A primer probe combination, product and application thereof for detecting alzheimer's disease

Abstract

The present application relates to the technical field of gene detection, in particular to a primer probe combination for detecting Alzheimer's disease, a product and application thereof. The primer probe combination and kit provided by the present application can simultaneously detect Alzheimer's disease gene mutation and methylation, can effectively distinguish Alzheimer's disease patients from non-Alzheimer's disease subjects, realize low-invasive, low-cost, high-sensitivity, high-specificity detection of Alzheimer's disease, the total sensitivity of the blood sample for detecting Alzheimer's disease can reach 88.89%, the specificity of the blood sample for detecting non-Alzheimer's disease can reach 95.74%, the detection probability of false negative results can be reduced, and the detection accuracy is high.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a primer-probe combination, product, and application for detecting Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease and a major cause of disability in the elderly. The disease has an insidious onset and is difficult to diagnose, often causing many patients to miss the optimal intervention window. Therefore, developing effective early screening and diagnostic methods is of great significance.

[0003] Current treatment and prevention strategies primarily focus on mild cognitive impairment (MCI), which is the preclinical stage of AD. However, the diagnosis of MCI requires cognitive function testing, positron emission tomography (PET), and magnetic resonance imaging (MRI). These diagnostic methods have certain limitations.

[0004] The U.S. Food and Drug Administration has approved cerebrospinal fluid (CSF) biomarker tests for Alzheimer's disease, which are included in the National Institute for Health and Clinical Excellence in Dementia (NIHA) guidelines. However, these tests involve invasive lumbar punctures, which can be invasive and harmful to patients. Furthermore, Alzheimer's disease detection kits currently available in China, based on biomarkers such as β-amyloid protein (Aβ) and phosphorylated tau protein (p-tau) in human serum or CSF, all suffer from low sensitivity and insufficient accuracy.

[0005] Compared to protein biomarkers, changes in nucleic acid biomarkers appear earlier, making them more suitable for early diagnosis. Among the many risk factors for Alzheimer's disease (such as aging, genetic factors, and lifestyle), genetic factors are a crucial aspect. The genetic factors of AD are complex and diverse, with approximately 70% of Alzheimer's cases related to genetics. The ApoEε4 allele is a major genetic risk factor for late-onset Alzheimer's disease, with carriers of this gene having a significantly increased risk. Early-onset Alzheimer's disease is mostly autosomal dominant, associated with mutations in the PSEN1, PSEN2, and APP genes. These gene mutations lead to abnormal deposition of amyloid protein in the brain, subsequently causing neurodegenerative changes. Methylation not only reflects an individual's current physiological state but may also predict disease progression, making it a promising dynamic biomarker.

[0006] In conclusion, promoting early screening and diagnosis of Alzheimer's disease not only helps differentiate it from other types of dementia and improves diagnostic accuracy, but also provides patients with earlier intervention opportunities, slows disease progression, improves quality of life, and reduces the burden of healthcare. Developing non-invasive, sensitive, and reliable detection methods has become an important research direction in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a primer-probe combination, product, and application for detecting Alzheimer's disease, in order to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a primer-probe combination for detecting Alzheimer's disease, including primer pairs and probes for detecting gene mutation sites and methylation sites; The gene mutation sites include the PSEN1 gene mutation site and the APOE gene mutation site; the methylation sites include the CHI3L1 gene methylation site, the TUBA1B gene methylation site, and the EXOC2 gene methylation site; The nucleotide sequences of the primer pairs for detecting the PSEN1 gene mutation site are shown in SEQ ID NO.1-2, and the nucleotide sequences of the probes are shown in SEQ ID NO.3. The nucleotide sequences of the primer pairs for detecting the APOE gene mutation sites are shown in SEQ ID NO.4-5, and the nucleotide sequences of the probes are shown in SEQ ID NO.6. The nucleotide sequences of the primer pairs for detecting the methylation sites of the CHI3L1 gene are shown in SEQ ID NO.13-14, and the nucleotide sequences of the probes are shown in SEQ ID NO.15. The nucleotide sequences of the primer pairs for detecting the methylation sites of the TUBA1B gene are shown in SEQ ID NO.10-11, and the nucleotide sequences of the probes are shown in SEQ ID NO.12. The nucleotide sequences of the primer pairs for detecting the methylation sites of the EXOC2 gene are shown in SEQ ID NO.16-17, and the nucleotide sequences of the probes are shown in SEQ ID NO.18.

