pM after the reaction of different miRNA involved in the test strip detection result graph.

[0050] Figure 11 For urine / blood samples of different concentrations of miRNA-1 involved in the reaction, the corresponding fluorescence value, and the error bar represents the standard deviation of three independent measurements. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0052] Example 1 The present application is based on the RCA-CRISPR technology combined with miRNA-1 and miRNA-208 single tube rapid detection method of double recognition probe trigger

[0053] Comprising the following steps:

[0054] (1) Synthesis of double recognition probe: 2 μL double recognition probe template (200 nM), 5 μL ddH2O, 1 μL T4 DNA ligase buffer were prepared into a mixture, and placed at 95°C for 5 minutes, and slowly cooled to room temperature; 2 μL T4 DNA ligase (2.7 x 10 4 units / ml) was added, and incubated at 37°C for 30 minutes to obtain the double recognition probe (D-probe); the double recognition probe contains recognition sequences complementary to two target miRNAs and a target sequence recognized by CRISPR / Cas12a, as shown in Table 1.

[0055] Table 1 DNA sequences used in the present application

[0056]

[0057]

[0058] "P" represents a phosphoric acid group modification, and the hatched area is complementary to miRNA-1 and miRNA-208, respectively.

[0059] Among them, the fluorescence reporter probe and the lateral flow reporter probe are purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0060] (2) RCA-CRISPR combined constant temperature single tube reaction: the double recognition probe obtained in step (1) was mixed with 2 μL target miRNA-1, 1 μL dNTP mixture (10 mM), 2 μL phi29 DNA polymerase reaction buffer, 2 μL phi29 DNA polymerase (6.7 x 102 units / ml) and 3 μL ddH2O were mixed and added to the bottom of the reaction tube; 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12a enzyme reaction buffer (buffer 1) and 1 μL of the reporter probe (10 μM) mixture were added to the cap of the reaction tube. Then, under the condition of constant temperature at 37°C, amplification was carried out for 20 minutes to obtain a large amount of long repeat single-stranded DNA, and then the CRISPR / Cas system in the cap was mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and the reaction was carried out at 37°C for 40 minutes;

[0061] (3) Fluorescent signal and visual signal collection: 170 μL ddH2O was added to the reaction product of step (2), and then the fluorescent signal released by the reporter probe was detected by a fluorescence spectrometer; the fluorescent reporter probe was replaced with a lateral flow reporter probe, 70 μL ddH2O was added to the reaction product of step (2), and after the lateral flow test strip was inserted into the reaction tube for 2 minutes, the visual detection result of miRNA-1 was obtained.

[0062] (4) PAGE analysis: 10% non-denaturing polyacrylamide gel was used for electrophoresis analysis of the reaction product. The analysis sample was mixed with DNA loading buffer at a volume ratio of 5:1, and was transferred into the gel well. The gel was placed in TBE buffer (1x) at a voltage of 120V for 40 minutes. Finally, the electrophoresis gel was placed in nucleic acid dye for 15 minutes, and the image was collected by a gel imager.

[0063] Example 2 Principle of the double-miRNA rapid detection method of the application

[0064] The principle of the detection method described in the application is shown in the accompanying Figure 1 The whole process only needs 1 hour, and the process is as follows: 37°C reaction for 20 minutes, mixing after centrifugation, 37°C reaction for another 40 minutes, and finally the detection results are output by a fluorescence spectrophotometer and a lateral flow test strip Figure 1 A). The principle of fluorescence detection is as follows:

[0065] The designed double recognition probe (D-probe) contains two miRNAs and a CRISPR / Cas12a recognition site, so in the presence of miRNA-1 or miRNA-208, it binds to the complementary region of the double recognition probe (D-probe) to start the rolling circle amplification reaction. Under the action of polymerase, long-chain DNA with repeated sequences is continuously synthesized with the double recognition probe as the template. The generated long-chain DNA contains

[0066] The DNA sequence recognizable by the CRISPR / Cas12a system, the Cas12a / crRNA complex specifically binds to the DNA sequence, and the cis and trans cleavage activities of Cas12a are activated. Among them, the cis cleavage product can act as a primer to further initiate cascade amplification, while the activated trans cleavage activity will non-specifically cleave the surrounding fluorescent reporter probe (DNA-FQ), separate the fluorescent group from the quenching group, produce a detectable fluorescent signal, and thus realize the detection of miRNA target Figure 1 B).

