Multiplex isothermal transcription amplification-based biological detection system for acute myocardial infarction, and application and method thereof

Abstract

The application belongs to the technical field of biological analysis detection, and specifically discloses an acute myocardial infarction biological detection system based on multiplex isothermal transcription amplification, application and method thereof, the system comprises a probe group, PBCV-1 DNA ligase, T7 RNA polymerase and thioflavin T, the probe group comprises probes P1, P2, double-stranded probe P2 / A1 and double-stranded probe A2 / A3, the double-stranded probe A2 / A3 is composed of probe A2 and probe A3, and the double-stranded probe P2 / A1 is composed of probe P2 and probe A1. The application innovatively combines target recognition based on PBCV-1 DNA ligase, double-cycle transcription signal amplification mediated by T7 RNA polymerase and split G-quadruplex / ThT fluorescence signal amplification system, and constructs a triple signal cycle amplification detection system, which has the characteristics of ultra-high sensitivity, excellent specificity and simple operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological analysis detection, in particular to an acute myocardial infarction biological detection system based on multiplex isothermal transcription amplification, and application and method thereof. BACKGROUND

[0002] Acute myocardial infarction (AMI) is a common acute condition in cardiovascular diseases, with high morbidity and mortality. Early diagnosis and timely treatment are crucial for reducing the mortality of acute myocardial infarction and improving prognosis. Currently, the diagnosis techniques for acute myocardial infarction mainly include electrocardiogram (ECG), myocardial enzyme spectrum detection (such as creatine kinase isoenzyme CK-MB), and troponin (cTnI) detection. These techniques have limitations in accurately predicting the occurrence and development of myocardial infarction, and are difficult to meet the needs of early diagnosis. For example, although electrocardiogram examination is rapid and non-invasive, its sensitivity for diagnosing non-ST segment elevation myocardial infarction (NSTEMI) is low, and it is easy to miss diagnosis. In addition, although myocardial enzyme spectrum and troponin detection have certain specificity, their release time is delayed, making it difficult to achieve ultra-early diagnosis, and the detection process is time-consuming, which cannot meet the needs of rapid diagnosis in clinical practice.

[0003] In recent years, with the continuous improvement of detection technology, a large number of studies have fully demonstrated that miRNA is a promising biomarker for acute myocardial infarction. For example, miR-499 has been proven to be closely related to myocardial injury, and its expression level in blood is expected to serve as an early diagnostic indicator for acute myocardial infarction. The detection of miRNA not only can be used for early screening and diagnosis of acute myocardial infarction, but also can be used for predicting treatment response and evaluating recurrence risk, becoming a new liquid biopsy analysis indicator in acute myocardial infarction. By detecting miRNA in patient blood samples, precise diagnosis and treatment of acute myocardial infarction can be achieved, and the application value in early diagnosis, treatment monitoring, and prognosis evaluation is extremely high.

[0004] G-quadruplex (G4) is a unique nucleic acid secondary structure that has attracted significant attention due to its unique advantages in biosensing and molecular diagnostics. G4 is a four-stranded structure formed by folding of guanine (G)-rich nucleic acid sequences, with high stability and specificity. In the field of biosensing, G4 structures can bind to specific small molecule fluorescent dyes such as thioflavin T (ThT), significantly enhancing the fluorescence signal, thereby enabling high-sensitivity detection of target molecules. This G4-based fluorescence sensing technology is hailed as an important component of the "next-generation molecular diagnostic technology." However, traditional G4-based detection methods still face some challenges in practical applications. For example, the formation of G4 requires specific sequences and environmental conditions, and is easily disturbed in complex biological samples, resulting in high background signals. In addition, the formation process of G4 is usually slow, making it difficult to meet the demand for rapid detection.

