Alzheimer disease related gene methylation detection method based on E-ice-COLD-PCR technology and application thereof

Abstract

The application relates to the technical field of genetic engineering, in particular to an Alzheimer disease related gene methylation detection method based on E-ice-COLD-PCR technology and application thereof, which comprises the following steps: S1, extracting cfDNA in a to-be-detected sample; S2, screening a target gene and performing bisulfite conversion treatment on the extracted cfDNA, the target gene being at least one of PSEN1, BDNF and ABCA7; S3, taking the converted DNA as a template, and performing amplification by adopting an E-ice-COLD-PCR amplification system, the amplification system containing amplification primers for the target gene and a blocking probe containing a locked nucleic acid, and the BDNF probe sequence being shown in SEQ ID NO: 7, the PSEN1 probe sequence being shown in SEQ ID NO: 8, and the ABCA7 probe sequence being shown in SEQ ID NO: 9; and S4, analyzing the methylation degree by high-resolution melting curve analysis, and establishing a standardized E-ice-COLD-PCR methylation detection system for PSEN1, BDNF and ABCA7 genes.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology and its application. Background Technology

[0002] Alzheimer's disease (AD) is a highly prevalent neurodegenerative disease with complex pathological mechanisms. While its etiology remains incompletely understood, its core pathological features include intracellular neurofibrillary tangles and extracellular amyloid-β peptide (Aβ protein) deposition. With the continued increase in the global patient population, early and accurate diagnosis of AD has become a pressing medical challenge.

[0003] DNA methylation, as a core mechanism of epigenetic regulation, plays a crucial role in the pathogenesis and progression of Alzheimer's disease (AD). Its abnormal patterns are closely related to the core pathological features of the disease and are potential early diagnostic biomarkers. However, AD-related methylation abnormalities are mostly characterized by low methylation levels. Whether it is specific methylation changes in local nerve cells in brain tissue or methylation signals in trace circulating cfDNA (cfDNA) in non-invasive samples such as peripheral blood and cerebrospinal fluid, the methylation ratio is often less than 5%. Conventional detection techniques are difficult to detect accurately due to insufficient sensitivity, which greatly limits the research and clinical translation of related biomarkers.

[0004] DNA cytosine methylation is the most widely characterized epigenetic modification. Recent studies have found that abnormal DNA methylation patterns are associated with the development of various diseases. Abnormal methylation changes in tumor tissue can be detected in a variety of cancers with high sensitivity and specificity. However, commonly used pathological tissue examinations are highly invasive, and the tissue biopsies obtained are only a small portion of the lesion, which may lead to an inability to fully assess heterogeneity. Compared with tissue biopsies, liquid biopsies are not limited by heterogeneity and the problem of repeated sampling. During apoptosis and necrosis, DNA fragments are released into the circulation to form circulating cfDNA (cfDNA), which provides the possibility for non-invasive detection of DNA methylation. cfDNA methylation characterization based on liquid biopsies has opened up a new avenue for non-invasive detection, but it faces difficulties in practical applications, such as insufficient sensitivity and difficulty in detecting low-abundance methylation signals.

[0005] Therefore, there is an urgent need to invent a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology and its application to solve the above-mentioned technical problems. Summary of the Invention

[0006] This invention relates to a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology. By using a blocking probe containing locked nucleic acid to specifically bind to unmethylated CpG sites, the method selectively inhibits the amplification of unmethylated templates at a critical temperature. A standardized E-ice-COLD-PCR methylation detection system has been established for PSEN1, BDNF, and ABCA7 genes, effectively solving the aforementioned technical problems.

[0007] This invention proposes a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology, comprising the following steps:

[0008] S1. Extract cfDNA from the sample to be tested;

[0009] S2. Screen for target genes and perform bisulfite conversion on the extracted cfDNA, wherein the target gene is at least one of PSEN1, BDNF and ABCA7;

[0010] S3. Using the transformed DNA as a template, amplification is performed using the E-ice-COLD-PCR amplification system. The amplification system contains amplification primers targeting the target gene and blocking probes containing locked nucleic acids. The BDNF probe sequence is shown in SEQ ID NO:7, the PSEN1 probe sequence is shown in SEQ ID NO:8, and the ABCA7 probe sequence is shown in SEQ ID NO:9.

[0011] S4. Analyze the degree of methylation using high-resolution melting curves.

[0012] Preferably, the LNA blocking probe in S3 is designed to have the following characteristics: length 40–65 nt, 3' end overlap with primer 5–6 nt, and LNA bases introduced at all unmethylated CpG sites.

[0013] Preferably, the detection limit of the E-ice-COLD-PCR system in step S3 is 4.27 × 10⁻⁶. -7 -6.03×10 - 6 The concentration of ng / μL corresponds to a copy number concentration of 136-992 copies / μL, which can distinguish samples with methylation gradients of 1%-100%.

[0014] Preferably, the concentration of the blocking probe containing locked nucleic acid is 10 nM, and the 3′ end of the probe is modified with a phosphate group to specifically bind to unmethylated CpG sites and block their amplification.

[0015] Preferably, the amplification primers have the following sequences:

[0016] The forward primer for the BDNF gene is shown in SEQ ID NO:1;

[0017] The reverse primer for the BDNF gene is shown in SEQ ID NO:2;

[0018] The forward primer for the PSEN1 gene is shown in SEQ ID NO:3;

[0019] The reverse primer for the PSEN1 gene is shown in SEQ ID NO:4;

[0020] ABCA7 gene forward primer: as shown in SEQ ID NO:5;

[0021] ABCA7 gene reverse primer: as shown in SEQ ID NO:6.

[0022] Preferably, the E-ice-COLD-PCR amplification system further includes EvaGreen fluorescent dye for real-time monitoring of amplification and melting curves.

[0023] Preferably, the degree of methylation is determined by comparing the high-resolution melting curves with pre-constructed fully methylated and fully unmethylated plasmid standards; the fully methylated and fully unmethylated plasmid standards have sequences as shown in SEQ ID NO:10 to SEQ ID NO:15.

[0024] Preferably, the fully methylated and fully unmethylated plasmid standards are constructed by enzymatic modification cloning or artificial synthesis, wherein:

[0025] The sequence of the fully methylated PSEN1 standard is shown in SEQ ID NO:10, and the sequence of the fully unmethylated PSEN1 standard is shown in SEQ ID NO:11;

[0026] The sequence of the fully methylated ABCA7 standard is shown in SEQ ID NO:12, and the sequence of the fully unmethylated ABCA7 standard is shown in SEQ ID NO:13;

[0027] The sequence of the fully methylated BDNF standard is shown in SEQ ID NO:14, and the sequence of the fully unmethylated BDNF standard is shown in SEQ ID NO:15.

[0028] Preferably, the kit is prepared using this method and is suitable for the combined detection of methylation of PSEN1, BDNF, and ABCA7 genes in serum cfDNA.

[0029] Preferably, the application of an Alzheimer's disease-related gene methylation detection method based on E-ice-COLD-PCR technology in the preparation of early screening products for Alzheimer's disease is proposed.

