IncRNA biomarker for diagnosis of acute myocardial infarction and kit and application thereof

Abstract

The invention discloses an lncRNA biomarker for diagnosis of acute myocardial infarction as well as a kit and application of the lncRNA biomarker, belongs to the technical field of biology, and relates to an lncRNA marker related to diagnosis of acute myocardial infarction as well as a kit and application of the lncRNA marker. The biomarker is long-chain non-coding RNASETBP1-DT, the gene sequence of the biomarker is shown as SEQIDNO: 1, and the gene sequence of the biomarker is shown as SEQIDNO: 2. Compared with healthy people, the lncRNASETBP1-DT is remarkably and highly expressed in blood of patients with acute myocardial infarction, an ROC curve shows that AUC can reach 0.750, the sensitivity is 0.60, and the specificity is 0.90, it is prompted that the lncRNASETBP1-DT can serve as a biomarker to be applied to diagnosis and screening of acute myocardial infarction, research on pathogenesis of acute myocardial infarction is further enriched, and the lncRNASETBP1-DT can be used as a biomarker for diagnosis and screening of acute myocardial infarction. A new molecular marker and a treatment target are provided for early diagnosis and prognosis monitoring of acute myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lncRNA biomarker for diagnosing acute myocardial infarction, a kit and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is an acute, hypoxic and ischemic disease characterized by invasion / rupture of coronary atherosclerotic plaques, followed by complete or incomplete thrombosis and vascular occlusion. Myocardial cell necrosis occurs rapidly after blood supply is interrupted, resulting in a sharp decline and damage to cardiac function. This is also the reason why the mortality rate of AMI is still high worldwide. As the basic disease of AMI, AMI patients are usually accompanied by the formation / rupture of lipid plaques in the coronary arteries, thrombosis / embolism, and lesions of vascular smooth muscle cells and endothelial cells. Therefore, a deep understanding of the pathological mechanisms of intravascular lipid plaques, thrombosis, and vascular cell lesions is the key to the clinical treatment of AMI. At present, the diagnosis of AMI still mainly depends on medical history, clinical manifestations, electrocardiogram (ECG), and changes in myocardial injury markers. With the rapid development of biomedical information, biomarkers have made significant progress in the diagnosis and treatment of diseases, including in a variety of diseases such as cancer, neurological and cardiovascular diseases. Therefore, it is particularly important to find new biomarkers for the early diagnosis and treatment of AMI.

[0003] Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length of more than 200 nt in exosomes. It does not have the function of coding proteins and has a low expression level in the human body. However, compared with protein-coding genes, lncRNA has stronger tissue specificity, poor sequence conservation, rapid evolution, and high abundance. At the epigenetic and transcriptional levels, lncRNA activates, decoys, guides, or scaffolds effect effector proteins; at the post-transcriptional level, lncRNA can act as RNA editing regulators, miRNA blockers, RNA shearing and decomposition regulators, etc. Zhang et al. found that regulating the KDM3A axis through miR-22-3p-dependent lncRNAH19 can reduce myocardial injury and ventricular remodeling after AMI; not only that, Zhou found that the level of lncRNAMIAT was significantly increased in the peripheral blood of AMI patients, and was positively correlated with the levels of cardiac biomarkers cTnT, CK, and CK-MB, and promoted the apoptosis of cardiomyocytes. This shows that lncRNA is a biomarker with great potential in AMI disease. The proposal of lncRNA as a new biomarker for AMI can help people detect and prevent AMI at the genetic level, and even delay the progression of AMI, providing new ideas for the diagnosis and treatment of cardiovascular diseases. Summary of the invention

[0004] The main purpose of the present invention is to propose and solve the problems of the shortcomings of existing diagnostic technologies.

[0005] To solve the above problems, the present invention proposes a lncRNA biomarker for the diagnosis of acute myocardial infarction, which is characterized by comprising:

[0006] lncRNA markers;

[0007] The lncRNA marker is lncRNASETBP1-DT, which is differentially expressed in the blood of normal people and patients with acute myocardial infarction. Its nucleotide sequence is shown in SEQ ID NO: 1.

