A method for detecting SNP loci related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 and its application

Abstract

This invention relates to the field of biodetection technology, specifically to a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, and its application. The detection method includes the following steps: S1, obtaining genomic DNA of the sample to be tested; S2, LDR ligase detection reaction: providing three sets of primers for the target SNP site, including a first ligation primer P1, a second ligation primer P3, and a third ligation primer P2, wherein the 3' end base of P1 is complementary to the wild-type base of the target SNP site; the 3' end base of P3 is complementary to the mutant base of the target SNP site; the 5' end of P2 is phosphorylated; using genomic DNA as a template, under the action of DNA ligase, the 3' hydroxyl group of P1 or P3 is covalently linked to the 5' phosphate group of P2 to form a ligation product; S3, PCR polymerase chain reaction: using the ligation product obtained in step S2 as a template, PCR amplification is performed using amplification primers to obtain the amplification product; S4, detecting the amplification product, determining the genotype, and applying the product to AD genetic risk screening or early auxiliary assessment.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive development, mainly manifested as memory loss, cognitive impairment, and behavioral abnormalities. With the increasing aging of the global population, the prevalence of AD is rising year by year, becoming a major public health problem that seriously threatens the health of the elderly and increases the social medical burden. Statistics show that the number of AD patients worldwide has exceeded 55 million. Although early-onset Alzheimer's disease (EOAD, onset age <65 years) accounts for about 5%-10%, its early onset, rapid progression, and significant genetic predisposition make it a key breakthrough for understanding the pathogenesis of AD and implementing early intervention.

[0003] The PSEN1 and PSEN2 genes are core pathogenic genes for early-onset AD (EOAD), encoding γ-secretase subunits that directly participate in the processing and metabolism of β-amyloid protein (Aβ). Single nucleotide polymorphisms (SNPs) in these two genes alter γ-secretase activity, affecting the Aβ42 / Aβ40 ratio, thereby promoting Aβ deposition and neurofibrillary tangles formation, ultimately driving the pathological progression of AD. Therefore, detecting SNP sites in the PSEN1 and PSEN2 genes has significant clinical value for early risk assessment of AD, screening of high-risk populations, and personalized intervention.

[0004] Existing SNP detection technologies have many limitations in clinical applications of Alzheimer's disease (AD). Traditional PCR methods rely on DNA polymerase to recognize bases, but DNA polymerase has a high tolerance for single-base mismatches, easily leading to non-specific amplification and false positives, resulting in insufficient detection specificity, especially with significant errors when detecting low-frequency mutations. While high-resolution melting curve (HRM) methods are relatively simple to operate, their results are highly dependent on instrument accuracy and operator skill; even small deviations in melting temperature (e.g., 0.5℃) can cause genotyping errors, resulting in poor repeatability and stability, and difficulty in distinguishing heterozygotes differing by only 1-2 bases. Probe methods (such as TaqMan) require fluorescently labeled specific probes, which are complex to design and expensive, and lack sufficient sensitivity for low-concentration samples and low-frequency mutations (e.g., mutation frequency <5%), failing to meet the needs of detecting small clinical samples.

[0005] Therefore, there is an urgent need to invent a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 and its application to solve the above-mentioned technical problems. Summary of the Invention

[0006] This invention relates to a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, specifically targeting the rs362344 and rs165934 sites of the PSEN1 gene and / or the rs8383 and rs12123818 sites of the PSEN2 gene, effectively solving the aforementioned technical problems.

[0007] This invention proposes a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, comprising the following steps:

[0008] S1. Obtain the genomic DNA of the sample to be tested;

[0009] S2, LDR ligase detection reaction: Three sets of primers targeting the target SNP site are provided, including wild-type ligation primer P1, mutant ligation primer P3, and shared ligation primer P2, which are designed and synthesized.

[0010] The 3' end base of P1 is complementary to the wild-type base of the target SNP site;

[0011] The 3' end base of P3 is complementary to the mutant base of the target SNP site;

[0012] The 5' end of P2 is modified by phosphorylation;

[0013] Using the genomic DNA as a template, under the action of DNA ligase, the 3' hydroxyl group of P1 or P3 is covalently linked to the 5' phosphate group of P2 to form a ligation product;

[0014] S3. PCR polymerase chain reaction: Using the ligation product obtained in step S2 as a template, PCR amplification is performed using amplification primers to obtain the amplification product.

[0015] S4. Detect the amplification product and determine the genotype.

[0016] Preferably, the ligation product in S2 is a single-stranded nucleic acid or a double-stranded nucleic acid, and its structure includes:

[0017] The first nucleic acid segment has a nucleotide sequence that at least partially overlaps with the nucleotide sequence of P1 or P3, and its 3' end contains the discriminant base of the target SNP site;

[0018] The second nucleic acid segment has a nucleotide sequence that at least partially overlaps with the nucleotide sequence of P2, and its 5' end is phosphorylated.

[0019] The first nucleic acid segment and the second nucleic acid segment are covalently linked by a phosphodiester bond; the length of the ligation product is 80-100 nt.

[0020] Preferably, the 5' ends of P1 and P3 also contain a universal sequence that does not contain the genomic sequence of the target SNP site; the 3' end of P2 also contains a universal sequence; the amplification primers in step S3 specifically recognize and bind to the universal sequence.

[0021] Preferably, the amplification primers include forward amplification primer F1 and reverse amplification primer R1; wherein the sequence of F1 is selected from any one of the sequences shown in SEQ ID NO:1, 3, 5, 7, and the sequence of R1 is selected from any one of the sequences shown in SEQ ID NO:2, 4, 6, 8.

[0022] Preferably, the three sets of primers mentioned in step S2 are designed for the rs362344 and rs165934 sites of the PSEN1 gene and / or the rs8383 and rs12123818 sites of the PSEN2 gene.

[0023] The primer sequences for P1, P3, and P2 targeting the rs362344 site are shown in SEQ ID NO:9, 11, and 10, respectively; the primer sequences for P1, P3, and P2 targeting the rs165934 site are shown in SEQ ID NO:18, 20, and 19, respectively; the primer sequences for P1, P3, and P2 targeting the rs12123818 site are shown in SEQ ID NO:15, 17, and 16, respectively; and the primer sequences for P1, P3, and P2 targeting the rs8383 site are shown in SEQ ID NO:12, 14, and 13, respectively.

[0024] Preferably, the reaction conditions for step S2 are: ligation temperature 65-68℃, ligation time 3 min, number of cycles 30, primer concentration ratio P1 to P2 concentration ratio 2:1, and P3 to P2 concentration ratio 2:1.

[0025] Preferably, the detection in step S4 includes high-resolution melting curve analysis and / or agarose gel electrophoresis. For PSEN1-rs362344, high-resolution melting curve analysis determines that the wild type genotype shows a specific melting peak at 81.0℃, and the mutant genotype shows a specific melting peak at 79.6℃. Agarose gel electrophoresis shows that the wild type amplification product is 80 bp in length, and the mutant amplification product is 79 bp in length.

[0026] Preferably, the method has a detection limit of 0.02 pg / μL genomic DNA and can detect mutation frequencies as low as 0.5%.

[0027] Preferably, a kit is provided, comprising:

[0028] P1, a set of ligation primers for the LDR reaction, the primer set being designed for at least one SNP site selected from PSEN1-rs362344, PSEN1-rs165934, PSEN2-rs12123818, and PSEN2-rs8383, including wild-type ligation primer P1, mutant ligation primer P3, and a 5' phosphorylated common ligation primer P2; and amplification primers F1 and R1 that are paired with the primers;

[0029] P2, Taq DNA ligase;

[0030] P3, DNA polymerase;

[0031] P4, a universal primer pair for PCR amplification, wherein the universal primers are complementary to the 5' universal sequences of P1 / P3.

[0032] Preferably, the method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 is applied to Alzheimer's disease genetic risk screening or early auxiliary assessment.

