SNP (Single Nucleotide Polymorphism) site and kit related to diabetes mellitus and cognitive function impairment combined therewith

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to diabetes mellitus and cognitive impairment combined with diabetes mellitus and a kit, belongs to the technical field of SNP detection, and particularly relates to the SNP site related to diabetes mellitus and cognitive impairment combined with diabetes mellitus and the kit. The kit comprises at least one of an amplification primer for detecting the SNP molecular marker of the ELAVL1 gene, a modified polyesteramide material and modified magnetic beads. The SNP (Single Nucleotide Polymorphism) molecular marker of the ELAVL1 gene comprises at least one SNP molecular marker of rs12985234, rs14394, rs35986520 and rs10402477, and the SNP molecular marker of the ELAVL1 gene comprises at least one of the following SNP molecular markers. According to the detection reagent provided by the invention, the amplification yield and the specificity of PCR amplification can be effectively and remarkably improved, and the PCR effect is improved, so that the detection effect of SNP sites is optimized.

Description

Technical Field

[0001] This invention relates to the field of SNP detection technology, specifically to SNP sites and kits related to diabetes and its associated cognitive impairment. Background Technology

[0002] Type 2 diabetes (T2D), one of the most common metabolic diseases worldwide, is characterized not only by persistent hyperglycemia and the typical clinical manifestations of polyuria, polydipsia, polyphagia, and weight loss, but also by frequent complications such as cardiovascular and cerebrovascular diseases, diabetic nephropathy, and retinopathy. Simultaneously, T2D patients often experience comorbidities such as emotional and cognitive impairment (e.g., depression, anxiety, and dementia), leading to a significant decline in their quality of life and a markedly increased risk of disability and death. Against the backdrop of a rapidly aging global population and insufficient social care resources, T2D patients with comorbid emotional and cognitive impairment face even more severe health challenges. Although some progress has been made in the prevention and treatment of T2D, its pathogenesis remains incompletely understood, and existing early screening and intervention methods have limitations in precision. In particular, there is a lack of reliable biomarkers that can simultaneously predict both T2D and emotional and cognitive impairment, which has become a critical issue that urgently needs to be addressed in clinical and public health fields.

[0003] The role of RNA-binding proteins in the regulation of metabolism and neurological diseases is receiving increasing attention. Among them, those derived from… ELAVL1 Human antigen R (HuR), encoded by the gene, is an important RNA-binding protein widely involved in cell proliferation, differentiation, stress response, and mRNA stability regulation. Previous studies have suggested that HuR expression is upregulated in complications such as diabetic nephropathy and retinopathy, and that it promotes disease progression by stabilizing related factor mRNAs. Simultaneously, HuR plays a crucial role in neurodevelopment and plasticity, and its expression changes can affect neuronal function and depressive-like behaviors. Single nucleotide polymorphisms (SNPs), as a common form of genetic variation, have been proven to be associated with susceptibility to various diseases. Currently, genome-wide association studies (GWAS) have identified hundreds of susceptibility loci for type 2 diabetes (T2D), but systematic research on HuR gene SNPs in T2D and its emotional and cognitive disorders remains very limited. Previous studies have shown that some HuR gene SNPs (such as rs12983784) are associated with cancer risk, and abnormal mutation rates of HuR gene SNPs are also observed in T2D patients, suggesting that HuR genetic variations may be involved in the development of T2D and its emotional and cognitive disorders, but its specific mechanisms of action and clinical application value remain unclear.

[0004] Although existing technologies have identified several SNP biomarkers associated with T2D risk, no studies have systematically explored the feasibility of HuR gene SNPs—especially their association with emotional and cognitive disorders—as targets for early screening and intervention of T2D. Therefore, how to provide a biomarker based on HuR gene SNPs and its detection technology to fill the gap in its application in the prediction, diagnosis, and prevention of T2D and its emotional and cognitive disorders has become an urgent scientific problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide SNP loci and kits related to diabetes and its associated cognitive impairment, not only based on ELAVL1 The gene provides SNP molecular markers associated with diabetes and associated cognitive impairment, and by optimizing key components of its detection kit, it significantly improves the amplification yield and specificity of PCR amplification, thereby effectively improving PCR reaction performance and achieving efficient and accurate detection of target SNP sites.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: The application of reagents for detecting SNP sites associated with diabetes and its associated cognitive impairment in the preparation of reagents for predicting diabetes and its associated cognitive impairment, wherein the SNP site is at least one of rs12985234, rs14394, rs35986520, and rs10402477.

[0007] Preferably, the reagents for detecting SNP sites associated with diabetes and its associated cognitive impairment are selected from one or more of the following: DNA microarrays or chips containing combinations of genetic markers for detecting SNP sites, specific PCR primers or probes, and targeted high-throughput sequencing reagents.

[0008] More preferably, the nucleotide sequence of the pre-primer for detecting rs12985234 is shown in SEQ ID No. 1, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 2; and / or The nucleotide sequence of the pre-primer for rs14394 amplification is shown in SEQ ID No. 3, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 4; and / or The nucleotide sequence of the pre-primer for rs35986520 amplification is shown in SEQ ID No. 5, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 6; and / or The nucleotide sequence of the pre-primer for rs10402477 amplification is shown in SEQ ID No. 7, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 8.

[0009] This invention also discloses a detection kit for SNP sites associated with diabetes and its associated cognitive impairment, comprising: Amplification primers for detecting at least one of the following sites in the ELAVL1 gene: rs12985234, rs14394, rs35986520, and rs10402477; The nucleotide sequence of the pre-primer for amplification of rs12985234 is shown in SEQ ID No. 1, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 2. The nucleotide sequence of the pre-primer for rs14394 amplification is shown in SEQ ID No. 3, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 4. The nucleotide sequence of the pre-primer for rs35986520 amplification is shown in SEQ ID No. 5, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 6. The nucleotide sequence of the pre-primer for rs10402477 amplification is shown in SEQ ID No. 7, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 8.

[0010] This invention discloses ELAVL1 Single nucleotide polymorphisms (SNPs) at the rs12985234, rs14394, rs35986520, and rs10402477 sites in the gene are associated with type 2 diabetes, and corresponding detection kits have been developed based on these SNP markers. This kit enables efficient and accurate detection of target SNP sites, providing an effective molecular tool for early screening and risk assessment of type 2 diabetes and its associated cognitive impairment.

[0011] Preferably, the test kit also includes a modified polyamide ester material.

[0012] More preferably, the preparation method of the modified polyamide ester material includes dissolving diethylenetriamine in methanol under low temperature and nitrogen atmosphere conditions, adding ethylene glycol diacrylate, stirring and mixing, reacting at room temperature, rotary evaporating under reduced pressure, adding 2-octenyl succinic anhydride, and reacting under reduced pressure to obtain the modified polyamide ester material. This invention, by using the modified polyamide ester material in the PCR system, may effectively improve PCR reaction performance through interactions with DNA templates, DNA primers, and proteins. It not only effectively increases the amplification yield of the target product but also enhances the specificity of primer and template binding, reduces the formation of non-specific products and primer dimers, thereby enhancing the specificity of PCR amplification.