[0009] This invention designs special primer and probe structures. For mutant DNA template sequences, under suitable extension temperature conditions, the forward-specific primer binding to the target sequence is unaffected by the probe and retains its extension ability. For wild-type sequences, under set extension temperature conditions, the probe binds to the DNA template before the forward-specific primer, and its extension ability is inhibited. This reaction process enriches and amplifies the mutant template DNA and significantly reduces non-specific amplification, improving the ability to distinguish between wild-type and mutant sequences, with a detection sensitivity as low as 0.01%.

[0010] Optionally, the probes for detecting the PSEN1 gene mutation site, the APOE gene mutation site, the CHI3L1 gene methylation site, the TUBA1B gene methylation site, and the EXOC2 gene methylation site are all labeled with a fluorescent group at their 5' end and a quencher group at their 3' end.

[0011] Optionally, the fluorescent group is one of FAM, VIC, ROX and CY5; the quenching group is one of BHQ1, BHQ2 and MGB.

[0012] This invention provides the application of the above-described primer-probe combination in the preparation of products for detecting Alzheimer's disease.

[0013] Optionally, the product includes reagents, reagent kits, and chips.

[0014] This invention provides a product for detecting Alzheimer's disease, comprising the primer-probe combination described above.

[0015] This invention provides the application of the above-described primer-probe combination in the preparation of products for early screening of Alzheimer's disease.

[0016] This invention provides a product for early screening of Alzheimer's disease, comprising the primer-probe combination described above.

[0017] This invention provides the application of the above-mentioned primer-probe combination in constructing an Alzheimer's disease risk assessment model.

[0018] This invention provides an Alzheimer's disease risk assessment model, which uses the above-mentioned primer and probe combination to perform fluorescence amplification detection on the test sample to obtain the melting curve Rm of the PSEN1 gene mutation site and the APOE gene mutation site, the Ct value of the CHI3L1 gene methylation site, the TUBA1B gene methylation site and the EXOC2 gene methylation site. Using the melting Rm values ​​of the PSEN1 and APOE gene mutation sites, and the Ct values ​​of the CHI3L1, TUBA1B, and EXOC2 gene methylation sites as input variables, an Alzheimer's disease risk assessment model is constructed; the Alzheimer's disease risk assessment model calculates the risk value P according to the following equation: Risk value P = -4.2155 - 0.0280 The Rm value of the PSEN1 gene mutation site is +0.1403. The Rm value of the APOE gene mutation site is -0.0150. Ct value of the CHI3L1 gene methylation site: -0.0266 The Ct value of the methylation site in the TUBA1B gene is +0.1382. Ct value of EXOC2 gene methylation site; When P ≥ 1.04936, the sample test result is positive; when P < 1.04936, the sample test result is negative. The present invention discloses the following technical effects: The primer-probe combination and product provided by this invention can simultaneously detect Alzheimer's gene mutations and methylation, effectively distinguishing between Alzheimer's patients and non-Alzheimer's subjects. It achieves low-invasiveness, low-cost, high-sensitivity, and high-specificity detection of Alzheimer's disease. The overall sensitivity for detecting Alzheimer's blood samples can reach 88.89%, and the specificity for detecting non-Alzheimer's blood samples can reach 95.74%, reducing the detection of false negative results and achieving high detection accuracy.