[0030] This invention proposes a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology, which has the following beneficial effects:

[0031] The E-ice-COLD-PCR detection system established in this invention utilizes a blocking probe containing locked nucleic acid to specifically bind to unmethylated CpG sites, selectively inhibiting the amplification of unmethylated templates at a critical temperature, and efficiently enriching methylated DNA as low as 1%. The detection limit of this system has been verified to be as low as 4.27 × 10⁻⁻⁻⁶. 7 ~6.03×10⁻ 6 The concentration is ng / μL, corresponding to a copy number concentration of 136~992 copies / μL; it can clearly distinguish samples with methylation gradients of 1%~100%, with significantly better specificity than traditional detection techniques, and reliable results can be obtained without secondary amplification. This technology has high sensitivity and high specificity for detecting low-abundance methylation signals.

[0032] This invention establishes a standardized E-ice-COLD-PCR methylation detection system for the PSEN1, BDNF, and ABCA7 genes for the first time, and verifies that the methylation level of these genes in the serum cfDNA of AD patients is significantly different from that in healthy individuals (P<0.05 or P<0.001). This system can serve as a valuable clinical biomarker for the early non-invasive diagnosis of AD. Furthermore, using serum cfDNA as the detection sample offers advantages over cerebrospinal fluid puncture and brain tissue biopsy, including being non-invasive, readily available, and dynamically monitorable, making it more suitable for large-scale early AD screening in primary healthcare institutions.

[0033] This invention optimizes and determines the optimal annealing temperature (BDNF 51℃, PSEN 156℃, ABCA 751℃) and critical temperature (BDNF 77℃, PSEN 172℃, ABCA 774℃) for three target genes, and determines 10nM as the optimal probe concentration through gradient probe concentration experiments. This method is simple to operate, and effective results can be obtained in one amplification, without the need for subsequent verification steps. It has good reproducibility and promotion value. Attached Figure Description

[0034] Figure 1 A diagram showing the CpG islands in the gene promoter region;

[0035] Figure 2 Figure 1 shows the modeling of SH-SY5Y cells before and after drug administration under a fluorescence microscope;

[0036] Figure 3 A bar chart showing drug concentration versus apoptosis rate;

[0037] Figure 4 The results of differentially expressed gene screening are shown in the following diagrams: (A: Volcano plot of differentially expressed genes; B: Heatmap of differentially expressed genes; C: Venn plot).

[0038] Figure 5 This is a relative quantitative graph of the transcriptional levels of each gene in the experimental group and the control group;

[0039] Figure 6 Diagram of differential expression pathways for each gene;

[0040] Figure 7 Sequencing diagrams of methylation sites in the control and experimental groups of three genes;

[0041] Figure 8 Electrophoresis images of Touchdown PCR and E-ice-COLD-PCR gradient dilutions.

[0042] Figure 9 The result of agarose gel electrophoresis at the optimal annealing temperature;

[0043] Figure 10 The image shows the results of agarose gel electrophoresis at the optimal critical temperature.

[0044] Figure 11 The image shows the PCR results of bacterial culture containing the BDNF gene.

[0045] Figure 12 Image of BDNF plasmid sequencing results (white represents unmethylated, black represents fully methylated);

[0046] Figure 13A Sequencing diagram of PSEN1-0 synthesized plasmid standard;

[0047] Figure 13B Sequencing diagram of PSEN1-100 synthesized plasmid standard;

[0048] Figure 13C Sequencing diagram of the ABCA7-0 synthesized plasmid standard;

[0049] Figure 13D Sequencing diagram of the ABCA7-100 synthetic plasmid standard;

[0050] Figure 14 The graph shows the methylated standards (AI represents the melting curves, HRM curves, and difference curves of the standards for the BDNF, PSEN1, and ABCA7 genes, respectively).

[0051] Figure 15 The concentrations, Ct values, and standard curves of the three gene standards are shown.

[0052] Figure 16 The amplification curves for the three genes are shown.

[0053] Figure 17 Melting curves for the three genes;

[0054] Figure 18 Melting curves, HRM, and HRM difference curves for the three genes (ABC: BDNF gene; DEF: PSEN1 gene; GHI: ABCA7 gene).

[0055] Figure 19 Line graphs showing the methylation level of different genes versus ct values;

[0056] Figure 20A The graphs show the 10nM, 50nM, and 100nM hrm curves and the hrm difference curves for the BDNF gene.

[0057] Figure 20B The graphs show the hrm curves and hrm difference curves for the PSEN1 gene at 10nM, 50nM, and 100nM.

[0058] Figure 20C The graphs show the 10nM, 50nM, and 100nM hrm curves and the hrm difference curves for the ABCA7 gene.

[0059] Figure 21 The difference in HRM and HRM of standard cfDNA samples from the healthy group and the difference in HRM and HRM of 10 samples.

[0060] Figure 22 The difference in HRM and HRM of standard samples of cfDNA samples in AD group and the difference in HRM and HRM of 10 samples;

[0061] Figure 23 This is a statistical chart showing the methylation levels of samples from the healthy group and the AD group in this invention;

[0062] in, Figure 1 , 10 In 15, 16, 17, 21, and 22, A is the BDNF gene, B is the PSEN1 gene, and C is the ABCA7 gene. Detailed Implementation

[0063] like Figures 1-23 As shown, this invention proposes a method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology, comprising the following steps:

[0064] S1. Extract cfDNA from the sample to be tested;

[0065] S2. Screen for target genes and perform bisulfite transformation on the extracted cfDNA. The target genes are at least one of PSEN1, BDNF and ABCA7.

[0066] S3. Using the transformed DNA as a template, amplification was performed using the E-ice-COLD-PCR amplification system. The amplification system contained amplification primers targeting the target gene and blocking probes containing locked nucleic acids. The BDNF probe sequence is shown in SEQ ID NO:7, the PSEN1 probe sequence is shown in SEQ ID NO:8, and the ABCA7 probe sequence is shown in SEQ ID NO:9.

[0067] S4. Analyze the degree of methylation using high-resolution melting curves.

[0068] This invention was verified through the following experiments, and the main reagents used in this invention include DMEM solution, 0.25% trypsin solution, 1×TBE solution, 2% agarose gel, Aβ cell treatment solution, IPTG solution, Amp solution, LB solid medium and LB liquid medium.

[0069] Experimental example:

[0070] First, the cryopreserved SH-SY5Y cells were thawed and passaged until they grew well and adhered to the culture medium at approximately 80%. The medium was discarded, and the cells were gently washed with 1 mL of PBS. After discarding the PBS, 1 mL of trypsin was added for 60 seconds. Digestion was stopped by adding DMEM medium containing FBS, and the cells were detached by pipetting. The cells were transferred to sterile 15 mL centrifuge tubes and centrifuged at 1000 rpm for 5 minutes to separate the cells. The supernatant was discarded by pipetting. The cells were resuspended in DMEM medium containing 10% FBS, gently mixed, and passaged at a 1:3 ratio. Cells were transferred to 60 mm culture dishes, and an appropriate amount of levofloxacin was added. The mixture was shaken in a cross-shaped manner, and the cells were observed under a microscope. The cells were then cultured in a normal oxygen incubator. The cell pellet was mixed with 700 µL DMEM, 200 µL FBS, and 100 µL LDMSO and transferred to cryovials, labeled with the time and name, and stored at -80°C.

