Primer probe group and kit for evaluating recurrence risk or prognosis of cerebral apoplexy patient

Abstract

The invention designs a primer probe group and a kit for evaluating the recurrence risk or prognosis of a cerebral apoplexy patient, the primer probe group aims at genes including U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2 and SF3B1, and mutation sites of the genes are determined. The method has the remarkable advantages of simplicity, rapidness, high accuracy and the like, is particularly convenient for detecting a plurality of SNP loci in the same sample, and is expected to provide guidance suggestions for prognosis of stroke patients.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and particularly relates to a primer probe set and a kit for evaluating the recurrence risk of stroke patients and providing guidance and suggestions for stroke prognosis. Background Art

[0002] Stroke is a disease with complex etiology, high mortality and recurrence rates. In the past 30 years, the incidence of stroke in my country has risen sharply and has become the leading cause of death among Chinese people. Stroke includes ischemic stroke (IS) and hemorrhagic stroke, of which ischemic stroke accounts for about 65%-85% of all strokes. Acute IS patients often have sudden onset, severe condition and rapid progression, which can cause permanent and irreversible damage to the nervous system function in a short period of time, seriously affecting their prognosis and quality of life. Patients who have had IS in the past or have a history of transient ischemic attack (TIA) have a significantly increased risk of IS recurrence, and the condition after recurrence is more severe, with higher disability and mortality rates. Taking ischemic stroke as an example, the 3-month recurrence rate, disability rate, and mortality rate data are as follows:

[0003]

[0004] Clonal haematopoiesis (CH) broadly refers to the presence of a cell population derived from mutant multipotent stem / progenitor cells that has a selective growth advantage in the absence of unexplained cytopenias, hematological cancers, or other clonal disorders.

[0005] In 2023, the WHO Classification of Hematopoietic Tumors formally introduced the concept of "Clonal Haematopoiesis", which is defined as the presence of blood cell populations in the bone marrow and / or peripheral blood that are derived from mutated multipotent stem cells / progenitor cells and have a dominant proliferation capacity. This population does not have unexplained blood cell reductions, hematopoietic tumors, and other clonal diseases, and its incidence increases with age. Some scholars also call it Clonal Haematopoiesis of Indeterminate Potential (CHIP).

[0006] Research by Bhattacharya R (Clonal Hematopoiesis Is Associated With Higher Risk of Stroke. Stroke. 2022 Mar; 53(3): 788-797. doi: 10.1161 / STROKEAHA.121.037388. Epub 2021 Nov 8.) et al. showed that clonal hematopoiesis is significantly associated with a high risk of stroke, but the authors only made a general statement based on the existing data and did not conduct in-depth research. Xin Qiu (Qiu X, Dai Y, Cheng S, et al. Somatic mutation contributing to clonal haematopoiesis is a risk factor of recurrent stroke in first-ever acute ischaemic stroke: a prospective cohort study. Stroke & Vascular Neurology 2023; 8: e001756. doi: 10.1136 / svn-2022-001756) et al. based on a study of 6016 patients with first stroke, believed that somatic mutation (CHIP) increased the risk of stroke recurrence, but the authors used whole-genome sequencing (WGS) technology for detection, which has high detection cost and long process, and is not conducive to large-scale promotion and popularization.

[0007] Currently, the strongest predictors of stroke prognosis are stroke severity and patient age. The severity of stroke can be clinically judged based on the degree of neurological deficits (such as changes in consciousness, language, behavior, visual field loss, and movement disorders) and the size and location of the infarct on MRI or CT neuroimaging. Other important factors affecting stroke outcomes include ischemic stroke mechanisms, comorbidities, epidemiological factors, and stroke complications. Currently, there is still a lack of effective IS efficacy monitoring and prognostic hematological indicators in clinical practice, and genetic detection indicators are not included in the existing stroke prognosis indicators. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a primer probe set and a kit for evaluating the recurrence risk or prognosis of stroke patients. In particular, a detection kit based on gene mutations related to hematopoiesis is designed for the prognosis evaluation of stroke.