[0019] Furthermore, the primer-probe combination provided by this invention, based on the independently developed J-STAR technology platform, enables rapid and accurate detection of liquid samples using this breakthrough molecular diagnostic method. This technology can be used to monitor the occurrence and progression of diseases in real time, thus providing a reliable basis for disease diagnosis. In addition, gene-level detection precedes protein detection, allowing for earlier disease detection and timely treatment. Moreover, quantitative real-time PCR requires less sample volume, and blood tests can improve patient compliance. Therefore, using gene methylation and gene mutation sites highly correlated with Alzheimer's disease as biomarkers for early screening of Alzheimer's disease has high application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1The results are shown in the graphs for positive and negative controls. A: Positive control from the first tube of reaction solution; 1: Positive peak of APOE gene mutation, i.e., the base of rs429358 in the APOE gene mutates from T to C, with the horizontal axis representing temperature (°C); 2: Positive peak of PSEN1 gene mutation; 3: Melting peak of the internal control GAPDH gene, i.e., the base of rs63751037 in the PSEN1 gene mutates from A to G; B: Negative control from the first tube of reaction solution, with the horizontal axis representing temperature (°C); C: Positive control from the second tube of reaction solution; a: Amplification curve of EXOC2 gene methylation site; b: Amplification curve of TUBA1B gene methylation site; c: Amplification curve of internal control ACTB gene site; d: Amplification curve of CHI3L1 gene methylation site, with the horizontal axis representing cycle number; D: Negative control from the second tube of reaction solution, with the horizontal axis representing cycle number. Figure 2 This is for evaluating the detection limit of the kit; where A: positive peak graph for PSEN1 mutation gradient detection (1: 1% MT, 2: 0.5% MT, 3: 0.1% MT, 4: 0.05% MT), with the horizontal axis representing temperature (°C); B: positive peak graph for APOE mutation gradient detection (1: 1% MT, 2: 0.5% MT, 3: 0.1% MT, 4: 0.05% MT). MT: C: Internal control GAPDH gene melting peak, x-axis is temperature (°C); D: TUBA1B gene methylation site gradient amplification curve (1: 5% methylation, 2: 1% methylation, 3: 0.5% methylation, 4: 0.1% methylation), x-axis is cycle number; E: CHI3L1 gene methylation site gradient amplification curve (5% methylation, 2: 1% methylation, 3: 0.5% methylation, 4: 0.1% methylation), x-axis is cycle number; F: EXOC2 gene methylation site gradient amplification curve (1: 5% methylation, 2: 1% methylation, 3: 0.5% methylation, 4: 0.1% methylation), x-axis is cycle number; G: Internal control ACTB amplification curve, x-axis is cycle number. Figure 3 ROC curves for the validation set. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: A methylation detection kit for identifying Alzheimer's disease This embodiment provides a methylation detection kit for identifying Alzheimer's disease. The methylation detection genes involved are CHI3L1, TUBA1B, and EXOC2, and the mutation detection sites are PSEN1 and APOE. Specific mutation site information is shown in Table 1, and specific methylation detection site information is shown in Table 2. The kit provided by this invention also uses internal reference genes to indicate the quality of DNA extraction and transformation; the internal references include, for example, the human ACTB gene and the human GAPDH gene.

[0028] Table 1. Information on gene mutation sites for Alzheimer's disease detection Table 2. Alzheimer's disease gene methylation detection site information Note: "+" indicates the sense strand; "-" indicates the antisense strand; CpG: abbreviation for Cytosine-phosphate-Guanine, that is: cytosine (C)-phosphate (p)-guanine (G). In the genome, the pattern in which cytosine (C) and guanine (G) are linked by a phosphodiester bond (p) is called CpG dinucleotide.

[0029] Based on the detection region sequences of each gene in Tables 1 and 2, the primers and probes used in Table 3 were synthesized by Shanghai Langjing Biotechnology Co., Ltd.

[0030] Table 3 Primer and probe sequences Note: Primer names containing "F" indicate upstream primers, "R" indicate downstream primers, and "P" indicate probes; VIC indicates probes are labeled with VIC fluorescent markers; ROX indicates probes are labeled with ROX fluorescent markers; FAM indicates probes are labeled with FAM fluorescent markers; CY5 indicates probes are labeled with CY5 fluorescent markers; ACTB and GAPDH are internal reference genes.

[0031] The kit provided in this embodiment contains a first tube of reaction solution primer-probe buffer components, a second tube of reaction solution primer-probe buffer components, and a primer-probe combination. The components and concentrations of the first tube of reaction solution primer-probe buffer are shown in Table 4; the components and concentrations of the second tube of reaction solution primer-probe buffer are shown in Table 5.

[0032] Table 4. Components and preferred concentrations of the primer / probe buffer in the first reaction tube. Table 5. Components and preferred concentrations of the primer / probe buffer in the second reaction tube. Example 2 Sensitivity Detection 1. Cell line gDNA extraction: Using a blood / cell / tissue genomic DNA extraction kit (Tiangen Biotech, DP304), gDNA was extracted from the HFF-1 cell line (FGD017) that is non-Alzheimer's disease-positive and PSEN1 and APOE mutation-negative, as a negative control. PSEN1 (Sinochem, HG16272-ACGLN), APOE (Sinochem, HG10817-M) mutation standard DNA were prepared as positive controls, along with CHI3L1 (Sinochem, HG11227-M), TUBA1B (Sinochem, HG14070-G), and EXOC2 (Fenghui Biotechnology, G120788) gene methylation standard DNA (as positive controls). Sample quantification was performed using the Qubit™ dsDNA quantification kit.

