Kit for detecting nucleic acid and protein markers of acute myocardial infarction and detection method

Abstract

The invention relates to a kit and a detection method for synchronously detecting nucleic acid and protein markers of acute myocardial infarction, and the kit is used for carrying out specific modifier marking on a target nucleic acid amplification product, so that a'modifier 1-dsDNA-modifier 2 'nucleic acid amplification product is formed; nucleic acid amplification products and target protein are captured through different functionalized nano sensors, and the content of nucleic acid markers and protein markers in a sample is detected through fluorescence values of the nano sensors. According to the kit disclosed by the invention, complex steps such as nucleic acid extraction and purification required in a conventional nucleic acid marker detection process can be omitted; nucleic acid quantitative detection can be carried out without professional nucleic acid detection experiment equipment, personnel or site requirements, and home detection can be realized; the synchronous quantitative detection of the nucleic acid marker and the protein marker can be realized in a single reaction, the advantages of the nucleic acid marker and the protein marker are complementary, and quicker, more comprehensive, more accurate and more specific diagnosis information is provided for AMI diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to a kit and a detection method for detecting nucleic acid and protein markers of acute myocardial infarction. Background Art

[0002] Acute myocardial infarction (AMI) is a life-threatening disease caused by acute obstruction of the coronary artery, which leads to insufficient blood supply to the corresponding myocardial area and myocardial necrosis. It is one of the leading causes of morbidity and mortality worldwide. In my country, the incidence of AMI has also shown a significant upward trend due to the aging of the population and the increase in the proportion of high-risk groups such as heart disease, smoking, diabetes and hyperlipidemia. Studies have shown that the mortality rate can be reduced to 3% if intervention is carried out within 1 hour of the onset of AMI, while the mortality rate is about 15% if intervention is carried out within 3-4 hours of the onset of AMI. Therefore, rapid diagnosis and appropriate treatment can effectively reduce the mortality rate and improve the prognosis of patients. The early diagnosis of AMI is mainly based on patient symptoms, electrocardiogram examination, imaging examination and serological tests. However, some patients do not have typical symptoms of acute myocardial infarction in the early stage of AMI, such as chest pain, back pain, shoulder pain, neck pain and arm pain; at the same time, about 30% of patients do not have typical abnormal electrocardiogram changes such as ST-T segment elevation, which poses a serious obstacle to the early diagnosis of AMI and delays the best treatment time. Therefore, AMI serological testing - the detection of myocardial markers plays a vital role in the early diagnosis of AMI.

[0003] At present, the main targets of clinical AMI serological tests include myoglobin (Myosin, Myo), cardiac troponin (cardiac troponin, cTn) and creatine phosphokinase-MB (creatine phosphokinase-MB, CK-MB). However, traditional myocardial markers have certain limitations in terms of sensitivity, specificity and timeliness: for example, their higher molecular weight leads to lower plasma concentration levels, and the presence of skeletal muscle-derived subtypes leads to reduced specificity. Therefore, the development of highly sensitive and specific marker combinations and their detection technologies is currently an effective means to improve the accuracy of AMI diagnosis, improve treatment effects and reduce mortality.