[0009] The present invention provides a primer probe set for evaluating the recurrence risk or prognosis of stroke patients, wherein the genes targeted by the primer probe set include U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2, and SF3B1 genes, and the mutation sites of the genes are determined.

[0010] Preferably, the mutation sites of the genes are: site c.470A>G of the U2AF1 gene, site c.233A>G of the GNB1 gene, site c.3088C>T of the TET2 gene, site c.2098C>T of the DNMT4A gene, site c.848G>A of the TP53 gene, site c.1849G>T of the JAK2 gene, and site c.1996A>C of the SF3B1 gene.

[0011] Preferably, the nucleotide sequences of the amplification primer pair of c.470A>G of the U2AF1 gene are shown as SEQ ID NO 1 and SEQ ID NO 2;

[0012] Preferably, the nucleotide sequence of the amplification primer pair of c.233A>G of the GNB1 gene is shown as SEQ ID NO 3 and SEQ ID NO 4;

[0013] Preferably, the nucleotide sequences of the amplification primer pair for the TET2 gene c.3088C>T are shown in SEQ ID NO 5 and SEQ ID NO 6;

[0014] Preferably, the nucleotide sequences of the amplification primer pair for the DNMT4A gene c.2098C>T are shown in SEQ ID NO 7 and SEQ ID NO 8;

[0015] Preferably, the nucleotide sequence of the amplification primer pair for the TP53 gene c.848G>A is shown in SEQ ID NO 9 and SEQ ID NO 10;

[0016] Preferably, the nucleotide sequences of the amplification primer pair for the JAK2 gene c.1849G>T are shown in SEQ ID NO 11 and SEQ ID NO 12;

[0017] Preferably, the nucleotide sequence of the amplification primer pair for the SF3B1 gene c.1996A>C is shown as SEQ ID NO 13 and SEQ ID NO 14.

[0018] Preferably, the nucleotide sequence of the c.470A>G mutant probe of the U2AF1 gene is as shown in SEQ ID NO 15, and the nucleotide sequence of the wild-type probe is as shown in SEQ ID NO 16;

[0019] Preferably, the nucleotide sequence of the c.233A>G mutant probe of the GNB1 gene is as shown in SEQ ID NO 17, and the nucleotide sequence of the wild-type probe is as shown in SEQ ID NO 18;

[0020] Preferably, the nucleotide sequence of the mutant probe of the TET2 gene c.3088C>T is shown in SEQ ID NO 19, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 20;

[0021] Preferably, the nucleotide sequence of the mutant probe of the DNMT4A gene c.2098C>T is shown in SEQ ID NO 21, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 22;

[0022] Preferably, the nucleotide sequence of the mutant probe of the TP53 gene c.848G>A is shown in SEQ ID NO 23, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 24;

[0023] Preferably, the nucleotide sequence of the mutant probe of the JAK2 gene c.1849G>T is shown in SEQ ID NO 25, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 26;

[0024] Preferably, the nucleotide sequence of the mutant probe of the SF3B1 gene c.1996A>C is shown as SEQ ID NO 27, and the nucleotide sequence of the wild-type probe is shown as SEQ ID NO 28.

[0025] Preferably, the 5' end of the probe nucleotide sequence is labeled with FAM or VIC, and the 3' end is labeled with MGB.

[0026] The present invention also provides a kit for evaluating the recurrence risk or prognosis of stroke patients, wherein the kit comprises any one of the primer probe sets.

[0027] More preferably, the 5' end of the mutant probe nucleotide sequence is labeled with FAM; and the 5' end of the wild-type probe nucleotide sequence is labeled with VIC.

[0028] Preferably, the kit further comprises PCR amplification reagents, positive quality control products and negative quality control products.

[0029] Preferably, the positive quality control product includes a first positive quality control product, a second positive quality control product and a third positive quality control product; wherein the first positive quality control product is a site homozygous wild-type plasmid mixture; the second positive quality control product is a site heterozygous wild-type and mutant plasmid mixture; the third positive quality control product is a site homozygous mutant plasmid mixture; and the negative quality control product is water.

[0030] The present invention also provides a method for detecting SNP sites, comprising: (1) extracting human genomic DNA of a sample to be tested; (2) performing a real-time fluorescent PCR amplification reaction; and (3) determining the results.

