Micro ribonucleic acid for detecting cerebral arterial thrombosis and application thereof

Abstract

The invention provides a micro ribonucleic acid for detecting cerebral arterial thrombosis and an application of the micro ribonucleic acid. The micro ribonucleic acid is selected from one or a combination of more of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p. The micro ribonucleic acid can be used for detecting cerebral arterial thrombosis. The invention also provides a kit for detecting cerebral apoplexy. The kit and the detection method can be used for non-invasively, rapidly and accurately carrying out auxiliary screening detection on ischemic cerebral apoplexy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a microRNA for detecting ischemic stroke and an application thereof. Background Art

[0002] Ischemic stroke (IS) is the disease with the highest disability rate among cerebrovascular diseases. In China, with the development of society and major changes in people's lifestyles, the exposure level and burden of risk factors for cerebrovascular diseases are increasing. At present, the incidence of cerebrovascular diseases has increased year by year, surpassing heart disease and becoming the leading cause of death and disability in adults. The main diagnostic method for detecting and evaluating ischemic stroke is neuroimaging, but this conventional method is not suitable for continuous dynamic monitoring of acute patients, lacks high sensitivity and specificity, and has weak differential diagnosis ability for early cerebral infarction and transient ischemic attack. Therefore, it is urgent to determine and establish new methods for clinical evaluation and onset prediction of ischemic cerebrovascular disease.

[0003] Extracellular vesicles are an important form of intercellular communication. They are nanoscale vesicles secreted by cells and contain biological molecules such as proteins, transcription factors, and various RNAs. They are ubiquitous in various body fluids. Extracellular vesicles play a role in a variety of physiological and pathological processes, including intercellular signaling, energy metabolism, immune regulation, tissue repair, etc.

[0004] MicroRNA (miRNA) is a type of endogenous single-stranded non-coding small RNA with a length of 19 to 22 nucleotides, which plays a vital role in the pathophysiological process of various diseases. Relevant studies have shown that some miRNAs are significantly expressed in the central nervous system and are closely related to the occurrence and development of various neurological diseases. They can be used as potential new indicators for risk assessment, disease monitoring and prognosis assessment of patients with ischemic stroke. Summary of the invention

[0005] In view of this, the object of the present invention is to provide the use of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p as miRNA molecular markers in the preparation of diagnostic reagents or kits for ischemic stroke, as well as related applications of primers for respectively detecting the expression levels of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p, and also to provide a diagnostic kit for detecting ischemic stroke.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides miRNA molecular markers for detecting ischemic stroke patients, wherein the miRNA is selected from a combination of one or more of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p.

[0008] The preliminary treatment of the present invention is combined with small molecule RNA sequencing technology to measure the miRNA expression profile in the patient's serum extracellular vesicles, screen the differentially expressed miRNAs in the serum extracellular vesicles of patients with ischemic stroke, and verify the sequencing results at the level of single-sample serum extracellular vesicles using the stem-loop PCR method. The expression levels of the target differential miRNAs are analyzed to obtain three differential serum extracellular vesicle miRNA markers.

[0009] The miRNA markers related to ischemic stroke screened according to the present invention are hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p, which are differentially expressed in at least one target serum and at least one healthy control serum, thus reflecting the differential expression in ischemic stroke patients and healthy people, embodying the high sensitivity and high specificity of ischemic stroke diagnosis.

[0010] In a specific embodiment, the gene sequence of the serum extracellular vesicle miRNA marker involved in the present invention is that the sequence of hsa-let-7f-5p is a single-stranded RNA as shown in SEQ ID NO.1, the sequence of hsa-miR-486-5p is a single-stranded RNA as shown in SEQ ID NO.2, and the sequence of hsa-let-7b-5p is a single-stranded RNA as shown in SEQ ID NO.3.

[0011] In a second aspect, the present invention also protects the use of a reagent or kit for detecting the expression level of miRNA in a biological sample in the preparation of a product for screening or auxiliary screening, diagnosis or auxiliary diagnosis, detection or auxiliary detection of ischemic stroke, wherein the miRNA is selected from a combination of one or more of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p.

[0012] In a specific embodiment, the sequence of hsa-let-7f-5p is a single-stranded RNA as shown in SEQ ID NO.1, the sequence of hsa-miR-486-5p is a single-stranded RNA as shown in SEQ ID NO.2, and the sequence of hsa-let-7b-5p is a single-stranded RNA as shown in SEQ ID NO.3.