[0008] In one embodiment, the reagent box comprises a reagent box body and a reagent box cover arranged outside the top of the reagent box body, wherein a plurality of reagent bottles are inserted into the interior of the reagent box body;

[0009] The reagent bottles inside the kit include RNA extraction reagent, specific primer pair, reverse transcription reaction reagent, fluorescent quantitative PCR reaction solution, and RNase-Free water.

[0010] In one embodiment, the RNA extraction reagent includes Trizol reagent, chloroform, isopropanol, and anhydrous ethanol.

[0011] In one embodiment, the specific primer pair includes a specific primer for lncRNA SETBP1-DT, and the sequence of the specific primer is an upstream primer as shown in SEQ ID NO: 2, and a downstream primer as shown in SEQ ID NO: 3.

[0012] In one embodiment, the specific primer pair further includes an internal reference primer (GAPDH), the sequence of the internal reference primer is an upstream primer: as shown in SEQ ID NO: 4, and a downstream primer: as shown in SEQ ID NO: 5.

[0013] In one embodiment, the reverse transcription reaction reagents include gDNARemover and a reverse transcription reaction premix.

[0014] In one embodiment, the reverse transcription reaction premix includes reverse transcriptase, RNase inhibitor, Oligo (dT) primer, Random6mers, reverse transcription buffer, and dNTP mixed solution.

[0015] In one embodiment, the fluorescent quantitative PCR reaction solution includes SYBR Green I dye, Taq enzyme, dNTP mixed solution, Mg2+, PCR buffer, and ROX.

[0016] In one embodiment, the reagent box cover can be completely covered on the outside of the top opening of the reagent box body, and a non-slip bottom pad is attached and fixed to the outer wall of the bottom end of the reagent box body, and the non-slip bottom pad is made of medical grade soft rubber, characterized in that: a storage protection device is arranged inside the reagent box cover;

[0017] The storage protection device includes a disinfection protection box, an adhesive fixing sheet and a moisture absorption protection box. The disinfection protection box and the moisture absorption protection box are both located inside the reagent kit cover. The tops of the disinfection protection box and the moisture absorption protection box are both provided with adhesive fixing sheets, and the adhesive fixing sheets are adhesively fixed to the inner wall of the top of the reagent kit cover.

[0018] In one embodiment, the storage protection device further comprises disinfection cotton, ventilation holes and hygroscopic silica gel particles. Ventilation holes are densely distributed around the upper sides of the center of the disinfection protection box and the hygroscopic protection box. Disinfection cotton is arranged inside the disinfection protection box, and hygroscopic silica gel particles are arranged inside the hygroscopic protection box.

[0019] The interiors of the disinfection protection box and the moisture absorption protection box are both connected to the outside through ventilation holes, the interior of the disinfection cotton is filled with tea tree essential oil, and the diameter of the moisture absorption silica gel particles is larger than the aperture of the ventilation holes.

[0020] Application of a lncRNA marker and a kit in the diagnosis and treatment target detection of acute myocardial infarction.

[0021] Beneficial effects:

[0022] 1. The present invention first discovered the abnormal expression of lncRNASETBP1-DT in patients with acute myocardial infarction through the detection of a large number of clinical samples; statistical analysis was performed based on the test results, confirming that lncRNASETBP1-DT can be used as a newly discovered biomarker to play an important role in the diagnosis of acute myocardial infarction. The area under the ROC curve AUC has been generally recognized as an inherent accuracy indicator for the authenticity evaluation of diagnostic tests. With lncRNASETBP1-DT as a diagnostic marker, the area under the ROC curve is 0.750 (P<0.05), AUC>0.7, and the cut-off value is 9. At this time, the specificity is 0.90 and the sensitivity is 0.60, indicating that lncRNASETBP1-DT has excellent diagnostic value for AMI and can be used as a detection target for the preparation of auxiliary diagnostic reagents for acute myocardial infarction. In summary, the lncRNASETBP1-DT disclosed in the present invention is a novel, reliable, easy-to-obtain and easy-to-detect AMI biomarker, which is of great significance in the early diagnosis and treatment of AMI, and helps to establish a standardized detection method for AMI as soon as possible.