[0031] The specific method is as follows:

[0032] (1) Extracting DNA from peripheral blood samples, and using a nucleic acid extraction kit to extract human genomic DNA from the sample to be tested;

[0033] (2) Real-time fluorescence PCR amplification reaction: 4 μl of the primer-probe combination, 12.5 μl of the PCR reaction enzyme system, 2 μl of template DNA, and sterile deionized water to make up to 25 μl. The template is the sample DNA in (1) as the template for the sample to be tested; the purified water is the template for the negative control; the homozygous wild-type nucleic acid fragment, the heterozygous mutant nucleic acid fragment, and the homozygous mutant nucleic acid fragment are respectively used as templates for the positive control. The negative and positive controls are subjected to PCR amplification reaction with the sample to be tested according to the same reaction process to verify the effectiveness of the test.

[0034] (3) Real-time fluorescence PCR amplification: according to ① 60℃×1min, 95℃×10min (1cycle); ② 95℃×15sec, 60℃×30sec (50cycles, fluorescence collection at 60℃, FAM and VIC selected as fluorescence channels);

[0035] (4) Result judgment: Preferably, an instrument such as ABI 7500 is used to perform the amplification reaction and read the test results. The test results of the sample are interpreted according to the following interpretation criteria: the wild type is a red figure on the allele typing scatter plot (close to the X axis), the heterozygous mutant type is a green figure on the allele typing scatter plot (close to the diagonal line), and the homozygous mutant type is a blue figure on the allele typing scatter plot (close to the Y axis).

[0036] The present invention provides an application of a biomarker in preparing a reagent for evaluating the recurrence risk or prognosis of a stroke patient, characterized in that the biomarker comprises one or more of c.470A>G of U2AF1 gene, c.233A>G of GNB1 gene, c.3088C>T of TET2 gene, c.2098C>T of DNMT4A gene, c.848G>A of TP53 gene, c.1849G>T of JAK2 gene, and c.1996A>C of SF3B1 gene.

[0037] The present invention provides an application of any of the primer probe sets or any of the kits in preparing a method for evaluating the recurrence risk or prognosis of stroke patients.

[0038] The principle of the present invention is as follows:

[0039] In the first aspect, the present invention provides SNP sites of stroke-related genes. The gene sites are: c.470A>G site of U2AF1 gene, c.233A>G site of GNB1 gene, c.3088C>T site of TET2 gene, c.2098C>T site of DNMT4A gene, c.848G>A site of TP53 gene, c.1849G>T site of JAK2 gene, c.1996A>C site of SF3B1 gene, and the corresponding relationship between the gene detection sites and rs numbers is as follows:

[0040]

[0041]

[0042] In a second aspect, the present invention provides a primer and probe combination for detecting the above target. According to the selected stroke-related susceptibility gene sequence, upstream and downstream primers are designed to specifically amplify each site, and the positions thereof are selected to be about 20-100 bases before and after the mutation site, but not on the DNA sequence containing the mutation site. The upstream primer has a start codon at the 3' end and at least 15 bases that completely match the template sequence, and the downstream primer includes a stop codon and at least 15 reverse complementary sequences of the template sequence;

[0043] Select 3-25 bases before / after the mutation site, but not overlapping with the upstream and downstream primers. The probe length is between 13-30bp, the GC content is controlled between 40%-60%, avoid the appearance of continuous identical fragments, and the Tm value is between 65-70℃. The 5' end of the wild-type and mutant target probes of the same site are labeled with two different fluorescent groups. The mutant probe uses the FAM group to insert the fluorescent signal, and the wild-type probe uses the VIC group as the inserted fluorescent signal. Add MGB quenching fluorescent group at the 3' end of the probe;

[0044] In a third aspect, the present invention provides a kit containing a primer and probe combination for detecting SNP sites related to stroke prognosis. The kit can specifically, quickly and accurately detect multiple stroke-related susceptibility gene polymorphism sites, providing a clinical auxiliary diagnosis basis for stroke prognosis.