[0033] This invention proposes a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, which has the following beneficial effects:

[0034] This invention proposes a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2. Utilizing the stringent recognition ability of DNA ligase for single-base mismatches, this invention designs a ligation system comprising a wild-type specific P1 primer and a mutant-type specific P3 primer. Only when the 3' end of the primer perfectly matches the template SNP bases can the ligase catalyze the formation of a phosphodiester bond between P1 / P3 and P2, producing the ligation product; if a mismatch exists, the ligation reaction fails, and no template is amplified. Experiments show that the detection specificity of this invention for the PSEN1 rs362344 and PSEN2 rs12123818 sites is completely consistent with Sanger sequencing results and significantly superior to HRM technology.

[0035] This invention introduces universal sequence-mediated PCR amplification, transforming the ligation product into a stable amplification template, significantly improving detection sensitivity. Experimental data show that the detection limit of this invention reaches 0.02 pg / μL for wild-type templates and 0.015 pg / μL for mutant templates, stably detecting mutation frequencies as low as 0.5%. This sensitivity far exceeds traditional PCR and HRM techniques, accurately detecting low-frequency mutations in trace clinical samples such as peripheral blood. This invention requires no fluorescently labeled probes; detection can be completed using only a conventional PCR instrument and gel electrophoresis or melting curve analysis. The use of dual-tube parallel reactions (wild-type / mutant primer grouping) provides intuitive result interpretation, avoiding complex data analysis. Furthermore, the universal primer design reduces synthesis costs, making it suitable for promotion in primary healthcare institutions.

[0036] This invention designs a dedicated primer combination targeting four key SNP loci in PSEN1 (rs362344, rs165934) and PSEN2 (rs8383, rs12123818), enabling simultaneous detection of multiple targets. Clinical sample validation showed that the genotype distribution of PSEN1 rs362344 and PSEN2 rs12123818 loci differed significantly between the AD group and the normal group (P<0.05), serving as independent genetic risk markers for AD and providing reliable molecular targets for early AD screening. By replacing the 3' SNP recognition region of the connecting primers (P1 / P3), detection of any SNP locus can be achieved, demonstrating strong technical versatility and broad application prospects. Attached Figure Description

[0037] Figure 1 Figure 1 shows the results of SNP primer verification. M: 500bp marker. (A) shows the PCR amplification results verified by PSEN1-rs362344 primer; (B) shows the PCR amplification results verified by PSEN1-rs165934 primer; (C) shows the PCR amplification results verified by PSEN2-rs8383 primer; (D) shows the PCR amplification results verified by PSEN2-rs12123818 primer.

[0038] Figure 2 Sequencing diagrams of wild-type and mutant plasmid standards for each gene SNP site;

[0039] Figure 3 This is a schematic diagram of the LDR-PCR detection of SNPs in this invention;

[0040] Figure 4 The following is a feasibility diagram for LDR-PCR detection of rs362344 in this invention: (A) a graph of LDR-PCR product gel electrophoresis verification, and (B) melting curves of wild-type and mutant plasmids and genomic DNA for LDR-PCR.

[0041] Figure 5 The following is a feasibility diagram for LDR-PCR detection of rs8383 in this invention: (A) Electrophoresis results of LDR-PCR products, (B) Melting curve of LDR-PCR products.

[0042] Figure 6 The following is a feasibility diagram for LDR-PCR detection of rs165934 in this invention: (A) Electrophoresis results of LDR-PCR products, (B) Melting curve of LDR-PCR products.

[0043] Figure 7 The following is a feasibility diagram for LDR-PCR detection of rs12123818 in this invention: (A) Electrophoresis results of LDR-PCR products, (B) Melting curve of LDR-PCR products.

[0044] Figure 8 The following are measurement views of the bonding products of PSEN1-rs362344 at different bonding temperatures: (A) is the electrophoresis result, (B) is the melting curve result, and (C) is the peak result statistics.

[0045] Figure 9 The diagram shows the optimized connection temperature of the remaining SNP sites in this invention, where (A) rs8383 connection temperature is optimized; (B) rs12123818 connection temperature is optimized; and (C) rs165934 connection temperature is optimized.

[0046] Figure 10 This is a diagram showing the optimization of the ligation time of PSEN1-rs362344 in this invention. The diagram shows the electrophoresis results of wild-type and mutant ligation products at different ligation times; (A) melting curve results; and (B) peak result statistics.

[0047] Figure 11 This is an optimization diagram of the number of iterations for PSEN1-rs362344 in this invention;

[0048] Figure 12 This is a diagram showing the optimized concentration ratio of the PSEN1-rs362344 ligation primers in this invention.

[0049] Figure 13 This is a diagram showing the optimized amplification and annealing temperature for the LDR product of this invention.

[0050] Figure 14 The optimal annealing temperature for the remaining sites of the LDR product of this invention is shown in the following diagram: (A) rs8383; (B) rs8383; (C) rs12123818; (D) rs12123818; (E) rs165934; (F) rs165934.

[0051] Figure 15 This is a sensitivity diagram of the LDR-PCR detection of the wild-type rs362344 template in this invention;

[0052] Figure 16 This is a sensitivity diagram of the LDR-PCR detection of the rs362344 mutant template in this invention;

[0053] Figure 17 This is a sensitivity diagram of LDR-PCR detection of the rs8383 mutant template in this invention;

[0054] Figure 18 This is a sensitivity diagram of LDR-PCR detection of the wild-type rs8383 template in this invention;

[0055] Figure 19 This is a sensitivity diagram of LDR-PCR detection of the rs12123818 mutant template in this invention;

[0056] Figure 20 This is a sensitivity diagram of LDR-PCR detection of the wild-type template rs12123818 in this invention;

[0057] Figure 21 This is a sensitivity diagram of the LDR-PCR detection of the rs165934 wild-type template in this invention;

[0058] Figure 22 This is a sensitivity diagram of LDR-PCR detection of the rs165934 mutant template in this invention;

[0059] Figure 23 This is a graph showing the mutation frequency of rs362344 detected by LDR-PCR in this invention.

[0060] Figure 24 This is a graph showing the mutation frequency of rs8383 detected by LDR-PCR in this invention.

[0061] Figure 25 This is a graph showing the mutation frequency of rs12123818 detected by LDR-PCR in this invention.

[0062] Figure 26 This is a graph showing the mutation frequency of rs165934 detected by LDR-PCR in this invention.

[0063] Figure 27 The images show partial clinical sample results of LDR-PCR detection of rs362344 in this invention. (A) Wild-type and mutant samples showed the target peak when using matched primers. (B) The sample with the melting peak at 79.6℃ in Figure A was detected with mismatched primers (P1 / P2). (C) The sample with the melting peak at 81℃ in Figure A was detected with mismatched primers (P3 / P2). (D) The sample was detected as heterozygous. (E) Partial sequencing results of the corresponding sample.

[0064] Figure 28 This is a partial image of clinical sample results for LDR-PCR detection of rs8383 according to the present invention;

[0065] Figure 29 This is a graph showing the results of LDR-PCR detection of rs12123818 in some clinical samples according to the present invention.

[0066] Figure 30 This is a partial image of clinical sample results for LDR-PCR detection of rs165934 according to the present invention;

[0067] Figure 31 This is a graph showing the HRM detection results for four types of SNPs in this invention;

[0068] Figure 32 This is a diagram showing the distribution of the four SNP genotypes in this invention. Detailed Implementation

[0069] This invention proposes a method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, targeting the rs362344 and rs165934 sites of the PSEN1 gene and / or the rs8383 and rs12123818 sites of the PSEN2 gene.

[0070] As per the instruction manual Figures 1-32 As shown, a method for preparing SNP loci related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 is proposed, including the following steps:

[0071] S1. Obtain the genomic DNA of the sample to be tested;

[0072] S2, LDR ligase detection reaction: Three sets of primers targeting the target SNP site are provided, including wild-type ligation primer P1, mutant ligation primer P3, and shared ligation primer P2, which are designed and synthesized.