[0004] MicroRNA (miRNA) is a class of endogenous small RNAs with a length of about 20-24 nt, which has a variety of important regulatory effects in cells. In recent years, studies have shown that in the early stage of AMI, damaged myocardial cells can release specific miRNAs, which participate in the pathophysiological process of AMI and its complications at the post-transcriptional level. The expression profile of peripheral blood miRNA in AMI patients can change with the progression of AMI, and play a role in regulating cell damage or protection after myocardial infarction, myocardial fibrosis and ventricular remodeling, and promote the repair and regeneration of myocardial tissue. Therefore, the expression pattern of miRNA can be used as a biomarker for early diagnosis and prognosis of AMI patients. Compared with traditional protein markers, miRNA has the following advantages in the diagnosis of AMI: 1. Early diagnosis: miRNA in peripheral blood will change early after AMI occurs, and protein markers may take a longer time (about 3 to 6 hours) to reach the detection level; 2. Stability: miRNA is relatively stable in the blood and is not easily affected by external factors. In contrast, protein markers may be interfered by factors such as sample processing, storage and transportation; 3. Diversity: There are rich types of miRNAs, and multiple miRNAs can be detected at the same time, thereby improving the accuracy of diagnosis. Protein markers can usually only detect one or a few proteins; 4: Changes in miRNAs reflect the biological state of cells and tissues, which helps to understand the pathogenesis of the disease, while the information provided by protein markers is not as comprehensive as that of miRNAs. At the same time, miRNAs have some limitations as markers of AMI: Lack of specificity: Although some miRNAs are abnormally expressed after AMI, they may also show similar changes in other heart diseases. Therefore, there is a certain risk of misdiagnosis when miRNAs are used as specific markers of AMI. Dynamic changes: The expression level of miRNA is affected by many factors, including time, disease stage and individual differences. This makes it difficult for miRNA detection results to stably reflect the status of AMI. Standardization issues: There is currently a lack of unified miRNA detection standards and clinical application guidelines. Different laboratories use different technologies and methods, which may lead to inconsistent results. The detection operation is cumbersome, relying on professional instruments and operators, and it is difficult to achieve rapid on-site detection (point-of-care testing, POCT). Therefore, given the shortcomings of existing methods, there is an urgent need to develop rapid and reliable synchronous multiple POCT detection and analysis technologies based on nucleic acid-protein markers in clinical practice. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a kit for detecting nucleic acid and protein markers of acute myocardial infarction.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a kit for synchronously detecting nucleic acid and protein markers of acute myocardial infarction, characterized in that the kit comprises:

[0008] 1) a probe that specifically hybridizes with an acute myocardial infarction nucleic acid marker and a nucleic acid ligase;

[0009] 2) Primers, probes and endonucleases for amplifying nucleic acid markers of acute myocardial infarction; the downstream primers of the primers are specifically modified, and the probes are specifically modified; the modifiers of the downstream primers are different from the modifiers of the probes;

[0010] 3) Functionalized nanosensors; the nanosensors include: protein marker nanosensors, nucleic acid marker nanosensors, and quality control nanosensors;

[0011] 4) A test strip for detecting acute myocardial infarction markers; the test strip is provided with a first test line, a second test line and a quality control line; the first test line is coated with an antibody that captures the probe modifier, the second test line is coated with an anti-protein marker antibody, and the quality control line is coated with a goat anti-rabbit antibody;

[0012] 5) Standard products of nucleic acid and protein markers of acute myocardial infarction.

[0013] The kit of the present invention realizes the synchronous detection of nucleic acid and protein markers of acute myocardial infarction, and can detect not only protein markers but also nucleic acid markers, especially miRNA markers. On the one hand, the kit of the present invention can design different amplification primers and detection probes for different target miRNAs, and only needs to satisfy that the downstream primers and detection probes of the primers for amplifying the target miRNA are respectively labeled with different specific modifiers, and when the target miRNA is isothermally amplified, the amplification product is labeled with two different modifiers. In subsequent detection, one of the modifiers is recognized by a nucleic acid marker nanosensor with recognition of the modifier, and the other modifier is captured by an antibody that can recognize the modifier coated by a test strip, and the content of the target miRNA is determined by the fluorescence value emitted by the nanosensor. On the other hand, for the detection of protein markers, antibodies are designed for the target protein, and the antibodies are combined with nanosensors. In subsequent detection, the content of the target protein is determined by the fluorescence value emitted by the protein marker nanosensor.

[0014] As a preferred embodiment of the first aspect, the protein marker nanosensor is connected to an anti-protein marker antibody, the nucleic acid marker nanosensor is connected to a substance that can be connected to a downstream primer modifier; and the quality control nanosensor is connected to a rabbit antibody.

[0015] As a preferred implementation of the first aspect, the downstream primer modifier is biotin, and the substance that can be linked to the downstream primer modifier is streptavidin.

[0016] As a preferred embodiment of the first aspect, the modifier of the downstream primer in 2) is biotin and is located at the 5' end of the primer; the modifier of the probe in 2) is digoxin and is located at the 5' end of the probe.