[0045] The kit comprises a first detection group and a second detection group; the first detection group comprises the primer-probe combination group described above in the present invention; and the second detection group comprises a PCR reaction solution enzyme system.

[0046] Preferably, the second detection group PCR reaction solution includes a buffer, Mg 2+ and dNTPs and hot-start Taq enzyme system;

[0047] The kit also includes a positive quality control and a negative quality control. The positive quality control includes positive quality control 1, positive quality control 2 and positive quality control 3; the positive quality control 1 is a mixture of homozygous wild-type plasmids at all sites, the positive quality control 2 is a mixture of heterozygous mutant plasmids at all sites, and the positive quality control 3 is a mixture of homozygous mutant plasmids at all sites. Preferably, the negative quality control is purified water.

[0048] In a fourth aspect, the present invention further provides a method for detecting a SNP site gene mutation kit related to stroke prognosis, the method comprising the following steps:

[0049] (1) Extracting DNA from peripheral blood samples, and using a nucleic acid extraction kit to extract human genomic DNA from the sample to be tested;

[0050] (2) Real-time fluorescence PCR amplification reaction: 4 μl of the primer-probe combination, 12.5 μl of the PCR reaction enzyme system, 2 μl of template DNA, and sterile deionized water to make up to 25 μl. The template is the sample DNA in (1) as the template for the sample to be tested; the purified water is the template for the negative control; the homozygous wild-type nucleic acid fragment, the heterozygous mutant nucleic acid fragment, and the homozygous mutant nucleic acid fragment are respectively used as templates for the positive control. The negative and positive controls are subjected to PCR amplification reaction with the sample to be tested according to the same reaction process to verify the effectiveness of the test.

[0051] (3) Real-time fluorescence PCR amplification: according to ① 60℃×1min, 95℃×10min (1cycle); ② 95℃×15sec, 60℃×30sec (50cycles, fluorescence collection at 60℃, FAM and VIC selected as fluorescence channels);

[0052] (4) Result judgment: Preferably, an instrument such as ABI 7500 is used to perform the amplification reaction and read the test results. The test results of the sample are interpreted according to the following interpretation criteria: the wild type is a red figure on the allele typing scatter plot (close to the X axis), the heterozygous mutant type is a green figure on the allele typing scatter plot (close to the diagonal line), and the homozygous mutant type is a blue figure on the allele typing scatter plot (close to the Y axis).

[0053] The invention discloses the SNP sites corresponding to stroke-related genes U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2, and SF3B1: c.470A>G, c.233A>G, c.3088C>T, c.2098C>T, c.848G>A, c.1849G>T, c.1996A>C, and primers and probe combinations for allele typing detection of the SNP sites, belonging to the field of gene detection technology. The method mainly adopts the TaqMan-MGB probe method to design a specific MGB probe for the allele of the SNP site. The wild-type and mutant target probes for the same site are labeled with two different fluorescent groups, and correspond to a set of universal upstream and downstream sequences, and typing detection is performed in the same tube. During real-time fluorescence quantitative PCR amplification, the product is scanned for fluorescence signals at the end point, and different fluorescence signals are classified by frequency and signal strength, so as to analyze the SNP target corresponding to the site. The detection using the primer and probe combination of the present invention has the advantages of simplicity, rapidity, high accuracy, etc. It is particularly convenient to detect multiple SNP sites in the same sample, providing evidence support for the recurrence risk or prognosis assessment of stroke patients.

[0054] Beneficial Effects

[0055] The present invention provides a combined detection kit based on clonal hematopoiesis-related gene mutations. The kit adopts Taqman-MGB probe and has the advantages of high sensitivity, good specificity, easy operation and popularization, and can be used for recurrence risk or prognosis assessment of stroke patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the PCR typing result of the rs371246226 site PCR reaction tube quality control product and the sample to be tested using ABI 7500 according to the embodiment of the present invention;

[0057] Figure 2 It is the PCR typing result of the rs869312823 site PCR reaction tube quality control product and the sample to be tested using ABI 7500 according to the embodiment of the present invention;

[0058] Figure 3It is the PCR typing result of the rs780043982 site PCR reaction tube quality control product and the sample to be tested using ABI 7500 according to the embodiment of the present invention.