[0013] In a specific embodiment, the product includes a system for detecting miRNA, the system includes reagents and / or instruments for detecting miRNA, and the reagents include formulations or kits.

[0014] In a specific embodiment, the stem-loop RT-qPCR technique is used to detect changes in miRNA expression levels.

[0015] Preferably, the kit further comprises a stem-loop reverse transcription reagent part and a stem-loop fluorescence quantitative detection reagent part.

[0016] Preferably, the stem-loop reverse transcription reagent consists of RNase-Free ddH2O, reverse transcriptase, dNTPs, reverse transcriptase buffer, and RT-Primer.

[0017] Preferably, the stem-loop fluorescence quantitative detection reagent consists of RNase-Free ddH2O, rTaq enzyme, 10X buffer (Mg2+plus), dNTPs, and Probe TM.

[0018] In the third aspect, the present invention protects a kit for screening or assisting screening, diagnosis or assisting diagnosis, detection or assisting detection of ischemic stroke, which contains a reagent for detecting the expression level of miRNA in a biological sample, and the miRNA is selected from one or more combinations of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p.

[0019] In a specific embodiment, the sequence of hsa-let-7f-5p is a single-stranded RNA as shown in SEQ ID NO.1, the sequence of hsa-miR-486-5p is a single-stranded RNA as shown in SEQ ID NO.2, and the sequence of hsa-let-7b-5p is a single-stranded RNA as shown in SEQ ID NO.3.

[0020] In a specific embodiment, the stem-loop RT-qPCR technique is used to detect changes in miRNA expression levels.

[0021] Preferably, the kit further comprises a stem-loop reverse transcription reagent part and a stem-loop fluorescence quantitative detection reagent part;

[0022] Preferably, the stem-loop reverse transcription reagent part consists of RNase-Free ddH2O, reverse transcriptase, dNTPs, reverse transcriptase buffer, and RT-Primer;

[0023] Preferably, the stem-loop fluorescence quantitative detection reagent part consists of RNase-Free ddH2O, rTaq enzyme, 10X buffer (Mg2+plus), dNTPs, and Probe TM.

[0024] In specific embodiments, the biological sample is serum.

[0025] In a fourth aspect, the present invention also protects a system for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke, the system comprising:

[0026] (1) Sample amplification module: extract the extracellular vesicle miRNA of the serum sample to be tested, use any of the kits described above to perform stem-loop reverse transcription on the target miRNA, and then perform real-time fluorescence quantitative reaction to detect the fluorescence signal.

[0027] (2) Result analysis module: The absolute content of the target miRNA is calculated by making a standard curve absolute quantification method, and the absolute quantitative content of the target miRNA is compared with the absolute content of the target miRNA in the normal healthy control serum.

[0028] In a fourth aspect, the present invention also protects the use of a system for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke in the preparation of a product for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke, the system comprising:

[0029] (1) Sample amplification module: extract the extracellular vesicle miRNA of the serum sample to be tested, use any of the kits described above to perform stem-loop reverse transcription on the target miRNA, and then perform real-time fluorescence quantitative reaction to detect the fluorescence signal.

[0030] (2) Result analysis module: The absolute content of the target miRNA is calculated by making a standard curve absolute quantification method, and the absolute quantitative content of the target miRNA is compared with the absolute content of the target miRNA in the normal healthy control serum.

[0031] In a specific embodiment, the ischemic stroke is selected from transient ischemic attack and / or cerebral infarction.

[0032] The beneficial effects of the present invention are as follows: the present invention analyzed the expression profiles of miRNA in serum extracellular vesicles of ischemic stroke patients and healthy controls, and found that the expression levels of 38 miRNAs in serum extracellular vesicles of ischemic stroke patients were significantly higher than those of healthy controls, and further tested whether the expression levels of 6 miRNAs were consistent with the sequencing results; it was found that 3 miRNAs can be used as molecular markers for clinical application in diagnostic reagents or kits for ischemic stroke.