[0005] Existing miR-499 detection methods mainly include real-time fluorescence quantitative PCR (qRT-PCR) and in situ hybridization. qRT-PCR is currently the most commonly used method for miRNA detection, but it requires complex reverse transcription steps, precise temperature control, and expensive equipment, making it cumbersome and costly to operate, and difficult to achieve rapid detection. In situ hybridization has high spatial resolution, but its sensitivity is low, making it difficult to detect low-abundance miR-499, and it is not suitable for large-scale clinical applications. Therefore, developing a high-sensitivity, simple-to-operate, and low-cost miR-499 detection method is of great significance for the early diagnosis and treatment of acute myocardial infarction. SUMMARY

[0006] Traditional miRNA detection methods rely on intact DNA probes, which are easily disturbed by non-specific binding when identifying target miRNAs, resulting in high background signals and low detection sensitivity. To overcome these limitations, the T7 transcription system is introduced innovatively in this application, significantly improving the formation efficiency of G4 structures and detection sensitivity through isothermal transcription amplification technology. The T7 transcription system is used to achieve efficient amplification of miRNAs and rapid formation of G4 structures. Compared with traditional intact DNA probes, the split DNA probes each carry functional sequences with dual functional properties. The split DNA probes with T7 promoters and other functional sequences only activate the T7 transcription function when they accurately recognize and bind to the target, thereby initiating an efficient transcription amplification process. This design not only achieves accurate recognition of the target, effectively avoiding false positive signals caused by non-target sequence binding, but also specifically activates functional sequences, significantly enhancing the amplification of downstream signals, thereby greatly improving the sensitivity and specificity of detection.

[0007] The present application is directed to the above problems, and aims to develop a precise detection reagent for acute myocardial infarction markers. A signal output mechanism based on split G4 merging into complete G4 is designed and constructed, and a new miRNA constant temperature detection technology combining T7 transcription amplification is developed. The technology takes the acute myocardial infarction related biomarker miRNA as the analysis object, takes T7 transcription amplification as the signal amplification means, takes the development of non-invasive diagnosis technology of circulating miRNA in blood as the target, takes the specially designed split DNA primer probe as the target specific molecular recognition element, and takes the specially designed split DNA auxiliary probe as the signal output element for constructing complete G4 structure. On this basis, a new G4-ThT fluorescence sensing technology is developed, and a new method for early diagnosis of acute myocardial infarction with high sensitivity, high specificity and rapid detection is established. Relying on the signal output mechanism of split G4 merging into complete G4, the specific combination with thioflavin T (ThT) induces fluorescence enhancement, significantly improves the sensitivity, solves the technical problems caused by the extremely low concentration of miRNA, and restricts the universal screening of acute myocardial infarction. It provides a promising solution for the field of acute myocardial infarction diagnosis and universal screening, and has important significance for improving the accuracy of acute myocardial infarction diagnosis and early detection and treatment.

[0008] In order to achieve the above-mentioned purpose, the present application first provides an acute myocardial infarction biological detection system based on multiple isothermal transcription amplification, which comprises a probe set, PBCV-1 DNA ligase, T7 RNA polymerase and thioflavin T. The probe set comprises probe P1, probe P2, double-stranded probe P2 / A1 and double-stranded probe A2 / A3. The double-stranded probe A2 / A3 is composed of probe A2 and probe A3. The double-stranded probe P2 / A1 is composed of probe P2 and probe A1.

[0009] The sequence of the probe P1 is shown in SEQ ID NO. 1, the sequence of the probe P2 is shown in SEQ ID NO. 2, the sequence of the probe A1 is shown in SEQ ID NO. 3, the sequence of the probe A2 is shown in SEQ ID NO. 4, and the sequence of the probe A3 is shown in SEQ ID NO. 5.

[0010] As a preferred embodiment, the molar ratio of the probe P1, the probe P2, the double-stranded probe P2 / A1 and the double-stranded probe A2 / A3 is 1:1.1:1:1.

[0011] As a preferred embodiment, the biological detection system further comprises an RNAase inhibitor, an NTP Mixture and potassium ions.

[0012] Based on one general inventive concept, the application further provides an application of the biological detection system in preparing a kit for detecting an acute myocardial infarction target, wherein the acute myocardial infarction target is miR-499, and the sequence of the miR-499 is shown as SEQ ID NO. 6.