[0013] More preferably, the ratio of diethylenetriamine to methanol is 1 mol: 50-200 mL.

[0014] More preferably, the molar ratio of diethylenetriamine to ethylene glycol diacrylate is 1:0.3-0.5.

[0015] More preferably, the molar ratio of diethylenetriamine to 2-octenylsuccinic anhydride is 1:1-3.

[0016] More preferably, the reduced pressure reaction temperature is 140-160℃.

[0017] Preferably, the detection kit for SNP sites associated with diabetes and its co-occurring cognitive impairment includes: a method for detecting... ELAVL1 At least one component of the following: amplification primers for SNP molecular markers of genes, modified polyamide ester material, and modified magnetic beads. ELAVL1 The SNP molecular markers of the gene include at least one of rs12985234, rs14394, rs35986520 and rs10402477.

[0018] More preferably, the pre-primer for amplifying rs12985234 is 5'-GCCGACTTTTATGAGACAC-3', the nucleotide sequence of which is shown in SEQ ID No.1.

[0019] More preferably, the back primer for amplifying rs12985234 is 5'-CTTTCCCTGATCTTTTACTG-3', the nucleotide sequence of which is shown in SEQ ID No.2.

[0020] More preferably, the front primer for amplifying rs14394 is 5'-GCGGATCACTTTCACATTGG-3', the nucleotide sequence of which is shown in SEQ ID No. 3.

[0021] More preferably, the back primer for amplifying rs14394 is 5'-TTTCATCTACAACCTGGGGC-3', the nucleotide sequence of which is shown in SEQ ID No. 4.

[0022] More preferably, the front primer for amplifying rs35986520 is 5'-AAGGCTTCTTTTTCCAGGA-3', the nucleotide sequence of which is shown in SEQ ID No. 5.

[0023] More preferably, the back primer for amplifying rs35986520 is 5'-GGGTCCCTGCTCTGTTG-3', the nucleotide sequence of which is shown in SEQ ID No. 6.

[0024] More preferably, the front primer for amplifying rs10402477 is 5'-GAGCAGGGACCCTCTTTT-3', the nucleotide sequence of which is shown in SEQ ID No.7.

[0025] More preferably, the back primer for amplifying rs10402477 is 5'-CTCCCACAGAATCCCAGA-3', the nucleotide sequence of which is shown in SEQ ID No. 8.

[0026] More preferably, the concentration of the modified polyamide ester material used is 0.022-0.03 μg / mL.

[0027] More preferably, the preparation method of the modified polyamide ester material is as follows: Diethylenetriamine was dissolved in methanol at 0-10℃ under a nitrogen atmosphere, and ethylene glycol diacrylate was added and stirred until homogeneous. The mixture was then reacted at room temperature for 2-6 hours, followed by rotary evaporation under reduced pressure. 2-Octenylsuccinic anhydride was added, and the mixture was reacted under reduced pressure at 140-160℃ for 2-6 hours to obtain the modified polyamide ester material.

[0028] More preferably, the ratio of diethylenetriamine to methanol is 1 mol: 50-200 mL.

[0029] More preferably, the molar ratio of diethylenetriamine to ethylene glycol diacrylate is 1:0.3-0.5.

[0030] More preferably, the molar ratio of diethylenetriamine to 2-octenylsuccinic anhydride is 1:1-3.

[0031] More preferably, the reaction temperature is 20-30℃.

[0032] More preferably, the preparation method of the modified magnetic beads is as follows: Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, and then an alkenyl monomer, methylenebisacrylamide, and azobisisobutyronitrile were added and ultrasonically homogenized. The mixture was reacted under a nitrogen atmosphere at 90-110℃ for 10-20 min, and then reacted at 80-100℃ for 6-12 h. The mixture was magnetically separated and washed 1-5 times with ethanol to obtain modified magnetic beads.

[0033] More preferably, the ratio of silicon dioxide@iron oxide nanoparticles to acetonitrile is 1g:100-300mL.

[0034] More preferably, the alkenyl monomer includes at least one of methacrylic acid, allyl malonic acid and 3-allyloxy-1,2-propanediol, wherein the mass ratio of methacrylic acid to allyl malonic acid is 1:0.5-2 and the mass ratio of methacrylic acid to 3-allyloxy-1,2-propanediol is 1:0.08-0.23.

[0035] More preferably, the alkenyl monomer includes methacrylic acid and allyl malonic acid, with a mass ratio of methacrylic acid to allyl malonic acid of 1:0.5-2. This invention uses methacrylic acid and allyl malonic acid to modify the surface of magnetic beads, which may efficiently capture DNA through the electrostatic and hydrophobic interactions of the surface carboxyl groups, improving DNA template quality and helping to reduce non-specific amplification in PCR, thereby significantly improving amplification yield and specificity.

[0036] More preferably, the alkenyl monomer includes methacrylic acid, allyl malonic acid, and 3-allyloxy-1,2-propanediol, with the mass ratio of methacrylic acid to allyl malonic acid being 1:0.5-2, and the mass ratio of methacrylic acid to 3-allyloxy-1,2-propanediol being 1:0.08-0.23. This invention further utilizes 3-allyloxy-1,2-propanediol for surface modification, which may, by introducing hydrophilic groups, help to further improve the quality of the DNA template, reduce non-specific amplification background, and thus further improve amplification yield and specificity.

[0037] More preferably, the mass ratio of silicon dioxide@iron tetroxide nanoparticles to alkenyl monomers is 1:2-6.

[0038] More preferably, the mass ratio of silica@iron tetroxide nanoparticles to methylenebisacrylamide is 1:0.5-2.

[0039] More preferably, the mass ratio of silicon dioxide@iron oxide nanoparticles to azobisisobutyronitrile is 1:0.05-0.2.

[0040] Preferably, the method for detecting SNP sites associated with diabetes and its co-occurring cognitive impairment is as follows: S1. DNA Extraction: Centrifuge the blood sample at 1000-5000g for 10-20 minutes, and extract genomic DNA using a whole blood DNA extraction kit. Follow the kit's instructions for extraction.

[0041] S2. DNA purification: Disperse the modified magnetic beads in ethanol to a concentration of 5-20 mg / mL using ultrasonication to obtain a magnetic bead solution; mix the genomic DNA with the binding solution by inversion, add the magnetic bead solution, let stand for 3-10 min, perform magnetic separation and discard the supernatant, add washing buffer and wash, repeat washing 1-5 times, perform magnetic separation and discard the supernatant, let stand at room temperature for 5-20 min, add elution buffer and incubate for 2-10 min, perform magnetic separation and retain the supernatant to obtain the purified DNA sample.