[0071] Furthermore, ten control groups and ten experimental groups were established using the CCK-8 assay. Cell concentration was measured using a cell counter, and based on the counting results, an appropriate amount of DMEM medium containing 10% FBS was added to dilute the cell suspension to 2 × 10⁻⁶ cells / mL. 5 Cells / mL. Seed 100 μL of cell suspension per well in a 96-well plate and incubate until cells are fully adherent. After confirming cell adhesion under a microscope, discard the culture medium and gently wash 2-3 times with 100 μL PBS (to avoid FBS interference with the drug). Add 100 μL DMEM to each well in the control group and 100 μL of different concentrations of Aβ diluted in DMEM to each well in the model group. 25-35 Solution culture for 24 h (Aβ) 25-35Cells were pre-aged and tangled in a 37°C cell culture incubator for 72 hours. After culture, the liquid in the wells was discarded, and the cells were gently washed three times with 100 μL PBS. Then, a mixture of DMEM medium and CCK8 reagent at a 9:1 ratio was prepared, and 100 μL of the mixture was added to each well. The wells were incubated in a normal oxygen incubator for 3 hours. After incubation, the OD value of each well was measured using a microplate reader, and the apoptosis rate was calculated. Based on the calculation results, the optimal reaction conditions were selected for subsequent model construction.

[0072] Furthermore, after extracting cellular DNA, cellular RNA was extracted and reverse transcribed. A set of experimentally validated Alzheimer's disease-related core genes was obtained by searching and screening the AlzData database. Simultaneously, Aβ... 25-35 SH-SY5Y cells were induced for transcriptome sequencing, and differentially expressed genes were screened. Intersection analysis of the two gene datasets was performed, and Venn diagrams were used to visually represent the gene overlap distribution. To verify the reliability of the transcriptome sequencing data, 10 significantly upregulated and 10 significantly downregulated genes were selected from the overlapping genes. qRT-PCR specific primers for the corresponding genes were downloaded from the Origene database. Real-time quantitative PCR was used to detect the expression levels of each gene, and the results were compared with the sequencing results to confirm the accuracy and reproducibility of the transcriptome data. Subsequently, combined with the results of overlapping gene functional enrichment analysis, the degree of association with Alzheimer's disease pathophysiological regulatory pathways, and the support of existing research literature, the overlapping genes were further linked to Aβ. 25-35 Intersection analysis was performed on differentially expressed genes in the transcriptome of SH-SY5Y cells to obtain candidate genes. After verifying the consistency of expression trends by qRT-PCR, PSEN1, BDNF, and ABCA7 were selected as key candidate genes. The specific qRT-PCR primer sequences, qRT-PCR reaction system, and qRT-PCR reaction parameters are shown in Table 1 and Table 2, respectively.

[0073] Table 1: qRT-PCR primer sequences

[0074] Table 2: qRT-PCR reaction system

[0075] Table 3: qRT-PCR reaction parameters

[0076] As shown in Table 3, the melting curve reaction parameters are: 60℃ for 30s, then rising from 60℃ to 95℃ at a rate of 0.5℃ / s, and finally dropping to 20℃ and holding for 10s.

[0077] In some embodiments, methylation primers and probes were synthesized and tested. Promoter methylation primers were designed using MethyPrimer online software and submitted to Shanghai Sangon Biotech Co., Ltd. for synthesis. Specific annealing temperatures (Tm) and primer sequences are shown in Table 4.

[0078] Table 4: Methylation Primer Information

[0079] Furthermore, LNA blocking probes were manually designed and Tc was calculated using the LNAoligoTm prediction tool. The unmethylated CpG site region at the center of the probe must contain at least two closely adjacent LNA bases; simultaneously, a phosphate group must be modified at the 3' end of the probe to prevent its extension by DNA polymerase during E-ice-COLD-PCR; the probe length should be controlled between 40-65 bases, and the 3' end must have a 5-6 nucleotide overlap with one of the primers in a pair of amplification primers to ensure amplification specificity. If it is impossible to cover all CpG sites in the amplification product, priority should be given to covering the largest number of consecutive CpG sites; and the probe must introduce LNA bases at each unmethylated bisulfite-converted CpG site corresponding to its sequence (i.e., the T site corresponding to unmethylation after bisulfite treatment), and be submitted to BGI Genomics in Beijing for synthesis. Specific probe sequences are shown in Table 5.

[0080] Table 5: Designed BDNF, PSEN1, and ABCA7 probe sequences

[0081] In some embodiments, an E-ice-COLD-PCR reaction system was established. The optimal annealing temperature was determined using gradient PCR with 2 ng of genomic BSP-DNA. The system and procedure are shown in Tables 6 and 7. 5 µL of the amplification product was mixed with 1 µL of 6× Loading Buffer and spotted onto a 2% agarose gel. Electrophoresis was performed at 120 V for 25 min.

[0082] Table 6: Gradient PCR reaction system for plasmid standards

[0083] Table 7 Reaction Procedure for Annealing Temperature

[0084] The critical temperature for 2 ng of genomic BSP-DNA was determined between 70°C and 90°C. The E-ice-COLD-PCR system and procedure are shown in Tables 8 and 9. 5 µL of amplification product was mixed with 1 µL of 6× Loading Buffer and spotted onto a 2% agarose gel. Electrophoresis was performed at 120°C for 25 min, and the temperature corresponding to the brightest band was selected as the critical temperature.

[0085] Table 8: E-ice-COLD-PCR reaction system

[0086] Table 9: E-ice-COLD-PCR reaction procedure

[0087] Furthermore, an E-ice-COLD-PCR standard was established. Cellular genomic DNA was treated with methyltransferase, and the system was prepared strictly according to the instructions as shown in Table 10. The mixture was thoroughly mixed and repeatedly pipetted at least 6 times; the mixture was incubated at 37°C for 1 hour; and the reaction was terminated by heating at 65°C for 20 minutes.

[0088] Table 10: Methyltransferase System

[0089] The DNA treated with methyltransferase and the untreated genomic DNA were simultaneously subjected to bisulfite conversion. Depending on the DNA concentration, different volumes of DNA and an appropriate amount of ddH2O were added to prepare 100 μl DNA solutions containing 500-2000 ng. The conversion was performed according to the instructions of the bisulfite conversion kit. The product was eluted with 40 μl of the solution, and the product concentration was determined by a micro spectrophotometer. The time name and concentration were marked, and the solution was stored at -80℃.

[0090] BSP-DNA was amplified by gradient PCR. The system and procedure are shown in Tables 11 and 12. 5 µL of the amplification product was mixed with 1 µL of 6× Loading Buffer and spotted onto a 2% agarose gel. Electrophoresis was performed at 120 V for 25 min.