[0033] Continuing through experimental studies, it was found that the expression levels of these three miRNAs: hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p in the extracellular vesicles of the serum of patients with ischemic stroke were significantly and stably changed; the ROC curve showed that hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p can be used alone or in combination as molecular markers for auxiliary identification of ischemic stroke, providing a variety of new application directions for the diagnosis and treatment of ischemic stroke. Studies have reported that miRNA has good specificity and sensitivity for early diagnosis and prediction of disease, good stability, and reliable test results. The specimen of the present invention is peripheral blood of the subject, the sample specimen is easy to obtain, the clinical operability is strong, and it is non-invasive to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0035] Figure 1 Based on the sequencing results, stem-loop RT-qPCR was used to determine the differential expression of six serum extracellular vesicle miRNAs in ischemic stroke and control groups.

[0036] Figure 2 To expand the sample size for 3 significantly differentially expressed miRNAs, RT-qPCR assays were performed.

[0037] Figure 3 ROC analysis of hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p in the initial screening group, cerebral infarction group and healthy control group, as well as ROC analysis of the combination of three miRNAs.

[0038] Figure 4 To expand the sample size, ROC analysis of hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p in the cerebral infarction group, ischemic stroke group and healthy control group, as well as ROC analysis of the three miRNAs combined, were performed.

[0039] Figure 5 The invention provides a scheme for the discovery and validation of the markers. DETAILED DESCRIPTION

[0040] 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.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The quantitative tests in the following examples were all performed three times, and the data are the average values ​​or the average values ​​± standard deviations of the three repeated experiments.

[0044] Main instruments: Cobas Z 480 real-time fluorescence quantitative PCR instrument was from Nanjing Weishi Biotechnology Co., Ltd., Axygen MaxyGeneⅡ gradient PCR amplifier was from Corning Incorporated, USA, and high-speed refrigerated centrifuge was from Changzhou Jintan Liangyou Instrument Co., Ltd.

[0045] Example 1

[0046] This embodiment provides a method for detecting serum extracellular vesicle miRNA in patients with ischemic stroke, the method comprising:

[0047] 1. Sample collection

[0048] Study subjects: 36 patients with ischemic stroke admitted to the Department of Neurology, Eastern Theater General Hospital from July 2023 to December 2023. All patients were diagnosed by cranial tomography (CT) and magnetic resonance imaging (MRI), and patients with other histories of hemorrhagic infarction, peripheral arterial occlusive disease, chronic liver and kidney disease, and primary / metastatic malignant diseases were excluded. During the same period, 36 healthy controls were selected from the Health Examination Center of the Eastern Theater General Hospital. This study was approved by the Medical Ethics Committee of the Eastern Theater General Hospital, and all participants gave written informed consent.

[0049] 2. Serum sample preparation

[0050] The venous blood of 3-5 mL was collected from patients before treatment and healthy controls who had fasted for more than 12 hours in a procoagulant vacuum blood collection tube containing separation gel. After the blood sample was collected, it was quickly centrifuged at 3500r / m for 5 minutes at room temperature with a centrifugal radius of 13.5cm. The upper serum was separated and stored at -80 degrees. All samples must avoid severe lipemia and hemolysis.

[0051] 3. Extraction of serum extracellular vesicle miRNA

[0052] After the serum is thawed, shake and mix on a vortexer, take 200μL into a new EP tube, centrifuge at 25℃2000g for 30min, and then draw about 100μL of the supernatant into another tube, add 20μL of extracellular vesicle extraction reagent, shake and mix, and then place in a 4℃ refrigerator for 30 minutes. After standing, centrifuge at 25℃10000g for 10min, and remove the supernatant. Add 100μL PBS, mix and place in a 4℃ refrigerator for 30 minutes. Add 1mL Trizol, shake and mix immediately, and place in a 4℃ refrigerator for 1 hour. Add 200μL of chloroform substitute Pal, shake and mix, and then let it stand for 5min. Centrifuge at 4℃12000g for 20min, and then draw about 480μL of the supernatant into another tube, add 480μL of isopropanol, invert and mix to form a homogeneous system, and place in a -20℃ refrigerator to stand overnight for precipitation. After centrifugation at 4℃12000g for 20min, discard the supernatant, add 1mL of 75% ethanol solution prepared with DEPC water, turn the EP tube upside down until the precipitate at the bottom of the tube floats up, and centrifuge again at 4℃12000g for 20min. Discard the supernatant again, turn it upside down on the table and dry it at room temperature for 10-15min. Add 15μLDEPC water to dissolve RNA, and store it in a -80℃ refrigerator or proceed to the next experiment.