[0013] Based on one general inventive concept, the application further provides a method for detecting an acute myocardial infarction target by using the biological detection system, which is not for the purpose of disease diagnosis and treatment, and the method comprises the following steps:

[0014] S1, probe sequence pretreatment: DEPC water is used to configure the freeze-dried powder of the probe P1, the probe P2, the probe A1, the probe A2 and the probe A3 into 100 µM solutions, respectively;

[0015] The probe P2 and the probe A1 are mixed with the second annealing buffer solution, incubated at 95°C for 5 min, slowly cooled to room temperature to form double-stranded probe P2 / A1, and stored at 4°C for standby;

[0016] The probe A2 and the probe A3 are mixed with the second annealing buffer solution, incubated at 95°C for 5 min, slowly cooled to room temperature to form double-stranded probe A2 / A3, and stored at 4°C for standby;

[0017] The probe P1, the probe P2 and the sample to be tested are mixed with the first annealing buffer solution, incubated at 95°C for 5 min, slowly cooled to room temperature to form a sample pretreatment solution, and stored at 4°C for standby;

[0018] S2, ligation reaction: PBCV-1 DNA ligase is added to the sample pretreatment solution, and the mixture is reacted at 37°C for 30 min to form a ligation product;

[0019] S3, transcription reaction: the ligation product, RNAase inhibitor Recombinant RNase inhibitor, T7 RNA polymerase, NTP Mixture, double-stranded probe P2 / A1 and double-stranded probe A2 / A3 are mixed in an ep tube and diluted to 20 ul, and the mixture is reacted at 37°C for 60 min to obtain a transcription reaction product;

[0020] S4, fluorescence reaction: the transcription reaction product is mixed with the second annealing buffer solution, thioflavin T and potassium ions, diluted to 150 uL with DEPC water, and the fluorescence is measured by a fluorescence spectrophotometer.

[0021] Preferably, the mixing molar ratio of the probe P1 and the probe P2 in the step S1 is 1:1.1; the first annealing buffer solution contains 100 mM Tris and 400 mM KCl, and the pH value is 7.5; and the second annealing buffer solution contains 100 mM Tris and 50 mM MgCl2.

[0022] As preferred, the final concentration of the PBCV-1 DNA ligase in step S2 is 1.3 U.

[0023] As preferred, the final concentration of the RNA enzyme inhibitor Recombinant RNase inhibitor in step S3 is 1 U, the final concentration of the T7 RNA polymerase is 4 U, the final concentration of the double-stranded probe P2 / A1 and the double-stranded probe A2 / A3 is 0.1 uM, and the final concentration of the NTP Mixture is 2.5 mM.

[0024] As preferred, the final concentration of the thioflavin T in step S4 is 20 mM, and the final concentration of the potassium ion is 40 mM.

[0025] As preferred, the excitation wavelength of the fluorescence spectrophotometer is 450 nm, and the maximum peak is measured at the emission wavelength EM=488 nm.

[0026] The detection principle and process of the acute myocardial infarction biological detection system based on multiplex isothermal transcription amplification provided in the application are specifically as follows:

[0027] As shown in Figure 1 On the basis of T7 transcription amplification, the application utilizes the specific binding properties of G-quadruplex (G4) structure and fluorescent dye thioflavin T (ThT), and realizes high-sensitivity detection of acute myocardial infarction biomarker miRNA through precise recognition of split DNA probes and efficient amplification of T7 transcription system. The split DNA probe includes a primer probe and an auxiliary probe.

[0028] In the molecular recognition stage, the primer probe is composed of two parts, P1 and P2. Probe P1 contains a complete T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') and a 12 nt recognition sequence (5'-TGCAAGTCTTAA-3') complementary to the 5' end of the target miR-499; probe P2 contains a 9 nt recognition sequence (5'-AAACATCAC-3') complementary to the 3' end of miR-499 and a transcription template for downstream signal amplification. When the target miR-499 is present, it binds to the complementary regions of probes P1 and P2 simultaneously, forming a stable triple-stranded complex. Probes P1 and P2 are connected to form a complete transcription template under the action of PBCV-1 DNA ligase, and then the first signal amplification, i.e. Transcription I, is mediated by T7 RNA polymerase, producing the corresponding RNA product (L strand). This transcription product not only amplifies the signal, but also triggers the amplification and output of the downstream signal.

