A primer-probe set and kit for evaluating the recurrence risk or prognosis of stroke patients

Abstract

The present invention designs a primer-probe set and a kit for assessing the recurrence risk or prognosis of stroke patients. The genes targeted by the primer-probe set include U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2, and SF3B1 genes, and the mutation sites of these genes are determined. The present invention has significant advantages such as simplicity, rapidity, and high accuracy, and is particularly convenient for detecting multiple SNP sites in the same sample, and is expected to provide guiding suggestions for the 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 guiding suggestions for stroke prognosis. Background Art

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

[0003]

[0004] Clonal haematopoiesis (CH) generally refers to the presence of cell populations derived from mutated multipotent / progenitor cells that have a selective growth advantage in the absence of unexplained cytopenia, blood cancer or other clonal diseases.

[0005] In 2023, the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues officially introduced the concept of "clonal haematopoiesis", which is defined as the presence of a blood cell population in the bone marrow and / or peripheral blood derived from mutated multipotent stem cells / progenitor cells with the ability of preferential proliferation. This population has no unexplained cytopenia, hematological malignancies and other clonal diseases, and its incidence increases with age. Some scholars also call it clonal haematopoiesis of indeterminate potential (CHIP).

[0006] Studies 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. have shown that clonal hematopoiesis is significantly associated with a high risk of stroke. However, the authors only made a general statement based on 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. believed that the increase in somatic mutations (CHIP) increased the risk of stroke recurrence based on a study of 6,016 first-ever stroke patients. However, the authors used Whole-genome sequencing (WGS) technology for detection, which has a high detection cost and a long process, making it not conducive to large-scale promotion and popularization.

[0007] The strongest predictors of stroke prognosis currently are stroke severity and patient age. The severity of stroke can be clinically judged based on the degree of neurological deficit (such as changes in consciousness, language, behavior, visual field defects, and motor disorders) and the size and location of infarcts on MRI or CT neuroimaging. Other important factors affecting stroke outcomes include the mechanism of ischemic stroke, co-existing diseases, epidemiological factors, and stroke complications. Currently, there is still a lack of effective hematological indicators for monitoring the efficacy and judging the prognosis of IS in clinical practice, and genetic testing 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 mutations in genes related to clonal hematopoiesis is designed for the prognosis evaluation of stroke.

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

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

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

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

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

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

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

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

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

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

[0019] Preferably, the nucleotide sequence of the mutant probe for the c.233A>G mutation 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 for the c.3088C>T mutation of the TET2 gene is as shown in SEQ ID NO 19, and the nucleotide sequence of the wild-type probe is as shown in SEQ ID NO 20;

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

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

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

[0024] Preferably, the nucleotide sequence of the mutant probe for the c.1996A>C mutation of the SF3B1 gene is as shown in SEQ ID NO 27, and the nucleotide sequence of the wild-type probe is as shown in 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, and the kit includes any one of the primer-probe sets.

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

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

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

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

[0031] The specific method is as follows:

[0032] (1) Extract DNA from a peripheral blood sample, and use a nucleic acid extraction kit to extract genomic DNA of the sample to be tested from a human.

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

[0034] (3) Amplification by a real-time fluorescence PCR instrument: according to ① 60 °C × 1 min, 95 °C × 10 min (1 cycle); ② 95 °C × 15 Sec, 60 °C × 30 Sec (50 cycles, fluorescence is collected at 60 °C, and the fluorescence channel is selected as FAM, VIC).

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

[0036] The present invention provides an application of a biomarker in the preparation of a reagent for assessing the recurrence risk or prognosis of stroke patients, characterized in that the biomarker comprises one or more of c.470A>G 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 of the SF3B1 gene.