[0042] S3. The PCR system consisted of: 0.5-1.5 μL of modified polyamide ester material, 2-3 μL of 10× buffer, 1-3 μL of dNTPs, 0.2-0.3 μL of Taq DNA polymerase, 0.5-2 μL of genomic DNA, 0.2-1 μL of the first primer and 0.2-1 μL of the second primer, containing at least six of the following: ultrapure water. The nucleotide sequence of the first primer for amplifying rs12985234 was shown in SEQ ID No. 1, and the nucleotide sequence of the second primer was shown in SEQ ID No. 2. The nucleotide sequence of the first primer for amplifying rs14394 was shown in SEQ ID No. 3, and the nucleotide sequence of the second primer was shown in SEQ ID No. 4. The nucleotide sequence of the first primer for amplifying rs35986520 was shown in SEQ ID No. 5, and the nucleotide sequence of the second primer was shown in SEQ ID No. 6. The nucleotide sequence of the front primer for amplifying rs10402477 is shown in SEQ ID No. 7, and the nucleotide sequence of the back primer is shown in SEQ ID No. 8. The PCR reaction conditions were as follows: pretreatment at 94-98℃ for 4-6 min, denaturation at 94-98℃ for 20-40 s, annealing at 50-60℃ for 20-40 s, extension at 70-75℃ for 20-40 s, for a total of 32-36 cycles, and a final extension at 70-75℃ for 2-10 min.

[0043] More preferably, in step S2, the volume ratio of genomic DNA to binding solution is 1:0.5-2.

[0044] More preferably, in step S2, the volume ratio of genomic DNA to magnetic bead solution is 1:0.05-0.2.

[0045] More preferably, the volume ratio of genomic DNA to washing solution in step S2 is 1:2-3.

[0046] More preferably, in step S2, the volume ratio of genomic DNA to elution buffer is 1:0.2-1.

[0047] More preferably, the concentration of the modified polyamide ester material used in step S3 is 0.022-0.03 μg / mL.

[0048] This invention discloses ELAVL1Single nucleotide polymorphisms (SNPs) at the rs35986520, rs10402477, rs12985234, and rs14394 loci of the gene are significantly associated with type 2 diabetes (T2D). Among them, rs35986520 and rs12985234 play a core role. At least one SNP marker among rs35986520, rs10402477, rs12985234, and rs14394 can be used to prepare reagents or kits for detecting type 2 diabetes. In the detection system for SNP loci associated with diabetes and its comorbid cognitive impairment, genomic DNA is first purified using the modified magnetic beads prepared in this invention, and then amplified using modified polyamide ester material as a PCR enhancer. This significantly improves the amplification yield and specificity of PCR, enhances PCR performance, and thus optimizes the detection effect of SNP loci. Attached Figure Description

[0049] Figure 1 The results are for homozygous and heterozygous sequencing of rs35986520.

[0050] Figure 2 The results are for homozygous and heterozygous sequencing of rs10402477.

[0051] Figure 3 The results are for homozygous and heterozygous sequencing of rs12985234.

[0052] Figure 4 The results are for homozygous and heterozygous sequencing of rs14394.

[0053] Figure 5 The ROC curve for rs10402477 is shown.

[0054] Figure 6 The ROC curve for rs35986520 is shown.

[0055] Figure 7 The ROC curve is 12985234.

[0056] Figure 8 The ROC curve for rs14394 is shown.

[0057] Figure 9 To increase the yield.

[0058] Figure 10 For specificity.

[0059] Figure 11 The image shows the infrared spectrum of the modified polyamide ester material.

[0060] Figure 12 This is a TEM image of the modified magnetic beads. Detailed Implementation

[0061] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] Example 1: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The gene rs12985234. In the reference genome GRCh38.p14, rs12985234 is located at position 7960607 on human chromosome 19, with a polymorphism of A>G.

[0064] Example 2: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The gene rs10402477. In the reference genome GRCh38.p14, rs10402477 is located at position 7961562 on human chromosome 19, with a polymorphism of C>T.

[0065] Example 3: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The gene rs14394. In the reference genome GRCh38.p14, rs14394 is located at position 7963660 on human chromosome 19, with polymorphisms of A>G or A>T.

[0066] Example 4: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The gene rs35986520. In the reference genome GRCh38.p14, rs35986520 is located at position 7961534 on human chromosome 19, with a polymorphism of G>A.

[0067] Example 5: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1The genes rs35986520 and rs12985234. In the reference genome GRCh38.p14, rs35986520 is located at position 7961534 on human chromosome 19, with a polymorphism of G>A; rs12985234 is located at position 7960607 on human chromosome 19, with a polymorphism of A>G.

[0068] Example 6: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The genes rs10402477 and rs14394. In the reference genome GRCh38.p14, rs10402477 is located at position 7961562 on human chromosome 19, with a polymorphism of C>T; rs14394 is located at position 7963660 on human chromosome 19, with a polymorphism of A>G or A>T.

[0069] Example 7: SNP loci associated with diabetes and its associated cognitive impairment include, ELAVL1 The genes rs12985234, rs14394, rs35986520, and rs10402477. In the reference genome GRCh38.p14, rs12985234 is located at position 7960607 on human chromosome 19, with a polymorphism of A>G; rs14394 is located at position 7963660 on human chromosome 19, with a polymorphism of A>G or A>T; rs35986520 is located at position 7961534 on human chromosome 19, with a polymorphism of G>A; and rs10402477 is located at position 7961562 on human chromosome 19, with a polymorphism of C>T.

[0070] Example 8: A detection kit for SNP sites associated with diabetes and its associated cognitive impairment, including, for detecting... ELAVL1Primers for amplifying genes rs12985234, rs14394, rs35986520, and rs10402477. The amplification primers include the front and back primers for amplifying rs12985234, rs14394, rs35986520, and rs10402477. The front primer for amplifying rs12985234 is 5'-GCCGACTTTTATGAGACAC-3', with its nucleotide sequence shown in SEQ ID No. 1; the back primer is 5'-CTTTCCCTGATCTTTTACTG-3', with its nucleotide sequence shown in SEQ ID No. 2. The primer for amplifying rs14394 was 5'-GCGGATCACTTTCACATTGG-3', with its nucleotide sequence shown in SEQ ID No. 3; the primer for amplifying rs35986520 was 5'-AAGGCTTCTTTTTCCAGGA-3', with its nucleotide sequence shown in SEQ ID No. 5; the primer for amplifying rs35986520 was 5'-AAGGCTTCTTTTTCCAGGA-3', with its nucleotide sequence shown in SEQ ID No. 5; the primer for amplifying rs10402477 was 5'-GAGCAGGGACCCTCTTTT-3', with its nucleotide sequence shown in SEQ ID No. 7; the primer for amplifying rs10402477 was 5'-CTCCCACAGAATCCCAGA-3', with its nucleotide sequence shown in SEQ ID No. 8. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0071] Methods for detecting SNP sites associated with diabetes mellitus and its associated cognitive impairment include, S1. DNA Extraction: Centrifuge the blood sample at 3000g for 15 minutes. Extract genomic DNA using a whole blood DNA extraction kit, following the kit's instructions. The whole blood DNA extraction kit used was the SE Blood DNA Kit manufactured by OMEGA.