[0023] 2. The present invention adds a novel storage protection device inside the reagent kit cover of the atherosclerotic plaque stability detection kit. The existence of the storage protection device effectively controls the internal environment of the reagent kit and reduces the adverse effects of external factors on reagents and consumables. The disinfection protection box and the hygroscopic protection box work together to reduce the risks of bacterial growth and moisture erosion, extend the service life of the detection reagents and consumables in the kit, and reduce the cost of frequent replacement of the kit. The disinfection gas emitted by the tea tree essential oil disinfection cotton in the disinfection protection box of the storage protection device is harmless to the human body and can effectively inhibit bacterial growth, thereby improving the safety of the kit during use. When operating the kit, the tester does not have to worry about the threat to his own health due to bacterial contamination. The hygroscopic silica gel particles are non-toxic and odorless, and will not have any adverse effects on the detection reagents and consumables. Even in unexpected circumstances, the silica gel particles will not cause harm to the human body, thereby ensuring the safety of the use of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The results of ultrasound examination of cardiac function in normal patients and AMI patients;

[0026] Figure 2 Heat map of differentially expressed lncRNA clustering;

[0027] Figure 3 Differential expression of lncRNASETBP1-DT in blood samples of 10 normal subjects and 10 patients with acute myocardial infarction;

[0028] Figure 4 ROC curve of lncRNASETBP1-DT;

[0029] Figure 5 This is a schematic diagram of the external three-dimensional structure of a kit for the diagnosis of acute myocardial infarction using lncRNA biomarkers according to the present invention;

[0030] Figure 6 This is a schematic diagram of the internal three-dimensional structure of a kit for the diagnosis of acute myocardial infarction using lncRNA biomarkers according to the present invention;

[0031] Figure 7 It is a schematic diagram of the three-dimensional structure of the storage protection device of the present invention.

[0032] The following are the descriptions of the reference numerals:

[0033] 1. Test kit cover; 2. Storage protection device; 3. Anti-slip bottom mat; 4. Test kit body; 5. Pipette tip; 6. Test tube; 7. Thickened fixed bottom cotton; 8. Reagent bottle; 9. Anti-slip strip; 10. Disinfection protection box; 11. Adhesive fixing sheet; 12. Disinfection cotton; 13. Ventilation hole; 14. Moisture absorption protection box; 15. Moisture absorption silica gel particles. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In order to achieve the above-mentioned invention object, the present invention provides a lncRNA biomarker for the diagnosis of acute myocardial infarction and a kit and application thereof

[0036] Example 1 A lncRNA marker for diagnosing acute myocardial infarction

[0037] In this embodiment, a lncRNA marker for diagnosing acute myocardial infarction is provided, characterized in that the lncRNA marker is lncRNASETBP1-DT, and its nucleotide sequence is shown in SEQ ID NO:1.

[0038] In this example, lncRNA SETBP1-DT is differentially expressed in the blood of normal subjects and patients with acute myocardial infarction.

[0039] Example 2 A kit for detecting lncRNA biomarkers for the diagnosis of acute myocardial infarction

[0040] The kit body 4 comprises a kit cover 1 arranged on the outside of the top of the kit body 4, the kit cover 1 can be completely densely covered on the outside of the top opening of the kit body 4, a non-slip bottom pad 3 is pasted and fixed on the outer wall of the bottom end of the kit body 4, the non-slip bottom pad 3 is made of medical-grade soft rubber, the left and right outer walls of the kit body 4 are provided with anti-slip strips 9, the two anti-slip strips 9 are close to the top opening of the kit body 4, the anti-slip strips 9 and the kit cover 1 are interference fit, a thickened fixed bottom cotton 7 is arranged on the bottom of the inside of the kit body 4, a plurality of suction tips 5 and a plurality of test tubes 6 are plugged on the left side of the center of the thickened fixed bottom cotton 7, the suction tips 5 are located on the front side of the test tube 6, and a plurality of reagent bottles 8 are plugged on the right side of the center of the thickened fixed bottom cotton 7;

[0041] In this embodiment, the test tube 6 is first taken out, and then the pipette tip 5 is taken out to suck a quantitative blood sample and drip it into the inside of the test tube 6. At this time, the reagent in the reagent bottle 8 is correspondingly sucked by the pipette tip 5 (the specific taking-out operation is shown in the following embodiment) and dripped into the inside of the test tube 6. Then, the test tube 6 mixed with the reagent and the blood sample is fully mixed and placed in the Q-PCR instrument LineGene9640 for detection.