[0067] Example 3 Verification of the feasibility of miRNA-1 and miRNA-208 detection of the application

[0068] (1) Experimental method:

[0069] 2 μL double recognition probe template (200 nM), 5 μL ddH2O, 1 μL T4 DNA ligase buffer were mixed to prepare a mixture, which was annealed at 95°C for 5 minutes and slowly cooled to room temperature; 2 μL T4 DNA ligase (2.7×10 4 units / ml) was added, and incubated at 37°C for 30 minutes to obtain the double recognition probe (D-probe).

[0070] miRNA-1 and miRNA-208 constant temperature single tube detection method: the synthesized double recognition probe was mixed with 2 μL target miRNA (miRNA-1 or miRNA-208), 1 μL dNTP mixture (10 mM), 2 μL phi29 DNA polymerase reaction buffer, 2 μL phi29 DNA polymerase (6.7×10 2 units / ml) and 3 μL ddH2O, and added to the bottom of the reaction tube; 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12a enzyme reaction buffer (buffer 1) and 1 μL fluorescent reporter probe (10 μM) were mixed and added to the cover of the reaction tube. Then, amplification was carried out at 37°C for 20 minutes to obtain a large amount of long repeated single-stranded DNA, and then the CRISPR / Cas system in the tube cover was mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and reacted at 37°C for 40 minutes. The fluorescence signal acquisition process is as follows: 170 μL ddH2O was added to the above reaction product, and the fluorescence intensity was measured by fluorescence spectrometer under 490 nm excitation.

[0071] One-pot detection method: the synthesized dual recognition probe was mixed with 2 μL target miRNA-1, 1 μL dNTP mixture (10 mM), 2 μL phi29 DNA polymerase reaction buffer, 2 μL phi29 DNA polymerase (6.7 x 10 2 units / ml), 6 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12 enzyme reaction buffer (buffer 1), and 1 μL of fluorescent reporter probe (10 μM), and reacted for 1 hour. The fluorescence signal acquisition process is as follows: 170 μL of ddH2O was added to the above reaction product, and the fluorescence intensity was measured by a fluorescence spectrometer under 490 nm excitation.

[0072] The reaction product was analyzed by electrophoresis using a 10% non-denaturing polyacrylamide gel. The analysis sample was mixed with DNA loading buffer at a volume ratio of 5:1 and transferred to the gel well. The gel was placed in TBE buffer (1x) at a voltage of 120V for 40 minutes. Finally, the electrophoresis gel was placed in nucleic acid dye for 15 minutes and the image was collected by a gel imager.

[0073] (2) Experimental results:

[0074] With miRNA-1 as the target, the feasibility of miRNA rapid detection based on the combination of dual recognition probe triggered rolling circle amplification and CRISPR / Cas12a was verified by electrophoresis and fluorescence detection. Figure 2 A is a schematic diagram of the dual recognition probe, where the arrow indicates the complementary region of the crRNA, and the two sides are the miRNA recognition region. Figure 2 B and Figure 2 C is an electrophoresis image. From Figure 2 As can be seen from B, the dual recognition probe template is successfully connected into a closed dual recognition probe under the action of T4 DNA ligase, where lane 1 is the dual recognition probe template (linear template), which is a rolling circle template band that remains linear without cyclization. Since linear templates will hybridize to each other, two bands appear. After linear annealing, lane 2 is the inactivated sample after the linear template is connected into a dual recognition probe by T4 DNA ligase. The electrophoresis band changes position compared to the linear rolling circle band of lane 1, indicating that the dual recognition probe is successfully formed and the probe structure is stable after inactivation. Lanes 3 and 4 are the products after rolling circle amplification with the dual recognition probe in the absence of target and in the presence of target miRNA-1, respectively. The faster moving electrophoresis band is similar to lane 2, which is a dual recognition probe, but there is also a clear band at the well, and the band in lane 4 is more obvious, indicating that miRNA-1 has successfully triggered the rolling circle amplification process and generated a large amount of long repetitive DNA sequences. Since the front-end amplification time is short, the overall band brightness is low. FromFigure 2 C whether the rolling circle amplification product can be recognized by