[0033] 2. Sulfite conversion: 1000 ng of gDNA extracted from non-Alzheimer's disease HFF-1 cell line that is PSEN1 mutation negative and APOE mutation negative, and CHI3L1, TUBA1B, and EXOC2 methylated standard DNA were taken and sulfite-converted using the EZ DNA Methylation Kit (ZYMO). After purification and recovery, the samples were quantified using the Qubit™ dsDNA quantification kit.

[0034] 3. Preparation of reference material for detection limit: Take PSEN1 and APOE mutant standard DNA and HFF-1 cell line gDNA respectively, and dilute them to 10 ng / μL using TE. Then, use 10 ng / μL HFF-1 cell line gDNA to dilute the above PSEN1 and APOE mutant standard DNA to 10 ng / μL 5% MT, 10 ng / μL 1% MT, 10 ng / μL 0.5% MT and 10 ng / μL 0.1% MT respectively. Take sulfite-converted CHI3L1, TUBA1B, EXOC2 methylated standard DNA and HFF-1 g DNA, and dilute them to 10 ng / μL using TE buffer. Then, use 10 ng / μL of sulfite-converted HFF-1 g DNA to dilute the 100% methylated sulfite-converted CHI3L1, TUBA1B, EXOC2 methylated standard DNA to 10 ng / μL 5%, 10 ng / μL 1%, 10 ng / μL 0.5%, and 10 ng / μL 0.1%.

[0035] 4. Amplification analysis: Using unconverted sample DNA as a template, add it to the PCR tube containing the first reaction solution, primer, and probe buffer components; using bisulfite-converted sample DNA as a template, add it to the PCR tube containing the second reaction solution, primer, and probe buffer components. Tightly cap the tubes, transfer them to the detection area, and perform fluorescence amplification analysis. The amplification results are shown below. Figure 1 and Figure 2 As shown in Table 6, the PCR reaction system and PCR reaction conditions are shown in Table 7.

[0036] Table 6 Specific Reaction System Table 7 Specific Reaction Procedures Note: FAM / ROX / CY5 fluorescence signals were collected during the gradient temperature increases at 50℃ in the third step of the PCR cycle and in the melting curve analysis step; instrument compatibility required. SLAN®-96P / SLAN®-96S features temperature control via a temperature mode selection module.

[0037] The results showed that, under the condition of 20 ng / reaction spiking, the kit provided by the present invention, when repeated 10 times, achieved 100% detection (Rm≥4.90) of PSEN1 mutation sites with 0.1% MT and 85% detection of PSEN1 mutation sites with 0.05% MT. Therefore, the detection sensitivity of PSEN1 mutation sites of the kit provided by the present invention is 0.1% mutation frequency under the condition of 20 ng / reaction spiking.

[0038] Under the condition of 20 ng / reaction spiking, the kit provided by the present invention was repeated 10 times. The detection of APOE mutation sites with 0.1% MT achieved 100% detection (Rm≥4.69), and the detection of APOE mutation sites with 0.05% MT achieved 80% detection. Therefore, the detection sensitivity of APOE mutation sites of the kit provided by the present invention is 0.1% mutation frequency under the condition of 20 ng / reaction spiking.

[0039] Under the condition of 20 ng / reaction spiking, the kit provided by this invention was repeated 10 times. The methylation sites of 0.5% TUBA1B, CHI3L1 and EXOC2 genes could be detected normally. When detecting 0.1% CHI3L1, TUBA1B and EXOC2, the methylation site of TUBA1B gene could be detected at 75% (Ct≤39.16), the methylation site of CHI3L1 gene could be detected at 55% (Ct≤39.45), and the methylation site of EXOC2 gene could be detected at 50% (Ct≤39.56). Therefore, the above methylation site sensitivity is the 0.5% methylation frequency under the condition of 20 ng / reaction spiking.

[0040] Example 3 Clinical Sample Testing The specific operating steps of this invention are as follows: DNA is extracted from the plasma sample of the subject, a portion of the DNA is transformed using a methylation conversion reagent, the transformed DNA is then purified, and qPCR amplification is performed on the original DNA sample and the transformed DNA sample using the kit provided by this invention. The methylation level and mutation status of the target region in the sample are detected by the STAR2 method, thereby determining whether the plasma sample to be tested has Alzheimer's disease. The specific steps are as follows: 1. Collection of blood samples A total of 61 blood samples were collected, including blood samples from 32 subjects pathologically diagnosed with Alzheimer's disease and blood samples from 29 subjects without Alzheimer's disease. The collected blood samples were centrifuged at room temperature, and the supernatant was collected to obtain plasma samples. These 61 samples were used as the test set.