[0017] As a preferred embodiment of the first aspect, the acute myocardial infarction nucleic acid marker is miRNA, and the nucleotide sequence of the specific hybridization probe is shown in SEQ ID NO: 1 to 2. More preferably, the miRNA is mi-499.

[0018] As a preferred embodiment of the first aspect, the nucleotide sequences of the primers for amplifying the acute myocardial infarction nucleic acid marker are shown as SEQ ID NOs:3-4; the nucleotide sequence of the probe is shown as SEQ ID NO:5.

[0019] As a preferred implementation manner of the first aspect, the amplified acute myocardial infarction protein marker is heart-type fatty acid binding protein (H-FABP).

[0020] As a preferred embodiment of the first aspect, the nanosensor is a nanosphere with a fluorescent material; the fluorescent material includes: lanthanide metal chelates, aggregation-induced emission materials, and oil-soluble CdSe / ZnS quantum dots; the nanosphere is a polystyrene nanosphere with surface carboxyl functionalization.

[0021] As a preferred implementation of the first aspect, the method for preparing the nanosensor comprises the following steps:

[0022] a. Add the fluorescent material to the suspension of nano-microspheres with functional groups on the surface to obtain a mixture, add the mixture to a flask, evacuate the mixture, react at 120-150° C. for 15-25 minutes under the protection of an inert gas, and centrifuge to obtain a nanosensor;

[0023] b. Connect the nanosensor in step a to streptavidin, anti-protein marker antibody, and rabbit antibody, respectively, to obtain a nucleic acid marker nanosensor, a protein marker nanosensor, and a quality control nanosensor, respectively.

[0024] The nucleic acid marker nanosensor is obtained by combining streptavidin with the nanosensor in step a; the protein marker nanosensor is obtained by combining anti-protein marker antibody with the nanosensor in step a; and the quality control nanosensor is obtained by combining rabbit antibody with the nanosensor in step a.

[0025] As a preferred implementation of the first aspect, the step b is specifically:

[0026] b-1 Take the nanosensor and resuspend it in the activation buffer, mix it evenly, and activate and wash it at room temperature to obtain an activated nanosensor;

[0027] b-2 Add the activated nanosensor to the streptavidin solution or the anti-protein marker antibody solution or the rabbit antibody solution respectively, incubate the solution at 4°C with shaking, wash by centrifugation, and collect the precipitate;

[0028] b-3 adding the precipitate from step b-2 to a bovine serum albumin solution, incubating with shaking at 37° C., and then washing and purifying to obtain a nucleic acid marker nanosensor, a protein marker antibody nanosensor, and a quality control nanosensor, respectively.

[0029] As a preferred embodiment of the first aspect, the test strip for detecting acute myocardial infarction markers includes: a sample pad, a conjugation pad, a cellulose acetate film, an absorption pad, and a base liner; the sample pad, the conjugation pad, the nitrocellulose membrane and the absorption pad are sequentially bonded to the base liner in an overlapping manner; a functionalized nanosensor is sprayed on the conjugation pad, and a first detection line coated with anti-digoxin antibodies, a second detection line of anti-protein marker antibodies and a quality control line of goat anti-rabbit antibodies are respectively provided on the nitrocellulose membrane, and the first detection line, the second detection line and the quality control line are spaced apart from each other.

[0030] In a second aspect, the present invention provides a method for detecting the content of nucleic acid and protein markers of acute myocardial infarction, comprising the following steps:

[0031] (1) taking a plasma sample;

[0032] (2) adding a specific hybridization probe of an acute myocardial infarction nucleic acid marker and a nucleic acid ligase, and incubating at 40 to 42° C. for 10 to 15 minutes;

[0033] (3) Add amplification primers, probes and nuclease, and incubate at 35-38° C. for 10-15 minutes; the downstream primer of the primer is specifically modified, and the probe is specifically modified; the modifier of the downstream primer is different from the modifier of the probe; the modifier of the downstream primer is biotin (Biotein), and is located at the 5' end of the primer; the modifier of the probe is digoxin (Digoxin) and is located at the 5' end of the probe.