[0075] CRISPR / Cas12a system. As can be seen from the figure, lane 1 is a sample subjected to rolling circle amplification and Cas12a cleavage without a target. Since there is no target miRNA-1, rolling circle amplification cannot be initiated, and no long-chain repeat DNA product is generated. At this time, the Cas12a / crRNA complex is not activated by the target long-chain DNA, and the cis and trans cleavage activities are not triggered, and there is no obvious product. The electrophoresis band is mainly the band of small molecule nucleic acids such as double recognition probes, and there is no characteristic band of long-chain DNA after cleavage. Lane 2 is a sample subjected to rolling circle amplification and Cas12a cleavage in the presence of target miRNA-1. miRNA-1 initiates rolling circle amplification to generate long-chain DNA containing the target sequence recognized by the CRISPR / Cas12a system. After the Cas12a / crRNA complex specifically binds to the target sequence, the cis and trans cleavage activities are activated to produce a large amount of small fragment nucleic acids. As can be seen from lane 2, the electrophoresis band near the loading well presents a high bright band, which is in sharp contrast to lane 1, proving that when the target miRNA-1 exists, rolling circle amplification is successfully performed, and the band dispersion phenomenon appears, indicating that the cis and trans cleavage activities of Cas12a are effectively activated to produce a large amount of small fragment nucleic acids. Lane 3 is a sample containing only crRNA. crRNA is a guide RNA in the CRISPR / Cas12a system, which is a linear small molecule RNA and can form an R loop structure, so lane 3 has two crRNA bands with different structures.

[0076] Figure 3 For the detection principle and fluorescence detection results of one-pot method and single-tube method, different experimental methods are optimized, such as Figure 3 As shown in C and 3D, the one-pot method has the same fluorescence value as the single-tube method, but the background signal of the one-pot method is higher, and the single-tube method can be ignored. High background signal is easy to produce false positive results, so we choose the single-tube method as the optimal method.

[0077] Subsequently, the feasibility of the method for detecting miRNA-208 was verified. As shown in Figure 4 A, using miRNA-1 or miRNA-208 as a signal input, the signal output is 0 only when there is no target, so an OR logic gate can be constructed. Figure 4 B confirms the fluorescence feasibility of the OR logic gate detection. When one or two miRNA targets exist, there is a strong fluorescence signal, and when there is no target, the fluorescence signal is very low, which confirms the feasibility of the method for detecting two miRNAs.

[0078] Example 4 Optimization experiment of parameters in the detection method of the application

[0079] (1) Experimental method:

[0080] Probe concentration optimization: Different concentrations of double recognition probes were synthesized and mixed with 2 μL target miRNA-1, 1 μL dNTP mixture (10 mM), 2 μL phi29 DNA polymerase reaction buffer, 2 μL phi29 DNA polymerase (6.7 x 10 2 units / ml) and 3 μL ddH2O, and added to the bottom of the reaction tube; 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12a enzyme reaction buffer (buffer 1) and 1 μL of fluorescent reporter probe (10 μM) mixture were added to the cover of the reaction tube. Then, amplification was carried out at 37°C for 20 minutes to obtain a large amount of long repeated single-stranded DNA, and then the CRISPR / Cas system in the tube cover was mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and the reaction was carried out at 37°C for 40 minutes. The fluorescence signal acquisition process is as follows: 170 μL ddH2O was added to the above reaction product, and the fluorescence intensity was measured by fluorescence spectrometer under 490 nm excitation.

[0081] Time ratio concentration optimization: Different concentrations of double recognition probes were mixed with 2 μL target miRNA-1, 1 μL dNTP mixture (10 mM), 2 μL phi29 DNA polymerase reaction buffer, 2 μL phi29 DNA polymerase (6.7 x 10 2 units / ml) and 3 μL ddH2O, and added to the bottom of the reaction tube; 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12a enzyme reaction buffer (buffer 1) and 1 μL of fluorescent reporter probe (10 μM) mixture were added to the cover of the reaction tube. Then, centrifugation was carried out according to different time ratios, and the fluorescence signal was collected as described above.

[0082] (2) Experimental results:

[0083] In order to improve the sensitivity of the method, the probe concentration and the time ratio were optimized. As shown in Figure 5 A, when the ratio of amplification time to shearing time is 1:2, the fluorescence value is significantly higher than that of other ratios, so we choose the ratio of amplification time to shearing time of 1:2 as the optimal condition. The concentration of double recognition probe is an important parameter, which is closely related to the sensitivity, as shown in Figure 5 B, the fluorescence response reaches the strongest at 200 nM. Therefore, we choose 200 nM of double recognition probe as the optimal condition.