[0072] S2. The PCR system consisted of: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 13.25 μL of ultrapure water. The 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd. The nucleotide sequence of the pre-primer for amplifying rs12985234 is shown in SEQ ID No. 1; the nucleotide sequence of the post-primer is shown in SEQ ID No. 2. The nucleotide sequence of the pre-primer for amplifying rs14394 is shown in SEQ ID No. 3; the nucleotide sequence of the post-primer is shown in SEQ ID No. 4. The nucleotide sequence of the pre-primer for amplifying rs35986520 is shown in SEQ ID No. 5; the nucleotide sequence of the post-primer is shown in SEQ ID No. 6. The nucleotide sequence of the first primer for amplifying rs10402477 is shown in SEQ ID No. 7; the nucleotide sequence of the second primer is shown in SEQ ID No. 8. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction conditions were: pretreatment at 96℃ for 5 min, denaturation at 96℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min. The PCR amplification products were sent to Suzhou Genewiz Biotechnology Co., Ltd. and Shanghai Maipu Biotechnology Co., Ltd. for sequencing.

[0073] Example 9: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes a modified polyamide ester material.

[0074] Methods for preparing modified polyamide ester materials include, Diethylenetriamine was dissolved in methanol at 4°C under a nitrogen atmosphere. Ethylene glycol diacrylate was added and stirred until homogeneous. The mixture was then reacted at 25°C for 4 hours. After rotary evaporation under reduced pressure, 2-octenyl succinic anhydride was added, and the mixture was reacted again at 150°C under reduced pressure for 4 hours to obtain the modified polyamide ester material. The molar ratio of diethylenetriamine to methanol was 1 mol: 100 mL; the molar ratio of diethylenetriamine to ethylene glycol diacrylate was 1:0.4; and the molar ratio of diethylenetriamine to 2-octenyl succinic anhydride was 1:2.

[0075] The method for detecting SNP sites in diabetes mellitus and its associated cognitive impairment was modified from Example 8, except that the PCR system was changed to: 1 μL of modified polyamide ester material with a final concentration of 0.026 μg / mL, 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 12.25 μL of ultrapure water; the modified polyamide ester material with a final concentration of 0.026 μg / mL was prepared by mixing the modified polyamide ester material with 10× buffer; 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd., and other conditions were the same as in Example 8.

[0076] Example 10: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified polyamide ester material and modified magnetic beads.

[0077] The preparation method of the modified polyamide ester material is the same as in Example 9.

[0078] Methods for preparing modified magnetic beads include, Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, followed by the addition of an alkenyl monomer, methylenebisacrylamide, and azobisisobutyronitrile (AIBN). The mixture was ultrasonically homogenized and reacted at 100°C for 15 min under a nitrogen atmosphere, then at 90°C for 8 h. The mixture was magnetically separated and washed three times with ethanol to obtain modified magnetic beads. The ratio of silica@Fe3O4 nanoparticles to acetonitrile was 1 g:200 mL; the alkenyl monomers included methacrylic acid and allyl malonic acid, with a mass ratio of 1:1; the mass ratio of silica@Fe3O4 nanoparticles to alkenyl monomers was 1:4; the mass ratio of silica@Fe3O4 nanoparticles to methylenebisacrylamide was 1:1; and the mass ratio of silica@Fe3O4 nanoparticles to AIBN was 1:0.1.

[0079] Methods for detecting SNP sites associated with diabetes mellitus and its associated cognitive impairment include, S1. DNA Extraction: Centrifuge the blood sample at 3000g for 15 minutes. Extract genomic DNA using a whole blood DNA extraction kit, following the kit's instructions. The whole blood DNA extraction kit used was the SE Blood DNA Kit manufactured by OMEGA.

[0080] S2. DNA Purification: Modified magnetic beads were ultrasonically dispersed in ethanol to a concentration of 10 mg / mL to obtain a magnetic bead solution. Genomic DNA was mixed with the binding buffer by inversion, the magnetic bead solution was added, and the mixture was allowed to stand for 5 min. After magnetic separation, the supernatant was discarded. Washing buffer was added, and the washing was repeated three times. After magnetic separation, the supernatant was discarded, and the mixture was allowed to stand at room temperature for 10 min. Elution buffer was added and incubated for 5 min. After magnetic separation, the supernatant was retained to obtain the purified DNA sample. The binding buffer, washing buffer, and elution buffer were all purchased from Shanghai Beyotime Biotechnology Co., Ltd. The volume ratio of genomic DNA to binding buffer was 1:1; the volume ratio of genomic DNA to magnetic bead solution was 1:0.1; the volume ratio of genomic DNA to washing buffer was 1:2.5; and the volume ratio of genomic DNA to elution buffer was 1:0.5.

[0081] S3. The PCR system consisted of: 1 μL of modified polyamide ester material with a final concentration of 0.026 μg / mL, 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of purified DNA sample, 0.5 μL of the first primer, 0.5 μL of the second primer, and 12.25 μL of ultrapure water. The modified polyamide ester material with a final concentration of 0.026 μg / mL was prepared by mixing the modified polyamide ester material with 10× buffer. 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd. The nucleotide sequence of the first primer for amplifying rs12985234 is shown in SEQ ID No. 1; the nucleotide sequence of the second primer is shown in SEQ ID No. 2. The nucleotide sequence of the first primer for amplifying rs14394 is shown in SEQ ID No. 3; the nucleotide sequence of the second primer is shown in SEQ ID No. 4. The nucleotide sequence of the first primer for amplifying rs35986520 is shown in SEQ ID No. 5; the nucleotide sequence of the second primer is shown in SEQ ID No. 6. The nucleotide sequence of the first primer for amplifying rs10402477 is shown in SEQ ID No. 7; the nucleotide sequence of the second primer is shown in SEQ ID No. 8. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction conditions were: pretreatment at 96℃ for 5 min, denaturation at 96℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 5 min. The PCR products were sequenced by Suzhou Genewiz Biotechnology Co., Ltd. and Shanghai Maipu Biotechnology Co., Ltd.

[0082] Example 11: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified polyamide ester material and modified magnetic beads.

[0083] The preparation method of the modified polyamide ester material is the same as in Example 9.

[0084] Methods for preparing modified magnetic beads include, Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, followed by the addition of alkenyl monomers, methylenebisacrylamide, and azobisisobutyronitrile, and then ultrasonically homogenized. The mixture was reacted at 100°C for 15 min under a nitrogen atmosphere, and then at 90°C for 8 h. After magnetic separation, the mixture was washed three times with ethanol to obtain modified magnetic beads. The ratio of silica@Fe3O4 nanoparticles to acetonitrile was 1 g: 200 mL. The alkenyl monomers included methacrylic acid, allyl malonic acid, and 3-allyloxy-1,2-propanediol. The mass ratio of methacrylic acid to allyl malonic acid was 1:1, and the mass ratio of methacrylic acid to 3-allyloxy-1,2-propanediol was 1:0.17. The mass ratio of silica@Fe3O4 nanoparticles to alkenyl monomers was 1:4; the mass ratio of silica@Fe3O4 nanoparticles to methylenebisacrylamide was 1:1; and the mass ratio of silica@Fe3O4 nanoparticles to azobisisobutyronitrile was 1:0.1.