[0034] (4) adding the reaction product obtained in step (3) dropwise onto the sample pad of the test strip, and leaving the test strip horizontally for 10 minutes;

[0035] (5) Performing fluorescence scanning detection on the test strip of step (4), the detection line on the test strip detects the strip with fluorescence signal, and calculating the content of nucleic acid and protein markers in the sample based on the fluorescence signal.

[0036] As a preferred embodiment of the second aspect, the acute myocardial infarction nucleic acid markers include mi-499 and heart-type fatty acid binding protein.

[0037] As a preferred embodiment of the second aspect, the nucleotide sequence of the probe in step (2) is shown as SEQ ID NO:1-2; the nucleotide sequence of the amplification primer in step (3) is shown as SEQ ID NO:3-4, and the nucleotide sequence of the probe is shown as SEQ ID NO:5.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: the kit of the present invention can eliminate the complex steps of nucleic acid extraction and purification required in the conventional nucleic acid marker detection process; nucleic acid quantitative detection can be performed without the need for professional nucleic acid detection laboratory equipment, personnel or site requirements, and home detection can be realized; and nucleic acid and protein markers can be simultaneously quantitatively detected in a single reaction, and the advantages of nucleic acid markers and protein markers complement each other, providing faster, more comprehensive, more accurate and more specific diagnostic information for AMI diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the components of the test strip of the present invention;

[0040] Figure 2 This is a flow chart of the method for synchronous analysis of nucleic acid-protein markers of acute myocardial infarction of the present invention;

[0041] Figure 3 Figure 1 is a schematic diagram of the relationship between the fluorescence signal ratio and the concentration of the analyte. Figure A is a schematic diagram of the fluorescence appearance of the test strip after ultraviolet excitation after the gradient concentration calibrator is tested; Figure B is a schematic diagram of the original fluorescence signal measurement of the test strip; Figure C is a schematic diagram of the relationship between miR-499 concentration and signal; Figure D is a schematic diagram of the correlation between H-FABP concentration and signal. DETAILED DESCRIPTION

[0042] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] Example 1

[0044] This embodiment takes the AMI nucleic acid marker mi-499 and the protein marker H-FABP as examples to provide a kit for rapid and simultaneous detection of nucleic acid-protein markers of acute myocardial infarction, the kit comprising:

[0045] 1. Probes D1 and D2 that specifically hybridize with mi-499 and SplintR ligase; the nucleotide sequences of probes D1 and D2 are shown in SEQ ID NOs: 1 to 2, respectively;

[0046] 2. Primers, probes and nfo endonuclease for amplifying mi-499; the nucleotide sequence of the upstream primer for amplifying mi-499 is shown in SEQ ID NO:3; the nucleotide sequence of the downstream primer for amplifying mi-499 is shown in SEQ ID NO:4, and the 5' end of the downstream primer is labeled with biotin; the nucleotide sequence of the probe is shown in SEQ ID NO:5, and the 5' end of the probe is labeled with digoxigenin ( Figure 2 shown);

[0047] 3. Functionalized nanosensors, including protein marker nanosensors, nucleic acid marker nanosensors, and quality control nanosensors;

[0048] The protein marker nanosensor is connected to an H-FABP antibody for detecting H-FABP; the nucleic acid marker nanosensor is connected to streptavidin for detecting mi-499; the quality control nanosensor is connected to a rabbit antibody for quality control;

[0049] 4. A test strip for detecting AMI markers, the test strip being provided with a first test line, a second test line and a quality control line; the first test line being coated with anti-digoxin antibody, the second test line being coated with anti-H-FABP antibody, and the quality control line being coated with goat anti-rabbit antibody;

[0050] The anti-H-FABP antibody coated on the second detection line and the H-FABP antibody on the protein marker nanosensor have different binding sites with H-FABP;

[0051] 5. AMI marker standard; the standard comprises both mi-499 nucleic acid marker standard and H-FABP protein marker standard.