[0084] Example 5: Detection performance of the reaction system in the detection method of the present application

[0085] (1) Experimental method:

[0086] The target miRNA-1 or miRNA-208 was gradiently diluted (concentration range of 2 fM to 200 pM), and 2 μL of the target solution at different concentrations was mixed with 1 μL of dNTP mixture (10 mM), 2 μL of phi29 DNA polymerase reaction buffer, 2 μL of phi29 DNA polymerase (6.7 x 10 2 units / ml) and 3 μL of ddH2O to fully mix, and then added to the bottom of the reaction tube; at the same time, 3 μL of ddH2O, 2 μL of crRNA (1 μM), 2 μL of Cas12a (1 μM) protein, 2 μL of Cas12a enzyme reaction buffer (buffer 1) and 1 μL of fluorescent reporter probe (10 μM) mixture were added to the cover of the reaction tube. Subsequently, the reaction system was placed in a constant temperature condition of 37°C for 20 minutes to perform amplification reaction, so that the target triggered the generation of a large amount of long repeated single-stranded DNA; the CRISPR / Cas system in the tube cover was fully mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and the reaction was continued at 37°C for 40 minutes. The fluorescence signal acquisition process was as follows: 170 μL of ddH2O was added to the above reaction product, and the fluorescence intensity of the system was measured by a fluorescence spectrometer at an excitation wavelength of 490 nm.

[0087] (2) Experimental results:

[0088] In the concentration range of 2 fM to 200 pM, as the concentration of miRNA-1 increased, the fluorescence response of the system showed a gradually increasing trend Figure 6 A). Further analysis showed that the fluorescence intensity difference (ΔF, i.e. the value after subtracting the fluorescence intensity of the blank group from the fluorescence intensity of the experimental group) and the logarithm of the miRNA-1 concentration showed a good linear relationship in the range of 2 fM to 200 pM Figure 6 B inset), and the linear fitting equation was ΔF = 1387.37 + 535.29x (R 2 = 0.99), where x was the concentration of miRNA-1 (fM). Similarly, in the concentration range of 2 fM to 200 pM, as the concentration of miRNA-208 increased, the fluorescence response of the system showed a gradually increasing trend Figure 6 C), and the linear fitting equation for miRNA-208 detection was ΔF = 1338.84 + 887.88x (R 2 = 0.99), where x was the concentration of miRNA-208 (fM) Figure 6D). The actual detection limit of this method is as low as 2 fM. Compared with existing miRNA detection technologies (Table 2), the miRNA detection method based on rolling circle amplification coupled with CRISPR / Cas12a established in the present application has a lower detection limit and a wider detection range.

[0089] The excellent performance of this method is mainly due to the following synergistic mechanisms: (1) specific recognition ability of the double recognition probe: the probe design contains a recognition sequence completely complementary to the miRNA, which can specifically bind to the target miRNA-1 and initiate the rolling circle amplification reaction, effectively avoiding the interference caused by non-specific amplification; (2) high-efficiency signal amplification effect of rolling circle amplification coupled with CRISPR / Cas12a: the miRNA is used as a primer to trigger the amplification reaction, and a large amount of long-chain DNA containing repeated target sequences is generated under the catalysis of phi29 DNA polymerase, which realizes significant amplification of the initial signal, and then the product is recognized by CRISPR / Cas12a, and under the action of Cas12a cis cleavage, fragments of different sizes are generated, which can further act as primers to realize cascade amplification; (3) high specificity activation characteristics of CRISPR / Cas12a: the target sequence in the long-chain DNA can be specifically recognized by the Cas12a / crRNA complex, and the trans-cleavage activity of Cas12a can be activated without relying on the PAM site; (4) high sensitivity characteristics of signal conversion: activated Cas12a releases fluorescent signals by specifically cleaving fluorescent reporter molecules, and the inherent high specificity and signal amplification capability of the CRISPR system further improve the sensitivity and accuracy of the detection.

[0090] Example 6 Comparison of the detection method of the present application and other detection methods

[0091] (1) Experimental method:

[0092] First, the target miRNA is transcribed into cDNA by stem-loop method, and the reaction process is as follows. In order to perform sensitivity detection, different concentrations of miRNA-1 are added to the mixture, which includes 2*miRNA L-RT solution mixture, miRNA L-RT enzyme mixture and stem-loop primer (10 μM), and the total solution volume is 20 μL. Then, the above solution is reacted at 16℃ for 30 minutes, at 37℃ for 30 minutes, and finally inactivated at 85℃ for 5 minutes. The prepared cDNA is used for further fluorescent quantitative PCR reaction. The reaction system of the fluorescent quantitative PCR reaction is as follows: 12.5 μL TB Green Premix EX Taq II, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2.5 μL above-mentioned cDNA, 8 μL ddH2O. The PCR program is as follows: 95℃ for 30 seconds; then 40 cycles: 95℃ for 3 seconds, 60℃ for 30 seconds.