[0053] Six miRNAs with differential expression were selected from the sequencing results. Thirty-six patients with ischemic stroke and 36 healthy controls were used as independent samples, and quantitative RT-PCR was used to verify whether their expression was consistent with the sequencing results. The sequences of the six miRNAs are shown in Table 1.

[0054] Table 1: 6 miRNA sequences

[0055]

[0056] 4. Stem-loop reverse transcription reaction and real-time fluorescence quantitative reaction

[0057] The target miRNA was reverse transcribed using the stem-loop reverse transcription method. The specific system is shown in Table 2.

[0058] Table 2: Stem-loop reverse transcription system

[0059]

[0060] The reaction conditions of reverse transcription were 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and 4°C for stopping, and then the subsequent PCR reaction was carried out.

[0061] The target miRNA was amplified using the stem-loop method real-time fluorescence quantitative reaction. The specific system is shown in Table 3.

[0062] Table 3: Real-time fluorescence quantitative reaction system of stem-loop method

[0063]

[0064]

[0065] The instrument used for the amplification reaction was TL988-Ⅳ96 real-time fluorescence quantitative PCR instrument. The reaction program settings are shown in Table 4, and other settings are all system default values. The reverse transcription primers and qPCR amplification forward primers of the stem-loop method real-time fluorescence quantitative kit were from TaqMan TM MicroRNA Assay, the rest of the reagents are from Takara.

[0066] Table 4: Sample amplification conditions for the stem-loop method.

[0067]

[0068] Three replicate wells were made for each sample, and the average was taken for statistics.

[0069] 5. Expression level analysis

[0070] The results were analyzed using GraphPad Prism 10 software. It was found that among the six miRNAs in serum extracellular vesicles of patients with ischemic stroke, the expression level of hsa-let-7f-5p was significantly lower than that of healthy controls, while the expression levels of hsa-miR-486-5p and hsa-let-7b-5p were significantly higher than those of healthy controls, and the differences were statistically significant (P < 0.05). Figure 1 .

[0071] Furthermore, we used ROC curve and area under the curve (AUC) analysis to verify the potential of hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p as molecular markers for the diagnosis of ischemic stroke. The analysis showed that compared with the healthy control group, the area under the curve of serum extracellular vesicle marker hsa-let-7f-5p was 0.6651 (95% CI: 0.5378 to 0.7925, P = 0.0159), the area under the curve of hsa-miR-486-5p was 0.6479 (95% CI: 0.5187 to 0.7771, P = 0.0347), and the area under the curve of hsa-let-7b-5p was 0.6912 (95% CI: 0.5634 to 0.8189, P = 0.006). Furthermore, the combined markers formed by the three miRNAs were used to analyze the ROC curves of the cerebral infarction group and the healthy control group, showing that its AUC reached 0.7964 (95% CI: 0.6866 to 0.9062, P < 0.0001), which was greater than the AUC of a single miRNA. The results showed that the combined detection of the three miRNAs was helpful to improve the effect of identifying patients with cerebral infarction. Figure 3 shown.

[0072] Example 2

[0073] In this example, 64 ischemic stroke patients, 40 transient ischemic attack patients and 64 healthy control samples were collected according to the method described in Example 1. The 64 cerebral infarction patients, 40 transient ischemic attack patients and 64 normal control samples were tested to further verify the use effect of the kit described in the present invention.