[0042] like Figure 5 and Figure 7 As shown, a storage protection device 2 is arranged inside the reagent box cover 1, and the storage protection device 2 includes a disinfection protection box 10, an adhesive fixing sheet 11 and a hygroscopic protection box 14. The disinfection protection box 10 and the hygroscopic protection box 14 are both located inside the reagent box cover 1, and adhesive fixing sheets 11 are arranged on the top of the disinfection protection box 10 and the hygroscopic protection box 14. The adhesive fixing sheets 11 are adhesively fixed to the inner wall of the top of the reagent box cover 1. The storage protection device 2 also includes disinfection cotton 12, ventilation holes 13 and hygroscopic silica gel particles 15. The disinfection protection box 10 and the hygroscopic protection box 14 are densely covered with ventilation holes 13 on the upper side around the center. The disinfection protection box 10 is provided with disinfection cotton 12, and the hygroscopic protection box 14 is provided with hygroscopic silica gel particles 15. The hygroscopic silica gel particles 15 in the hygroscopic protection box 14 can absorb moisture in the reagent box to maintain a dry internal environment, so that the detection reagents and consumables can better maintain their stability in a dry environment. and activity, to prevent deterioration due to moisture, thereby ensuring the accuracy of the test results. The interior of the disinfection protection box 10 and the moisture absorption protection box 14 are connected to the outside through the vent 13. The interior of the disinfection cotton 12 is filled with tea tree essential oil. The disinfection cotton 12 in the disinfection protection box is filled with tea tree essential oil, and the disinfection gas is emitted through the vent 13, which can effectively inhibit the growth of bacteria and prevent the test kit from being contaminated by bacteria during storage and transportation. This is very important for testing reagents and consumables, and avoids inaccurate test results caused by bacterial contamination. The diameter of the hygroscopic silica gel particles 15 is larger than the aperture of the vent 13. The setting of the vent 13 can ensure that the disinfection protection box 10 and the moisture absorption protection box 14 play a role without affecting the overall sealing of the test kit. When the test kit needs to be used, after the test kit cover 1 is opened, the storage protection device 2 will not interfere with the operation, so that the test personnel can quickly and accurately take the reagents and consumables.

[0043] Example 3 Preparation of a kit for in vitro detection of lncRNA markers according to Example 1 in the blood of patients with acute myocardial infarction

[0044] In this embodiment, a kit for in vitro detection of lncRNASETBP1-DT markers in the blood of patients with acute myocardial infarction is provided. The kit includes an RNA extraction reagent, a specific primer pair, a reverse transcription reaction reagent, and a fluorescent quantitative PCR reaction solution.

[0045] In this embodiment, the RNA extraction reagent includes Trizol reagent, chloroform, isopropanol, and anhydrous ethanol. The specific configuration is shown in Table 1:

[0046] Table 1 RNA extraction reagent specifications

[0047]

[0048] In this example, a specific primer pair for lncRNASETBP1-DT and an internal reference gene (GAPDH) was designed, including an upstream primer and a downstream primer. The primer design is shown in Table 2:

[0049] Table 2 Specific primer pairs for lncRNASETBP1-DT and internal reference gene (GAPDH)

[0050] Primers sequence Serial number SETBP1-DT-F 5'-TGGCTGCTGGTTTGAGTTCCTTC-3' SEQ ID NO:2 SETBP1-DT-R 5'-CCCAGTCTCTTTCACTCCACTTCAC-3' SEQ ID NO:3 GAPDH-F 5'-AGAAGGCTGGGGCTCATTTG-3' SEQ ID NO:4 GAPDH-R 5'-AGGGGCCATCCACAGTCTTC-3' SEQ ID NO:5

[0051] In this embodiment, the reverse transcription reaction reagents include gDNARemover and reverse transcription reaction premix. The specific configuration is shown in Table 3:

[0052] Table 3 Reverse transcription reagent specifications

[0053] Reagents Specification Quantity(tube) gDNARemover 50μL / tube 1 Reverse transcription reaction master mix 500μL / tube 1

[0054] In this embodiment, the reverse transcription reaction premix includes reverse transcriptase, RNase inhibitor, Oligo (dT) primer, Random6mers, reverse transcription buffer, and dNTP mixed solution.