[0093] (2) Experimental results:

[0094] As shown in Figure 7 Figure 6, the actual detection limit of RT-qPCR for miRNA-1 was 200 fM. Compared with the method described in the present application, the high detection limit of RT-qPCR might be caused by low reverse transcription efficiency and degradation of miRNA during reverse transcription. Although the stem-loop reverse transcription method for miRNA has high specificity, the homologous region of the single stem-loop primer complementary to the corresponding miRNA is short, which will lead to low reverse transcription efficiency. In contrast, the actual detection limit of the method described in the present application is two orders of magnitude higher than that of RT-qPCR. Compared with other reported methods (Table 2), the proposed strategy also showed excellent performance in detection time, detection limit and detection range.

[0095] Table 2 Comparison with other miRNA detection methods

[0096]

[0097] [1]. Zeng D, Wang Z, Meng Z, et al. DNA Tetrahedral Nanostructure-Based Electrochemical miRNA Biosensor for Simultaneous Detection of Multiple miRNAs in Pancreatic Carcinoma. ACS Appl Mater Interfaces. 2017; 9(28): 24118-24125.

[0098] [2]. Gao J, Liu L, Liu A, et al. Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction. Microchim Acta. 2022; 189(3): 133.

[0099] [3]. Shi H, Yang L, Zhou X, et al. A gold nanoparticle-based colorimetric strategy coupled to duplex-specific nuclease signal amplification for the determination of microRNA

[0100] [4]. Zhou T, Huang M, Lin J, Huang R, Xing D. High-Fidelity CRISPR / Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA. Anal Chem. 2021; 93(4): 2038-2044.

[0101] [5]. Ai Z, Wang W, Li X, et al. A CRISPR / Cas12a-coupled multiplexed amplification system for ultrasensitive detection of miRNA-155. Anal Methods. 2025; 17(19): 4044-4050.

[0102] [6]. Li Y, Zeng Z, Lv X, et al. A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR / Cas12a. Microchim Acta. 2025; 192(2): 73.

[0103] Example 7 Selectivity and repeatability test of the detection method of the present application

[0104] (1) Experimental method:

[0105] Selectivity experiment: 2 μL of different miRNAs (30 pM), 1 μL of dNTP mixture (10 mM), 2 μL of phi29 DNA polymerase reaction buffer, 2 μL of phi29 DNA polymerase (6.7 x 10 2A mixture of 3 μL of ddH2O and 3 μL of ddH2O was added to the bottom of the reaction tube. A mixture of 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a protein (1 μM), 2 μL Cas12a enzyme reaction buffer (buffer 1), and 1 μL of fluorescent reporter probe (10 μM) was added to the cap of the reaction tube. Amplification was then performed at 37°C for 20 minutes to obtain a large amount of long repeat single-stranded DNA. The CRISPR / Cas system in the cap was then mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and the reaction was continued at 37°C for 40 minutes. Fluorescence signal acquisition was performed as follows: 170 μL of ddH2O was added to the above reaction products, and the fluorescence intensity was measured using a fluorescence spectrometer under 490 nm excitation.

[0106] Repeatability experiment: Five parallel experimental groups and a control group were set up, and the experimental method was as described above.

[0107] (2) Experimental Results

[0108] Selectivity and reproducibility are crucial for sensing systems. Therefore, we first used DNA such as miRNA-21 and miRNA-96 as interfering agents to study the selectivity of the method described in this invention for site-specific miRNA-1 detection. Taking miRNA-1 as an example, from... Figure 8 A and Figure 8 As shown in Figure B, a significant fluorescence value was only observed in the presence of miRNA-1. Other miRNAs at the same concentration (20 pM) exhibited weaker fluorescence values. Furthermore, repeatability was assessed by testing the fluorescence responses of five parallel experimental groups and a control group. Figure 8 B), the target concentration was 200 pM for all groups. There was no significant fluctuation in fluorescence values ​​among the groups, and the relative standard deviation (RSD) was 4.42%. These results demonstrate that the detection method described in this invention has good anti-interference ability and repeatability.

[0109] Example 8: Application of the lateral flow test strip in the detection method of the present invention

[0110] (1) Experimental method:

[0111] The obtained dual-recognition probe was mixed with 2 μL of target miRNA-1, 1 μL of dNTP (10 mM), 2 μL of phi29 DNA polymerase reaction buffer, and 2 μL of phi29 DNA polymerase (6.7 × 10⁻⁶ mM). 2units / ml) and 3 μL ddH2O were mixed and added to the bottom of the reaction tube; 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a (1 μM) protein, 2 μL Cas12a enzyme reaction buffer and 1 μL of lateral flow reporter probe (10 μM) mixture were added to the cover of the reaction tube. Then, amplification was carried out at 37°C for 20 minutes to obtain a large amount of long repeat single-stranded DNA, and then the CRISPR / Cas system in the cover was mixed with the rolling circle amplification product at the bottom of the tube by centrifugation, and the reaction was carried out at 37°C for 40 minutes; then 70 μL ddH2O was added to the reaction product, and after the lateral flow test strip was inserted into the reaction tube for 2 minutes, the visual detection result of miRNA-1 was obtained.