[0074] The expression levels of hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p in the serum of 64 patients with cerebral infarction, 40 patients with transient ischemic attack and 64 normal controls were analyzed to evaluate their detection effect on ischemic stroke. The results showed that the relative expression level of hsa-let-7f-5p in serum extracellular vesicles of patients with cerebral infarction was 4.348 (3.8774-4.8186), the relative expression level of hsa-let-7f-5p in serum extracellular vesicles of patients with transient ischemic attack was 3.453 (3.1017-3.8043), and the relative expression level of hsa-let-7f-5p in serum extracellular vesicles of the healthy control group was 6.804 (6.2102-7.3978). The statistical data showed that the expression difference between the cerebral infarction group and the healthy control group was statistically significant (P=0.0004), which was significantly different; the expression difference between the transient ischemic attack group and the healthy control group was statistically significant (P<0.0001). The relative expression level of hsa-miR-486-5p in serum extracellular vesicles of patients in cerebral infarction group was 1.335 (1.1834-1.4866), the relative expression level of hsa-miR-486-5p in serum extracellular vesicles of patients in transient ischemic attack group was 1.023 (0.9048-1.1412), and the relative expression level of hsa-miR-486-5p in serum extracellular vesicles of healthy control group was 0.8792 (0.8044-0.9540). The statistical data showed that the expression difference between cerebral infarction group and healthy control group was statistically significant (P=0.004), which was statistically significant. The relative expression of hsa-let-7b-5p in serum extracellular vesicles of patients with cerebral infarction was 33.36 (33.7756-33.9444), the relative expression of hsa-let-7b-5p in serum extracellular vesicles of patients with transient ischemic attack was 31.84 (30.9751-32.7049), and the relative expression of hsa-let-7b-5p in serum extracellular vesicles of healthy control group was 30.84 (30.5147-31.1653). The statistical data showed that the expression difference between cerebral infarction group and healthy control group was statistically significant (P=0.0087). The relative expression of hsa-let-7f-5p in serum extracellular vesicles of patients with ischemic stroke was lower than that of healthy control group, while the relative expression of hsa-miR-486-5p and hsa-let-7b-5p were higher than those of healthy control group. Figure 2 .

[0075] Furthermore, we used ROC curve and area under the curve (AUC) analysis to verify the potential of hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p as molecular markers for the diagnosis of ischemic stroke. The analysis showed that compared with the healthy control group, the area under the curve of the serum extracellular vesicle marker hsa-let-7f-5p in the cerebral infarction group was 0.6888 (95% CI: 0.6080-0.7579, P < 0.001), the area under the curve of hsa-miR-486-5p was 0.6681 (95% CI: 0.5721 to 0.7640, P = 0.0012), and the area under the curve of hsa-let-7b-5p was 0.6839 (95% CI: 0.5818 to 0.7861, P = 0.0014); compared with the healthy control group, the area under the curve of the serum extracellular vesicle marker hsa-let-7f-5p in the transient ischemic attack group was 0.7627 (95% CI: 0.6712 to 0.8542, P < 0.0001) as shown in Table 5. Furthermore, the combined markers formed by the three miRNAs were used to analyze the ROC curves of the cerebral infarction group and the healthy control group, showing that its AUC reached 0.8027 (95% CI: 0.7183 to 0.8871, P < 0.0001), which was greater than the AUC of a single miRNA. The results showed that the combined detection of the three miRNAs was helpful to improve the effect of identifying patients with cerebral infarction. Figure 4 As shown. The above results show that hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p can not only distinguish patients with cerebral infarction from healthy patients, but also can be used as diagnostic biomarkers for cerebral infarction. The combination of these three markers can also be used as a distinguishing marker between patients with cerebral infarction and healthy patients, so as to screen patients with ischemic stroke early. In addition, hsa-let-7f-5p can not only distinguish patients with transient ischemic attack from healthy patients, but also can be used as a diagnostic biomarker for transient ischemic attack and ischemic stroke. The blood specimens for the test are easy to obtain, clinical operability is strong and it is a non-invasive operation. In addition, the circulating miRNA is stable and easy to detect. Therefore, hsa-let-7f-5p, hsa-miR-486-5p and hsa-let-7b-5p are worthy of promotion and clinical application as non-invasive biomarkers for ischemic stroke.

[0076] Table 5: AUC values ​​of three miRNAs

[0077]

[0078]

[0079] In summary, the present invention screened and discovered three serum extracellular vesicle markers associated with ischemic stroke, prepared a kit for diagnosing ischemic stroke based on these markers, developed a method for diagnosing ischemic stroke based on these markers, and verified the method (e.g. Figure 4 ), the results showed that the present invention provided three serum extracellular vesicle markers, among which the expression level of hsa-let-7f-5p was significantly downregulated in the serum extracellular vesicles of ischemic stroke disease, and the expression levels of hsa-miR-486-5p and hsa-let-7b-5p were significantly upregulated in the serum extracellular vesicles of ischemic stroke disease. The combined detection of the three miRNAs has good sensitivity and specificity for the diagnosis of ischemic stroke disease, which is helpful for the diagnosis of ischemic stroke disease and has potential clinical application value.