[0085] The method for detecting SNP sites of diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the modified magnetic beads are replaced with the modified magnetic beads prepared in this example.

[0086] Example 12: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified polyamide ester material and modified magnetic beads.

[0087] The preparation method of the modified polyamide ester material is the same as in Example 9.

[0088] Methods for preparing modified magnetic beads include, Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, followed by the addition of alkenyl monomers, methylenebisacrylamide, and azobisisobutyronitrile, and then ultrasonically homogenized. The mixture was reacted at 100°C for 15 min under a nitrogen atmosphere, and then at 90°C for 8 h. After magnetic separation, the mixture was washed three times with ethanol to obtain modified magnetic beads. The ratio of silica@Fe3O4 nanoparticles to acetonitrile was 1 g:200 mL. The alkenyl monomers included methacrylic acid, allyl malonic acid, and 3-allyloxy-1,2-propanediol. The mass ratio of methacrylic acid to allyl malonic acid was 1:1, and the mass ratio of methacrylic acid to 3-allyloxy-1,2-propanediol was 1:0.23. The mass ratio of silica@Fe3O4 nanoparticles to alkenyl monomers was 1:4; the mass ratio of silica@Fe3O4 nanoparticles to methylenebisacrylamide was 1:1; and the mass ratio of silica@Fe3O4 nanoparticles to azobisisobutyronitrile was 1:0.1.

[0089] The method for detecting SNP sites of diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the modified magnetic beads are replaced with the modified magnetic beads prepared in this example.

[0090] Example 13: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified polyamide ester material and modified magnetic beads.

[0091] The preparation method of the modified polyamide ester material is the same as in Example 9.

[0092] Methods for preparing modified magnetic beads include, Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, followed by the addition of alkenyl monomers, methylenebisacrylamide, and azobisisobutyronitrile, and then ultrasonically homogenized. The mixture was reacted at 100°C for 15 min under a nitrogen atmosphere, and then at 90°C for 8 h. After magnetic separation, the mixture was washed three times with ethanol to obtain modified magnetic beads. The ratio of silica@Fe3O4 nanoparticles to acetonitrile was 1 g:200 mL; the alkenyl monomers included methacrylic acid, allyl malonic acid, and 3-allyloxy-1,2-propanediol, with a mass ratio of methacrylic acid to allyl malonic acid of 1:1 and a mass ratio of methacrylic acid to 3-allyloxy-1,2-propanediol of 1:0.08; the mass ratio of silica@Fe3O4 nanoparticles to alkenyl monomers was 1:4; the mass ratio of silica@Fe3O4 nanoparticles to methylenebisacrylamide was 1:1; and the mass ratio of silica@Fe3O4 nanoparticles to azobisisobutyronitrile was 1:0.1.

[0093] The method for detecting SNP sites of diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the modified magnetic beads are replaced with the modified magnetic beads prepared in this example.

[0094] Comparative Example 1: The detection kit for SNP sites associated with diabetes and cognitive impairment, compared with Example 8, also includes the fifth-generation polyamide-amine dendritic polymer G5.NH2; G5.NH2 is a polymer formed by extending molecular units linked by amino groups with ethylenediamine as the core; G5.NH2 was purchased from Xi'an Qiyue Biotechnology Co., Ltd., and other conditions are the same as in Example 8.

[0095] The method for detecting SNP sites in diabetes mellitus and its associated cognitive impairment was modified from Example 8, except that the PCR system was changed to: 1 μL of G5.NH2 with a final concentration of 0.026 μg / mL, 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA, 0.5 μL of the front primer, 0.5 μL of the back primer, and 12.25 μL of ultrapure water; the G5.NH2 with a final concentration of 0.026 μg / mL was prepared by mixing G5.NH2 with 10× buffer; 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd., and other conditions were the same as in Example 8.

[0096] Comparative Example 2: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified magnetic beads.

[0097] Methods for preparing modified magnetic beads include, Silica@Fe3O4 nanoparticles were ultrasonically dispersed in acetonitrile, followed by the addition of an alkenyl monomer, methylenebisacrylamide, and azobisisobutyronitrile (AIBN). The mixture was ultrasonically homogenized and reacted at 100°C for 15 min under a nitrogen atmosphere, then at 90°C for 8 h. The mixture was magnetically separated and washed three times with ethanol to obtain modified magnetic beads. The ratio of silica@Fe3O4 nanoparticles to acetonitrile was 1 g:200 mL; the alkenyl monomers included methacrylic acid and allyl malonic acid, with a mass ratio of 1:1; the mass ratio of silica@Fe3O4 nanoparticles to alkenyl monomers was 1:4; the mass ratio of silica@Fe3O4 nanoparticles to methylenebisacrylamide was 1:1; and the mass ratio of silica@Fe3O4 nanoparticles to AIBN was 1:0.1.

[0098] The method for detecting SNP sites in diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the PCR system is changed to: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA, 0.5 μL of front primer, 0.5 μL of back primer, and 13.25 μL of ultrapure water; 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd., and other conditions are the same as in Example 10.

[0099] Comparative Example 3: The detection kit for SNP sites associated with diabetes and cognitive impairment, compared with Example 8, includes carboxyl magnetic beads purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the other conditions are the same as in Example 10.

[0100] The method for detecting SNP sites of diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the modified magnetic beads are replaced with carboxyl magnetic beads purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0101] Comparative Example 4: The detection kit for SNP sites associated with diabetes and cognitive impairment is the same as that in Example 8, except that it also includes modified polyamide ester material and modified magnetic beads.

[0102] The preparation method of the modified polyamide ester material is the same as in Example 9.

[0103] The preparation method of the modified magnetic beads is the same as that of Example 11, except that allylmalonic acid was not used.

[0104] The method for detecting SNP sites of diabetes mellitus and its associated cognitive impairment is the same as in Example 10, except that the modified magnetic beads are replaced with the modified magnetic beads prepared in this comparative example.

[0105] Experimental Example 1: Blood samples from 961 patients with type 2 diabetes at Yuhuan People's Hospital were used as the type 2 diabetes patient group, and blood samples from 167 healthy volunteers at Yuhuan People's Hospital were used as the healthy control group. Blood samples from both groups were collected, centrifuged at 3000g for 15 minutes, and genomic DNA was extracted using a whole blood DNA extraction kit. The extraction procedure was performed according to the kit's instructions. The whole blood DNA extraction kit was the SE Blood DNA Kit manufactured by OMEGA.