[0073] The 3' end base of P1 is complementary to the wild-type base of the target SNP site;

[0074] The 3' end base of P3 is complementary to the mutant base of the target SNP site;

[0075] The 5' end of P2 is modified by phosphorylation;

[0076] Using the genomic DNA as a template, under the action of DNA ligase, the 3' hydroxyl group of P1 or P3 is covalently linked to the 5' phosphate group of P2 to form a ligation product;

[0077] S3. PCR polymerase chain reaction: Using the ligation product obtained in step S2 as a template, PCR amplification is performed using amplification primers to obtain the amplification product.

[0078] S4. Detect the amplification product and determine the genotype.

[0079] In the specific experiment, the following steps are further included:

[0080] The main reagents prepared include: 200 mg / mL IPTG (isopropyl-β-D-thiogalactoside), 5×TBE electrophoresis buffer, 100 mg / mL ampicillin sodium, 2% agarose gel, 30% acrylamide solution, 1×TBE electrophoresis buffer, 20 mg / mL X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside), LB solid medium, and LB liquid medium.

[0081] This invention selects patients with cognitive impairment or dementia who visited the Affiliated Hospital of Beihua University between January 2025 and November 2025, as well as members of the general population who underwent health checkups at the same hospital during the same period, as research subjects. The invention protocol has been reviewed and approved by the Ethics Committee of Beihua University, and all selected subjects signed written informed consent forms after fully understanding the research process and related matters.

[0082] All patients with cognitive impairment or dementia were diagnosed by neurologists according to recognized clinical diagnostic procedures, and standardized cognitive function assessments were completed using the MMSE and MoCA scales. Patients with other types of dementia, such as vascular dementia, frontotemporal dementia, and Lewy body dementia, as well as those with severe mental illness or unable to cooperate with the examination, were excluded. The healthy control group consisted of individuals who underwent cognitive assessments to exclude those with subjective cognitive decline and had no family history of dementia or neurological diseases. Based on pre-defined inclusion and exclusion criteria, blood samples from 25 eligible cases and 40 from the healthy control group were included.

[0083] Common susceptibility SNP sites for Alzheimer's disease (AD) were identified, specifically two SNP sites with relatively high mutation frequencies in the AD susceptibility genes PSEN1 and PSEN2. Simultaneously, the corresponding base sequences upstream and downstream of each gene site were collected, yielding the following results: rs362344 genotype: wild-type C, mutant T; rs8383 genotype: C, mutant T; rs165934 wild-type genotype: C, mutant A; rs12123818 wild-type genotype: G, mutant A.

[0084] DNA was extracted from the test subjects, and PCR amplification and product sequencing were performed. Two mL of fasting venous blood was collected from each subject and placed in an anticoagulant tube for temporary storage at -20°C. Total DNA was isolated from the blood samples using a genomic DNA extraction kit. The concentration and purity of the obtained DNA were detected using a UV-Vis spectrophotometer. Qualified samples were then transferred to an ultra-low temperature freezer at -80°C for subsequent experiments. Sequence information of two SNP sites each of the human AD-related susceptibility genes PSEN1 and PSEN2 was retrieved from the NCBI database. Site-specific PCR primers were designed using the NCBI Primer online tool, and the secondary structure and binding energy of each primer oligonucleotide were analyzed and predicted using the Yunzhou Bio platform.

[0085] The primer sequences, PCR reaction system, and amplification procedure used in this invention are shown in Tables 1 and 2, respectively. After amplification, the PCR products were subjected to 2% agarose gel electrophoresis. The fragment size was determined based on the migration position of the target band to verify whether the specificity and length of the amplified products met expectations.

[0086] Table 1 PCR primer sequences

[0087] Table 2 PCR reaction system DNA high-fidelity polymerase 10 template 2 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 Ultrapure water 6 Total volume 20

[0088] Table 3 PCR reaction procedure

[0089] Cloning and sequencing were performed using the following procedure:

[0090] ① Take the PCR products of different genes from different individuals obtained above, take 1.5 ml of EP tube and add 1 μl of T-vector PMD, 4 μl of PCR product and 5 μl of DNA Ligation Kit-Ⅰ, mix thoroughly and react in a metal bath at 16℃ for 30-40 min.

[0091] ② Add 100 μL of competent cells to an EP tube, gently pipette to mix, and then place it in an ice bath for 30 minutes. Then heat shock it in a 42°C water bath for 1 minute, and then quickly transfer it to ice to cool for 1-2 minutes.

[0092] ③ Add 940 μL of ampicillin-free LB medium to the system and incubate with shaking at 37°C and 150 r / min for 1 h. Then centrifuge at 8000 r / min for 3 min, remove the supernatant, and resuspend and mix the precipitate.

[0093] ④ Mix the remaining 100ul of bacterial culture with a pipette, and place 50ul on each of two LB solid media that have been coated with X-gal, IPTG and ampicillin. Repeat the spreading until the bacterial culture is dry. Incubate the culture media in a 37°C incubator in the dark. Keep the culture upright for the first 30 minutes, and then invert it for 16-18 hours.

[0094] ⑤ Take out the liquid culture medium that has been autoclaved and stored at 4℃, add 100-150ul Amp, and shake thoroughly to mix. Dispense the mixed liquid culture medium into test tubes (3ml / tube).

[0095] ⑥ Remove the pre-prepared culture medium and select plump, pure white single colonies. If the number of white single colonies on the plate is small, the plate can be placed in a 4°C refrigerator for 10 to 20 minutes to promote colony development. Subsequently, the selected white single colonies are inoculated into LB liquid medium supplemented with ampicillin and cultured at a constant temperature of 37°C with shaking at a speed of 210 r / min. After overnight incubation, the bacterial cells are amplified.

[0096] ⑦ Perform bacterial PCR using the same reaction systems as in Tables 2 and 3, and observe the presence of bright bands by 2% agarose gel electrophoresis to determine whether the cloning was successful. After the bacterial culture that amplified the target band, the plasmid was isolated and extracted using a plasmid extraction kit. The extracted plasmid samples were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing direction was clearly marked as forward.

[0097] ⑧ The sequences obtained from sequencing were imported into Chromas software, and specific analyses were conducted on the detection sites required for the study. Finally, wild-type and mutant recombinant plasmids corresponding to the four SNP sites were obtained.

[0098] This invention employs LDR technology to detect and analyze SNP sites related to Alzheimer's disease (AD). The established method requires two types of primers for SNP site detection: ligation primers and ligation product amplification primers. The ligation temperature of the designed LDR primers was predicted using NEB, specific primers for PCR amplification were designed using the NCBI Primer website, and the bond energies of the oligonucleotide secondary structures of each primer were predicted using Yunzhou Biotechnology. The primer sequences used for ligation and the lengths of the ligation products are shown in Table 3, and the primer lengths and sequences used for ligation product amplification are shown in Table 4. The design principles for ligation primers are: a Tm value of 60-70℃ (the suitable temperature for Taq DNA ligase is 35-70℃); a GC content between 40% and 60%; a ligation product size of 80-100 nt; and a universal sequence at the 5' end of primers P1 and P3. This universal sequence is not similar to or complementary to the target sequence or primer base sequence. The presence of the universal sequence facilitates subsequent PCR amplification and maximizes the length of the LDR product, allowing for identification by horizontal gel electrophoresis after ligation product amplification. Primers P1 and P3 have SNP sites at their 3' ends, with P1 containing wild-type bases and P3 containing mutant bases. Primer P2 has a phosphate group modified at its 5' end and a universal sequence at its 3' end. Based on the template SNP sequence, DNA ligase links the 3' hydroxyl group of P1 / P3 to the phosphate group at the 5' end of P2 via a phosphodiester bond, forming the ligation product. If there are mismatch sites between the double-stranded DNA template and primer bases, DNA ligase cannot complete the ligation of two adjacent primers. The design principles for amplification primers are: primer length 15-30 bp; amplification product length 70-90 bp; and a Tm value of 50-60℃.

