Primer group and kit for detecting APOE gene locus polymorphism

Abstract

The invention discloses a primer group and a kit for detecting APOE gene locus polymorphism as well as a method and application for detecting the APOE gene locus polymorphism. The primer group comprises a multiple PCR amplification primer and a UEP primer used for a single base extension reaction, multiple PCR amplification is firstly carried out, SAP purification is then carried out, the extension reaction is then carried out, nucleic acid mass spectrometry detection is carried out on an extension product, and related genotype information is obtained. The method has the advantages of high accuracy, high flux, low cost and short period, 96 samples or 384 samples can be detected in one batch, only 8-10 hours are needed, the time is short, and the price is low. The kit can detect two gene loci related to APOE genotyping, the sensitivity is high, a detection result can be obtained only through a trace amount of genome DNA, and the detection result can guide health management, assist clinicians in evaluating the disease and progress risks of AD and related dementia and clinically select proper drugs, so that the purpose of precise medical treatment is achieved.

Description

Technical Field

[0001] The present invention relates to a primer set and a kit for detecting polymorphism of APOE gene loci. Background Art

[0002] With the aging of the population, dementia has become a common disease among the elderly, among which Alzheimer's disease (AD) dementia accounts for 60% to 80% and is the main cause of disability and death among the elderly. AD prevention and treatment is a global problem, and the primary reason is that it is difficult to diagnose early. The number of people with dementia in my country accounts for 1 / 4 of the world's total. Due to the differences in language and culture with the West, and the fact that early diagnostic technologies such as magnetic resonance imaging (MRI), positron emission tomography (PET) and blood biomarkers are not widely used, the early dementia diagnosis rate in general hospital outpatient clinics is only 0.1%.

[0003] Among the risk genes for Alzheimer's disease (AD), APOE is the most intensively studied and widely recognized. Apolipoprotein E (APOE) is an apolipoprotein present in chylomicrons and intermediate-density lipoproteins. It is mainly produced by the liver and macrophages and is involved in the transportation, storage and excretion of blood lipids. The human APOE gene is located on chromosome 19, 19q13.2. The two functional SNPs of this gene, rs429358 (c.388T>C, Cys130Arg) and rs7412 (c.526C>T, Arg176Cys), constitute three haplotypes, namely E2 (rs429358T-rs7412T), E3 (rs429358T-rs7412C), and E4 (rs429358C-rs7412C). The carrier rates in the population are 10% to 20%, 60%, and 20% to 30%, respectively. Studies have shown that the APOE E4 allele is involved in regulating the production of beta-amyloid (Aβ) and affects the clearance of Aβ by astrocytes and neurons, thereby affecting the formation and deposition of Aβ. At the same time, the APOE E4 gene cannot effectively maintain the stability of the connection between tau protein and tubulin and cannot inhibit the self-aggregation of tau protein, resulting in the formation of double helical fibers. The effects of different APOE alleles on the risk of AD vary. Among them, the risk of AD in APOE E4 allele carriers increases and the average age of onset decreases. Studies have shown that people carrying one APOE E4 allele have a risk of AD that is about 3.2 times that of normal people, while people carrying two APOE E4 alleles have a risk of AD that is 8 to 12 times that of normal people. Compared with non-carriers, people carrying the APOE E4 allele also have a significantly faster conversion rate from mild cognitive impairment (MCI) to AD.

[0004] In 2023, the FDA approved lecanemab (LEQEMBI) for the treatment of patients with early Alzheimer's disease with mild cognitive impairment or mild dementia. According to the FDA's lecanemab drug label, APOE E4 homozygous patients have a higher incidence of ARIA (amyloid related imaging abnormalities), including symptomatic and severe ARIA, when treated with this type of drug (including LEQEMBI) compared to heterozygotes and non-carriers. Therefore, APOE E4 mutation status should be tested before starting treatment to clarify the risk of ARIA.