[0037] The present invention provides an application of any one of the primer-probe sets or any one of the reagent kits in the preparation of an assessment of 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: the c.470A>G site of the U2AF1 gene, the c.233A>G site of the GNB1 gene, the c.3088C>T site of the TET2 gene, the c.2098C>T site of the DNMT4A gene, the c.848G>A site of the TP53 gene, the c.1849G>T site of the JAK2 gene, and the c.1996A>C site of the SF3B1 gene. The corresponding relationship between the gene detection sites and rs numbers is shown as follows:

[0040]

[0041]

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

[0043] Select a position 3-25 bases before / after the mutation site, but not overlapping with the upstream and downstream primers. The probe length is between 13-30 bp, the GC content is controlled between 40%-60%, the appearance of continuous identical fragments is avoided, the Tm value is between 65-70°C, and the 5' ends of the wild-type and mutant target probes at the same site are labeled with 2 different fluorescent groups. The mutant probe selects the FAM group to insert the fluorescent signal, and the wild-type probe selects the VIC group as the inserted fluorescent signal. An MGB quenching fluorescent group is added to the 3' end of the probe;

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

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

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

[0047] The kit further includes a positive control product and a negative control product. The positive control product includes positive control product 1, positive control product 2 and positive control product 3; positive control product 1 is a mixed solution of plasmids homozygous for all sites in the wild type, positive control product 2 is a mixed solution of plasmids heterozygous for all sites in the mutant type, and positive control product 3 is a mixed solution of plasmids homozygous for all sites in the mutant type. Preferably, the negative control product is purified water.

[0048] In a fourth aspect, the present invention also provides a detection method for a SNP site gene mutation kit related to stroke prognosis. The method includes the following steps:

[0049] (1) Extract the DNA of the peripheral blood sample, and use a nucleic acid extraction kit to extract the genomic DNA of the sample to be tested.

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

[0051] (3) Amplification by a real-time fluorescence PCR instrument: according to ① 60 °C × 1 min, 95 °C × 10 min (1 cycle); ② 95 °C × 15 Sec, 60 °C × 30 Sec (50 cycles, fluorescence is collected at 60 °C, and the fluorescence channel is selected as FAM, VIC);

[0052] (4) Result judgment: Preferably, instruments such as ABI 7500 are used to perform the amplification reaction and read the test results. The test results of the samples are judged according to the following judgment criteria: The wild type is the red graph (close to the X-axis) on the allelic typing scatter plot, the heterozygous mutant type is the green graph (close to the diagonal) on the allelic typing scatter plot, and the homozygous mutant type is the blue graph (close to the Y-axis) on the allelic typing scatter plot.

[0053] The present invention discloses SNP sites corresponding to stroke-related genes U2AF1, GNB1, TET2, DNMT4A, TP53, JAK2, SF3B1: c.470A>G, c.233A>G, c.3088C>T, c.2098C>T, c.848G>A, c.1849G>T, c.1996A>C, and primer and probe combinations for allelic typing detection of the SNP sites, belonging to the technical field of gene detection. This method mainly uses the TaqMan-MGB probe method to design specific MGB probes for the alleles of the SNP sites. The wild-type and mutant target probes for the same site are labeled with two different fluorescent groups, and correspond to a set of common upstream and downstream sequences, and typing detection is carried out in the same tube. During real-time fluorescence quantitative PCR amplification, the fluorescence signal of the product is scanned at the end point, and the different fluorescence signals are classified according to the frequency and signal strength, so as to analyze the SNP target corresponding to this site. Using the primer and probe combinations of the present invention for detection has the advantages of simplicity, rapidity, high accuracy, etc., and is particularly convenient for detecting multiple SNP sites for 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 mutations of genes related to clonal hematopoiesis. The kit uses Taqman-MGB probes and has the advantages of high sensitivity, good specificity, easy operation and popularization and use, etc., and can be used for the recurrence risk or prognosis assessment of stroke patients. Description of the Drawings

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

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

[0058] Figure 3It is the PCR genotyping results of the rs780043982 site PCR reaction tube quality control product and the sample to be tested using ABI 7500 in the embodiments of the present invention.