[0106] Using the extracted genomic DNA as a template, polymerase chain reaction (PCR) was performed to amplify molecular markers rs12985234, rs14394, rs35986520, and rs10402477, respectively. Homozygous and heterozygous sequencing results for each SNP locus were statistically analyzed. The PCR system consisted of: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA template, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 13.25 μL of ultrapure water. The 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Beyotime Biotechnology Co., Ltd. The reaction conditions were as follows: pretreatment at 96℃ for 5 min, denaturation at 96℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles (96℃ high temperature to dissociate double-stranded DNA into single strands, annealing at 55℃ to allow primers to bind to the complementary regions of the template DNA, and a medium temperature of 72℃ to allow DNA polymerase to catalyze the DNA strand extension reaction starting from the primers), and a final extension at 72℃ for 5 min. The PCR amplification products were sent to Suzhou Genewiz Biotechnology Co., Ltd. and Shanghai Maipu Biotechnology Co., Ltd. for sequencing and analysis.

[0107] Figure 1 The results are for homozygous and heterozygous sequencing of rs35986520. Figure 2 The results are for homozygous and heterozygous sequencing of rs10402477. Figure 3 The results are for homozygous and heterozygous sequencing of rs12985234. Figure 4 The results are for homozygous and heterozygous sequencing of rs14394.

[0108] Table 1 Number of heterozygous and homozygous mutations at SNP sites

[0109] The results are shown in Table 1. At the four single nucleotide polymorphism (SNP) sites rs12985234, rs14394, rs35986520, and rs10402477, heterozygous genotype mutations were higher in patients with type 2 diabetes than in healthy individuals. Specifically, heterozygous mutations at rs35986520 and rs10402477 were significantly increased in patients with type 2 diabetes. This suggests a possible association between heterozygous mutations at rs12985234, rs14394, rs35986520, and rs10402477 and type 2 diabetes.

[0110] Experimental Example 2: Blood samples from 961 patients with type 2 diabetes at Yuhuan People's Hospital were used as the type 2 diabetes patient group, and blood samples from 167 healthy volunteers at Yuhuan People's Hospital were used as the healthy control group. Blood samples from both groups were collected, centrifuged at 3000g for 15 min, and genomic DNA was extracted using a whole blood DNA extraction kit. The extraction procedure was performed according to the kit's instructions. The whole blood DNA extraction kit was the SE Blood DNA Kit manufactured by OMEGA. Polymerase chain reaction (PCR) was performed using the extracted genomic DNA as a template to amplify the molecular markers rs12985234, rs35986520, and rs10402477, respectively.

[0111] Blood samples from 943 patients with type 2 diabetes at Yuhuan People's Hospital were used as the type 2 diabetes patient group, and blood samples from 94 healthy volunteers at Yuhuan People's Hospital were used as the healthy control group. Blood samples from both groups were collected, centrifuged at 3000g for 15 min, and genomic DNA was extracted using a whole blood DNA extraction kit. The extraction procedure was performed according to the kit's instructions. The whole blood DNA extraction kit was the SE Blood DNA Kit manufactured by OMEGA. Polymerase chain reaction (PCR) was performed using the extracted genomic DNA as a template to amplify the rs14394 molecular marker.

[0112] The PCR system consisted of: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA template, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 13.25 μL of ultrapure water. The 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd. The reaction conditions were: 96℃ pretreatment for 5 min, 96℃ denaturation for 30 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles (96℃ high temperature to dissociate double-stranded DNA into single strands, 55℃ annealing to allow primers to bind to the complementary regions of the template DNA, and 72℃ medium temperature to allow DNA polymerase to catalyze the DNA strand extension reaction starting from the primers), and a final extension at 72℃ for 5 min. The PCR products were sent to Suzhou Genewiz Biotechnology Co., Ltd. and Shanghai Maipu Biotechnology Co., Ltd. for sequencing and analysis.

[0113] Chi-square test was used to compare the differences in allele frequencies and genotype frequencies of rs12985234, rs14394, rs35986520 and rs10402477 between patients with type 2 diabetes and healthy controls, and to analyze the correlation between each SNP and type 2 diabetes.

[0114] Table 2. Differences in Genotype Frequency Distribution

[0115] * indicates a statistically significant difference.

[0116] Table 3. Differences in allele frequency distribution

[0117] * indicates a statistically significant difference.

[0118] Table 4. Differences in genotype and allele frequency distributions of rs14394

[0119] The results are shown in Tables 2 to 4. The A allele of rs35986520 and the G allele of rs12985234 were significantly associated with susceptibility to type 2 diabetes.

[0120] Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated to evaluate the efficacy of the SNP biomarker in distinguishing between patients with type 2 diabetes and healthy individuals. An AUC in the range of 0.5–1 indicates that the SNP biomarker has a certain degree of discriminative accuracy.

[0121] Figure 5 The ROC curve for rs10402477 is shown. Figure 6 The ROC curve for rs35986520 is shown. Figure 7 The ROC curve is 12985234. Figure 8 The ROC curve for rs14394 is shown.

[0122] The results are as follows Figures 5 to 8 As shown, the area under the receiver operating characteristic (ROC) curve for rs35986520 is above 0.5211, for rs10402477 it is above 0.5022, for rs12985234 it is above 0.5216, and for rs14394 it is above 0.5058. Therefore, rs35986520, rs10402477, rs12985234, and rs14394 are conservative SNP markers with potential screening capabilities.

[0123] Experimental Example 3: Cognitive impairment in patients with type 2 diabetes was assessed using the Montreal Cognitive Assessment Scale. Patients with an assessment score ≥26 were classified as having normal cognitive function, while those with a score <26 were classified as having abnormal cognitive function. Blood samples were collected from both groups and centrifuged at 3000g for 15 min. Genomic DNA was extracted using a whole blood DNA extraction kit, following the kit's instructions. The whole blood DNA extraction kit used was the SE Blood DNA Kit manufactured by OMEGA. Polymerase chain reaction (PCR) was performed using the extracted genomic DNA as a template to amplify the molecular markers rs12985234, rs14394, rs35986520, and rs10402477.

[0124] The PCR system consisted of: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA template, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 13.25 μL of ultrapure water. The 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd. The reaction conditions were: 96℃ pretreatment for 5 min, 96℃ denaturation for 30 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles (96℃ high temperature to dissociate double-stranded DNA into single strands, 55℃ annealing to allow primers to bind to the complementary regions of the template DNA, and 72℃ medium temperature to allow DNA polymerase to catalyze the DNA strand extension reaction starting from the primers), and a final extension at 72℃ for 5 min. The PCR products were sent to Suzhou Genewiz Biotechnology Co., Ltd. and Shanghai Maipu Biotechnology Co., Ltd. for sequencing and analysis. Statistical analysis was performed to determine the significance of genotype frequencies in rs12985234, rs14394, rs35986520, and rs10402477 between the cognitively normal and cognitively abnormal groups.