[0099] Table 4. Ligation primer sequences and ligation product lengths Note: Underlined bases in the primer sequences are universal fragments; yellow highlighted bases in the sequences have undergone phosphorylation modification; red marked bases are key sites for Taq DNA ligase to recognize SNPs; P1 can pair complementaryly with wild-type gene sequences, while P3 is complementary to mutant sequences.

[0100] Table 5. Primers and annealing temperatures for LDR product amplification

[0101] Plasmid standards and genomic DNA with clearly defined genotypes after sequencing were used as templates for the ligation reaction. Following the reaction system shown in Table 5, all components were added and mixed thoroughly. To facilitate genotype determination, two groups were prepared using the same template: one group with wild-type ligation primers and the other with mutant ligation primers. All other reagents were prepared identically. All operations were performed on ice to avoid repeated freeze-thaw cycles, which could affect ligase activity. The ligation reaction was carried out in a standard PCR instrument according to the reaction procedure shown in Table 6.

[0102] Table 6 LDR Reaction System Taq DNA ligase 1 <![CDATA[ddH2O]]> 13 P2 (Shared Primer) 1 P1 (wild-type primer) / P3 (mutant primer) 1 template 2 10×Ligasebuffer 2 Total volume 20

[0103] Table 7 LDR Reaction Procedure

[0104]

[0105] Based on the reaction system given in Table 7, using the LDR amplification product as a template, the components were thoroughly mixed and the amplification reaction was carried out. The reaction was completed on a conventional PCR amplification instrument with reference to the program parameters in Table 8.

[0106] Table 8. Ligation product amplification reaction system DNA high-fidelity polymerase 10 DNA template 2 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 20×Evagreen 1 <![CDATA[ddH2O]]> 5 Total volume 20

[0107] Table 9. Ligation Product Amplification Reaction Procedure

[0108] Subsequently, melting curves and electrophoresis were performed on the LDR-PCR reaction products. Two tubes of LDR-PCR reaction products, one containing wild-type primers and the other containing mutant primers, were added to the same sample. The reaction system was placed in a real-time quantitative PCR instrument. The melting curve parameters were set as follows: temperature range 4–95℃, with a uniform temperature increase from 4℃ to 95℃ at a rate of 0.1℃ every 0.2 seconds, while simultaneously acquiring fluorescence signals. Finally, the temperature was lowered to 20℃ and held for 10 seconds to complete the melting curve analysis. Genotype was determined based on the shape, position, and occurrence of the melting peaks in both tubes: if only one tube showed a specific melting peak, the wild-type was identified if only tubes P1 and P2 showed melting peaks; if only tubes P3 and P2 showed melting peaks, the mutant was identified; if both tubes showed clear and specific melting peaks, the genotype was identified as heterozygous.

[0109] The melting curve results were verified using 2% agarose gel electrophoresis. 5 μL of PCR product was mixed with 1 μL of 6× loading buffer and electrophoresed at 110V for 23 min. The target band was analyzed using a gel imaging analyzer. The presence or absence of bands with different ligation primers was observed to determine whether the ligation reaction was successful. No band was observed if there was a mismatch. The position of the marker and the band was compared to determine whether it was the target band.

[0110] To optimize the experimental conditions for LDR-PCR, wild-type and mutant plasmid standards should be used as reaction templates. The focus should be on investigating the specific effects of ligation temperature (set to 62℃, 64℃, 66℃, 68℃, and 70℃, respectively) on the formation of reaction products. By adjusting different temperature parameters, the differences in LDR-PCR product formation at each temperature can be clarified to ensure the accuracy and reliability of experimental results.

[0111] Wild-type and mutant plasmid standards were selected as reaction substrates to optimize the LDR-PCR system. The LDR-PCR system was optimized by setting 20, 25, 30 and 35 cycle gradients respectively, and the effects of different cycle numbers on the LDR-PCR product generation were compared and analyzed.

[0112] Using wild-type and mutant plasmid standards as templates, the P1, P3, and P2 ligation primers were diluted to 10 μM, 5 μM, and 2.5 μM, respectively. The effects of primer combinations P1 / P3 and P2 at concentration ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 on the generation of LDR-PCR amplification products were investigated.

[0113] Using wild-type and mutant plasmid standards as templates, we conducted an optimization experiment on LDR-PCR reaction conditions to investigate the effects of ligation times of 2 min, 2.5 min, 3 min, and 3.5 min on the formation of LDR-PCR products.

[0114] Using wild-type plasmid standards as templates, wild-type and mutant primers were added to optimize LDR-PCR reaction conditions. The effects of different annealing temperatures on the formation of amplified products during LDR-PCR were investigated. Annealing temperatures included 53.9℃, 55.3℃, 56.7℃, 58.1℃, and 59.4℃. The optimal annealing temperature was determined by the result where the band of the primer-matched group was the brightest and the band of the mismatched group primer dimer was the weakest.

[0115] Wild-type and mutant plasmids were diluted at concentration gradients (100 ng / ul, 10 ng / ul, 1 ng / ul, 0.1 ng / ul, 0.01 ng / ul) and used as templates for LDR-PCR amplification. Samples at each concentration gradient were ligated and amplified to obtain the corresponding CT values. A standard curve was constructed with log10C (C being the plasmid concentration) on the x-axis and CT values ​​on the y-axis. The limit of detection (LOD) was defined as the concentration corresponding to a CT value ≈ 30 with a specific melting peak; values ​​> 30 without a specific melting peak were considered undetectable. The plasmid concentration corresponding to this CT value was derived by reverse calculation using the standard curve formula, which represents the theoretical sensitivity of LDR-PCR for detecting this SNP. The theoretical LOD concentration was then validated to determine the minimum detection concentration for this method.

[0116] Wild-type and mutant plasmids were thoroughly mixed in different proportions, accounting for 1%, 10%, 50%, 75%, and 100% of the total system, respectively. LDR-PCR amplification was then performed. Since only mutations were detected, only mutant primers were added, and no grouping was performed. A CT value ≈ 30 with a mutation-specific melting peak was considered the lowest detectable mutation frequency. When the mutation proportion decreased to a certain level, the Ct value rose above 30 without a significant mutation peak. This proportion is the limit of detection (LOD) for that SNP site.

[0117] Genomic DNA was extracted from blood samples from patients with Alzheimer's disease (AD) and healthy controls. Four SNP loci, PSEN1-rs362344, rs165934, PSEN2-rs12123818, and rs8383, were detected using LDR-PCR.

[0118] This invention utilizes HRM technology to detect AD-related SNPs. The HRM reaction system and procedure used in this experiment are shown in Tables 10 and 11. Wild-type and mutant plasmid standards were added sequentially and thoroughly mixed. Then, HRM amplification was performed using a real-time quantitative PCR instrument according to the procedure in Table 10. The melting curve parameters were set as follows: incubation at 60℃ for 30s, followed by a uniform temperature increase from 60℃ to 95℃ at a rate of 0.1℃ / s, and finally cooling to 20℃ and holding for 10s.

[0119] Table 10 HRM Reaction System DNA high-fidelity polymerase 10 DNA template 2 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 Ultrapure water 5 Evagreen fluorescent dye 1 Total volume 20

[0120] Table 11 HRM Reaction Procedure

[0121] This invention extracts genomic DNA from peripheral blood samples of Alzheimer's disease patients and healthy controls. Simultaneously, wild-type plasmids, mutant plasmids, and a standard mixture of both are prepared. HRM technology is then used to simultaneously detect and analyze four target SNP loci.

[0122] Statistical analysis was performed using SPSS 24.0 software. Genotype distribution and allele frequencies between the AD group and the normal group were expressed as count data. Chi-square test was used for comparisons between groups, and a p-value < 0.05 was considered statistically significant.