[0059] Figure 4 It is a schematic diagram comparing the detection results of samples of different concentrations detected by the detection kit of the present invention with the sequencing results. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0061] Example 1

[0062] Stroke-related genes and polymorphic loci

[0063] After extensive and in-depth research, the inventors have developed a stroke-related gene analysis and detection method based on a real-time fluorescence PCR platform. The sample type is automatically categorized according to the difference in the detected fluorescence value, thereby detecting the c.470A>G site polymorphism of the U2AF1 gene, c.233A>G of the GNB1 gene, c.3088C>T of the TET2 gene, c.2098C>T of the DNMT4A gene, c.848G>A of the TP53 gene, c.1849G>T of the JAK2 gene, and c.1996A>C site polymorphism of the SF3B1 gene in human peripheral whole blood samples.

[0064] The correspondence between the gene detection sites and RS numbers is shown in Table 1:

[0065] Table 1

[0066] Gene Location rs number U2AF1 c.470A>G rs371246226 GNB1 c.233A>G rs869312823 TET2 c.3088C>T rs780043982 DNMT4A c.2098C>T rs775823075 TP53 c.848G>A rs371409680 JAK2 c.1849G>T rs77375493 SF3B1 c.1996A>C rs754688962

[0067] Example 2

[0068] Primers and probes for detecting stroke-related genes and polymorphic sites

[0069] In this example, the primer nucleotide sequences used in each amplification site can be found in Table 2:

[0070] Table 2

[0071]

[0072]

[0073] In this embodiment, the mutant probe nucleotide sequence used in each amplification site is labeled with FAM at the 5' end; the wild-type probe nucleotide sequence is labeled with VIC at the 5' end; and the 3' end is labeled with MGB. The probe nucleotide sequence can be seen in Table 3:

[0074] Table 3

[0075]

[0076] Example 3

[0077] One aspect of the present invention relates to a primer-probe mixture and a PCR kit for polymorphism detection of human stroke-related gene typing. The basic composition of the kit is shown in Table 4:

[0078] Table 4

[0079]

[0080]

[0081] How to use the kit:

[0082] 1. Detection method

[0083] 1. Preparation before PCR system preparation:

[0084] Take out the reagent kit from the refrigerator, equilibrate to room temperature, fully dissolve all components, vortex to mix, and centrifuge for 10 seconds to prepare the PCR system.

[0085] Table 5 is the PCR reaction amplification system:

[0086] Reagents concentration Volume / reaction (μL) Upstream primer 10uM 1.5 Downstream primer 10uM 1.5 Mutant MGB probe 10uM 0.75 Wild-type MGB probe 10uM 0.75 PCRMasterMix / 12.5 ddH2O / 6 gDNA / 2 total / 25

[0087] 2. Extraction of DNA from stroke samples:

[0088] In this embodiment, the steps of extracting DNA from stroke samples are:

[0089] Step 1. Take 500ul of blood sample and add 2 times volume (1000ul) of Buffer TBP, mix thoroughly, and place at room temperature for 1min until the red blood cells are completely lysed. Centrifuge at 8000rpm for 1min and discard the supernatant. Resuspend the precipitate with 500ulTE Buffer (enzyme-free water), centrifuge at 8000rpm for 1min, discard the supernatant, and wash once more with TE Buffer (enzyme-free water) until the precipitate is white, and then add 200ul PBS solution.

[0090] Step 2: Add 30ul Proteinase K and mix well. Then add 200ul Buffer DL, shake and mix well, and incubate in a 56° water bath for 20 minutes.

[0091] (solution clear)

[0092] Step 3: Add 200ul of anhydrous ethanol and mix thoroughly by inversion.

[0093] Step 4: Place the adsorption column into the collection tube, add all the solution in the centrifuge tube into the adsorption column, let it stand for 2 minutes, then centrifuge at 10,000 rpm at room temperature for 1 minute, and discard the waste liquid.

[0094] Step 5. Place the adsorption column into the collection tube, add 500ul GW Solution to the adsorption column, centrifuge at 10000rpm for 30s and discard the waste liquid.