[0029] In the second amplification stage of the downstream signal, auxiliary probes play an important role, including auxiliary single strand (Auxiliary single strand, probe A1) and auxiliary double strand (Auxiliary double strand, double strand probe A2 / A3). Auxiliary double strand probe A2 / A3 is formed by two DNA strands of probe A2 and A3. The 5' end of probe A2 contains a complete single strand of split T7 promoter (5'-CTAATACGACTCACTATAGGG-3'), and the 3' end contains a transcription template of L chain (5'-TTAAGACTTGCAGTGATGTTTTGGGTAGGGCGGGTCAAGCAATCGGAGTTTATTAGG-3'); the 5' end of probe A3 contains a sequence complementary to the L chain transcription template (5'-CCTAATAAACTCCGATTGCTTGACCCGCCCTACCCAAAACATCACTGCAAGTCTTAACCC-3'), which is complementary to the 3' end of probe A2 and forms a double strand. The 3' end of probe A3 contains a sequence complementary to A1 (5'-ATTGCTTG-3'), a part of split G4 structure (5'-TGGGT-3'), and only the latter half of the split T7 promoter complementary to the 5' end of probe A2 (5'-TATAGTGAGT-3'). Probe A1 is an auxiliary single strand, and its 3' end contains the remaining part of the split T7 promoter (5'-CGTATTAGG-3') and the sequence complementary to probe A3, and its 5' end contains the remaining part of the split G4 structure (5'-TGGGTAGGGCGGGT-3'). The auxiliary single strand probe A1 is locked by probe P2 to form double strand probe P2 / A1, so that it cannot bind to the auxiliary double strand to form a triple helix structure without target, and thus cannot form a complete T7 promoter and a complete G4 structure; only when the L chain exists, through displacement reaction (Displacement reaction), waste double chain (Waste chains) P2 / L is produced, and probe A1 is released. Free probe A1 binds to the pre-existing auxiliary double strand in the system to form a complete T7 promoter and a complete G4 structure. The complete T7 promoter initiates a new round of transcription, i.e. Transcription II, to produce L chain again, and the cycle continues to amplify the signal.

[0030] Finally, the third heavy signal amplification is formed by the cycle of split G-quadruplex. The complete G4 structure binds with fluorescent dye ThT, significantly enhancing the fluorescence signal.

[0031] Through this split DNA probe, split G4 structure based T7 transcription amplification mediated double signal amplification system, the application realizes high sensitivity detection of miRNA-499, while effectively avoiding false positive signals caused by non-target sequence binding.

[0032] The application innovatively combines target recognition based on PBCV-1 DNA ligase, T7 RNA polymerase mediated double cycle transcription signal amplification and split G-quadruplex / ThT fluorescence signal amplification system, and constructs a novel triple signal cycle amplification detection system. The system has ultra-high sensitivity, excellent specificity and simple operation, and exhibits excellent performance in actual clinical sample detection, providing a novel detection method for early diagnosis of acute myocardial infarction. The system is the first detection platform combining split DNA probe design and T7 transcription amplification technology, and the beneficial effects mainly include:

[0033] 1. A triple signal amplification system is constructed: the first amplification relies on T7 mediated transcription cycle, the initial target triggers the continuous catalysis of T7 RNA polymerase to generate a large amount of RNA transcription product, and the initial cascade amplification of the signal is completed; the second amplification is achieved by the specific binding of released probe A1 and double-stranded probe A2 / A3 complex, which starts a new round of T7 transcription cycle for further amplification, so that the signal intensity is increased twice; the third amplification utilizes a large amount of L generated by the double transcription cycle, which produces a large amount of split G4 structure through its specific cycle amplification, and finally forms a complete G4 structure and specifically binds with ThT to form a strong fluorescence signal. The system fully utilizes the high specificity of nucleic acid molecule recognition and the high efficiency of enzymatic reaction, and develops a precise quantitative detection technology based on circulating miRNA, which can realize early and rapid diagnosis of acute myocardial infarction. The core innovation of the system is that through the design of three-level progressive signal amplification, the signal is exponentially enhanced from the target to the detection signal, breaking through the sensitivity bottleneck of traditional single or double amplification strategy. Moreover, this multi-dimensional cascade amplification design further converts the nucleic acid signal into a directly detectable optical signal through the cycle generation of G4, and the three-level amplification mechanism makes the detection limit as low as 0.86 aM, which is two orders of magnitude higher than the sensitivity of traditional qRT-PCR method, and significantly reduces the detection limit.

[0034] 2. Split probe (split DNA probe P1 / P2) realizes specific recognition of target miR-499: through the synergistic recognition mechanism of split probe, the target specificity of miR-499 is greatly improved, and the limitation of traditional single probe being easily disturbed by homologous sequences is overcome. This two-component synergistic recognition mode significantly reduces the cross-reaction probability with homologous miRNA, and makes the recognition accuracy of the system for miR-499 increase by one order of magnitude.

[0035] 3. The design of split T7 promoter greatly improves the controllability and specificity of the whole detection system: in this system, the fragments of split T7 promoter are associated with the auxiliary chain respectively. When the L chain exists, the A1 chain is released in the system, forming a triplex with the double-stranded probe A2 / A3, and the split T7 promoter fragments also approach and reassemble into a complete promoter structure that can be recognized by T7 RNA polymerase. This design achieves strict control of transcription initiation, effectively avoids background transcription caused by spontaneous activation of the promoter, and greatly reduces the probability of non-specific transcription.

[0036] 4. The use of split G-quadruplex structure significantly improves the signal-to-noise ratio of signal output: by regulating the formation conditions of G-quadruplex through split design, the dual effects of "background suppression-signal enhancement" are achieved, which significantly improves the signal-to-noise ratio of the system and provides stable signal output guarantee for high specificity detection. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0038] Figure 1 Schematic diagram of the detection principle of the acute myocardial infarction biological detection system based on multiple isothermal transcription amplification of the present application;

[0039] Figure 2 Graph of the feasibility analysis results of the acute myocardial infarction biological detection system in Experimental Example 1 of the present application for detecting miR-499;

[0040] Figure 3 Graph of the specificity selection analysis results of the acute myocardial infarction biological detection system in Experimental Example 2 of the present application for detecting different disease biomarker specific target sequences;

[0041] Figure 4 Graph of the specificity selection analysis results of the acute myocardial infarction biological detection system in Experimental Example 2 of the present application for detecting single base mutant chain of miR-21 and miR-499;

[0042] Figure 5 Fluorescence intensity curve graph of the detection of different concentrations of acute myocardial infarction biomarker miR-499 in Experimental Example 3 of the present application;

[0043] Figure 6 Trend graph of the fluorescence intensity of the detection of different concentrations of acute myocardial infarction biomarker miR-499 in Experimental Example 3 of the present application;

[0044] Figure 7 The response calibration curve of the acute myocardial infarction biomarker miR-499 at different concentrations was detected in Experimental Example 3 of the present application. DETAILED DESCRIPTION

[0045] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or substitutions of the methods, steps or conditions of the present application, which do not depart from the spirit and essence of the present application, are intended to fall within the scope of the present application.

[0046] The technical means used in the examples, if not specifically indicated, are the conventional means known to those skilled in the art; the reagents used in the examples, if not specifically indicated, are commercially available.

[0047] The percentage sign "%" involved in the present application, if not specifically indicated, refers to the mass percentage; but the percentage of the solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0048] The weight parts described in the present application can be the weight units commonly known in the art such as μg, mg, g, kg, etc., or multiples thereof such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0049] The probes involved in the present application are purchased from Shanghai Generay Biotech Co., Ltd. The probe sequences used in the following examples and experimental examples are shown in Table 1.