[0041] 2. Sample extraction, transformation, and purification. 2.1 Extracting DNA samples Centrifuge 10 mL of whole blood at room temperature to obtain plasma samples. Extract cfDNA from the plasma using the QIAamp Circulating Nucleic Acid Kit (50) (Cat. No. 55114), using 4-5 mL of plasma. Refer to the kit instructions for specific procedures. After extraction, elute with 40 μL of purified water. Quantify the sample using the Qubit™ dsDNA quantification kit.

[0042] 2.2. Bisulfite Conversion and Purification Treatment The extracted free DNA was divided into two equal portions. One portion was then converted to bisulfite using the EZ DNA Methylation-Gold™ Kit (Cat.No.D5005). For specific experimental procedures, please refer to the kit instructions. After conversion, the DNA was eluted with 10 μL of purified water and stored at -20℃ for later use.

[0043] 3. PCR amplification Using unconverted cell-free plasma DNA as a template, add it to the PCR tube containing the first reaction solution, primer, and probe buffer components; using bisulfite-converted tissue sample DNA as a template, add it to the PCR tube containing the second reaction solution, primer, and probe buffer components. Tightly cap the tubes and transfer them to the detection area. The PCR reaction system is shown in Table 6, and the PCR reaction conditions are shown in Table 7.

[0044] After the reaction, the baseline and appropriate threshold need to be manually adjusted. The baseline is typically the fluorescence signal over 3-15 cycles. Samples that failed the test are discarded according to quality control requirements, and the Ct values ​​of the qualified samples are read. The quality control standards are shown in Table 8. If all samples meet the requirements, the results of the samples to be tested can be analyzed.

[0045] Table 8. Amplified Data Quality Control Note: All of the above conditions must be met in the same experiment; otherwise, the results of this experiment will be invalid. The above experiment tested 61 clinical samples. The PSEN1 and APOE gene mutations were positive, and the TUBA1B, CHI3L1 and EXOC2 methylation was positive. The proportions of these samples in the clinical diagnosis of non-Alzheimer's disease and Alzheimer's disease are shown in Table 9.

[0046] For the test results of the above 61 clinical samples, logistic regression was performed with the melting Rm of PSEN1 and APOE gene mutations, and the Ct values ​​of methylation sites of TUBA1B, CHI3L1, and EXOC2, and the clinical pathological diagnosis results to construct the predictive risk value of Alzheimer's disease based on gene mutations and gene methylation, i.e., the P value, P = -4.2155 - 0.0280. The Rm value of the PSEN1 gene mutation site is +0.1403. The Rm value of the APOE gene mutation site is -0.0150. Ct value of the CHI3L1 gene methylation site: -0.0266 The Ct value of the methylation site in the TUBA1B gene is +0.1382. Ct value of EXOC2 gene methylation site.

[0047] The above sample test results were converted to risk values. Using MedCalc software, ROC curves were constructed and the optimal cut-off risk values ​​were determined based on the melting Rm values ​​of PSEN1 and APOE gene mutations, and the Ct values ​​of methylation sites in TUBA1B, CHI3L1, and EXOC2 genes, combined with clinicopathological results. A p-value ≥ 1.04936 was considered a positive result, and a p-value < 1.04936 was considered a negative result.

[0048] Table 9. Detection sensitivity and specificity of the test set samples Example 4: Clinical Validation 1. Collection of blood samples A total of 267 blood samples from Alzheimer's patients were collected from hospitals in three different regions. Among them, 126 cases were pathologically diagnosed with Alzheimer's disease, and 141 cases were pathologically diagnosed without Alzheimer's disease. These samples were blinded and used as the validation set. This study met the criteria for conduct as approved by the hospital's ethics committee, and all patients signed informed consent forms.

[0049] 2. The sample extraction, transformation, and purification process is as follows: 2.1 Extracting DNA samples Centrifuge 10 mL of whole blood at room temperature to obtain plasma samples. Extract cfDNA from the plasma using the QIAamp Circulating Nucleic Acid Kit (50) (Cat. No. 55114), using 4-5 mL of plasma. Refer to the kit instructions for specific procedures. After extraction, elute with 40 μL of purified water. Quantify the sample using the Qubit™ dsDNA quantification kit.