[0125] Table 5. Genotype frequency distribution of rs35986520

[0126] The results are shown in Table 5. The rs35986520 genotype was significantly associated with cognitive impairment and can serve as an effective screening marker for diabetes and cognitive impairment comorbidities. Molecular markers rs12985234, rs14394, and rs10402477 were not significantly associated with cognitive impairment.

[0127] Experiment Example 4: PCR amplification using rs35986520 labeling was performed in a blank control group, six experimental groups, and six control groups. The PCR reaction system for the blank control group consisted of: 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of genomic DNA template, 0.5 μL of front primer, 0.5 μL of back primer, and 13.25 μL of ultrapure water. The 10× buffer, dNTPs, and Taq DNA polymerase were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0128] The PCR reaction systems for the six experimental groups were as follows: 1 μL of modified polyamide ester material with a final concentration of 0.022, 0.024, 0.026, 0.028, 0.03 or 0.032 μg / mL, 2.5 μL of 10× buffer, 2 μL of dNTPs, 0.25 μL of Taq DNA polymerase, 1 μL of purified DNA sample, 0.5 μL of pre-primer, 0.5 μL of post-primer, and 12.25 μL of ultrapure water.

[0129] The PCR reaction system for the six control groups consisted of: 1 μL of G5.NH2 at final concentrations of 0.022, 0.024, 0.026, 0.028, 0.03, or 0.032 μg / mL; 2.5 μL of 10× buffer; 2 μL of dNTPs; 0.25 μL of Taq DNA polymerase; 1 μL of purified DNA sample; 0.5 μL of pre-primer; 0.5 μL of post-primer; and 12.25 μL of ultrapure water.

[0130] The reaction conditions for each group were as follows: pretreatment at 96℃ for 5 min, denaturation at 96℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles (96℃ high temperature to dissociate double-stranded DNA into single strands, annealing at 55℃ to allow primers to bind to the complementary regions of the template DNA, and annealing at 72℃ to allow DNA polymerase to catalyze the DNA strand extension reaction starting from the primers), and a final extension at 72℃ for 5 min. After the PCR reaction, the PCR products were analyzed by 1% agarose gel electrophoresis, and the electrophoresis images were obtained using a gel imaging system. The brightness of the target band and non-target bands in each group was detected using ImageJ software. The brightness of the target band and non-target bands in the blank group were used as the benchmark. The relative yield of the target product in other experimental groups was calculated as: target band brightness in the experimental group / target band brightness in the blank group. The relative yield of the non-target product was calculated as: non-target band brightness in the experimental group / non-target band brightness in the blank group. The signal-to-noise ratio was calculated as: relative yield of the target product / relative yield of the non-target product. The amplification yield was assessed by the relative yield of the target product, and the amplification specificity was assessed by the signal-to-noise ratio.

[0131] Figure 9 To increase the yield. Figure 10 For specificity. Results as follows Figure 9 and Figure 10 As shown, compared with Comparative Example 1, the PCR enhancer prepared in Example 9 can effectively improve the PCR amplification yield. When the concentration of the PCR enhancer is higher than 0.03 μg / mL, the amplification yield of both Comparative Example 1 and Comparative Example 2 is inhibited. Compared with Comparative Example 1, the PCR enhancer prepared in Example 10 can effectively improve the specificity of the target product. When the concentration is 0.024-0.03 μg / mL, the amplification specificity is approximately 1, indicating that the amplification result is basically a single target band, and non-specific amplification is effectively inhibited. Therefore, when the concentration of the PCR enhancer prepared in this invention is 0.022-0.03 μg / mL, the effect of improving amplification yield and amplification specificity is better.

[0132] Experimental Example 5: Blood samples were collected from patients with type 2 diabetes at Yuhuan People's Hospital. Following the detection methods for SNP loci in diabetes and its associated cognitive impairment provided in Examples 8-13 and Comparative Examples 1-4, rs35986520 labeling was performed to obtain the corresponding PCR products of Examples 8-13 and Comparative Examples 1-4. The PCR products were analyzed by 1% agarose gel electrophoresis, and electrophoresis images were obtained using a gel imaging system. ImageJ software was used to detect the brightness of the target band and non-target bands in each group. Using the brightness of the target band and non-target bands in Example 8 as a benchmark, the relative yield of the target product in other experimental groups was calculated as: (brightness of the target band in the experimental group / brightness of the target band in the blank group); the relative yield of the non-target product was calculated as: (brightness of the non-target band in the experimental group / brightness of the non-target band in the blank group). The signal-to-noise ratio was calculated as: relative yield of the target product / relative yield of the non-target product. The amplification yield was assessed using the relative yield of the target product, and the amplification specificity was assessed using the signal-to-noise ratio.

[0133] Table 6 Amplification Yield

[0134] The results are shown in Table 6. Compared with Example 8, the amplification yields of Examples 9-13 were significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 9-13 additionally used modified polyamide ester material as a PCR enhancer, while Example 8 did not. Compared with Comparative Example 1, the amplification yields of Examples 9-13 were significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 9-13 used modified polyamide ester material as a PCR enhancer, while Comparative Example 1 used G5.NH2 as a PCR enhancer. This indicates that using the modified polyamide ester material prepared in Examples 9-13 of this invention as a PCR enhancer can effectively improve the PCR amplification yield and enhance the PCR effect.

[0135] Compared to Example 9, the amplification yields of Examples 10-13 were significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 10-13, in addition to using modified polyamide ester material as a PCR enhancer, also used modified magnetic beads to purify the genomic DNA; while Example 9 did not undergo purification. Compared to Comparative Examples 2 and 3, the amplification yields of Examples 10-13 were significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 10-13, in addition to using modified polyamide ester material as a PCR enhancer, further used modified magnetic beads to purify the genomic DNA; while Comparative Example 2 only used modified magnetic beads to purify the genomic DNA without using modified polyamide ester material as a PCR enhancer, and Comparative Example 3, in addition to using modified polyamide ester material as a PCR enhancer, further used carboxyl magnetic beads to purify the genomic DNA. This indicates that in the detection method of SNP sites in diabetes and its associated cognitive impairment, first purifying genomic DNA using the modified magnetic beads prepared in this invention, and then using modified polyamide ester material as a PCR enhancer for PCR amplification, can effectively improve the PCR amplification yield and improve the PCR effect.

[0136] Compared to Example 10 and Comparative Example 4, the amplification yields of Examples 11-13 were further improved. This is because, in the preparation of the modified magnetic beads, Examples 11-13 used allylmalonic acid and 3-allyloxy-1,2-propanediol in combination for magnetic bead modification, while Example 10 used only allylmalonic acid, and Comparative Example 4 used only 3-allyloxy-1,2-propanediol. Compared to Example 11, the amplification yield of Example 12 was improved, while the amplification yield of Example 13 was reduced. This is due to the different amounts of 3-allyloxy-1,2-propanediol used in the preparation of the modified magnetic beads. This indicates that using appropriate amounts of allylmalonic acid and 3-allyloxy-1,2-propanediol in combination for magnetic bead modification helps to further improve the PCR amplification yield; excessively high or low amounts will affect the improvement of PCR amplification.