[0052] The preparation method of the kit of this embodiment comprises the following steps:

[0053] Preparation of S1 functionalized nanosensor:

[0054] a. Disperse the purified oil-soluble quantum dots and polystyrene nanospheres functionalized with surface carboxyl groups in a reaction solvent to obtain a mixture, add the mixture into a flask, evacuate the flask, react at 120-150°C for 15 minutes under inert gas protection, cool naturally, collect the product, centrifuge and collect the precipitate for multiple washings to obtain a nanosensor. At this time, the surface of the nanosensor contains carboxyl groups, which can be conveniently chemically coupled with streptavidin or antibody molecules.

[0055] b. Connect the nanosensor in step a to streptavidin, anti-H-FABP antibody, and rabbit antibody, respectively. The specific steps are:

[0056] b-1 Activated Nanosensor:

[0057] Take 10 mg of nanosensor and resuspend it in 1 ml of activation buffer, mix it by gentle inversion, activate it at room temperature for 30 minutes and then wash it to obtain the activated nanosensor solution;

[0058] The activation buffer is: 0.05M phosphate buffer (pH 5.0), which is used to activate the carboxyl groups on the surface of the nanosensor to form unstable reaction intermediates.

[0059] b-2 The activated nanosensor was chemically coupled with streptavidin, anti-H-FABP antibody, and rabbit antibody, respectively, as follows:

[0060] Diluting the streptavidin solution or the anti-H-FABP antibody solution or the rabbit antibody solution to 2 mg / ml with a connection buffer, and adding the activated nanosensor solution to the above solutions, respectively, to obtain a streptavidin nanosensor solution, an anti-H-FABP antibody nanosensor solution, and a rabbit antibody nanosensor solution;

[0061] The connection buffer is: 0.05M phosphate buffer (pH 6.4-7.0, depending on the isoelectric point of the target protein), which is used to destroy the protein hydration membrane under mild conditions, making it easy to connect with the nanosensor.

[0062] b-3 The above-mentioned various nanosensor solutions were placed in 4°C shaking incubation overnight, centrifuged and washed, and the precipitate was added to a bovine serum albumin solution with a concentration of 10 mg / ml, and incubated at 37°C shaking for 30 minutes to block the residual sites and obtain functionalized nanosensors. The different functionalized nanosensors were named: AMI nucleic acid marker nanosensor, AMI protein marker antibody nanosensor, and quality control nanosensor.

[0063] Preferably, in step S1a, the reaction solvent is a mixture of ethylene glycol, hexadecane and water, and the proportions thereof are 70-80% (v / v) of ethylene glycol, 10-20% (v / v) of hexadecane and 10% (v / v) of water. The reaction solvent has a great influence on the morphology of the prepared nanosensor. When the oil-soluble quantum dots are dissolved in the reaction solvent, the quantum dots are dissolved in hexadecane. When the hexadecane content is higher than 30%, the morphology uniformity and water dispersibility of the prepared nanosensor will deteriorate. The role of the reaction solvent is to protect the polystyrene nanospheres and prevent them from excessive swelling, resulting in poor morphology uniformity and water dispersibility.

[0064] Preferably, the appropriate heating temperature in step S1 is 120-150°C. Too high a temperature can easily cause non-specific aggregation of the nanosensor. Preferably, the reaction temperature is not greater than 180°C.

[0065] Preferably, the oil-soluble quantum dots added in step S1 may be oil-soluble CdSe / ZnS quantum dots, but are not limited to oil-soluble CdSe / ZnS quantum dots, and may be other fluorescent materials, such as lanthanide metal chelates (europium chelates), aggregation-induced emission (AIE) materials, etc.

[0066] Preferably, the nanospheres used in step S1 are not limited to polystyrene nanospheres functionalized with surface carboxyl groups, and nanospheres made of other materials or with surfaces modified with other functional groups, such as epoxy-modified silica nanospheres, may be used.

[0067] Preparation of S2 test strips:

[0068] The test strip of the present invention is mainly composed of a backing, a sample pad, a conjugate pad, a cellulose acetate film, and an absorbent pad attached in sequence. Figure 1 As shown, the sample pad and the conjugate pad are pre-treated with sample binding buffer for 30 minutes, and are fully squeezed and air-dried for use. After air-drying, the conjugate pad is sprayed with the functionalized nanosensor prepared in step S1 and then vacuum-dried for use; the functionalized nanosensor includes: AMI nucleic acid marker nanosensor, AMI protein marker nanosensor, and quality control nanosensor. The AMI nucleic acid marker nanosensor is used to detect mi-499 in the sample; the AMI protein marker antibody nanosensor is used to detect the AMI protein marker in the sample, such as H-FABP; the quality control nanosensor is used for quality control and verification of the reaction.