[0091] Table 11: PCR amplification system for BSP-DNA

[0092] Table 12: PCR reaction procedure for BSP-DNA

[0093] Further, the target band was precisely excised from the agarose gel, removing as much impurity as possible. The excised gel piece was placed in a sterile EP tube, labeled, and weighed. DNA recovery was performed strictly according to the instructions of the standard agarose gel DNA recovery kit. Finally, 30 µL of double-distilled water was added dropwise to the CA2 adsorption column, incubated at room temperature for 2 min, followed by centrifugation at 12000 rpm for 2 min. The centrifuged liquid was collected, sealed, labeled, and stored at -80℃ for later use. The subsequent ligation vector included:

[0094] a. Prepare according to the system in Table 13 using the PMD19-TVector kit.

[0095] b. Place the EP tube containing the above solution into a metal bath at 16°C for 30 minutes.

[0096] Table 13: Carrier Linkage System

[0097] Further, conversion and blue-white screening are performed:

[0098] a. Transfer 10µL of the above liquid into an EP tube using a pipette, add 50µL of LDH5α competent cells, gently mix by pipetting, and then place in ice and let stand for 30 minutes.

[0099] b. Immediately transfer the EP tube to a 42℃ metal bath for heat shock for 45 seconds, then quickly bury it in ice and let it cool down for 2 minutes.

[0100] c. Remove the EP tube and add 940µL of LLB liquid culture medium (without Amp). Shake at 37°C (150rpm / min) for 1 hour to allow the bacteria to regain activity. Centrifuge at 8000rpm for 3 minutes, discard 850µL of supernatant, and mix thoroughly by pipetting.

[0101] d. Using a disposable L-stick, spread the above mixture (40µL / plate) evenly on LB solid medium containing X-gal, IPTG, and Amp. Incubate upright in a 37°C incubator for 30 min, then invert for 16 h.

[0102] e. Pick a single, plump white colony from the solid medium and inoculate it into LB liquid medium containing Amp. Incubate at 37°C with shaking (210 rpm / min) for 20 h.

[0103] Centrifuge 4 mL of the above-mentioned bacterial culture at 12000 rpm for 1 min, discard the supernatant, collect the bacterial cells, and strictly follow the instructions in the plasmid miniprep kit for recovery. Finally, suspend the CP3 adsorption column and add 50 µL ddH2O, incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 2 min, collect the liquid, aliquot, seal, label, and store at -80℃ for later use. Use 2 µL of the plasmid solution as a template for drop PCR amplification using the same system and program. After electrophoresis, observe the target band. Select positive plasmids with the target band and send them to BGI Genomics Co., Ltd. for sequencing identification. Analyze the sequencing results using online QUMA software.

[0104] Furthermore, standard plasmids for the ABCA7 and PSEN1 genes were constructed. Treatment with bisulfite caused unmethylated cytosine (C) in DNA to undergo specific deamination, converting it to uracil (U). This U then pairs complementaryly with thymine (T) in subsequent PCR amplification. Methylated cytosine (5mC), due to the steric hindrance of the methyl group (-CH3), could not undergo deamination and remained C. Based on this, corresponding sequences simulating the "fully methylated" (target sites are all C) and "fully unmethylated" (target sites are all T) states were artificially synthesized to construct plasmid standards for methylation detection. The plasmid standard sequences are shown in Table 14.

[0105] Table 14: Plasmid Standard Sequences

[0106] Prepare 2 ng of standard quality particles with 0% and 100% methylation according to the table below, and perform E-ice-COLD-PCR reaction in 20 μl systems. Take three replicates for each concentration and obtain HRM curves and difference graphs.

[0107] Table 15: Mixing Ratio of Standards with Different Degrees of Methylation

[0108] Further, E-ice-COLD-PCR reaction sensitivity testing was performed. 2 ng of standard plasmids with 0% and 100% methylation were mixed 1:1 to obtain a plasmid with 50% methylation. 1 μl of this plasmid was added to 9 μl of dH2O, gently mixed 25 times with a pipette tip, vortexed for 5 seconds, and then briefly centrifuged. This was standard solution 1. 1 μl of standard solution 1 was added to 9 μl of dH2O, gently mixed 25 times with a pipette tip, vortexed for 5 seconds, and then briefly centrifuged. This process was repeated seven times to obtain serially diluted 10⁻⁶ ppm. 7 Using double the standard, perform E-ice-COLD-PCR reaction according to the system in Table 16, take five consecutive points, plot the negative logarithm of concentration-CT value standard curve, substitute the CT value of 30 to calculate the minimum detection concentration and verify it.

[0109] Table 16: E-ice-COLD-PCR Reaction System

[0110] Furthermore, the specificity of the E-ice-COLD-PCR reaction was tested. 2 ng of standard plasmids with methylation levels of 0% and 100% were mixed in different proportions to prepare plasmids with methylation levels of 0%, 1%, 5%, 10%, 25%, 50%, 75%, and 100%. The standards are shown in the table below. E-ice-COLD-PCR reaction was performed in 20 μl systems, and HRM curves and difference graphs were obtained.

[0111] Table 17: Standard Product Mixing Volume Table

[0112] Furthermore, suitable probe concentrations were tested. Real-time quantitative PCR was performed using templates with methylation levels of 0%, 25%, 50%, 75%, and 100%. The optimal concentration was determined using gradient probe concentration experiments of 0 nM, 10 nM, 50 nM, and 100 nM. A line graph was plotted with probe concentration as the abscissa (x) and the corresponding Ct value of each sample as the ordinate (y) to determine the optimal probe concentration for the reaction.

[0113] Example 1:

[0114] This invention tests the clinical application of E-ice-COLD-PCR in serum cfDNA.

[0115] Peripheral blood samples were collected from 40 healthy individuals and 30 Alzheimer's patients at the affiliated hospital between January and December 2025. The study was reviewed and approved by the ethics committee, and all participants fully understood the study content and signed written informed consent forms.

[0116] Blood samples were centrifuged at 2500×g for 12 min, and the clear serum supernatant was transferred to an enzyme-free centrifuge tube, labeled, and sealed for storage. 200 μl of serum was equilibrated to room temperature, and extraction was performed strictly according to the serum / plasma cfDNA extraction kit instructions. The CR2 adsorption column was transferred to a clean centrifuge tube, and 50 μl of elution buffer TB was added dropwise to the center of the adsorption membrane. The tube was incubated at room temperature for 2-5 min, then centrifuged at 12,000 rpm for 2 min. The cfDNA was collected in the centrifuge tube and stored at -20℃ for later use. Bsp-cfDNA was extracted from the above cfDNA and amplified with plasmid standards of different methylation levels according to the E-ice-COLD-PCR system and program. Melting curves, HRM curves, and HRM difference curves were obtained, and the results were plotted statistically.