[0080] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of a reagent or kit for detecting the expression level of miRNA in a biological sample in the preparation of a product for screening or auxiliary screening, diagnosis or auxiliary diagnosis, detection or auxiliary detection of ischemic stroke, characterized in that: The miRNA is selected from a combination of one or more of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p.

2. The use according to claim 1, characterized in that: The sequence of the hsa-let-7f-5p is a single-stranded RNA as shown in SEQ ID NO.1, the sequence of the hsa-miR-486-5p is a single-stranded RNA as shown in SEQ ID NO.2, and the sequence of the hsa-let-7b-5p is a single-stranded RNA as shown in SEQ ID NO.

3.

3. The use according to claim 1, characterized in that: The product includes a system for detecting miRNA, the system includes reagents and / or instruments for detecting miRNA, and the reagents include preparations or kits.

4. The use according to claim 1, characterized in that: The stem-loop RT-qPCR technique was used to detect changes in miRNA expression levels; Preferably, the kit further comprises a stem-loop reverse transcription reagent part and a stem-loop fluorescence quantitative detection reagent part; Preferably, the stem-loop reverse transcription reagent part consists of RNase-Free ddH2 O, reverse transcriptase, dNTPs, reverse transcriptase buffer, and RT-Primer; Preferably, the stem-loop fluorescence quantitative detection reagent consists of RNase-Free ddH2O, rTaq enzyme, 10X buffer (Mg2+plus), dNTPs, and Probe TM.

5. A kit for screening or assisting screening, diagnosis or assisting diagnosis, detection or assisting detection of ischemic stroke, characterized in that: Contains a reagent for detecting the expression level of miRNA in a biological sample, wherein the miRNA is selected from a combination of one or more of hsa-let-7f-5p, hsa-miR-486-5p and / or hsa-let-7b-5p.

6. The kit according to claim 4, characterized in that The sequence of the hsa-let-7f-5p is a single-stranded RNA as shown in SEQ ID NO.1, the sequence of the hsa-miR-486-5p is a single-stranded RNA as shown in SEQ ID NO.2, and the sequence of the hsa-let-7b-5p is a single-stranded RNA as shown in SEQ ID NO.

3.

7. The kit according to claim 5, characterized in that The change of miRNA expression level is detected by using stem-loop RT-qPCR technology; preferably, the kit also includes a stem-loop reverse transcription reagent part and a stem-loop fluorescence quantitative detection reagent part; Preferably, the stem-loop reverse transcription reagent part consists of RNase-Free ddH2O, reverse transcriptase, dNTPs, reverse transcriptase buffer, and RT-Primer; Preferably, the stem-loop fluorescence quantitative detection reagent part consists of RNase-Free ddH2O, rTaq enzyme, 10X buffer (Mg2+plus), dNTPs, and Probe TM.

8. The use according to any one of claims 1 to 4, or the kit according to any one of claims 5 to 7, characterized in that: The biological sample is serum.

9. A system for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke, characterized in that: The system comprises: (1) Sample amplification module: extracting serum extracellular vesicle miRNA from the sample to be tested, performing stem-loop reverse transcription on the target miRNA using the kit described in any one of claims 1 to 7, and then performing real-time fluorescence quantitative reaction to detect the fluorescence signal. (2) Result analysis module: The absolute content of the target miRNA is calculated by making a standard curve absolute quantification method, and the absolute quantitative content of the target miRNA is compared with the absolute content of the target miRNA in the normal healthy control serum.

10. Use of a system for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke in preparing a product for screening or assisting screening, diagnosing or assisting diagnosis, detecting or assisting detection of ischemic stroke, characterized in that: The system comprises: (1) Sample amplification module: extracting serum extracellular vesicle miRNA from the sample to be tested, performing stem-loop reverse transcription on the target miRNA using the kit described in any one of claims 1 to 7, and then performing real-time fluorescence quantitative reaction to detect the fluorescence signal. (2) Result analysis module: The absolute content of the target miRNA is calculated by making a standard curve absolute quantification method, and the absolute quantitative content of the target miRNA is compared with the absolute content of the target miRNA in the normal healthy control serum.

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