[0055] In this embodiment, the specific configuration of the fluorescent quantitative PCR reaction solution is shown in Table 4:

[0056] Table 4 PCR reaction solution specifications

[0057] Reagents Specification Quantity(tube) Fluorescence quantitative PCR reaction solution 1000μL / tube 1

[0058] In this embodiment, the specific configuration specifications of RNase-Free water are shown in Table 5.

[0059] Table 5 RNase-Free Water Specifications

[0060] Reagents Specification Quantity(tube) RNase-Free water 2000μL / tube 1

[0061] In this embodiment, the fluorescent quantitative PCR reaction solution includes SYBR Green I dye, Taq enzyme, dNTP mixed solution, Mg2+, PCR buffer, and ROX.

[0062] It should be noted that in this embodiment, the specifications and configurations of all reagents are only for illustrative purposes and are not intended to limit the specific contents of this application.

[0063] Example 4 An application of the lncRNA marker and the kit described in Examples 1 and 2 in the diagnosis and treatment of acute myocardial infarction.

[0064] 1. Sample collection: Peripheral blood samples were collected from 10 normal subjects and 10 patients with acute myocardial infarction. All patients gave informed consent. The acquisition of all the above specimens was approved by the ethics committee.

[0065] 2. Preparation and analysis of RNA samples: Total RNA was extracted using the Trizol method.

[0066] ① Add 1 mL Trizol to the sample and lyse for 5 minutes at room temperature;

[0067] ② For phase separation, add chloroform at a rate of 200 μL chloroform / mL Trizol, shake and mix for 15 seconds, place at room temperature for 3 minutes, and centrifuge at 4°C, 12,000 g for 15 minutes; aspirate the upper colorless aqueous phase into a new RNase-free EP tube;

[0068] ③ Add an equal volume of isopropanol, gently invert up and down 10 times to mix, leave at room temperature for 10 minutes to precipitate RNA, centrifuge at 4°C, 12000g for 15 minutes, and discard the supernatant;

[0069] ④ Add 1 mL of 75% ethanol prepared with RNase-Free water, shake gently to wash the RNA precipitate, centrifuge at 7500 g for 5 min at 4°C, and discard the supernatant;

[0070] ⑤ Dry the RNA precipitate in a 37°C oven for about 15 minutes; dissolve the RNA precipitate in 20 μL RNase-Free water and promote dissolution in a 60°C metal bath for 10 minutes;

[0071] ⑥Take 2 μL of extracted RNA and use a nucleic acid protein detector to detect the concentration and purity of the extracted RNA. The purity standard is OD260 / 280 between 1.7-2.0.

[0072] 3. Q-PCR verification of differential expression of lncRNASETBP1-DT

[0073] ① Reverse transcription of total RNA, the reaction system is 20 μL, the components are as follows:

[0074]

[0075] After thorough mixing, place in PCR instrument T100TM Thermal Cycler, set the program as: 50℃15min, 85℃5sec, the obtained product is cDNA.

[0076] ② Real-time quantitative PCR was used to detect the expression of lncRNA SETBP1-DT. The primers were designed as follows: SETBP1-DT-F5'-TGGCTGCTGGTTTGAGTTCCTTC-3' (SEQ ID NO: 2)

[0077] SETBP1-DT-R5'-CCCAGTCTCTTTCACTCCACTTCAC-3'(SEQIDNO:3)

[0078] GAPDH-F5'-AGAAGGCTGGGGCTCATTTG-3'(SEQIDNO:4)GAPDH-R5'-AGGGGCCATCCACAGTCTTC-3'(SEQIDNO:5)

[0079] The PCR reaction system is 20 μL, and the components are as follows:

[0080]

[0081] After thorough mixing, place in Q-PCR instrument LineGene9640, and set the program as: 95℃5min; [95℃30sec; 60℃1min; 72℃30sec; 95℃15sec] and repeat 45 cycles.