[0050]

[0051] Example 1: The target gene miR-499 was detected by the acute myocardial infarction biomarker detection system based on multiplex isothermal transcription amplification, and the specific steps were as follows:

[0052] (1) Probe sequence pretreatment

[0053] The probe P1, the probe P2, the probe A1, the probe A2 and the probe A3 are respectively configured into a 100 µM solution with DEPC water. 5.5 uL of the probe P2 (10 uM), 5 uL of the probe A1 (10 uM) and 2.5 uL of a second annealing buffer solution (100 mM Tris, 50 mM MgCl2) are mixed, DEPC is added to 25 uL, incubated at 95°C for 5 min, slowly cooled to room temperature, and a double-stranded probe P2 / A1 is formed, which is stored at 4°C for standby; 5 uL of the probe A2 (10 uM), 5.5 uL of the probe A3 (10 uM) and 2.5 uL of the second annealing buffer solution (100 mM Tris, 50 mM MgCl2) are mixed, DEPC is added to 25 uL, incubated at 95°C for 5 min, slowly cooled to room temperature, and a double-stranded probe A2 / A3 is formed, which is stored at 4°C for standby. 5 uL of the probe P1 (1 uM), 5.5 uL of the probe P2 (1 uM), 5.5 uL of the sample to be tested and 2.5 uL of a first annealing buffer solution (100 mM Tris, pH 7.5, 400 mM KCl) are mixed, DEPC is added to 21.2 uL, incubated at 95°C for 5 min, slowly cooled to room temperature for 2 hours, and a sample pretreatment solution is formed, which is stored at 4°C for standby.

[0054] (2) Ligation reaction: 1.3 uL of PBCV-1 DNA ligase (25 U) and 2.5 uL of PBCV-1 DNA ligase buffer solution (500 mM Tris-HCl, pH 7.4, 100 mM MgCl2, 10 mM ATP, 100 mM DTT) are added to the sample pretreatment solution, the total volume is 25 uL, and the reaction is carried out at 37°C for 30 min. The target miR-499 in the sample to be tested is connected into a complete long chain by the split probes P1 and P2, and a ligation product is formed.

[0055] (3) Transcription reaction: 10 uL of the ligation product is mixed with 0.5 uL of RNAase inhibitor Recombinant RNase inhibitor (40 U), 1.6 uL of T7 RNA polymerase (50 U), 2 uL of NTP Mixture (25 mM), 1 uL of double-stranded probe P2 / A1 (2 uM), 1 uL of double-stranded probe A2 / A3 (2 uM), 2 uL of T7 RNA polymerase buffer solution (400 mM Tris-HCl, 460 mM MgCl2, 20 mM spermidine, 100 mM DTT, pH 7.9), and DEPC is added to 20 uL. The reaction is carried out at 37°C for 60 min to obtain a transcription reaction product. The transcription reaction product releases the auxiliary single-stranded probe A1 through displacement reaction, makes the split G4 structure complete, and starts transcription again, realizing signal amplification again.

[0056] (4) Fluorescence reaction: 20 uL of transcription reaction product was mixed with 6 uL of thioflavin T (500 mM), 15 uL of potassium ion (400 mM), 15 uL of second annealing buffer solution B(a) (100 mM Tris, 50 mM MgCl2), and DEPC was added to 150 uL. K ion and ThT stabilize G4 structure, at the same time, ThT embedded in the structure of complete G4 emits fluorescence, 150 uL of reaction solution was taken in a quartz cuvette, and fluorescence was measured by fluorescence spectrometer RF-6000, with excitation wavelength 450 nm, and the maximum peak was measured at emission wavelength EM=488 nm.

[0057] Experimental Example 1: Analysis of the feasibility of the acute myocardial infarction biological detection system.