[0137] Table 7 Amplification Specificity

[0138] The results are shown in Table 7. Compared with Example 8, the amplification specificity of Examples 9-13 was significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 9-13 additionally used modified polyamide ester material as a PCR enhancer, while Example 8 did not. Compared with Comparative Example 1, the amplification specificity of Examples 9-13 was significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 9-13 used modified polyamide ester material as a PCR enhancer, while Comparative Example 1 used G5.NH2 as a PCR enhancer. This indicates that using the modified polyamide ester material prepared in Examples 9-13 of this invention as a PCR enhancer can effectively improve PCR amplification specificity and improve PCR performance.

[0139] Compared to Example 9, the amplification specificity of Examples 10-13 was significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 10-13, in addition to using modified polyamide ester material as a PCR enhancer, also used modified magnetic beads to purify the genomic DNA; while Example 9 did not undergo purification. Compared to Comparative Examples 2 and 3, the amplification specificity of Examples 10-13 was significantly improved. This is because, in the detection method for SNP sites in diabetes and its associated cognitive impairment, Examples 10-13, in addition to using modified polyamide ester material as a PCR enhancer, further used modified magnetic beads to purify the genomic DNA; while Comparative Example 2 only used modified magnetic beads to purify the genomic DNA without using modified polyamide ester material as a PCR enhancer, and Comparative Example 3, in addition to using modified polyamide ester material as a PCR enhancer, further used carboxyl magnetic beads to purify the genomic DNA. This indicates that in the detection method of SNP sites in diabetes and its associated cognitive impairment, first purifying genomic DNA using the modified magnetic beads prepared in this invention, and then using modified polyamide ester material as a PCR enhancer for PCR amplification, can effectively improve the specificity of PCR amplification and improve the PCR effect.

[0140] Compared to Examples 10 and 4, the amplification specificity of Examples 11-13 was further improved. This is because, in the preparation of the modified magnetic beads, Examples 11-13 used allylmalonic acid and 3-allyloxy-1,2-propanediol in combination for magnetic bead modification, while Example 10 used only allylmalonic acid, and Comparative Example 4 used only 3-allyloxy-1,2-propanediol. Compared to Example 11, the amplification specificity of Example 12 was improved, while the amplification specificity of Example 13 was decreased. This is due to the different amounts of 3-allyloxy-1,2-propanediol used in the preparation of the modified magnetic beads. This indicates that using appropriate amounts of allylmalonic acid and 3-allyloxy-1,2-propanediol in combination for magnetic bead modification helps to further improve the PCR amplification specificity; excessively high or low amounts will affect the improvement of PCR amplification.

[0141] Experimental Example 6: The modified polyamide ester material was detected using Fourier transform infrared spectroscopy.

[0142] Figure 11 The image shows the infrared spectrum of the modified polyamide ester material. The results are as follows: Figure 11 As shown, 3350cm -1 An absorption peak for NH appears nearby, at 2940 cm⁻¹. -1 An absorption peak for CH appears nearby, at 1730 cm⁻¹. -1 An absorption peak appears near C=O, at 1690 cm⁻¹. -1 An absorption peak of C=C appears nearby, at 1250 cm⁻¹. -1 An absorption peak for COC appears nearby. At 1640 cm⁻¹ -1 The presence of a C=0 bond of an amide group (-CO-NH-) nearby indicates that 2-octenyl succinic anhydride reacts with an amino group to form a cross-linked amide bond.

[0143] The surface morphology of the modified magnetic beads prepared in Example 11 was observed using a transmission electron microscope.

[0144] Figure 12 The image shows a TEM image of the modified magnetic beads. The results are as follows: Figure 12 As shown, the modified magnetic beads prepared by this invention are regular spherical in shape, and an organic layer can be observed on the surface of the modified magnetic beads, forming a core-shell structure.

[0145] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0146] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of reagents for detecting SNP sites associated with diabetes mellitus and its associated cognitive impairment in the preparation of reagents for predicting diabetes mellitus and its associated cognitive impairment, characterized in that: The SNP site is at least one of rs12985234, rs14394, rs35986520, and rs10402477.

2. The application according to claim 1, characterized in that: The reagents for detecting SNP sites associated with diabetes and its associated cognitive impairment are selected from one or more of the following: DNA microarrays or chips containing combinations of genetic markers for detecting SNP sites, specific PCR primers or probes, and targeted high-throughput sequencing reagents.

3. The application according to claim 1 or 2, characterized in that: The nucleotide sequence of the pre-primer for detecting rs12985234 is shown in SEQ ID No. 1, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 2; and / or The nucleotide sequence of the pre-primer for rs14394 amplification is shown in SEQ ID No. 3, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 4; and / or The nucleotide sequence of the pre-primer for rs35986520 amplification is shown in SEQ ID No. 5, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 6; and / or The nucleotide sequence of the pre-primer for rs10402477 amplification is shown in SEQ ID No. 7, and the nucleotide sequence of the post-primer is shown in SEQ ID No.

8.

4. A kit for detecting SNP sites associated with diabetes mellitus and its associated cognitive impairment, characterized in that, include: Used for detection ELAVL1 Primers for amplification of at least one of the following loci in the gene: rs12985234, rs14394, rs35986520, and rs10402477; The nucleotide sequence of the pre-primer for rs12985234 amplification is shown in SEQ ID No. 1, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 2; The nucleotide sequence of the pre-primer for rs14394 amplification is shown in SEQ ID No. 3, and the nucleotide sequence of the post-primer is shown in SEQ ID No.

4. The nucleotide sequence of the pre-primer for rs35986520 amplification is shown in SEQ ID No. 5, and the nucleotide sequence of the post-primer is shown in SEQ ID No. 6; The nucleotide sequence of the pre-primer for rs10402477 is shown in SEQ ID No. 7, and the nucleotide sequence of the post-primer is shown in SEQ ID No.

8.

5. The detection kit according to claim 4, characterized in that, The test kit also includes modified polyamide ester material.

6. The detection kit according to claim 5, characterized in that, The method for preparing the modified polyamide ester material includes dissolving diethylenetriamine in methanol under low temperature and nitrogen atmosphere conditions, adding ethylene glycol diacrylate, stirring and mixing, reacting at room temperature, rotary evaporating under reduced pressure, adding 2-octenyl succinic anhydride and reacting under reduced pressure to obtain the modified polyamide ester material.

7. The detection kit according to claim 6, characterized in that, The ratio of diethylenetriamine to methanol is 1 mol: 50-200 mL.

8. The detection kit according to claim 6, characterized in that, The molar ratio of diethylenetriamine to ethylene glycol diacrylate is 1:0.3-0.

5.

9. The detection kit according to claim 6, characterized in that, The molar ratio of diethylenetriamine to 2-octenylsuccinic anhydride is 1:1-3.

10. The detection kit according to claim 6, characterized in that, The decompression reaction temperature is 140-160℃.

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