[0117] Statistical analysis and chart plotting were performed on the above experimental data. One-way ANOVA was used for comparisons between groups of continuous data, and Tukey's multiple comparison test was used for pairwise comparisons. Count data were expressed as rates (%), and the χ² test was used for comparisons between groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered extremely statistically significant. The figures are labeled as: ×P < 0.05, ××P < 0.01, ×××P < 0.001.

[0118] Furthermore, the results of CCK-8 assay showed that SH-SY5Y cells were exposed to 10, 20, 40, and 80 μmol / LAβ... 25-35 After 24 hours of treatment, cellular synapses shortened, and the apoptosis rate increased in a dose-dependent manner with increasing drug concentration. The 20 μmol / L group showed the closest apoptosis rate to 50%, exhibiting the best apoptosis-inducing effect; therefore, this concentration was determined as the optimal concentration for subsequent experiments. Figure 2 and 3 As shown.

[0119] Furthermore, the results of differentially expressed gene screening were tested and statistically analyzed, such as... Figure 4 As shown, an intersection analysis was performed on the AlzData dataset and the cell modeling sequencing results. Differentially expressed genes in the AD transcriptome were identified using Venn diagrams, with 334 upregulated genes and 909 downregulated genes. Volcano plots (blue marking downregulated genes, red marking upregulated genes) and differentially expressed gene heatmaps were further constructed to visually represent the gene expression differences. Ten upregulated and ten downregulated genes were selected for qRT-PCR validation. The results showed that PSEN1, SPSB1, ABCA7, FEZ1, FRY, DPF1, PLD1, CCNB2, CHST8, and CD9 genes were significantly upregulated; while C5AR1, VTCN1, BDNF, CUBN, TYMP, SPOCK3, B3GALT1, OPCML, RASD2, and ADARB2 genes were significantly downregulated, highly consistent with the sequencing results, confirming the reliability and accuracy of the sequencing data. Finally, BDNF, PSEN1, and ABCA7 genes were selected for further testing.

[0120] Based on the above analysis, differentially expressed gene pathways were examined. First, KEGG enrichment analysis was performed on differentially expressed BDNF genes. This invention analyzed 334 upregulated genes and 909 downregulated genes. In the PI3K-Akt signaling pathway, BDNF, as a key upstream neurotrophic factor, activates its high-affinity receptor TrkB, initiating the PI3K-Akt signaling cascade, thereby regulating key processes such as neuronal survival, synaptic plasticity, and metabolism. Results showed that BDNF transcription levels were significantly downregulated in the AD model, and this decreased expression may weaken the neuroprotective function of the PI3K-Akt pathway, thus exacerbating neurological damage.

[0121] The functions of the ABCA family in cholesterol metabolism pathways; the ABCA7 gene, through a function similar to ABCA1, represents cholesterol efflux, mediating the transport of cholesterol and phospholipids to apolipoproteins, and plays a role in Alzheimer's disease-related cholesterol metabolism disorders. Changes in its methylation level may affect lipid metabolism during AD disease development; PSEN1, as the core catalytic subunit of γ-secretase, not only participates in APP cleavage to produce Aβ protein, but is also responsible for the proteolytic activation of Notch receptors. In AD pathology, abnormal PSEN1 function or substrate competition may lead to impaired Notch signaling, thereby affecting neural development, synaptic plasticity, and cell survival. The significant enrichment of the Notch signaling pathway in this invention confirms that this pathway may undergo functional changes in AD models. Figure 6 ).

[0122] This invention extracted genomic DNA from control and experimental cells, respectively, and tested the methylation status of BDNF, PSEN1, and ABCA7 genes in an AD cell model. After bisulfite treatment, the target fragment containing the target CpG island was amplified by PCR. The amplification products were cloned into a plasmid vector, and single-clone sequencing analysis was performed on each sample.

[0123] Sequencing results as follows Figure 7The results showed that, compared with the control group, the experimental group cells exhibited varying degrees of methylation changes at specific CpG sites in the BDNF, PSEN1, and ABCA7 genes. In the AD cell model constructed in this study, the methylation profile of the BDNF gene promoter region was analyzed: the total methylation rate of BDNF in the normal cell control group (n=10) was 4%, while the total methylation rate of BDNF in the AD cell model group (n=10) significantly increased to 10%; the total methylation rate of PSEN1 in the normal cell control group (n=10) was 3.64%, while the total methylation rate in the AD cell model group (Treat group, n=10) was 8.18%; the total methylation rate of ABCA7 in the normal cell control group (n=10) was 12.00%; and the total methylation rate of ABCA7 in the AD cell model group (Treat group, n=10) was 17.33%.

[0124] In some embodiments, Touchdown PCR was used to test amplification conditions and to preliminarily evaluate its detection sensitivity. In the preliminary experimental stage, a single gene fragment of approximately 150 bp was used as the amplification template to rapidly complete the amplification system. In the subsequent formal methodology establishment, to ensure amplification stability, three gene fragments of approximately 200 bp were used for system validation. The same concentration (2 ng) of template was serially diluted in a 1+9 ratio. The amplification products were detected by agarose gel electrophoresis, and the results showed... Figure 8 Touchdown PCR shows specific amplification bands only in the first dilution gradient, with the bands becoming almost nonexistent with further dilutions. Touchdown PCR has certain advantages, with a shorter amplification program, requiring only 1.5 hours to complete the reaction, compared to 2.5 hours for E-ice-COLD-PCR. However, Touchdown PCR has significant limitations in sensitivity: it cannot accurately determine whether the template dilution is uniform or adequate based solely on electrophoresis bands, and is easily affected by factors such as sample mixing and operational errors. Furthermore, Touchdown PCR uses a 1°C cooling rate per cycle, making it impossible to obtain Ct values ​​and amplification curves in real time, hindering accurate quantitative analysis of amplification efficiency and initial template amount.

[0125] In contrast, E-ice-COLD-PCR does not require cycling-level cooling, allowing for complete amplification within the instrument and real-time acquisition of Ct values ​​and amplification curves. E-ice-COLD-PCR consistently amplifies clear bands at various dilution gradients, and the electrophoresis results clearly demonstrate that its sensitivity is significantly superior to Touchdown PCR. Therefore, E-ice-COLD-PCR is superior to Touchdown PCR in sensitivity evaluation.

[0126] Table 18: Comparison of Touchdown PCR and E-ice-COLD-PCR methods

[0127] Furthermore, the optimal annealing temperatures for PCR amplification of the target genes (PSEN1, ABCA7, and BDNF) were determined through testing. Gradient PCR was used to screen annealing temperatures, with the clarity, brightness, and interference of specific bands as evaluation indicators. Annealing temperature parameters were optimized based on agarose gel electrophoresis results. Specific primers for the three genes PSEN1, ABCA7, and BDNF were designed, with annealing temperature gradients of 49℃~53℃ for BDNF, 51℃~56℃ for PSEN1, and 47℃~55℃ for ABCA7, while maintaining other PCR reaction conditions. PCR products were subjected to 1.5% agarose gel electrophoresis (120V, 20min), stained with nucleic acid dyes, and the bands were observed using a gel imaging system.