[0123] Linkage disequilibrium (LD) analysis was performed on SNP loci in the target region using Haploview 4.2 software. Haploview is a visualization analysis software widely used in population genetics research. Its core functions include Hardy-Weinberg equilibrium test for SNP loci, genotype deletion rate screening, linkage disequilibrium analysis, haplotype construction and frequency estimation, etc. It can intuitively present the genetic association patterns of gene loci and provide reliable tool support for the association analysis of susceptibility gene SNP loci. In this invention, AD-related SNP genotyping data were first obtained by detecting AD using LDR-PCR technology. Preprocessing was performed first: low-quality SNP loci and samples with genotype deletion rate >5% and minimum allele frequency (MAF) <0.05 were removed to ensure the reliability of the analysis data. Then, the qualified genotyping data were converted into the Ped / Map format compatible with Haploview software. The Ped file contains family information, individual phenotype and genotype data (missing values ​​are represented by "0"), and the Map file contains basic information such as the chromosomal location and physical location of SNP loci. The analysis results are presented visually as a color-coded heatmap, showing the intensity of linkage disequilibrium (LD) between loci: dark red blocks represent strong linkage disequilibrium or high linkage between two loci, with high D' and r² values; light red and pink blocks indicate moderate linkage; white / light blue blocks indicate weak or no linkage. The values ​​within the blocks correspond to the quantified D' or r² values, which are used to determine the linkage relationships and haplotype block distribution between SNP loci. Analysis of two SNP loci each from PSEN1 and PSEN2 using Haploview software clarifies the linkage disequilibrium intensity and haplotype block division between the two gene SNPs, providing a basis for subsequent locus association and haplotype analysis.

[0124] Genomic DNA was obtained from blood samples. Using the genomic DNA as a template, specific primers targeting PSEN1-rs362344, rs165934, and PSEN2-rs12123818, rs8383 were designed. PCR amplification was performed on these four SNP sites. The optimal annealing temperature for each specific primer was investigated. The product fragment size and optimal annealing temperature were verified by 2% agarose gel electrophoresis. Figure 1 As shown, the PSEN1-rs362344 amplified fragment was 159 bp in size, and the selected annealing temperature was 56.2℃; the PSEN1-rs165934 amplified fragment was 192 bp in size, and the selected annealing temperature was 57.4℃; the PSEN2-rs12123818 amplified fragment was 149 bp in size, and the selected annealing temperature was 58.6℃; the PSEN1-rs8383 amplified fragment was 191 bp in size, and the selected annealing temperature was 60.0℃. Using the PCR products at these optimal annealing temperatures as cloning templates, cloning and sequencing were performed. Figure 2 Wild-type and mutant plasmid standards were successfully prepared at each locus.

[0125] LDR-PCR combined detection technology is a target nucleic acid detection technology that relies on the specific catalytic action of thermostable DNA ligase combined with the amplification capability of PCR signal. First, two primers, completely complementary to the target sequence, are designed targeting specific base sites of the target nucleic acid. Primers P1 and P3 have universal sequences at their 5' ends. These universal sequences are not similar to or complementary to the target sequence or primer base sequences. The presence of these universal sequences facilitates subsequent PCR amplification and maximizes the length of the LDR product, allowing for identification by horizontal gel electrophoresis after amplification. Primers P1 and P3 have SNP bases at their 3' ends, with P1 containing wild-type bases and P3 containing mutant bases. Primer P2 has a phosphate group modified at its 5' end and a universal sequence at its 3' end. Based on the template SNP sequence, the DNA ligase links the 3' hydroxyl groups of P1 / P3 to the phosphate group at the 5' end of P2 via a phosphodiester bond, forming the ligation product.

[0126] However, the ligation product from a single LDR is extremely small, making it difficult to identify the detection results. Therefore, the obtained ligation product is used as a new template for PCR amplification to achieve exponential product amplification. The amplified products are then detected by agarose gel electrophoresis, revealing a double-stranded target band of the same length as the primer ligation. Melting curve analysis shows melting peaks specific to different genotypes. For ease of interpretation, two parallel reactions were used: one tube containing wild-type primer P1 and a common primer P2, and the other containing a mutant primer P3. Only when primers corresponding to the genotype of the template were used did the electrophoresis and melting curves show the corresponding target band and melting peak. Mismatched primers failed to ligate, resulting in no ligation product or subsequent amplification product.

[0127] The core advantage of the LDR-PCR method is that it can amplify the target sequence with the high efficiency of PCR, making the results easy to interpret, and can also accurately distinguish subtle differences such as single base mutations through DNA ligase, thus combining high sensitivity and high specificity.

[0128] Taking PSEN1-rs362344 as an example, wild-type and mutant plasmids (100 ng / ul) were used as templates for LDR ligation, followed by PCR amplification. The amplification products were verified by 2% agarose gel electrophoresis and melting curve analysis. Figure 4M: 20bp gradient marker; Lane 1 is set as a blank control without template; Lanes 2 and 3: template is mutant plasmid, concentration is 100ng / ul; Lanes 4 and 5: template is wild-type plasmid, concentration is 100ng / ul; Lanes 6 and 7: template is wild-type genomic DNA; Lanes 8 and 9: template is mutant genomic DNA; Lanes 2, 4, 6, and 9: LDR primers are P1 and P2; Lanes 3, 5, 7, and 8: LDR primers are P3 and P2. (B) Melting curves of LDR-PCR for wild-type and mutant plasmids and genomic DNA, where (A) electrophoresis results show: compared with the blank group without LDR product template, the target band appears when primers matching the SNP site are added; Lanes 2-5 are the results using wild-type / mutant plasmids as templates. When primers matching the target site were added, the wild-type plasmid showed the target band at 80 bp and the mutant plasmid at 79 bp, consistent with the designed primer ligation length. Specifically, wild-type templates could only be successfully ligated and amplified with the addition of P1 (wild-type primer) and P2 (shared primer), while the P3 (mutant primer) and P2 primer sets could not be recognized by DNA ligase due to SNP base mismatch, and the results were consistent with the template-free blank control group. Mutant templates could only be successfully ligated and amplified with the addition of P3 (mutant primer) and P2 (shared primer), while ligation failed with the addition of P1 and P2, and the ligation failure results were consistent with the template-free blank control group. The plasmid group results were consistent with the genomic DNA results, demonstrating the applicability of this method in clinical samples. Figure 4 The melting curve results of B showed that: only when the added primers were P1 (wild-type primer) and P2, the wild-type template would undergo a ligation reaction and a specific melting peak would appear at 81℃ after amplification; only when the added primers were P3 (mutant primer) and P2, the mutant template would undergo a ligation reaction and a specific melting peak would form at 79.6℃ after amplification.

[0129] Furthermore, Figure 5 Lane M: 20bp DNA gradient marker; Lanes 1 and 3: Mutant plasmid template 100 ng / µl; Lanes 2 and 4: Wild-type plasmid template 100 ng / µl; Lanes 5 and 7: Mutant genomic DNA template; Lanes 6 and 8: Wild-type genomic DNA template; Lanes 2, 3, 6, and 7: LDR primers P1 and P2; Lanes 1, 4, 5, and 8: LDR primers P3 and P2. Figure 5 The feasibility test results of rs8383 are presented. The mutant product of rs8383 is 81bp with a melting peak of 81.4℃, and the wild-type product is 83bp with a melting peak of 80.3℃.

[0130] Furthermore, Figure 6Lane M: 20bp gradient marker; Lane 1: Mutant plasmid with template concentration of 100 ng / ul; Lane 2: Wild-type plasmid with template concentration of 100 ng / ul; Lane 3: Mutant genomic DNA as template; Lane 4: Wild-type genomic DNA as template; LDR primers added to lanes 1 and 4 are P2 and P3; LDR primers added to lanes 2 and 3 are P1 and P2. Figure 6 The feasibility test results of rs165934 were presented. The wild-type product of PSEN1-rs165934 has a size of 86 bp and a melting temperature of 81.2℃, while the mutant product has a size of 84 bp and a melting temperature of 80.5℃.