[0058] To verify the feasibility of the method for detecting acute myocardial infarction marker miR-499, nine groups of control experiments were designed, and the effects of various key components on the detection system were systematically investigated. Other conditions and methods were the same as in Example 1. The target miR-499 concentration of all experimental groups was 220 nM, and the remaining conditions were kept consistent. The fluorescence signal was measured at 489 nm (excitation wavelength 450 nm), and the results are shown in Table 1. Figure 2

[0059] ​Complete experiment group (group 9): when the system contains all the necessary components (PBCV-1 DNA ligase, T7 RNA polymerase, NTPs, target miR-499, split probe P1 / P2 and auxiliary double-stranded probe A2 / A3, P2 / A1), a significant fluorescence signal enhancement is observed, proving that the system can effectively achieve target-dependent triple signal amplification. Group 4 does not add target miR-499, and due to the lack of target-mediated probe ligation, the system cannot start transcription, proving that the detection is strictly dependent on the presence of the target. The fluorescence signals of other experimental groups are lower than those of group 4 blank experiment except groups 6 and 9. Group 1 does not add PBCV-1 DNA ligase, and due to the inability of P1 and P2 probes to connect to form a complete transcription template, the fluorescence signal is significantly reduced, close to the background level, indicating that the ligase is essential for starting transcription; group 2 does not add T7 RNA polymerase, and there is no transcription amplification, group 3 does not add NTPs, and the lack of nucleotide substrate leads to transcription termination, and the fluorescence signals of the two groups are the lowest, verifying the core role of T7 RNA polymerase and NTPs in signal amplification; group 5 does not add P1 probe, and lacks the T7 promoter region, and cannot form a functional transcription template, the signal is significantly reduced; group 7 does not add double-stranded probe A2 / A3, and cannot form a complete G4 structure, the ThT fluorescence signal is very low, indicating the necessity of G4 structure for signal output; group 8 does not add double-stranded probe P2 / A1, and lacks the release path of auxiliary chain A1, leading to the inability to start secondary transcription, and the signal is significantly lower than the complete system. Group 6 does not add P2 probe, and the transcription template part is missing, leading to no L chain generation, but P1 can still be partially transcribed, and there is some signal. This experiment shows that only when all the key components (target, enzyme, probe) coexist, can the cascade signal amplification be triggered, effectively avoiding non-specific signals (such as group 4 vs. group 9). Moreover, the data of each group clearly shows that each module of the system (ligation, transcription, signal output) is indispensable, proving the rigor of the design. This experiment fully confirms the feasibility and reliability of the method, and provides a better detection system for high-sensitivity detection of acute myocardial infarction targets.

[0060] Experimental Example 2 Specificity analysis of acute myocardial infarction biological detection system

[0061] To investigate the specificity of the detection system for screening the biomarker miR-499 of acute myocardial infarction, the specific target sequences of different disease biomarkers and the single-base mutant chain of miR-21 were selected and compared with miR-499 at a concentration of 1 uM to verify the specificity of the biomarker detection system for acute myocardial infarction. According to the reaction steps of Example 1, 10 groups of different target experiments were set up, and the designs were as follows: miRNAs related to other diseases (miR-21, cZNF292, miR-24, miR-155, miR-200b); single-base mutant sequences of miR-21 (smiR-21, tmiR-21, dmiR-21); blank control (Blank: buffer system without target). The concentration of the target and the interferent in all experimental groups was 1 uM, the fluorescence signal was measured at 489 nm (excitation wavelength 450 nm), each experimental group was repeated 3 times, and the results are shown in Figure 3 and Figure 4 The signal value (Δ fluorescence intensity) after adding the target miR-499 of acute myocardial infarction was significantly higher than that of other sequences with the same concentration (Δ fluorescence intensity). The experiment showed that the technology had high specificity for the biomarker miR-499 of acute myocardial infarction.

[0062] Experimental Example 3: Quantitative analysis of the biomarker detection system for acute myocardial infarction.