[0112] (2) Experimental results:

[0113] The method of the present application can also be applied to lateral flow test strips by changing the reporter probe (Bio-DNA-FAM) for real-time detection. As shown in Figure 9 A, Biotin and FAM groups are modified at both ends of the probe, which can be combined with AuNPs-anti-FAM. Because there is streptavidin in the control line area (C line), the

[0114] AuNPs-anti-FAM / Bio-DNA-FAM complex, so there is a red band on the C line. When there is a miRNA-1 target, they can start a series of reactions to produce a large amount of ssDNA. Then the transcleavage activity of Cas12a is activated, so that the Bio-DNA-FAM reporter gene is cut. The cut reporter probe containing AuNPs can be captured by the rabbit anti-human serum albumin antibody on the test line (T line), and a red stripe will appear on the T line. On the contrary, in the absence of a target, there is only one red band on the C line. Based on the detection principle of the sensor, it can be successfully distinguished whether it contains miRNA-1, and applied to the point-of-care platform. In the test strip detection, only the experimental group with miRNA-1 has two obvious bands on the test line, which is due to the coupling of AuNPs-anti-FAM. The above results show that the method can successfully detect miRNA-1, and has low background interference, high sensitivity and accuracy Figure 9 B).

[0115] Then, the sensitivity of the method based on the lateral flow test strip was studied. Figure 10 A is the schematic diagram of the negative and positive detection results of the lateral flow test strip. As shown in Figure 10As shown in Figure B, on line T, the band color gradually changes from colorless to dark as the miRNA-1 concentration increases from 200 fM to 2 nM. However, the color on line C shows the opposite trend. miRNA-1 at concentrations as low as 200 fM can be clearly identified. Furthermore, from... Figure 10 As shown in Figure C, only miRNA-1 induced a red color on the test line. Other miRNAs, such as miRNA-21, only showed red on the control line. These results indicate that the method employed in this invention has high sensitivity and selectivity in test strip detection applications, and shows good application prospects in resource-limited areas.

[0116] Example 9: The detection method of the present invention is used for real sample detection.

[0117] (1) Experimental method:

[0118] The target miRNA-1 was serially diluted 20-fold with artificial urine (concentrations of 0, 200 fM, 2 pM, and 20 pM). 2 μL of each concentration of the target solution was mixed with 1 μL of dNTP mixture (10 mM), 2 μL of phi29 DNA polymerase reaction buffer, and 2 μL of phi29 DNA polymerase (6.7 × 10⁻⁶). 2 After thoroughly mixing 3 μL of ddH2O (units / ml) and 2 μL of ddH2O, add the mixture to the bottom of the reaction tube. Simultaneously, add a mixture of 3 μL ddH2O, 2 μL crRNA (1 μM), 2 μL Cas12a protein (1 μM), 2 μL Cas12a enzyme reaction buffer (buffer 1), and 1 μL fluorescent reporter probe (10 μM) to the cap of the reaction tube. Then, incubate the reaction at 37°C for 20 minutes to amplify the target and generate a large amount of long repeat single-stranded DNA. Centrifuge to thoroughly mix the CRISPR / Cas system in the cap with the rolling circle amplification product at the bottom of the tube, and continue the reaction at 37°C for another 40 minutes. Fluorescence signal acquisition is as follows: add 170 μL ddH2O to the above reaction product and measure the fluorescence intensity of the system using a fluorescence spectrometer at an excitation wavelength of 490 nm.

[0119] (2) Experimental results:

[0120] The results show that... Figure 11 As shown, the fluorescence detection recovery rates of different target concentrations are all within a reasonable range. This result indicates that the detection method has a detection signal deviation of ≤±15% in sample matrices containing 5% urine or 5% serum. The signal sensing platform can still effectively resist matrix interference, verifying its reliability in real sample analysis.