[0069] The components and concentrations of the sample binding buffer are as follows: 0.1 M Na2B4O7·10H2O, 1% PVP, 0.2% casein sodium salt, 1% TritonTM X-100 and 0.1% NaN3.

[0070] S3 test strips coated with specific antibodies:

[0071] Anti-digoxin antibody, anti-AMI protein marker antibody, and goat anti-rabbit antibody were diluted to a concentration of 2 mg / mL using antibody coating buffer, and then "draw lines" on cellulose acetate film to form antibody binding strips, nucleic acid detection lines, protein detection lines, and quality control lines (such as Figure 1 shown).

[0072] Antibody coating buffer composition and concentration: 10 mM Na2HPO4·12H2O, 0.9% NaCl, 0.3% trehalose and 0.1% NaN3, pH 7.4.

[0073] The structure of the test strip in this embodiment is as follows Figure 1 As shown:

[0074] The test strip comprises a sample pad, a conjugation pad, a cellulose acetate film, an absorption pad and a backing. The sample pad, the conjugation pad, the nitrocellulose membrane and the absorption pad are sequentially overlapped and bonded to the backing, the conjugation pad is sprayed with a functionalized nanosensor, the nitrocellulose membrane is respectively provided with a first detection line coated with anti-digoxin antibody, a second detection line of anti-AMI protein marker antibody and a quality control line of goat anti-rabbit antibody, and the first detection line, the second detection line and the quality control line are spaced from each other.

[0075] The first detection line is the nucleic acid detection line, the second detection line is the protein detection line, and the third detection line is the quality control line. The test strip is 6 cm long, 3 mm wide, and the cellulose acetate film is 2.5 cm long.

[0076] Example 2

[0077] This embodiment provides a method for using the kit of embodiment 1 to simultaneously detect nucleic acid-protein markers of acute myocardial infarction, and the specific steps are as follows:

[0078] Taking AMI-related miRNA markers (mi-499) and protein markers (heart-type fatty acid binding protein, H-FABP) as examples, the following method was used:

[0079] 1. Preparation of standard curve:

[0080] The kit of the present invention contains a set of standard products (6 bottles, numbered a to f, prepared with hormone-free serum during the production process, and freeze-dried for standby use after packaging), wherein the standard products numbered b to f contain both miR-499 and h-FABP (concentrations are shown in Table 1). The standard products of each concentration have been pre-mixed, freeze-dried after packaging, and can be directly used for detection after reconstitution with pure water.

[0081] The fluorescence signal values ​​of mi-499 and h-FABP were obtained by fluorescence scanning detection, and a standard curve of the fluorescence signal value of the standard and its concentration was established with the concentration of the standard as the X-axis and the fluorescence signal value as the Y-axis.

[0082] Table 1: Gradient concentration standard concentration and fluorescence signal value

[0083]

[0084] 2. Take 15 μl of plasma sample from AMI patients.

[0085] 3. Add mi-499 specific hybridization probes D1 and D2 and SplintR ligase to the sample to be tested, and incubate at 42°C for 10 minutes to form miRNA-DNA hybrid chains. The sequences of hybridization probes D1 and D2 are shown in Table 1 below;

[0086] Table 2: mi-499 specific hybridization probes D1, D2

[0087]

[0088] 4. Add amplification primers, probes and nfo endonuclease in step 3 above, and incubate at 38°C for 15 minutes to achieve large-scale amplification of specific DNA fragments. The sequences of amplification primers and probes are shown in Table 2:

[0089] Table 3: mi-499 specific amplification primers and probes

[0090]

[0091] Load the reaction product onto the sample pad of the test strip, let it stand at room temperature for 10 minutes, and then use a mobile phone / fluorescence scanner to perform fluorescence scanning on the detection area to achieve simultaneous multiplex real-time rapid detection of nucleic acid-protein markers (such as Figure 2 As shown), the fluorescence signal values ​​in the blood sample in this embodiment are T / C 499 :2.3865,T / C fabp :0.8198. The detected content of mi-499 is 8.44×10 9 copies / μl, and the content of H-FABP was 41.10 ng / ml.