[0131] Furthermore, Figure 7 Lane M: 20bp DNA gradient band; Lanes 1 and 2: Wild-type plasmid template (100 ng / µl); Lanes 3 and 4: Wild-type genomic DNA template; Lanes 5 and 6: Mutant plasmid template (100 ng / µl); Lanes 7 and 8: Mutant genomic DNA template; Lanes 2, 4, 5, and 7: LDR primers P2 and P3; Lanes 1, 3, 6, and 8: LDR primers P1 and P2. Figure 7 The feasibility test results for rs12123818 were presented. The wild-type product of PSEN2-rs12123818 was 78 bp in size and had a melting temperature of 79.7℃, while the mutant product was 81 bp in size and had a melting temperature of 80.4℃. The melting curves and electrophoresis results corroborated each other, demonstrating that the LDR-PCR method can specifically and easily detect AD-related SNPs.

[0132] Furthermore, to optimize the ligation temperature of the LDR-PCR method, experiments were conducted using PSEN1-rs362344 as an example. The feasibility experiments demonstrated that the LDR-PCR method can accurately identify SNP sites. However, since the wild-type and mutant products are similar in size, further optimization of the reaction system is needed to find the conditions that maximize the peak difference between wild-type and mutant products, representing the optimal reaction conditions for LDR-PCR. The activity range of Taq DNA ligase is 35℃-70℃. In the LDR reaction system, keeping template concentration, primer concentration, and other conditions constant, changing the ligation temperature will alter the ligation efficiency and specificity of LDR. Therefore, finding the optimal ligation temperature is crucial for the accuracy of LDR-PCR detection.

[0133] For the rs362344 locus, the ligation temperature-time gradient was set to 62℃, 64℃, 66℃, 68℃, and 70℃. The optimal conditions were determined by combining electrophoresis results and melting curve results. The optimal reaction conditions were defined as follows: the brightest bands in the electrophoresis of wild-type and mutant cells with no non-specific bands, or the faintest non-specific bands; and the temperature corresponding to the most significant difference in fluorescence values ​​between the melting peaks of wild-type and mutant cells. Figure 8 As shown, the difference in fluorescence peak derivatives changes with increasing temperature. At 68℃, the two genotypes showed a significant difference in melting peak fluorescence values, with a bright single band. However, at 70℃, the mutant sample band became significantly lighter, presumably because the temperature was close to the upper limit of the ligase's optimal range, leading to a decrease in the enzyme's specific binding ability, resulting in non-specific ligation or degradation of the amplification product. Considering both reaction efficiency and result stability, 68℃ was ultimately selected as the optimal LDR ligation temperature. Since the greatest difference between different sites lies in the designed ligation primers, and different primers can cause changes in ligation temperature, the ligation temperatures for other sites were further investigated. Using the same method, comparing the peak differences and melting peak fluorescence derivative changes between the mutant and wild types under different conditions, the optimal ligation temperature for rs8383 was found to be 66℃. Figure 9 The optimal connection temperature for the RS12123818 is 65℃. Figure 9 The optimal connection temperature for the B, rs165934 is 67℃. Figure 9 C.

[0134] Furthermore, the ligation time of the LDR-PCR method was optimized, and the effects of ligation times of 2 min, 2.5 min, 3 min, and 3.5 min on the rs362344 LDR-PCR amplification products were tested. Figure 10 As shown, when the ligation time is 3 min, the difference between wild type and mutant is more significant. When the time is extended to 3.5 min, the wild type electrophoresis results show dimers and the melting curve results show non-specific peaks. Therefore, 3 min is selected as the optimal ligation time, and the remaining sites follow this ligation time.

[0135] Furthermore, the number of cycles for the LDR-PCR method was optimized, specifically for detecting rs362344. The effects of 20, 25, 30, and 35 cycles on the reaction results were observed. Figure 11 As shown, the difference between wild type and mutant is greatest when the number of cycles is 30. Therefore, the number of cycles of 30 is selected as the optimal reaction condition, and this condition is used for other sites.

[0136] Furthermore, the primer concentration ratio for the LDR-PCR method was optimized while keeping other conditions constant. The ligation efficiency of LDR changes with variations in primer concentration; therefore, finding an optimal primer concentration ratio (P1 / P3:P2) is crucial for the LDR reaction. We set five different primer concentration ratios: P1 / P3 (10 μM):P2 (2.5 μM) = 4:1; P1 / P3 (10 μM):P2 (5 μM) = 2:1; P1 / P3 (10 μM):P2 (10 μM) = 1:1; P1 / P3 (5 μM):P2 (10 μM) = 1:2; P1 / P3 (2.5 μM):P2 (10 μM) = 1:4. Figure 12 As shown, the greatest difference between wild-type and mutant occurred when the primer concentration ratio was 2:1. Therefore, the optimal primer concentrations for ligation were selected as 10 μM for P1 / P3 and 5 μM for P2. This primer concentration ratio was used for other sites.

[0137] Furthermore, the optimal annealing temperature for the PCR amplification of the ligation products was screened. Too low an annealing temperature significantly reduced the specificity of primer-template binding, easily leading to non-specific binding and primer dimer formation, resulting in the amplification of non-target bands and interfering with genotype identification. Conversely, too high a temperature might prevent primers from effectively binding to the template, causing a decrease in the amplification efficiency of the target fragment. Therefore, after optimizing the LDR reaction system and conditions, determining the optimal annealing temperature through gradient annealing experiments is a crucial step in ensuring the specificity and sensitivity of subsequent PCR amplification of the LDR products. Figure 13 As shown, taking the rs362344 site as an example, using wild-type plasmid (10 ng / ul) as a template, at an annealing temperature of 58.1℃, the amplification product bands were clear when the correct primer set (P1, P2) was added, while the dimer was faintest when the mismatched primer set (P3, P2) was added. Therefore, 58.1℃ is set as the optimal annealing temperature. The optimal annealing temperature for other sites can be obtained using the same method; the optimal annealing temperature for rs8383 is 58.8℃. Figure 14 For components A and B; rs12123818, the optimal annealing temperature is 55.3℃. Figure 14 For C and D; the optimal annealing temperature for rs165934 is 54.4℃. Figure 14 E and F in the middle.

[0138] Furthermore, the sensitivity of LDR-PCR was tested. Wild-type and mutant plasmid standards of rs362344 were serially diluted with ultrapure water to obtain wild-type and mutant plasmid standards of 100 ng / ul, 10 ng / ul, 1 ng / ul, 100 pg / ul, and 10 pg / ul. Ligation and amplification were performed according to the optimal reaction conditions obtained above. A standard equation was obtained by plotting a standard curve of Log10C (plasmid concentration) against the corresponding CT value. The theoretical limit of detection was obtained by substituting a CT value ≈ 30 into the equation, and then the limit of detection was verified. Figure 15 As shown, the results indicate that as the wild-type template is serially diluted, the peak value decreases and the CT value increases accordingly. Figure 15 In sections A and B, a strong linear negative correlation was observed between template concentration and CT value. Figure 15 The linear range for the plasmid E was 100 ng / ul to 10 pg / ul, and the standard curve equation was y = -2.924x + 16.16 (R² = 0.9966). When the CT value was 30, x = 1.86 × 10⁻². Using wild-type plasmids at concentrations of 0.2 pg / ul, 0.02 pg / ul, and 0.002 pg / ul as templates for LDR-PCR amplification, the obtained CT values ​​were 27.12, 29.79, and 32.25, respectively. The CT values, combined with the melting curve, determined that this method could detect wild-type plasmid concentrations as low as 0.02 pg / ul. Figure 15 As shown in C and D.