[0005] In addition, APOE participates in the regulation of lipid metabolism in the body through various pathways and is an important internal factor affecting the body's blood lipid level. APOE polymorphism detection can also indicate the individual's efficacy in statins and provide a basis for patients to use statins rationally and effectively. At present, there are many methods for detecting gene mutations and gene polymorphisms, but there are problems such as limited detection throughput, low detection efficiency, and high prices. For this reason, this field urgently needs to develop a high-throughput, low-cost, and highly accurate APOE gene polymorphism detection solution. Summary of the invention

[0006] The purpose of the present invention is to provide a primer set and a kit for detecting polymorphism of APOE gene loci. The APOE gene loci polymorphism detection kit of the present invention uses nucleic acid mass spectrometry technology to detect and determine SNP sites related to APOE typing, and is used for gene detection of Alzheimer's disease medication, while guiding the use of statins and the risk of Alzheimer's disease treatment drug Lencanerumab.

[0007] The technical solution adopted by the present invention is:

[0008] A primer set for detecting polymorphism of APOE gene loci, comprising multiple PCR amplification primers, the sequences of which are shown in SEQ NO. 1 to SEQ NO. 4, and used for amplifying two APOE gene loci:

[0009]

[0010] The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ NO5 to SEQ NO6:

[0011]

[0012]

[0013] The present invention also provides a kit for detecting polymorphism of APOE gene loci, wherein the kit comprises the above-mentioned multiple PCR amplification primers and UEP primers.

[0014] Furthermore, the kit includes a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase (SAP) purification reaction system, and a single base extension reaction system.

[0015] The multiplex PCR amplification reaction solution includes multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture (dNTP), DNA polymerase, MgCl 2 .

[0016] The SAP purification reaction system comprises SAP enzyme and SAP buffer.

[0017] SAP enzyme and SAP buffer can be purchased from Agena.

[0018] The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Terminationmix and iPLEX Pro Enzyme.

[0019] The iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme were purchased from Agena.

[0020] The present invention also provides the use of the kit in detecting APOE gene site polymorphism.

[0021] The application includes APOE genotyping for health management, lipid-lowering drug guidance, cardiovascular disease and cognitive impairment risk assessment. The lipid-lowering drugs include probuco ezetimibe, arovastatin, rosuvastatin, pravastatin or lovastatin.

[0022] The gene loci include r7412 and rs429358.

[0023] The present invention also provides a method for detecting APOE gene site polymorphism using the kit, the method comprising the following steps:

[0024] (1) Multiplex PCR amplification reaction:

[0025] Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products;

[0026] The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components:

[0027] Ultrapure water 0.8μL, 10xPCR Buffer 0.5μL, 25mM MgCl 2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCR Enzyme 0.2μL, DNA template 2μL;

[0028] The P mix is ​​the primer sequence of SEQ NO1 to SEQ NO4, and the concentration of each primer is 0.5 μM;

[0029] The reaction conditions for the multiplex PCR amplification reaction were 95°C for 2 min; 95°C for 30 s, 56°C for 30 s, and 72°C for 60 s, for 45 cycles; and then 72°C for 5 min.

[0030] (2) Shrimp alkaline phosphatase (SAP) purification reaction:

[0031] The multiplex PCR amplification products were purified with SAP enzyme. The SAP purification reaction system included the following components: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL amplification product, a total of 7 μL reaction system; after the reaction, the purified product was obtained;

[0032] The reaction conditions for the purification reaction were 37 degrees for 40 minutes and 80 degrees for 5 minutes.

[0033] (3) Single base extension reaction

[0034] The purified product of step (2) is subjected to a single base extension reaction, wherein the single base extension reaction system comprises the following components: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus, 0.2 μL of iPLEX Termination mix, 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme, and 7 μL of purified product, for a total of 9 μL of reaction system; the reaction is performed to obtain an extension product;

[0035] UEP Mix contained the primer sequences of SEQ NO5 and SEQ NO6, and the concentration of each primer was 0.7 μM;

[0036] The reaction procedure of the single base extension reaction is 94° C. for 30 s, {94° C. for 5 s, (41° C. for 5 s, 80° C. for 5 s) 5 internal cycles} 39 external cycles, 72° C. for 3 min.