[0121] In summary, the present application aims at the core clinical needs of "precise identification of characteristic markers, rapid results to guide first aid, and capture of early low-abundance signals" in myocardial infarction diagnosis, and successfully constructs a new method for rapid detection of miRNA-1 and miRNA-208 based on double recognition probe triggered rolling circle amplification (RCA) and CRISPR / Cas12a system coupling. The method has the following advantages: first, double target collaborative detection, targeted locking of key markers miRNA-1 (early ischemia sensitive) and miRNA-208 (myocardial specificity high) of myocardial infarction, covering the early diagnosis window and avoiding other tissue interference, solving the problem of missed diagnosis and misdiagnosis of traditional single target detection; second, fast response capability, eliminating the high temperature annealing step of traditional detection, achieving 37℃ constant temperature single tube operation throughout, greatly shortening the detection time, meeting the scene needs of myocardial infarction "time is myocardial"; third, ultra-high sensitivity characteristics, relying on the signal cascade amplification of RCA and the self-amplification effect of CRISPR / Cas12a, can accurately capture the trace release of miRNA in early myocardial infarction, effectively making up for the defects of traditional technology in low-abundance marker detection. Through the above advantages, the method successfully solves the key problems of existing miRNA detection technology such as complex operation, limited detection target, insufficient sensitivity, and long detection time, and provides a reliable technical solution for accurate, rapid and early diagnosis of myocardial infarction.

[0122] The method innovatively uses prefabricated double recognition probes to start the rolling circle amplification reaction by specifically recognizing double miRNA targets, eliminating the high temperature annealing step in traditional detection, and achieving constant temperature (37℃) single tube operation throughout. Experimental verification shows that the precise complementary combination of double recognition probes and miRNA can effectively avoid non-specific amplification interference, and the rolling circle amplification technology can efficiently generate long-chain DNA containing repeated target sequences with miRNA as primer, realizing significant amplification of the initial signal. Then the product is recognized by CRISPR / Cas12a, and under the action of Cas12a cis cleavage, fragments of different sizes are generated, which can further act as primers to realize cascade amplification. At the same time, the CRISPR / Cas12a system activates the transcleavage activity by specifically recognizing the target sequence in the long-chain DNA without relying on the PAM site. Transcleavage releases a detectable signal by cutting the fluorescent reporter probe, and further improves the detection sensitivity through the signal self-amplification characteristics of the CRISPR / Cas system.

[0123] Performance evaluation results show that the method exhibits excellent analytical performance in fluorescence detection mode: in the concentration range of 2fM to 200pM, the fluorescence intensity difference (ΔF) and the logarithm of miRNA concentration show a good linear relationship (R 2The actual detection limit is as low as 2 fM, which is two orders of magnitude higher than the sensitivity of the traditional RT-qPCR method (200 fM), and the detection range is wider. By optimizing the probe concentration (200 nM) and the amplification-cleavage time ratio (1:2), the stability and efficiency of the detection are further ensured. The selective experiment proves that the method has specific recognition ability for miRNA-1 and can effectively resist the interference of homologous sequences such as miRNA-21 and miRNA-96. The relative standard deviation (RSD = 4.42%) shows that it has good repeatability. In addition, by replacing the reporter probe with a biotin-fluorophore double-labeled lateral flow probe, the method can be combined with a lateral flow test strip to realize visual detection. As low as 200 fM of miRNA-1 can still be clearly judged by the naked eye, which is suitable for resource-limited on-site detection scenarios. The real sample detection results show that in complex matrices such as urine and blood, the method still has strong anti-interference ability, which verifies its practical value in clinical sample analysis.

[0124] Based on the programmable characteristics of the CRISPR / Cas system, the present application can be expanded into a universal detection platform for other miRNA species and nucleic acid markers, which has broad application prospects in the fields of early diagnosis and prognosis monitoring of diseases. Subsequent research can further optimize the reaction system and explore its detection performance in more types of biological samples, promoting the clinical transformation of the technology.

[0125] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the technical solutions. Those of ordinary skill in the art should understand that modifications or equivalent replacements to the technical solutions of the present application without departing from the spirit and scope of the technical solutions should be covered in the scope of the claims of the present application.

Claims

1. A dual-recognition probe template for diagnosing and / or assisting in the diagnosis of acute myocardial infarction, characterized in that: Its DNA sequence is shown in SEQ ID NO:

3.

2. Use of the dual-recognition probe template of claim 1 in the preparation of a diagnostic and / or auxiliary diagnostic kit for acute myocardial infarction.

3. A dual-recognition probe for diagnosing and / or assisting in the diagnosis of acute myocardial infarction, characterized in that: It involves first thermally annealing the dual recognition probe template described in claim 1 to allow it to self-assemble into a partially complementary double-stranded backbone, and then performing gap sealing at 37°C in the presence of T4 DNA ligase and its dedicated buffer to finally obtain a complete dual recognition probe (D-probe).