[0128] Agarose gel electrophoresis results Figure 9 The results showed that the quality of the PCR amplification products of the three genes differed significantly at different annealing temperatures:

[0129] BDNF gene: At an annealing temperature of 51℃, the target band is clear and sharp with the fewest impurities, and the amplification specificity is optimal. Therefore, the optimal annealing temperature for the BDNF gene is set at 51℃.

[0130] PSEN1 gene: At an annealing temperature of 56℃, the amplified bands were clear and bright, consistent with the expected fragment size, and without significant interference from other bands; when the temperature was below 56℃, multiple other bands appeared, indicating poor specificity. The optimal annealing temperature for PSEN1 gene PCR amplification was determined to be 56℃.

[0131] ABCA7 gene: When the annealing temperature is 51℃, the amplification product shows a single specific band with uniform brightness and no contamination from other bands. The optimal annealing temperature for the ABCA7 gene is determined to be 51℃.

[0132] Furthermore, the optimal critical temperature for E-ice-COLD-PCR amplification of (PSEN1, ABCA7, BDNF) was determined using a controlled variable method. The critical temperature parameters were adjusted gradients, with the clarity and brightness of the specific bands of the amplified products and the degree of interference from impurities / primer dimers as the core evaluation indicators, combined with agarose gel electrophoresis results.

[0133] Specific primers for the target genes (PSEN1, ABCA7, BDNF) were used, and critical temperature gradient ranges were designed based on the E-ice-COLD-PCR system: 70℃~80℃ for BDNF, 70℃~86℃ for PSEN1, and 70℃~90℃ for ABCA7; all other reaction conditions remained consistent. After E-ice-COLD-PCR amplification, the products were separated by 1.5% agarose gel electrophoresis (120V, 20min), stained with nucleic acid dyes, and the band characteristics were observed and recorded using a gel imaging system.

[0134] Agarose gel electrophoresis results Figure 10 The results showed that the band quality of the E-ice-COLD-PCR amplification products of the three genes differed significantly under different critical temperature conditions. The specific results are as follows:

[0135] BDNF gene: At a critical temperature of 77℃, the target band is clear and sharp with minimal interference from other bands, resulting in optimal amplification specificity. Therefore, the optimal critical temperature for E-ice-COLD-PCR amplification of the BDNF gene is determined to be 77℃.

[0136] PSEN1 gene: When the critical temperature is 72℃, the amplified bands are clear and bright, the fragment size is consistent with the expectation, and there are no obvious extraneous bands or primer dimer interference. When the temperature is higher than 72℃, primer non-specific binding is enhanced, multiple extraneous bands appear, and the specificity is significantly reduced. Therefore, the optimal critical temperature for PSEN1 gene amplification is determined to be 72℃.

[0137] ABCA7 gene: When the critical temperature is 74℃, the amplification product shows a single specific band, without any impurities, and with uniform brightness. The amplification efficiency and specificity are both at the optimal level. Therefore, the optimal critical temperature for the ABCA7 gene is set to 74℃.

[0138] Furthermore, a precise quantitative standard system was established for the three target genes BDNF, PSEN1, and ABCA7. Fully methylated (FM) and fully unmethylated (FU) plasmid standards were constructed using both enzymatic modification cloning and artificial synthesis methods. The verification results are as follows:

[0139] Enzymatic methylation modification was used: PCR products of BDNF gene amplification with / without methyltransferase treatment were used as templates and cloned into the pMD19-T vector, respectively. Fresh bacterial culture was used for PCR identification, and the PCR products were detected by 1.5% agarose gel electrophoresis. Figure 11 The results showed that the size of the amplified band was consistent with the expected fragment, indicating that the recombinant plasmid had been successfully transformed into the host bacteria and was stably expressed.

[0140] Sequencing verification: Single colonies that were positive for PCR were selected and expanded for culture. Plasmids were extracted and sent to Beijing BGI Co., Ltd. for Sanger sequencing. Figure 12 Sequencing results showed that cytosine at all CpG sites in the fully methylated plasmid standard did not undergo C→T conversion (maintaining methylation), while all CpG sites in the fully unmethylated plasmid standard underwent C→T conversion (unmethylated state), proving that the fully methylated and fully unmethylated plasmid standards of the BDNF gene were successfully constructed.

[0141] Furthermore, PSEN1 and ABCA7 gene plasmid standards were determined. Based on the core principle of bisulfite transformation, fully methylated (PSEN1-100, ABCA7-100) and fully unmethylated (PSEN1-0, ABCA7-0) gene sequences were designed and optimized. Standardized synthesis was commissioned to Shanghai Sangon Biotech Co., Ltd. The synthesized fragments were confirmed to be completely identical to the designed sequences, with no base mutations, deletions, or insertions, meeting the quality requirements for standards. Figure 13A , 13B 13C, 13D).

[0142] Furthermore, a standard system for the degree of full methylation is constructed. This invention uses fully methylated and unmethylated plasmids of the BDNF, PSEN1, and ABCA7 genes that have been verified to be qualified to be mixed in a gradient according to a preset ratio to prepare a series of standards with gradient methylation levels.

[0143] After E-ice-COLD-PCR amplification of the above-mentioned gradient methylation standards, high-resolution melting curve (HRM) technology was used for detection and analysis, obtaining melting curves and high-resolution melting curves for each standard. Based on the principle of bisulfite conversion, the base composition of the gene sequence of standards with different degrees of methylation differs significantly after treatment: in the unmethylated standards, all C at the CpG sites are converted to U, corresponding to T after subsequent PCR amplification; in the fully methylated standards, the cytosine at the CpG sites remains methylated, and remains C after PCR. The number of hydrogen bonds formed by C and G pairing (3) is greater than that between T and A pairing (2). This difference in the number of hydrogen bonds directly leads to different Tm values ​​in the DNA double strand, resulting in differences in the peak positions of the melting curves for standards with different degrees of methylation.

[0144] The results showed that as the degree of methylation of the standard gradually increased, the corresponding melting curve Tm value continuously increased.

[0145] Furthermore, sensitivity tests were performed on the BDNF, PSEN1, and ABCA7 genes. Starting with a standard concentration of 2 ng / μL (50% methylation), the concentrations were serially diluted tenfold each time to obtain a gradient concentration. Real-time quantitative PCR was used to measure the corresponding Ct values, and the effective concentrations of each gradient were converted to logarithmic form (log). 10 A standard curve was fitted using linear regression analysis with the logarithm of concentration as the X-axis and Ct value as the Y-axis. When the Ct value is ≥30, the amplified fragment efficiency cannot be guaranteed, so the concentration corresponding to Ct of 30 is the limit of detection.