[0082] ③ Result calculation: This experiment uses relative quantitative analysis method. Each sample is made into three replicates, and the result is the average CT value of the three replicates. △Ct = average CT value of target gene - average CT value of reference gene;

[0083] △△Ct=△Ct(AMI)-△Ct(Healthy); Relative expression level=2-△△Ct. GAPDH was used as an internal reference to calculate the relative expression level of lncRNASETBP1-DT.

[0084] 4. Binary logistic regression analysis and ROC curve analysis

[0085] Data were analyzed using SPSS 20.0 software. Binary Logistic regression was performed with the relative expression of target lncRNA as the independent variable and group as the dependent variable. The regression fit was tested using the likelihood ratio test, and the regression parameter estimates were tested using nonparametric tests. The sensitivity and specificity of target lncRNA diagnosis were evaluated based on the receiver operating characteristic (ROC) curve and the area under the curve (AUC).

[0086] 5. Results

[0087] ① Figure 1These are the results of cardiac function ultrasound examinations of normal patients and AMI patients. The results show that the cardiac ejection fraction EF% of AMI patients is less than 50%, indicating that AMI patients have varying degrees of cardiac function decline.

[0088] ② Figure 2 This is an expression profile analysis of 39 lncRNAs significantly expressed in AMI patients and normal subjects. According to the screening criteria of foldchange>2, P-value<0.05, it can be seen that compared with normal subjects, 29 lncRNAs in the peripheral blood RNA of AMI patients were upregulated and 10 lncRNAs were downregulated.

[0089] ③Compared with patients without acute myocardial infarction, the expression of lncRNASETBP1-DT in the blood of AMI patients was significantly increased relative to the internal reference, and the level in the AMI group was 11.38 times higher than that in the control group (P<0.005). Figure 3 shown.

[0090] ④ The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. The AUC of a completely worthless diagnostic test is 0.5, and the AUC of an ideal diagnostic test is 1. It is generally believed that AUC has a certain diagnostic value when it is between 0.7 and 0.9. Figure 4 As shown, the area under the ROC curve was 0.750 (P<0.05), the result was greater than 0.7, and the cut-off value was 9. At this time, the specificity was 0.90 and the sensitivity was 0.60.

[0091] In summary, lncRNASETBP1-DT has excellent diagnostic value for acute myocardial infarction, is a relatively reliable biomarker for acute myocardial infarction, and has the potential to serve as a new therapeutic target for acute myocardial infarction.

[0092] The technical solutions disclosed in the present invention are not limited to the technical means disclosed in the above embodiments. For ordinary technicians in this technical field, without departing from the principle of the present invention, making some improvements and modifications to the present invention, directly or indirectly applying the above technical solutions to other technical fields, are all included in the patent protection scope of the present invention.

[0093] Sequence Listing

[0094] <110> The First Affiliated Hospital of Kunming Medical University

[0095] <120> A new lncRNA biomarker for the diagnosis of acute myocardial infarction and its kit and application