[0139] The detection limit of the mutant plasmid was determined using the same method. The standard curve equation for the mutant plasmid was y = -3.099x + 20.974 (R² = 0.9709). When the CT value was 30, x = 1.22 pg / ul. The mutant plasmid was diluted to 1 pg / ul, 0.1 pg / ul, and 0.01 pg / ul for verification, and the corresponding CT values ​​were 29.24, 31.61, and 33.74, respectively. When CT > 30, the template concentration was extremely low, and excess primers were more likely to aggregate and form non-specific primer dimers. Therefore, a CT value of 29.24 was selected as the lowest detection concentration, and the detection limit of the mutant plasmid was determined to be 1 pg / ul. Specific data can be found in [link to data]. Figure 16 AE (Chinese AE)

[0140] Using the same detection procedure, the sensitivity of other sites was determined, enabling the detection of mutant plasmid standards of rs8383 down to 1 pg / ul. Figure 17 Wild-type plasmid standards as low as 0.2 pg / ul, such as Figure 18 It can detect mutant plasmid standards of rs12123818 down to 0.1 pg / ul, such as... Figure 19 Wild-type plasmid standards as low as 0.008 pg / ul, such as Figure 20Wild-type plasmid standards capable of detecting rs165934 at concentrations as low as 0.4 pg / ul, such as... Figure 21 Mutant plasmid standards as low as 0.15 pg / ul.

[0141] Furthermore, LDR-PCR was used to detect the mutation frequency of SNPs. In clinical samples, SNP mutations often appeared as a mixture of wild-type and mutant types, rather than simply homozygous mutations or homozygous wild-type. To simulate the heterogeneity of actual clinical samples and evaluate the detection capability of this method for different proportions of mutations, we constructed simulated samples with different mutation frequencies by gradient mixing of wild-type and mutant plasmids at concentrations of 0%, 10%, 25%, 50%, 75%, and 100%. The selected ligation primer sets were P2 and P3 (i.e., the mutant primer set, ensuring that the ligation reaction is only related to the mutated bases in the sample). A Ct value ≤ 30 with a clear mutation peak was used as the positive criterion, while a Ct value higher than 30 without a specific melting peak was considered undetectable. Taking rs362344 as an example, the results showed that as the proportion of mutant plasmid decreased, the detected Ct value gradually increased, and the mutation signal correspondingly weakened. Figure 12 The melting curves and amplification curves obtained after LDR-PCR amplification using mixed mutant and wild-type plasmids at different concentration ratios as templates are shown below. Figure 12 For samples A and B, when the mutation frequency was as low as 1%, the corresponding CT value was 26.76. Further reducing the mutation frequency to 1%, 0.5%, and 0.1% yielded CT values ​​of 25.31, 29.32, and 32.39, respectively. Figure 12 In C, the amplification curve results and melting curve are as follows: Figure 12 The results from the middle and lower bounds (D) mutually validated each other, showing that ligation failed and no target peak occurred when the mutant proportion was 0.1%, with CT = 32.39. Therefore, this method can detect mutation frequencies as low as 0.5%. The results confirm that this LDR-PCR system can effectively identify low proportions of mutations in mixed samples and is suitable for detecting SNP mutation frequencies in heterogeneous clinical samples.

[0142] Using the above detection method, the detection limit of LDR-PCR for mutation frequencies at other sites was explored. This method can detect mutation frequencies as low as 1% of rs8383. Figure 24 It can detect mutation frequencies as low as 1% for rs12123818. Figure 25 It can detect mutation frequencies as low as 1% for rs165934. Figure 26 .

[0143] Furthermore, genomic DNA was extracted from blood samples from 25 AD patients and 40 healthy individuals in clinical settings. LDR-PCR was used to detect four SNP loci: PSEN1 (rs362344, rs165934) and PSEN2 (rs12123818, rs83834). To facilitate result interpretation, samples from the same individual were divided into two groups, with wild-type primer P1 and mutant primer P3 added to one group respectively. The results from both groups were combined to determine the genotype. Figure 27 The results showed that this method can effectively distinguish three different genotypes: genotypes showing specific melting peaks in tubes P1 and P2, and in tubes P3 and P2 respectively, are homozygous wild-type; genotypes showing specific melting peaks in tubes P3 and P2, but not in tubes P1 and P3, are homozygous mutant; genotypes showing melting peaks in both tubes are heterozygous. Some test results are shown below. Figure 18-30 Specific melting peaks were observed at all four SNP loci. For PSEN1-rs362344, the wild-type template showed a specific melting peak at 81℃, and the mutant template showed a specific melting peak at 79.6℃. For PSEN1-rs165934, the wild-type melting peak was at 81.2℃, and the mutant melting peak was at 80.5℃. For PSEN2-rs12123818, the wild-type melting temperature was 79.7℃, and the mutant melting temperature was 80.4℃. For rs8383, the mutant melting peak was at 81.4℃, and the wild-type melting peak was at 80.3℃. These results are consistent with those obtained using Sanger sequencing.

[0144] Furthermore, HRM technology was used to detect AD-related SNPs. Genomic DNA was extracted from blood samples from the AD group and the normal group, and HRM was used to detect four SNP loci. The results of some samples are shown below. Figure 17 As shown, SNP genotyping (wild-type, mutant, and heterozygous) can be performed by determining the shape and position of the high-resolution melting curve. However, the results of some samples are inconsistent with the Sanger sequencing results, and the melting temperatures of different batches at the same locus vary, which may be due to errors caused by different sample loading procedures.

[0145] Furthermore, SNP loci were analyzed. This invention targets SNP loci related to Alzheimer's disease (AD). Clinical samples were tested using LDR-PCR and HRM technologies, respectively. The results of these two methods were then systematically compared with Sanger sequencing results (the gold standard). LDR-PCR results were completely consistent with Sanger sequencing, demonstrating extremely high accuracy, while HRM results showed partial discrepancies with Sanger. Therefore, LDR-PCR is more accurate than HRM in SNP detection. The obtained results were analyzed using GraphPadprism 8.0. Figure 18The genotype distribution of each locus is shown. Chi-square tests were performed on the genotypes and frequencies of the four SNPs using SPSS 24.0, as shown in Tables 12-15. PSEN1-rs362344 and PSEN2-12123818 showed statistically significant differences between the AD group and the normal group (P < 0.05), indicating a significant difference between the AD group and the normal group. However, there were no statistically significant differences in genotype and allele frequencies between the AD group and the normal group for PSEN1-rs165934 and PSEN1-rs8383 (P > 0.05). Therefore, we hypothesize that both PSEN1-rs362344 and PSEN2-12123818 are significantly associated with AD and can serve as independent risk loci for predicting AD. PSEN1-rs165934 and PSEN2-rs8383, however, may not be significantly associated with the genetic risk of AD in the population of this invention and cannot yet be considered independent genetic risk loci for AD. Simultaneously, Haploview software was used to supplement the analysis of linkage disequilibrium and haplotype distribution of SNP loci, to comprehensively verify the reliability of the detection method and the association between SNP loci and the disease. D' is an indicator of the degree of linkage disequilibrium (LD), ranging from 0 to 1; the LOD value is used to test the significance of LD, generally LOD ≥ 2 is considered significant LD; r² reflects the degree to which genotypes between two loci can predict each other, ranging from 0 to 1. The closer r² is to 1, the stronger the linkage between the two SNPs; the closer it is to 0, the more independent the two SNPs are. Figure 18 As shown, analysis of the PSEN1 locus results shows that D' = 0.041 indicates a very weak linkage disequilibrium between these two loci. LOD = 0.02, far below the critical value, further indicates no significant linkage disequilibrium between the two loci. r² = 0.001, also indicating a very weak association, meaning there is almost no co-inheritable tendency between them. This suggests that the PSEN1-rs362344 and rs165934 loci are independently inherited and have no predictive ability. Analysis of the PSEN2 locus results shows that r² = 0.032 (close to 0), indicating a very weak linkage relationship. This means that the PSEN2-rs12123818 and rs8383 loci are almost independently inherited in the population, with extremely low co-variation association of alleles. D' = 0.354, indicating a weak to moderate linkage disequilibrium, suggesting some co-inheritable tendency between these two loci, but the association is not strong.