[0050] 2.2. Bisulfite Conversion and Purification Treatment The extracted free DNA was divided into two equal portions. One portion was then converted to bisulfite using the EZ DNA Methylation-Gold™ Kit (Cat.No.D5005). For specific experimental procedures, please refer to the kit instructions. After conversion, the DNA was eluted with 10 μL of purified water and stored at -20℃ for later use.

[0051] Amplification Analysis: Using unconverted cell-free plasma DNA as a template, add it to a PCR tube containing the corresponding amplification reagent reaction solution 1 described above; using DNA from tissue samples converted with bisulfite as a template, add it to a PCR tube containing the corresponding amplification reagent reaction solution 2 described above. Tightly cap the tubes and transfer them to the detection area. Prepare the amplification reaction solution according to the amplification system setup in Table 6, and perform fluorescence amplification analysis according to the amplification reaction procedure in Table 7.

[0052] The above experiment tested 267 clinical samples. PSEN1 and APOE gene mutations were positive, and TUBA1B, CHI3L1, and EXOC2 methylation was positive. The proportions of these mutations in the clinical diagnoses of non-Alzheimer's disease and Alzheimer's disease are shown in Table 10. ROC curves are shown in [Figure 1]. Figure 3 As shown.

[0053] The reagent kit provided by this invention has a sensitivity of 88.89% (95% CI: 84.92%~95.56%), a specificity of 95.74% (95% CI: 90.04%~97.98%), and an overall concordance rate of 93.26% (95% CI: 89.55%~95.96%).

[0054] Table 10 Detection sensitivity and specificity of validation set samples The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer-probe combination for detecting Alzheimer's disease, characterized in that, This includes primer pairs and probes for the first tube (detecting gene mutation sites) and the second tube (detecting methylation sites); The gene mutation sites include the PSEN1 gene mutation site rs63751037 and the APOE gene mutation site rs429358; the methylation sites include the CHI3L1 gene methylation site cg07423149, the TUBA1B gene methylation site cg00973724 and the EXOC2 gene methylation site cg21987077; The nucleotide sequences of the primer pairs for detecting the PSEN1 gene mutation site rs63751037 are shown in SEQ ID NO.1-2, and the nucleotide sequences of the probes are shown in SEQ ID NO.

3. The nucleotide sequences of the primer pairs for detecting the APOE gene mutation site rs429358 are shown in SEQ ID NO.4-5, and the nucleotide sequence of the probe is shown in SEQ ID NO.

6. The nucleotide sequences of the primer pair for detecting the methylation site cg07423149 of the CHI3L1 gene are shown in SEQ ID NO.13-14, and the nucleotide sequence of the probe is shown in SEQ ID NO.

15. The nucleotide sequences of the primer pair for detecting the methylation site cg00973724 of the TUBA1B gene are shown in SEQ ID NO.10-11, and the nucleotide sequence of the probe is shown in SEQ ID NO.

12. The nucleotide sequences of the primer pair for detecting the EXOC2 gene methylation site cg21987077 are shown in SEQ ID NO.16-17, and the nucleotide sequence of the probe is shown in SEQ ID NO.

18.

2. The primer-probe combination according to claim 1, characterized in that, The probes used to detect the PSEN1 gene mutation site rs63751037, the APOE gene mutation site rs429358, the CHI3L1 gene methylation site cg07423149, the TUBA1B gene methylation site cg00973724, and the EXOC2 gene methylation site cg21987077 were all labeled with a fluorescent group at their 5' end and a quencher group at their 3' end.

3. The primer-probe combination according to claim 2, characterized in that, The fluorescent group is one of FAM, VIC, ROX and CY5; the quenching group is one of BHQ1, BHQ2 and MGB.

4. The use of the primer-probe combination according to any one of claims 1-3 in the preparation of products for detecting Alzheimer's disease.

5. The application according to claim 4, characterized in that, The products include reagents, reagent kits, and chips.

6. A product for detecting Alzheimer's disease, characterized in that, Includes the primer-probe combination as described in any one of claims 1-3.

7. The use of the primer-probe combination according to any one of claims 1-3 in the preparation of a product for early screening of Alzheimer's disease.

8. A product for early screening of Alzheimer's disease, characterized in that, Includes the primer-probe combination as described in any one of claims 1-3.

9. The application of the primer-probe combination according to any one of claims 1-3 in constructing an Alzheimer's disease risk assessment model.

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