[0146] BDNF gene: y = -3.38x + 8.46, the target fragment length of this gene is 212 bp, and the vector is pMD-19T (2692 bp). Substituting y = 30 into the above equation, the corresponding concentration logarithm is x = -6.37, which further converts to an effective concentration of 10 - 6.37 = 4.27 × 10⁻⁶. -7 ng / μL. Based on the total length of the target fragment-vector (212bp + 2692bp = 2904bp), and according to the double-stranded DNA molecular weight formula M = number of base pairs × 649 Da / bp, the molecular weight of a single copy is calculated to be approximately 2904 × 649 = 1.885 × 10⁶ Da. Using Avogadro's constant NA = 6.022 × 10⁻²³ mol / L, the calculated single-copy mass is 3.13 × 10⁻¹⁸ g / copy. Therefore, the copy number concentration corresponding to this detection limit is 136 copies / μL, and the detection limit concentration is 4.27 × 10⁻¹⁸ g / μL. -7 ng / μL.

[0147] Based on the above calculation method, for the PSEN1 gene: y = -5.783x - 1.7841, the length of the target fragment of this gene is 226bp, and the vector is PCDNA3.4 (5428bp). Finally, the copy number concentration corresponding to the detection limit is obtained as 518 copies / μl, and the detection limit concentration is 3.16×10-6ng / μL.

[0148] ABCA7 gene: y = -2.78x + 15.5, target fragment length is 215 bp, vector is PCDNA3.4 (5428 bp), the final copy number concentration corresponding to this detection limit is 992 copies / μl, and the detection limit concentration is 6.03 × 10⁻⁶. - 6 ng / μL.

[0149] Furthermore, specificity tests were conducted. Standard quality particles with different degrees of methylation were mixed according to the proportions in Table 14, and high-resolution melting curves were obtained. The results showed that E-ice-COLD-PCR could distinguish samples with methylation as low as 1%.

[0150] Furthermore, the optimal probe concentration was tested. Locked nucleic acid probes for E-ice-COLD-PCR specifically bound to and blocked the amplification of unmethylated sites (without affecting methylated sites). Gradient probe concentrations of 0 nM, 10 nM, 50 nM, and 100 nM were set, and real-time quantitative PCR was performed using templates with methylation levels of 0%, 25%, 50%, 75%, and 100%, respectively. A line graph was plotted with probe concentration as the x-axis and the corresponding Ct value for each sample as the y-axis. The optimal concentration was determined according to the following criteria:

[0151] (1) The Ct value of the unmethylated sample is >30 (no effective amplification);

[0152] (2) The Ct values ​​of the fully methylated samples were not significantly different from those of the probe-free control group;

[0153] (3) As the methylation ratio of the sample increases, the Ct value gradually decreases and approaches the Ct value of the sample without probe (the probe only specifically blocks unmethylated sites).

[0154] As shown in the figure, for all three genes, the blocking effect weakens as the methylation ratio of the sample increases when the probe concentration is 10 nM. Unmethylated genes are completely blocked, while fully methylated genes are unaffected. Therefore, the optimal probe concentration is 10 nM.

[0155] Table 19: BDNF gene methylation level - probe Ct value

[0156] Table 20: PSEN1 gene methylation level - probe Ct value

[0157] Table 21: ABCA7 gene methylation level - probe Ct value

[0158] In some embodiments, serum samples were collected from 40 healthy controls and 30 patients (AD group). cfDNA was separated from the serum using a cfDNA extraction kit. The extracted product was subjected to bisulfite conversion treatment. Based on the methylation characteristics of the target genes in the Methbank 4.0 database, a series of methylation ratio standards for each gene were prepared. Using the converted cfDNA samples and the series of methylation standards for each gene as templates, E-ice-COLD-PCR technology was used for amplification.

[0159] Standard product settings:

[0160] The BDNF gene was set with methylation gradients of 0%, 10%, 25%, 50%, 75%, and 100%.

[0161] The PSEN1 gene was set with methylation gradients of 0%, 5%, 10%, 25%, 50%, 75%, and 100%.

[0162] The ABCA7 gene was set with methylation gradients of 0%, 15%, 25%, 50%, 75%, and 100%.

[0163] BDNF gene methylation: In the healthy group (40 cases), 100% of the methylation gradient was concentrated in the 0%–10% range; in the AD group (30 cases), 93.33% were in the 0%–10% methylation gradient, and 6.67% were in the 10%–25% methylation gradient. The difference between the groups was P<0.05, indicating a statistically significant difference in BDNF methylation levels between the two groups.

[0164] PSEN1 gene methylation: In the healthy group (40 cases), 72.50% had a methylation gradient of 0%–5%, and 27.50% had a methylation gradient of 5%–10%; in the AD group (30 cases), 40.00% had a methylation gradient of 0%–5%, and 60.00% had a methylation gradient of 5%–10%. Intergroup difference: P<0.05, indicating a statistically significant difference in PSEN1 methylation levels between the two groups.

[0165] ABCA7 gene methylation: In the healthy group (40 cases), 82.50% had a methylation gradient of 0%–15%, and 17.50% had a methylation gradient of 15%–25%. In the AD group (30 cases), 10.00% had a methylation gradient of 0%–15%, 80.00% had a methylation gradient of 15%–25%, and 10.00% had a methylation gradient of 25%–50%. Intergroup difference: P<0.001, indicating a more significant difference in ABCA7 methylation levels between the two groups.

[0166] Overall, the methylation levels of BDNF, PSEN1, and ABCA7 genes in the AD group were all different from those in the healthy group, and the differences between the groups were statistically significant. See the results figures and statistical data below. Figure 21-23 Table 22-24.

[0167] Table 22: Intergroup comparison of BDNF gene methylation levels

[0168] Table 23: Intergroup comparison of PSEN1 gene methylation levels

[0169] Table 24: Intergroup comparison of ABCA7 gene methylation levels

[0170] This invention uses intersection analysis of the AlzData database and transcriptome sequencing results to screen for three disease-related genes: BDNF, PSEN1, and ABCA7. PSEN1 mutations associated with AD exhibit high clinical heterogeneity. Mutations or epigenetic abnormalities in PSEN1 can lead to dysregulation of γ-secretase activity, thereby promoting Aβ peptide aggregation, which is one of the core mechanisms of AD pathogenesis. Its DNA methylation pattern is significantly correlated with Alzheimer's disease pathology. Abnormal methylation of the ATP-binding cassette subfamily A member 7 (ABCA7) is significantly associated with imbalances in brain lipid metabolism and impaired Aβ clearance. Its peripheral blood methylation level can reflect pathological changes in the central nervous system and is one of the AD-related risk genes. From a neuroprotective perspective, brain-derived neurotrophic factor (BDNF) secretion in the synaptic cleft is regulated by activity-dependent changes and long-term enhancement, and it prevents neuronal cell death caused by the aggregation of Aβ and tau proteins in AD through neuronal regulation. As a neurotrophic factor, methylation silencing of BDNF leads to decreased synaptic plasticity and weakened neuronal survival, which is directly related to cognitive decline in AD patients. Methylation of the BDNF gene promoter in peripheral blood is crucial.

[0171] Most AD-related methylation abnormalities are subtle, and conventional detection techniques are difficult to detect accurately due to low sensitivity. This technical bottleneck severely limits the clinical application of methylation biomarker detection. E-ice-COLD-PCR technology uses LNA blocking probes to specifically inhibit the amplification of unmethylated templates, and can sensitively detect methylated DNA as low as 1%, meeting the basic requirements for clinical DNA methylation detection.