[0063] The biomarker miR-499 of acute myocardial infarction was prepared at concentrations of 0 aM, 1 aM, 10 aM, 50 aM, 500 aM, 1 fM, 10 fM, 50 fM, 500 fM and 1 pM. Under the optimal experimental conditions, the quantitative detection of the biomarker miR-499 standard sample of acute myocardial infarction was realized according to the steps of Example 1, and the results are shown in Figure 5 Figure 5 The fluorescence spectrum of the biomarker miR-499 of acute myocardial infarction with concentrations of 0 aM, 1 aM, 10 aM, 50 aM, 500 aM, 1 fM, 10 fM, 50 fM, 500 fM and 1 pM is shown in Figure 6 The fluorescence intensity of the biomarker miR-499 of acute myocardial infarction with different concentrations is shown in Figure 7 ​As shown, the acute myocardial infarction biomarker miR-499 concentration has a good linear trend between 10aM-10fM, and the curve regression equation is y=115x-59.7374, x is the concentration of miR-499 (aM), the linear correlation coefficient R²=0.9931, the detection limit is 0.86aM (LOD=3σ / S), the detection method realizes the ultra-sensitive detection of acute myocardial infarction biomarker miR-499, and the concentration of acute myocardial infarction biomarker miR-499 is parallelly repeated 3 times.

[0064] Experimental Example 4: Detection of acute myocardial infarction biomarker miR-499 in serum.

[0065] To investigate the detection performance of the biological detection system in actual sample detection, the standard addition method was used to simulate the actual sample detection. Different concentrations of acute myocardial infarction biomarker miR-499 were added to 10-fold diluted human serum samples, and the concentrations were 500aM, 1fM and 10fM, respectively, for recovery rate determination. The experimental results are shown in Table 2, the average recovery rate of actual sample detection is between 96% and 104%, and the relative standard deviation (RSD) is between 4.35% and 7.01%. It shows that the sensing technology has good detection performance for acute myocardial infarction biomarker miR-499 in human serum.

[0066]

[0067] Obviously, the above examples are only examples for clarity, and are not limited to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the scope of protection of the present application.

Claims

1. A multiplex isothermal transcription amplification based target bio-detection system for acute myocardial infarction, characterized in that, The probe set comprises a probe P1, a probe P2, a double-stranded probe P2 / A1, and a double-stranded probe A2 / A3, the double-stranded probe A2 / A3 is composed of a probe A2 and a probe A3, the double-stranded probe P2 / A1 is composed of the probe P2 and a probe A1; The sequence of the probe P1 is shown as SEQ ID NO. 1, the sequence of the probe P2 is shown as SEQ ID NO. 2, the sequence of the probe A1 is shown as SEQ ID NO. 3, the sequence of the probe A2 is shown as SEQ ID NO. 4, and the sequence of the probe A3 is shown as SEQ ID NO.

5. The acute myocardial infarction target is miR-499, and the sequence of the miR-499 is shown as SEQ ID NO.

6.

2. The biological detection system according to claim 1, characterized in that The molar ratio of the probe P1, the probe P2, the double-stranded probe P2 / A1, and the double-stranded probe A2 / A3 is 1:1.1:1:

1.

3. The biological detection system of claim 1, wherein, The biological detection system further comprises an RNAase inhibitor, an NTP Mixture, and potassium ions.

4. The use of the biological detection system according to any one of claims 1 to 3 for the preparation of a kit for the detection of the target of acute myocardial infarction, characterized in that, The acute myocardial infarction target is miR-499, and the sequence of the miR-499 is shown as SEQ ID NO.

6. The probe set comprises a probe P1, a probe P2, a double-stranded probe P2 / A1, and a double-stranded probe A2 / A3, the double-stranded probe A2 / A3 is composed of a probe A2 and a probe A3, the double-stranded probe P2 / A1 is composed of the probe P2 and a probe A1; The sequence of the probe P1 is shown as SEQ ID NO. 1, the sequence of the probe P2 is shown as SEQ ID NO. 2, the sequence of the probe A1 is shown as SEQ ID NO. 3, the sequence of the probe A2 is shown as SEQ ID NO. 4, and the sequence of the probe A3 is shown as SEQ ID NO.

5. The acute myocardial infarction target is miR-499, and the sequence of the miR-499 is shown as SEQ ID NO. 6.

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