[0096] <130>

[0097] <160> 5

[0098] <170>

[0099] <210>1

[0100] <211>2036<2

[0101] 12>DNA

[0102] <213>Homosapiens<40

[0103] 0>1

[0104] 1cttttcttcaaaggatgggttggagcgttaaaaaatgttggtgggtttgggctcgcagga

[0105] 61ggcggcgcgcggagggcgagggcgcttttgctgagctgtgcagttcggtccgctgagccc

[0106] 121ggcggccctgcccgcccgctcgtccgcccgcggtcgctagtgttgccgccgccgccgccg

[0107] 181ccgccgccgctgctgctccatggccgcgcccgccccgcgcccccgccgcgtcccgtcgcc

[0108] 241gcccgcccgcgtccccgagaattggacacttgatgaagggaaggacacggagagatgaca

[0109] 301aacgcagactggcatgcagttgttttcagggtgggaagaacaaagtggaatgtcttcagt

[0110] 361tcttacaaaatatggtggcatttgtaatctctgagtttttgtatttggcctacaaagggg

[0111] 421gtcttttcagggaaacacgaatggaacccttcagagacatgtctacattgtaaaacaata

[0112] 481taggatagatacggattcgggaaggtctgcaaatggttccgctcaaagaacttggggtaa

[0113] 541agttgaccgaggactcggattctgagaggactcctatcacgttcttcatgagagttttag

[0114] 601gctatttggactgaggaatgatttggaggtacgtcgggggcattccctcactgaggccat

[0115] 661cagtggctgcctccctgcattggctgctggtttgagttccttcacagaacttcttcgtca

[0116] 721agtttttcataggaacgctgcagtgaagtggagtgaaagagactggggattgaagggtga

[0117] 781accccttccagccccaatcccatcccgattcctgtctggatgcagattcagtctgactca

[0118] 841gaacggatagttcacccttttactgatccaagtgctctgtcctctttttttcagaggaat

[0119] 901gttctgactggctacaaacttttcttctgtcactgcaggacctcccgtggagcaattttc

[0120] 961gataggtcttctactatgcttttttaccgcgtcattgttattgctgagaaaataatgctt

[0121] 1021agttaggaaaaaaaatagcagagcatatgagctgatgatagaagtagagatcaaggcaca

[0122] 1081gaatacaatatcaaaacaactattctctgtaatcaaatgacctttctcatttgcttgtac

[0123] 1141acccagatttctgaaatcctacaccaaagtccacagagaactaagatagcatgtggccta

[0124] 1201ttccccgcgcttttccagtggagaaagtagccccagaaatcagaagggtttgtccacaag

[0125] 1081catatggctgtctgatgtcatccaaaccagaactaaagcctattgccctagtcgtttccc

[0126] 1141ttctataacctaggcgtatttcagagtcttccttcctgtggtgttgtgtggattagaaa

[0127] 1201agtagctaagaaaagcacataccttattaaaattcactgcaatagatgataagtctgtag

[0128] 1261aattatagtgcagtagaagtttacctgtgagatcatctattagaatcccatctttttaaa

[0129] 1321actgaggaaacgatgcactaccccaagccactaagttaattcaaagccaggaatagatgc

[0130] 1381tatgttccttgacttcttatccaacgttccttccttgtatgtagcccatctctgaatcat

[0131] 1441gagaggggagaggagagaaaggaaaggagggatattctcactctcttacatatatatgaa

[0132] 1501aaaatatatatgcctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtacacacacccataataa

[0133] 1561tgtttcagtgaacaacagaccacatattactgtgatcccatataataataatactgtat

[0134] 1621ttttgctgtacattttctgtgtttagatacacaaatacttaccattgtgttacatttgcc

[0135] 1681cacagtatttagtacagtaatattctgtacaggtttgcagcctagaagcaataggctacc

[0136] 1741ccatatagcctgagtgtgcagtagcctatatcatctagatttgtgtaagtgcactccatg

[0137] 1801atgtttgcacaaagtcgcctactgatgtatttctcagaacacctccccataagctaattg

[0138] 1861acgcatgattggagatatatatatatatatatatatatacacacacacatataaatatat

[0139] 1921atacatatgtaaaatagatacaataaatattttatacacattaatatttttggtatataa

[0140] 1981taatatattacaaatataaaaattttaatatatgataataaaggatttctaaaaac<210>2

[0141] <211>23<21

[0142] 2>DNA

[0143] <213>Artificial Sequence

[0144] <400>2

[0145] tggctgctggtttgagttccttc

[0146] <210>3

[0147] <211>25<21

[0148] 2>DNA

[0149] <213>Artificial Sequence

[0150] <400>3

[0151] cccagtctctttcactccacttcac

[0152] <210> 4

[0153] <211> 20<21

[0154] 2>DNA

[0155] <213> Artificial sequence

[0156] <400> 4

[0157] agaaggctggggctcatttg

[0158] <210> 5

[0159] <211> 20<21

[0160] 2>DNA

[0161] <213> Artificial sequence

[0162] <400> 5

[0163] aggggccatccacagtcttc.