[0059] Figure 4 It is a schematic diagram comparing the results of detecting samples with different concentrations by the detection kit of the present invention with the sequencing results. Detailed implementation manners

[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 not 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 also fall within the scope defined by the appended claims of this application.

[0061] Example 1

[0062] Stroke-related genes and polymorphic sites

[0063] Through extensive and in-depth research, the present inventor has developed a method for analyzing and detecting stroke-related genes based on a real-time fluorescence PCR platform. According to the differences in the detected fluorescence values, the sample types are automatically scatter-typed, so as to detect the c.470A>G site of the U2AF1 gene, the c.233A>G of the GNB1 gene, the c.3088C>T of the TET2 gene, the c.2098C>T of the DNMT4A gene, the c.848G>A of the TP53 gene, the c.1849G>T of the JAK2 gene, and the c.1996A>C site polymorphisms in human peripheral whole blood samples.

[0064] The corresponding relationship between the gene detection sites and rs numbers is shown in Table 1:

[0065] Table 1

[0066] Gene Locus 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 embodiment, the primer nucleotide sequences used for each amplification site can be seen in Table 2:

[0070] Table 2

[0071]

[0072]

[0073] In this embodiment, the 5' ends of the mutant probe nucleotide sequences used for each amplification site are labeled with FAM; the 5' ends of the wild-type probe nucleotide sequences are labeled with VIC; and the 3' ends are all labeled with MGB. The probe nucleotide sequences can be seen in Table 3 as follows:

[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 as follows:

[0078] Table 4

[0079]

[0080]

[0081] Usage method of the kit:

[0082] I. Detection method

[0083] 1. Preparation before PCR system preparation:

[0084] Take out the kit from the refrigerator, balance it to room temperature, fully dissolve each component, vortex and mix well, then centrifuge for 10 seconds to prepare the PCR system.

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

[0086] Reagent Concentration Volume per reaction (μL) Forward primer 10uM 1.5 Reverse primer 10uM 1.5 Mutant MGB probe 10uM 0.75 Wild-type MGB probe 10uM 0.75 PCR Master Mix / 12.5 ddH2O / 6 gDNA / 2 Total / 25

[0087] 2. Extract DNA from stroke samples:

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

[0089] Step 1. Add 500 μl of blood sample to 2-fold volume (1000 μl) of Buffer TBP, mix well, and let it stand at room temperature for 1 minute until the red blood cells are completely lysed. Centrifuge at 8000 rpm for 1 minute and discard the supernatant. Resuspend the precipitate with 500 μl of TE Buffer (enzyme-free water), centrifuge at 8000 rpm for 1 minute, discard the supernatant, and it can be washed once more with TE Buffer (enzyme-free water) until the precipitate is white, then add 200 μl of PBS solution.

[0090] Step 2. Add 30 μl of Proteinase K and mix well. Then add 200 μl of Buffer DL, mix by oscillation, and incubate in a water bath at 56 °C for 20 minutes.

[0091] (The solution is clear)

[0092] Step 3: Add 200 ul of absolute ethanol and invert thoroughly to mix well.

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

[0094] Step 5: Place the adsorption column into the collection tube, add 500 ul of GW Solution to the adsorption column, centrifuge at 10,000 rpm for 30 s, and discard the waste liquid.

[0095] Step 6: Place the adsorption column into the collection tube, add 700 ul of Wash Solution to the adsorption column, centrifuge at 10,000 rpm for 30 s, and discard the waste liquid.

[0096] Step 7: Repeat Step 6 once.

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

[0098] Step 9: Take out the adsorption column, place it into a new centrifuge tube, add 50 ul of CE Buffer, let it stand for 3 min, centrifuge at 12,000 rpm at room temperature for 2 min, and collect the DNA solution. Store at -20 °C.

[0099] Use the DNA extracted from the sample as a template for fluorescence PCR amplification and configure the PCR reaction amplification system. At the same time, set up positive and negative control products.