[0095] Step 6. Place the adsorption column into the collection tube, add 700ul Wash Solution to the adsorption column, centrifuge at 10000rpm for 30s and discard the waste liquid.

[0096] Step 7. Repeat step 6 once.

[0097] Step 8: Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes at room temperature to remove the remaining Wash Solution.

[0098] Step 9. Take out the adsorption column, put it into a new centrifuge tube, add 50ul CE Buffer and let it stand for 3min, centrifuge at 12000rpm for 2min at room temperature, collect the DNA solution and store at -20°.

[0099] The DNA extracted from the sample was used as a template for fluorescent PCR amplification, and the PCR reaction amplification system was configured. Positive quality control products and negative quality control products were also set.

[0100] 3. PCR amplification

[0101] ABIPrism 7500 instrument settings

[0102] (1) Open the "Set up" window, set the negative control, positive control allel1 / allel1, allel1 / allel2, allel2 / allel2, and unknown sample in the order of the samples, and set the sample name in the "sample name" column. Set the probe detection mode to: FAM / VIC.

[0103] (2) Open the RUN Method window and set the cycle conditions as shown in Table 6:

[0104] Table 6

[0105]

[0106] Pre-denaturation: one cycle, 95°C, 10 min;

[0107] Cyclic reaction: 50 cycles, 95°C, 10 sec, 60°C, 30 sec;

[0108] End point signal acquisition: 1 cycle, 60°C, 30 sec.

[0109] The final detection results of the fluorescence signals of the two groups FAM and VIC are as follows Figure 1-3 shown.

[0110] 2. Interpretation of results

[0111] The wild type is the red figure on the allele typing scatter plot (close to the X axis), the heterozygous mutant is the green figure on the allele typing scatter plot (close to the diagonal), and the homozygous mutant is the blue figure on the allele typing scatter plot (close to the Y axis). If none of the three are displayed, the sample loading is insufficient or missed, and the sample needs to be reloaded.

[0112] Example 4

[0113] In this study, peripheral blood samples were collected from 200 patients with first acute ischemic stroke (AIS), whose diagnosis of AIS was confirmed by brain imaging, and 200 healthy patients.

[0114] The peripheral blood was obtained, DNA was extracted, and the samples were tested using the kit of this patent. The data are as follows:

[0115]

[0116] The experimental results showed that the positive rate in the stroke group was significantly higher than that in the healthy group, indicating that CHIP is a significant marker for stroke patients.

[0117] For the stroke group, prospective follow-up was performed, and the primary outcome was recurrent stroke at 3 months, defined as a new ischemic or hemorrhagic stroke occurring at 3 months of follow-up. The data during the follow-up period were as follows:

[0118]

[0119] The experimental results showed that the recurrence rate of CHIP-positive patients was significantly higher than that of negative patients, indicating that CHIP is a good prognostic marker for stroke patients.

[0120] Example 5

[0121] Comparative experiment: Different concentrations of mutant plasmids and wild-type sample nucleic acids were mixed to prepare simulated heterozygous samples with mutation abundances of 40%, 20%, 10%, 1%, and 0%, respectively. The kit of the present invention was used for detection, and the results were displayed by amplification curves (such as Figure 4 A); and at the same time, the above simulated heterozygous samples were sequenced by SBT gold standard, and the sequencing results were displayed by peak diagram (such as Figure 4 B), in order to compare the experimental results of the present invention.

[0122] The SBT sequencing results were compared with the results of the detection method of the present invention (see Figure 4 ), it was found that the detection sensitivity of the kit of the present invention was 1% mutation abundance, while the sequencing result only detected 40% mutation abundance, thus indicating that the kit of the present invention has higher sensitivity than the gold standard sequencing and can detect 1% mutation abundance.

[0123] The embodiments of the present invention are only used to illustrate the present invention and do not constitute a limitation on the scope of the claims. Other substantially equivalent substitutes that can be thought of by those skilled in the art are all within the protection scope of the present invention.

Claims

1. A primer probe set for evaluating the recurrence risk or prognosis of stroke patients, characterized in that: The genes targeted by the primer probe set include U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2, and SF3B1 genes, and the mutation sites of the genes are determined.