[0146] Table 12 Genotyping and frequency of rs362344 detected by LDR, HRM, and Sanger sequencing

[0147] Table 13 Genotyping and frequency of rs8383 detected by LDR, HRM, and Sanger sequencing

[0148] Table 14 Genotyping and frequency of rs12123818 detected by LDR, HRM, and Sanger sequencing

[0149] Table 15 Genotyping and frequency of rs165934 detected by LDR, HRM, and Sanger sequencing

[0150] LDR (Ligation Reduction) has long been used to detect midpoint mutations in target gene sequences due to the precise recognition of SNP sites by ligases. Previously, most researchers opted for a PCR-first, LDR-later approach to detect disease mutations in genomic DNA samples. While this PCR-later technique allows for simultaneous detection of multiple SNP sites and is compatible with degraded DNA or trace samples, PCR amplification can generate byproducts such as primer dimers and nonspecific bands. If these products share some homology with the LDR probe, they can lead to misligation and false positives. Furthermore, PCR products require additional purification, increasing operational complexity and the risk of contamination. For low-copy-number trace samples, PCR amplification is prone to random effects, resulting in uneven target amplification and subsequent LDR reaction with allele loss, affecting accuracy. With advancements in molecular biology, more researchers are choosing to combine LDR with probes to advance its clinical application; however, probe design is stringent and complex, and fluorescent labeling is expensive.

[0151] The SNP loci selected in this invention are PSEN1-rs362344 and rs165934, and PSEN2-rs8383 and rs12123818. The association between each locus and the disease was analyzed using Haploview and statistical software. PSEN1-rs362344 and PSEN2-rs12123818 showed significant differences between normal individuals and the AD group, and can be considered independent risk loci for predicting AD. Neither of these loci is linked to another SNP locus in the same gene. While PSEN1-rs165934 and PSEN2-rs12123818 have high mutation rates in the population, the detection results for rs165934 and rs12123818 showed no significant difference between normal individuals and AD patients, and therefore cannot be used as indicators for predicting AD progression.

[0152] In summary, this invention establishes an LDR-PCR detection method for detecting PSEN1 and PSEN2-related SNPs in Alzheimer's disease (AD). Compared with existing AD detection methods, this method is simple to operate, requires less sophisticated equipment, and can achieve accurate SNP genotyping by using melting curves obtained from two tubes of the same sample with wild-type or mutant primers. The LDR-PCR detection has high sensitivity; even heterozygous samples with low mutation frequencies can be clearly detected; the results are accurate and reliable; and it has strong clinical applicability. Furthermore, significant differences were found between the PSEN1-rs362344 and PSEN2-rs12123818 loci in the AD group and the normal control group, indicating that these two loci can serve as independent risk sites for AD and have the potential to transform into AD. No differences were found between the PSEN1-rs165934 and PSEN2-rs8383 loci in the AD group and the normal control group, which is consistent with the Haploview analysis showing that the PSEN1 and PSEN2 SNP loci are independently inherited and not linked. In summary, this method provides an efficient detection scheme for rapid detection of AD-phase SNPs, suitable for early diagnosis of AD and large-scale clinical sample testing.

[0153] 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 SNP loci related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2, characterized in that, Includes the following steps: S1. Obtain the genomic DNA of the sample to be tested; S2, LDR ligase detection reaction: Three sets of primers targeting the target SNP site are provided, including wild-type ligation primer P1, mutant ligation primer P3, and shared ligation primer P2, which are designed and synthesized. The 3' end base of P1 is complementary to the wild-type base of the target SNP site; The 3' end base of P3 is complementary to the mutant base of the target SNP site; The 5' end of P2 is modified by phosphorylation; Using the genomic DNA as a template, under the action of DNA ligase, the 3' hydroxyl group of P1 or P3 is covalently linked to the 5' phosphate group of P2 to form a ligation product; S3. PCR polymerase chain reaction: Using the ligation product obtained in step S2 as a template, PCR amplification is performed using amplification primers to obtain the amplification product. S4. Detect the amplification product and determine the genotype.

2. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The ligation product in S2 is a single-stranded or double-stranded nucleic acid, and its structure includes: The first nucleic acid segment has a nucleotide sequence that at least partially overlaps with the nucleotide sequence of P1 or P3, and its 3' end contains the discriminant base of the target SNP site; The second nucleic acid segment has a nucleotide sequence that at least partially overlaps with the nucleotide sequence of P2, and its 5' end is phosphorylated. The first nucleic acid segment and the second nucleic acid segment are covalently linked by a phosphodiester bond; Preferably, the length of the connection product is 80-100 nt.

3. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The 5' ends of P1 and P3 also contain a universal sequence that does not contain the genomic sequence of the target SNP site; the 3' end of P2 also contains a universal sequence; the amplification primers in step S3 specifically recognize and bind to the universal sequence.

4. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The amplification primers include forward amplification primer F1 and reverse amplification primer R1; wherein the sequence of F1 is selected from any one of the sequences shown in SEQ ID NO:1, 3, 5, 7, and the sequence of R1 is selected from any one of the sequences shown in SEQ ID NO:2, 4, 6, 8.

5. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The three sets of primers mentioned in step S2 are designed for the rs362344 and rs165934 sites of the PSEN1 gene and / or the rs8383 and rs12123818 sites of the PSEN2 gene. The primer sequences for P1, P3, and P2 targeting the rs362344 site are shown in SEQ ID NO:9, 11, and 10, respectively; the primer sequences for P1, P3, and P2 targeting the rs165934 site are shown in SEQ ID NO:18, 20, and 19, respectively; the primer sequences for P1, P3, and P2 targeting the rs12123818 site are shown in SEQ ID NO:15, 17, and 16, respectively; and the primer sequences for P1, P3, and P2 targeting the rs8383 site are shown in SEQ ID NO:12, 14, and 13, respectively.

6. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The reaction conditions for step S2 are: ligation temperature 65-68℃, ligation time 3 min, number of cycles 30, primer concentration ratio P1 to P2 concentration ratio 2:1, and P3 to P2 concentration ratio 2:

1.

7. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that: The detection in step S4 includes high-resolution melting curve analysis and / or agarose gel electrophoresis. For PSEN1-rs362344, the genotype is determined by high-resolution melting curve analysis. The wild type shows a specific melting peak at 81.0℃, and the mutant type shows a specific melting peak at 79.6℃. The agarose gel electrophoresis shows that the length of the wild type amplification product is 80 bp, and the length of the mutant amplification product is 79 bp.

8. The method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 according to claim 1, characterized in that, The method has a detection limit of 0.02 pg / μL genomic DNA and can detect mutation frequencies as low as 0.5%.

9. An application of the method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 as described in any one of claims 1 to 8, characterized in that, A reagent kit is proposed, comprising: P1, a set of ligation primers for the LDR reaction, the primer set being designed for at least one SNP site selected from PSEN1-rs362344, PSEN1-rs165934, PSEN2-rs12123818, and PSEN2-rs8383, including wild-type ligation primer P1, mutant ligation primer P3, and a 5' phosphorylated common ligation primer P2; and amplification primers F1 and R1 that are paired with the primers; P2, Taq DNA ligase; P3, DNA polymerase; P4, a universal primer pair for PCR amplification, wherein the universal primers are complementary to the 5' universal sequences of P1 / P3.

10. An application of the method for detecting SNP sites related to the Alzheimer's disease susceptibility genes PSEN1 and PSEN2 as described in any one of claims 1 to 8, characterized in that, It can be used for genetic risk screening or early auxiliary assessment of Alzheimer's disease.

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