Claims

1. A lncRNA biomarker for the diagnosis of acute myocardial infarction, characterized in that: include: lncRNA markers; The lncRNA marker is lncRNASETBP1-DT, which is differentially expressed in the blood of normal people and patients with acute myocardial infarction. Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The kit for the detection of lncRNA biomarkers for the diagnosis of acute myocardial infarction according to claim 1, characterized in that: It comprises a reagent box body (4) and a reagent box cover (1) arranged outside the top of the reagent box body (4), and a plurality of reagent bottles (8) are inserted into the interior of the reagent box body (4); The reagent bottle (8) inside the reagent kit body (4) includes an RNA extraction reagent, a specific primer pair, a reverse transcription reaction reagent, a fluorescent quantitative PCR reaction solution, and RNase-Free water.

3. A kit for the diagnosis of lncRNA biomarkers for acute myocardial infarction according to claim 2, characterized in that: RNA extraction reagents include Trizol reagent, chloroform, isopropanol, and anhydrous ethanol.

4. A kit for lncRNA biomarkers for diagnosing acute myocardial infarction according to claim 2, characterized in that: The specific primer pair includes a specific primer for lncRNASETBP1-DT, and the sequence of the specific primer is an upstream primer as shown in SEQ ID NO: 2, and a downstream primer as shown in SEQ ID NO:

3.

5. A kit for the diagnosis of lncRNA biomarkers for acute myocardial infarction according to claim 2 or 4, characterized in that: The specific primer pair also includes an internal reference primer (GAPDH), the sequence of the internal reference primer is an upstream primer: as shown in SEQ ID NO:4, and a downstream primer: as shown in SEQ ID NO:

5.

6. A kit for lncRNA biomarkers for diagnosing acute myocardial infarction according to claim 2, characterized in that: Reverse transcription reaction reagents include gDNARemover and reverse transcription reaction premix.

7. A kit for lncRNA biomarkers for diagnosing acute myocardial infarction according to claim 6, characterized in that: The reverse transcription reaction premix includes reverse transcriptase, RNase inhibitor, Oligo (dT) primer, Random6mers, reverse transcription buffer, and dNTP mixed solution.

8. The kit for lncRNA biomarkers for diagnosing acute myocardial infarction according to claim 2, characterized in that: The fluorescent quantitative PCR reaction solution includes SYBR Green I dye, Taq enzyme, dNTP mixed solution, Mg2+, PCR buffer and ROX.

9. The kit for lncRNA biomarkers for diagnosing acute myocardial infarction according to claim 2, characterized in that: The reagent box cover (1) can be completely covered on the outside of the top opening of the reagent box body (4), and a non-slip bottom pad (3) is glued and fixed to the outer wall of the bottom end of the reagent box body (4), and the non-slip bottom pad (3) is made of medical grade soft rubber, characterized in that: a storage protection device (2) is arranged inside the reagent box cover (1); The storage protection device (2) comprises a disinfection protection box (10), an adhesive fixing sheet (11) and a moisture absorption protection box (14); the disinfection protection box (10) and the moisture absorption protection box (14) are both located inside the reagent kit cover (1); the tops of the disinfection protection box (10) and the moisture absorption protection box (14) are both provided with adhesive fixing sheets (11); the adhesive fixing sheets (11) are adhesively fixed to the inner wall of the top of the reagent kit cover (1); The storage protection device (2) further comprises disinfection cotton (12), ventilation holes (13) and hygroscopic silica gel particles (15); the ventilation holes (13) are densely distributed around the upper sides of the centers of the disinfection protection box (10) and the hygroscopic protection box (14); the disinfection cotton (12) is arranged inside the disinfection protection box (10); and the hygroscopic silica gel particles (15) are arranged inside the hygroscopic protection box (14); The interiors of the disinfection protection box (10) and the moisture absorption protection box (14) are both connected to the outside through the ventilation holes (13); the interior of the disinfection cotton (12) is filled with tea tree essential oil; and the diameter of the moisture absorption silica gel particles (15) is larger than the aperture of the ventilation holes (13).

10. Use of the lncRNA marker and kit according to any one of claims 1 and 2 in the diagnosis of acute myocardial infarction and the detection of therapeutic targets.

Images