2. The primer probe set according to claim 1, characterized in that: The mutation sites of the genes are: site c.470A>G of the U2AF1 gene, site c.233A>G of the GNB1 gene, site c.3088C>T of the TET2 gene, site c.2098C>T of the DNMT4A gene, site c.848G>A of the TP53 gene, site c.1849G>T of the JAK2 gene, and site c.1996A>C of the SF3B1 gene.

3. The primer probe set according to claim 2, characterized in that: The nucleotide sequence of the amplification primer pair of c.470A>G of the U2AF1 gene is shown in SEQ ID NO 1 and SEQ ID NO 2; the nucleotide sequence of the amplification primer pair of c.233A>G of the GNB1 gene is shown in SEQ ID NO 3 and SEQ ID NO 4; the nucleotide sequence of the amplification primer pair of c.3088C>T of the TET2 gene is shown in SEQ ID NO 5 and SEQ ID NO 6; the nucleotide sequence of the amplification primer pair of c.2098C>T of the DNMT4A gene is shown in SEQ ID NO 7 and SEQ ID NO 8; the nucleotide sequence of the amplification primer pair of c.848G>A of the TP53 gene is shown in SEQ ID NO 9 and SEQ ID NO 10; the nucleotide sequence of the amplification primer pair of c.1849G>T of the JAK2 gene is shown in SEQ ID NO 11 and SEQ ID NO 12; the nucleotide sequence of the amplification primer pair of c.1996A>C of the SF3B1 gene is shown in SEQ ID NO 13. NO 13 and SEQ ID NO 14.

4. The primer probe set according to claim 2, characterized in that: The nucleotide sequence of the mutant probe of the U2AF1 gene c.470A>G is shown in SEQ ID NO 15, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 16; the nucleotide sequence of the mutant probe of the GNB1 gene c.233A>G is shown in SEQ ID NO 17, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 18; the nucleotide sequence of the mutant probe of the TET2 gene c.3088C>T is shown in SEQ ID NO 19, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 20; the nucleotide sequence of the mutant probe of the DNMT4A gene c.2098C>T is shown in SEQ ID NO 21, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 22; the nucleotide sequence of the mutant probe of the TP53 gene c.848G>A is shown in SEQ ID NO 23, and the nucleotide sequence of the wild-type probe is shown in SEQ ID NO 24; the nucleotide sequence of the mutant probe of the JAK2 gene c.1849G>T is shown in SEQ ID NO NO25, the nucleotide sequence of the wild-type probe is shown as SEQ ID NO 26; the nucleotide sequence of the mutant probe of the SF3B1 gene c.1996A>C is shown as SEQ ID NO 27, and the nucleotide sequence of the wild-type probe is shown as SEQ ID NO 28.

5. The primer probe set according to claim 4, characterized in that: The 5' end of the probe nucleotide sequence is labeled with FAM or VIC, and the 3' end is labeled with MGB.

6. A kit for evaluating the recurrence risk or prognosis of stroke patients, characterized in that: The kit comprises the primer-probe set according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit also includes PCR amplification reagents, positive quality control products and negative quality control products.

8. The kit according to claim 7, characterized in that: The positive quality control product includes a first positive quality control product, a second positive quality control product and a third positive quality control product; wherein the first positive quality control product is a site homozygous wild-type plasmid mixture; the second positive quality control product is a site heterozygous wild-type and mutant plasmid mixture; the third positive quality control product is a site homozygous mutant plasmid mixture; and the negative quality control product is water.

9. Use of a biomarker in a reagent for assessing the risk of recurrence or prognosis of stroke patients, characterized in that: The biomarkers include one or more of c.470A>G of U2AF1 gene, c.233A>G of GNB1 gene, c.3088C>T of TET2 gene, c.2098C>T of DNMT4A gene, c.848G>A of TP53 gene, c.1849G>T of JAK2 gene, and c.1996A>C of SF3B1 gene.

10. A primer probe set according to any one of claims 1 to 5 or a kit according to any one of claims 6 to 8, for use in assessing the risk of recurrence in stroke patients and providing guidance and suggestions for their prognosis.

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