[0172] This invention optimizes key parameters such as annealing temperature, critical temperature, and probe concentration, and establishes a standard methylation detection system for three core genes: BDNF, PSEN1, and ABCA7. Results show that the detection limit of this system is as low as 4.27 × 10⁻⁻⁻⁶. 7 ~6.03×10⁻ 6 With a specificity of ng / μL, corresponding to copy number concentrations of 136–992 copies / μL, this method can clearly distinguish methylated samples with different gradients from 1% to 100%. Its specificity and sensitivity are significantly superior to traditional bisulfite sequencing and other techniques. Furthermore, the designed LNA probe can efficiently block the amplification of unmethylated templates without affecting the amplification of methylated templates, ensuring the accuracy and reliability of the detection results.

[0173] This invention applies the established E-ice-COLD-PCR technology to serum cfDNA detection, enabling the detection of different degrees of methylation in healthy individuals and AD patients. In the ABCA7 gene, ABCA7 methylation is increased in the AD group, but mRNA is upregulated. However, in terms of the distribution of methylation levels, in the healthy group, 82.50% are in the 0%–15% range, and 17.50% are in the 15%–25% range; in the AD group, 10.00% are in the 0%–15% range, 80.00% are in the 15%–25% range, and 10.00% are in the 25%–50% range. The difference between groups is P<0.001. This reflects a continuous change in epigenetic modifications during the transition from healthy individuals to AD patients, indicating a gradual shift in epigenetic modifications as the disease progresses. It is not a zero-sum game between health and disease, but rather a dynamic change that occurs with the development of diseases such as Aβ protein deposition and neuronal damage. Among them, ABCA7 is a key gene for Aβ protein clearance. When Aβ protein deposition increases in AD, cells will initiate compensation by upregulating the expression of ABCA7 gene mRNA to enhance the clearance of Aβ protein. At this time, the slight increase in methylation does not reach the threshold for inhibiting gene expression, so it ultimately results in a small increase in methylation and upregulation of mRNA. Increased methylation does not necessarily represent transcriptional repression. Moreover, this invention detects the methylation level of the promoter region, which reflects the role of the ABCA7 gene in AD pathology.

[0174] The results showed that the methylation levels of all three gene groups in the serum cfDNA of AD patients were significantly associated with the onset of AD (P<0.05 or P<0.001). This result confirms that the changes in methylation levels in the cell model are consistent with those in clinical samples. Compared with cerebrospinal fluid puncture and brain tissue biopsy in early AD screening, serum cfDNA-based methylation detection is safer, more convenient, and more suitable for early AD screening in primary healthcare institutions, providing a new method and approach for early detection and intervention of AD.

[0175] The above description is a further detailed explanation of the present invention in conjunction with specific preferred embodiments. For those skilled in the art, several simple deductions or substitutions can be made without departing from the present invention, and all such deductions or substitutions should be considered as falling within the scope of patent protection determined by the submitted claims.

Claims

1. A method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology, characterized in that, Includes the following steps: S1. Extract cfDNA from the sample to be tested; S2. Screen for target genes and perform bisulfite conversion on the extracted cfDNA, wherein the target gene is at least one of PSEN1, BDNF and ABCA7; S3. Using the transformed DNA as a template, amplification is performed using the E-ice-COLD-PCR amplification system. The amplification system contains amplification primers targeting the target gene and blocking probes containing locked nucleic acids. The BDNF probe sequence is shown in SEQ ID NO:7, the PSEN1 probe sequence is shown in SEQ ID NO:8, and the ABCA7 probe sequence is shown in SEQ ID NO:

9. S4. Analyze the degree of methylation using high-resolution melting curves.

2. The method for detecting Alzheimer's disease-related gene methylation based on E-ice-COLD-PCR technology according to claim 1, characterized in that, The LNA blocking probe in S3 is designed to meet the following requirements: length 40–65 nt, 3' end overlap with primer 5–6 nt, and LNA bases introduced at all unmethylated CpG sites.

3. The method for detecting Alzheimer's disease-related gene methylation based on E-ice-COLD-PCR technology according to claim 1, characterized in that, The detection limit of the E-ice-COLD-PCR system in S3 is 4.27 × 10⁻⁶. -7 -6.03×10 - 6 The concentration of ng / μL corresponds to a copy number concentration of 136-992 copies / μL, which can distinguish samples with methylation gradients of 1%-100%.

4. The method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology according to claim 1, characterized in that, The concentration of the blocking probe containing locked nucleic acid is 10 nM, and the 3′ end of the probe is modified with a phosphate group to specifically bind to unmethylated CpG sites and block their amplification.

5. The method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology according to claim 1, characterized in that, The amplification primers have the following sequences: The forward primer for the BDNF gene is shown in SEQ ID NO:1; The reverse primer for the BDNF gene is shown in SEQ ID NO:2; The forward primer for the PSEN1 gene is shown in SEQ ID NO:3; The reverse primer for the PSEN1 gene is shown in SEQ ID NO:4; ABCA7 gene forward primer: as shown in SEQ ID NO:5; ABCA7 gene reverse primer: as shown in SEQ ID NO:

6.

6. The method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology according to claim 1, characterized in that, The E-ice-COLD-PCR amplification system also contains EvaGreen fluorescent dye, which is used to monitor the amplification curve and melting curve in real time.

7. The method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology according to claim 1, characterized in that: The degree of methylation is determined by comparing the high-resolution melting curves with pre-constructed fully methylated and fully unmethylated plasmid standards; the fully methylated and fully unmethylated plasmid standards have sequences as shown in SEQ ID NO:10 to SEQ ID NO:

15.

8. The method for detecting methylation of Alzheimer's disease-related genes based on E-ice-COLD-PCR technology according to claim 7, characterized in that, The fully methylated and fully unmethylated plasmid standards were constructed through enzymatic modification cloning or artificial synthesis, wherein: The sequence of the fully methylated PSEN1 standard is shown in SEQ ID NO:10, and the sequence of the fully unmethylated PSEN1 standard is shown in SEQ ID NO:11; The sequence of the fully methylated ABCA7 standard is shown in SEQ ID NO:12, and the sequence of the fully unmethylated ABCA7 standard is shown in SEQ ID NO:13; The sequence of the fully methylated BDNF standard is shown in SEQ ID NO:14, and the sequence of the fully unmethylated BDNF standard is shown in SEQ ID NO:

15.

9. The application of the Alzheimer's disease-related gene methylation detection method based on E-ice-COLD-PCR technology according to any one of claims 1-8, characterized in that, A kit was prepared using this method, and the kit is suitable for the combined detection of methylation of PSEN1, BDNF, and ABCA7 genes in serum cfDNA.

10. The application of the Alzheimer's disease-related gene methylation detection method based on E-ice-COLD-PCR technology according to any one of claims 1-8 in the preparation of early screening products for Alzheimer's disease.

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