[0092] The principle of the detection method of this embodiment is as follows: First, the nucleic acid marker and the specific hybridization probe are complementary and form single-stranded DNA (ssDNA) under the action of ligase. Subsequently, the ssDNA is combined with the specifically modified upstream and downstream primers (the downstream primer is labeled with biotin) and the probe (the probe is labeled with digoxigenin), and amplified and cut under the action of nfo nuclease to form a "biotin-dsDNA-digoxigenin" nucleic acid amplification product ( Figure 2 As shown in Figure 2 ). Since hybridization and amplification are performed at a relatively mild temperature, the process will not affect the physiological activity of the protein marker in the sample. Therefore, after the amplification is completed, the nucleic acid marker amplification product and the protein marker are diluted with the detection buffer and directly loaded on the sample pad of the test strip. Under the capillary force, the liquid moves to the absorption pad and combines with the functionalized nanosensors (AMI nucleic acid marker nanosensor, AMI protein marker antibody nanosensor, quality control nanosensor) pre-sprayed on the binding pad, forming a complex 1 of biotin-dsDNA-digoxigenin nucleic acid amplification product combined with AMI nucleic acid marker nanosensor, and a complex 2 of AMI protein marker combined with anti-AMI protein marker antibody nanosensor; the complexes 1, 2 and the quality control nanosensor further flow through the detection line on the cellulose acetate film, and then are captured by the antibodies pre-coated on the detection line: the digoxigenin antibody coated on the first detection line captures the complex 1, the anti-AMI protein marker antibody coated on the second detection line captures the complex 2, and the goat anti-rabbit antibody coated on the third detection line captures the quality control nanosensor, thereby forming three strips with fluorescent signals. According to the strength of the detected fluorescent signal, the content of nucleic acid and protein markers in the sample can be analyzed and calculated.

[0093] The kit of the present invention realizes the simultaneous detection of nucleic acid and protein markers of acute myocardial infarction, and can detect not only protein markers but also nucleic acid markers, especially miRNA markers. On the one hand. The kit of the present invention can design different amplification primers and detection probes for different target miRNAs, and only needs to satisfy that the downstream primers and detection probes of the primers for amplifying the target miRNA are respectively labeled with different specific modifiers, and when the target miRNA is isothermally amplified, the amplification product is labeled with two different modifiers. In the subsequent detection, one of the modifiers is recognized by a nanosensor with a recognition of the modifier, and the other modifier is captured by an antibody that can recognize the modifier coated with a test strip, and the content of the target miRNA is determined by the fluorescence value emitted by the nanosensor. On the other hand, for the detection of protein markers, antibodies are designed for the target protein, and the antibodies are combined with nanosensors. In the subsequent detection, the content of the target protein is determined by the fluorescence value emitted by the antibody nanosensor. Therefore, the present invention is not only capable of detecting mi-499 and heart-type fatty acid binding protein (H-FABP), but also capable of detecting a variety of miRNAs and marker proteins, and the same function can be achieved by satisfying the above conditions.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A kit for detecting nucleic acid and protein markers of acute myocardial infarction, characterized in that: The kit comprises: 1) a probe that specifically hybridizes with an acute myocardial infarction nucleic acid marker and a nucleic acid ligase; 2) Primers, probes and endonucleases for amplifying nucleic acid markers of acute myocardial infarction; the downstream primers of the primers are specifically modified, and the probes are specifically modified; the modifiers of the downstream primers are different from the modifiers of the probes; 3) Functionalized nanosensors; the functionalized nanosensors include: protein marker nanosensors, nucleic acid marker nanosensors, and quality control nanosensors; 4) A test strip for detecting acute myocardial infarction markers; the test strip is provided with a first test line, a second test line and a quality control line; the first test line is coated with an antibody that captures the probe modifier, the second test line is coated with an anti-protein marker antibody, and the quality control line is coated with a goat anti-rabbit antibody; 5) Standard products of nucleic acid and protein markers of acute myocardial infarction.