4. The method for preparing the dual-recognition probe for diagnosing or / and assisting in the diagnosis of acute myocardial infarction according to claim 3, characterized in that: It includes the following steps: a. Prepare a mixture of dual recognition probe template, ddH2O, and T4 DNA ligase buffer, anneal at 95°C for 5 minutes, and then slowly cool to room temperature; b. Add T4 DNA ligase and incubate at 37°C to obtain a dual recognition probe.

5. A kit for diagnosing and / or assisting in the diagnosis of acute myocardial infarction, characterized in that: It includes the dual recognition probe as described in claim 3, CRISPR / Cas12a protein, dNTP mixture, crRNA, phi29 DNA polymerase reaction buffer, phi29 DNA polymerase, Cas12a enzyme reaction buffer, and reporter probe.

6. The kit for diagnosing and / or assisting in the diagnosis of acute myocardial infarction according to claim 3, characterized in that: The final concentrations of each raw material in the kit are as follows: Dual recognition probe template 0.05–0.2 μM, CRISPR / Cas12a protein, dNTP mixture 0.5 mM, crRNA 100 nM, phi29 DNA polymerase reaction buffer 1×, phi29 DNA polymerase 0.067 U / μL, Cas12a enzyme reaction buffer 1×, fluorescent reporter probe or lateral flow reporter probe 500 nM.

7. A single-tube detection method for dual-target miRNAs based on dual-recognition probe-triggered RCA-CRISPR technology, characterized in that: It uses the kit described in claim 3 or 4 to detect target miRNAs using a single-tube CRISPR-Cas12a fluorescence / lateral flow chromatography method; wherein the target miRNAs are miRNA-1 and / or miRNA-208.

8. The method for single-tube detection of dual-target miRNA based on dual-recognition probe-triggered RCA-CRISPR technology according to claim 5, characterized in that: It includes the following steps: a. Take the sample to be tested, pre-treat the target miRNA by diluting the miRNA target concentration with DEPC water. b. Tube bottom RCA: Using the dual recognition probe as a template, phi29 DNA polymerase isothermally amplifies the DNA in a rolling circle at 37℃ for 20 min to generate target-specific long ssDNA. After centrifugation and mixing, the Cas12a / crRNA complex pre-placed in the tube cap recognizes the amplification product and activates the trans-cleavage activity for 40 min, continuously cleaving the reporter probe. The released fluorescent or gold-labeled band signal is read by a spectrometer or a lateral flow test strip to achieve quantitative / qualitative detection of miRNA.

9. The method for single-tube detection of dual-target miRNA based on dual-recognition probe-triggered RCA-CRISPR technology according to claim 8, characterized in that: The specific method for step b is as follows: The dual recognition probe was mixed with 2 μL of target miRNA, 1 μL of dNTP (10 mM), 2 μL of phi29 DNA polymerase reaction buffer, and 2 μL of phi29 DNA polymerase (6.7 × 10⁻⁶ mM). 2 Mix 3 μL of ddH2O and 3 μL of ddH2O and add to the bottom of the reaction tube; add a mixture of 3 μL of ddH2O, 2 μL of crRNA (1 μM), 2 μL of Cas12a protein (1 μM), 2 μL of Cas12a enzyme reaction buffer (buffer 1), and 1 μL of fluorescent reporter probe or lateral flow reporter probe (10 μM) to the cap of the reaction tube; then, amplify at 37°C for 20 minutes to obtain a large amount of long repeat single-stranded DNA. Then, centrifuge to mix the CRISPR / Cas system in the cap with the rolling circle amplification product at the bottom of the tube, and react at 37°C for 40 minutes; detect the fluorescence signal released by the reporter probe using a fluorescence spectrometer, or observe the detection line signal visually using a lateral flow test strip to obtain the miRNA detection result; The concentration of the dual recognition probe is 200 nM.

10. The method for single-tube detection of dual-target miRNA based on dual-recognition probe-triggered RCA-CRISPR technology according to claim 8 or 9, characterized in that: The samples were derived from urine or serum; the fluorescent reporter probe was 5'-FAM-TTATTATT-BHQ1-3' or the lateral flow reporter probe 5'-FAM-TTATTATT-Biotin-3'; The fluorescence signal was collected using a fluorescence spectrometer with fluorescence intensity in the range of excitation wavelength 490 nm and emission wavelength 500 nm-700 nm.

11. A method for diagnosing or / and assisting in the diagnosis of acute myocardial infarction for non-therapeutic purposes, characterized in that: It uses the dual-target miRNA single-tube detection method based on dual recognition probe-triggered RCA-CRISPR technology as described in any one of claims 7-10 to detect the content of miRNA-1 and / or miRNA-208 in urine or blood.

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