[0100] 3. PCR Amplification

[0101] ABIPrism 7500 Instrument Settings

[0102] (1) Open the "Set up" window, set the negative control, positive control (positive control allel1 / allel1, allel1 / allel2, allel2 / allel2), and the sample to be tested (unknown) in the corresponding 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 cycling 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: One cycle, 60°C, 30 sec.

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

[0110] II. Result interpretation

[0111] The wild type is the red graph (close to the X-axis) on the allele typing scatter plot, the heterozygous mutant is the green graph (close to the diagonal) on the allele typing scatter plot, and the homozygous mutant is the blue graph (close to the Y-axis) on the allele typing scatter plot. If none of them are shown, the sample loading amount is insufficient or omitted, and reloading and running on the machine are required.

[0112] Example 4

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

[0114] Take the obtained peripheral blood, extract DNA, and use the kit of this patent to detect the samples. The data are as follows:

[0115]

[0116] The experimental results show that the positive rate of the stroke group is significantly higher than that of the healthy group. It can be seen that CHIP is a significant marker for stroke patients

[0117] For the stroke group, a prospective follow-up was conducted. The main outcome set was stroke recurrence at 3 months, defined as occurring at 3 months follow-up after the onset of new ischemic or hemorrhagic stroke. The data during the follow-up are as follows:

[0118]

[0119] The experimental results show that the recurrence rate of CHIP-positive patients is significantly higher than that of negative patients. It can be seen that CHIP is a good prognostic marker for stroke patients.

[0120] Example 5

[0121] Comparative experiment: Different concentrations of mutant plasmids were mixed with wild-type sample nucleic acids to prepare simulated heterozygous samples with mutant abundances of 40%, 20%, 10%, 1%, and 0%, respectively. The kit of the present invention was used for detection, and the results were shown by amplification curves (as shown in Figure 4 A); at the same time, the above simulated heterozygous samples were subjected to SBT gold standard sequencing, and the sequencing results were shown by peak diagrams (as shown in Figure 4 B) to compare with 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% mutant abundance, while the sequencing results only detected 40% mutant abundance. Therefore, it was shown that the kit of the present invention had higher sensitivity than the gold standard sequencing and could detect 1% mutant abundance.

[0123] The examples in the present invention are only used to illustrate the present invention and do not constitute a limitation to the scope of the claims. Other substantially equivalent alternatives that can be conceived by those skilled in the art are 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 consist of U2AF1, GNB1, TET2, DNMT3A, TP53, JAK2 and SF3B1 genes, and the mutation sites of the genes are determined; The mutation sites of the genes are: site c.470A>G of U2AF1 gene, site c.233A>G of GNB1 gene, site c.3088C>T of TET2 gene, site c.2098C>T of DNMT3A gene, site c.848G>A of TP53 gene, site c.1849G>T of JAK2 gene, site c.1996A>C of SF3B1 gene; 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 DNMT3A 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 and SEQ ID NO 14; 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 DNMT3A 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 25, 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.

2. The primer probe set according to claim 1, 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.

3. 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 1-2.

4. The kit according to claim 3, characterized in that: The kit also includes PCR amplification reagents, positive quality control products and negative quality control products.

5. The kit according to claim 4, 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.

6. Use of a reagent for detecting a biomarker in the preparation of a reagent for assessing the risk of recurrence or prognosis of a stroke patient, characterized in that: The biomarkers consist of c.470A>G of U2AF1 gene, c.233A>G of GNB1 gene, c.3088C>T of TET2 gene, c.2098C>T of DNMT3A gene, c.848G>A of TP53 gene, c.1849G>T of JAK2 gene, and c.1996A>C of SF3B1 gene.

7. A primer probe set according to any one of claims 1-2 or a kit according to any one of claims 3-5, for evaluating the risk of recurrence in stroke patients and providing guidance and suggestions for their prognosis.

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