2. The kit according to claim 1, characterized in that The acute myocardial infarction nucleic acid marker is miRNA, and the nucleotide sequence of the specific hybridization probe is shown in SEQ ID NO: 1-2.

3. The kit according to claim 2, characterized in that The nucleotide sequences of the primers for amplifying the nucleic acid marker for acute myocardial infarction are shown in SEQ ID NOs: 3-4; the nucleotide sequence of the probe is shown in SEQ ID NO:

5.

4. The kit according to claim 1, characterized in that The nanosensor is a nano-microsphere with a fluorescent luminescent material; the fluorescent luminescent material includes but is not limited to: quantum dots, lanthanide metal chelates, and aggregation-induced luminescence materials.

5. The kit according to claim 4, characterized in that The preparation method of the nanosensor comprises the following steps: a. Adding a fluorescent material to a suspension of nano-microspheres having functional groups on the surface to obtain a mixture, adding the mixture to a flask, evacuating the mixture, reacting at 120-150° C. for 15-25 minutes under inert gas protection, centrifuging, collecting the precipitate, and obtaining a nanosensor; b. Connect the nanosensor in step a to streptavidin, anti-protein marker antibody, and rabbit antibody, respectively, to obtain a nucleic acid marker nanosensor, a protein marker nanosensor, and a quality control nanosensor, respectively.

6. The kit according to claim 5, characterized in that The step b is specifically as follows: b-1. Resuspend the nanosensor prepared in step a in an activation buffer, mix well, activate at room temperature and wash to obtain an activated nanosensor; b-2 Add the activated nanosensor to the streptavidin solution or the anti-protein marker antibody solution or the rabbit antibody solution respectively, incubate the solution at 4°C with shaking, wash by centrifugation, and collect the precipitate; b-3 adding the precipitate from step b-2 to a bovine serum albumin solution, incubating with shaking at 37° C., and then washing and purifying to obtain a nucleic acid marker nanosensor, a protein marker antibody nanosensor, and a quality control nanosensor, respectively.

7. The kit according to claim 1, characterized in that The test strip for detecting acute myocardial infarction markers comprises: a sample pad, a conjugation pad, a cellulose acetate film, an absorption pad, and a base liner; the sample pad, the conjugation pad, the nitrocellulose membrane and the absorption pad are sequentially overlapped and bonded to the base liner; a functionalized nanosensor is sprayed on the conjugation pad, and a first detection line coated with anti-digoxin antibodies, a second detection line of anti-AMI protein marker antibodies and a quality control line of goat anti-rabbit antibodies are respectively arranged on the nitrocellulose membrane, and the first detection line, the second detection line and the quality control line are spaced apart from each other.

8. A method for detecting the content of nucleic acid and protein markers of acute myocardial infarction, characterized in that: The steps include: (1) taking a plasma sample; (2) adding a specific hybridization probe of an acute myocardial infarction nucleic acid marker and a nucleic acid ligase, and incubating at 40 to 42° C. for 10 to 15 minutes; (3) adding amplification primers, probes and endonucleases, and incubating at 35-38° C. for 10-15 minutes; the downstream primer of the primer is specifically modified, and the probe is specifically modified; the modification of the downstream primer is different from the modification of the probe; (4) adding the reaction product obtained in step (3) dropwise onto the sample pad of the test strip; (5) Performing fluorescence scanning detection on the test strip of step (4), the detection line on the test strip detects the strip with fluorescence signal, and calculating the content of nucleic acid and protein markers in the sample based on the fluorescence signal.

9. The method according to claim 8, characterized in that The acute myocardial infarction nucleic acid markers include mi-499 and heart-type fatty acid binding protein.

10. The method according to claim 9, characterized in that The nucleotide sequence of the probe in step (2) is shown as SEQ ID NO: 1-2; the nucleotide sequence of the amplification primer in step (3) is shown as SEQ ID NO: 3-4, and the nucleotide sequence of the probe is shown as SEQ ID NO: 5.

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