[0037] (4) Nucleic acid mass spectrometry analysis

[0038] 35 μL of deionized water was added to the extension product, which was then detected using a nucleic acid mass spectrometer to obtain the APOE gene site polymorphism.

[0039] The nucleic acid mass spectrometer is generally a MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer.

[0040] The nucleic acid mass spectrometer detector converts electrical signals into visual peak graphs. Each genotype has a unique molecular weight, and the software automatically analyzes the corresponding base type to obtain the final genotyping results.

[0041] The present invention firstly performs PCR amplification with self-designed primers, then SAP purification, and finally uses self-designed probes to perform extension reaction of target bases. The obtained extension products are subjected to nucleic acid mass spectrometry detection, and finally relevant genotype information is obtained.

[0042] The APOE gene site polymorphism detection kit of the present invention has the advantages of high accuracy, high throughput, low cost and short cycle. Compared with conventional Sanger sequencing, pyrophosphate sequencing and real-time fluorescence PCR technology, it has significant high throughput advantages and can detect 96 samples (96-well plate) or 384 samples (384-well plate) in one batch, and each sample can measure 6 gene sites; compared with the second-generation sequencing NGS, the present invention only needs 8-10 hours to complete a batch of samples, which is short in time, improves detection efficiency, and also has a more affordable price.

[0043] This kit can detect 2 APOE gene loci, involving 6 gene combinations and 3 genotypings. It has high sensitivity and only requires a trace amount of genomic DNA to obtain the test results. The test results can guide the clinic to select appropriate drugs, provide health guidance for the testers, and assist clinicians in assessing the risk of AD and related dementias and the risk of cardiovascular diseases, so as to truly achieve the goal of precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the mass spectrometry result of the rs7412 site of sample No. 13.

[0045] Figure 2 This is the Sanger sequencing result of the rs7412 site of sample No. 13.

[0046] Figure 3 This is the mass spectrometry result of the rs429358 site of sample No. 13.

[0047] Figure 4 This is the Sanger sequencing result of the rs429358 site of sample No. 13. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0049] In the present invention, SEQ NO1 to SEQ NO6 were all commissioned to be synthesized by Shanghai Bio-Tech Co., Ltd.

[0050] Example 1

[0051] Take one whole blood sample from each of the three volunteers and detect the six SNP sites of these three samples. First, extract the genomic DNA of the whole blood of the three volunteers, then perform multiple PCR reaction, SAP purification and single base extension reaction, and then place the product in the nucleic acid mass spectrometer for detection. Finally, after the offline data is analyzed by relevant software, the genotype of the two SNP sites of each sample can be obtained. At the same time, the mass spectrometry results are verified by the first-generation sequencing method. The verification results show that the results of the method of the present invention and the first-generation sequencing method have a 100% consistency rate.

[0052] Detection method of the kit of the present invention: 1. Multiplex PCR amplification reaction

[0053] Single sample PCR amplification system: ultrapure water 0.8 μL, 10xPCR Buffer 0.5 μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), 25 mM MgCl 2 0.4μL, dNTP0.1μL, P Mix 1μL, PCR Enzyme 0.2μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), DNA template 2μL, a total of 5μL reaction system. The reaction conditions are 95 degrees for 2 minutes; 95 degrees for 30 seconds, 56 degrees for 30 seconds, 72 degrees for 60 seconds, 45 cycles; 72 degrees for 5 minutes. The primers of P Mix are SEQ NO1 to SEQ NO4 in Table 1, and the concentration of each primer is 0.5μM.

[0054] The DNA template was extracted using the CWE960 Blood DNA Kit from Kangwei Century.

[0055] Each sample reaction yields an amplification product.

[0056] The multiplex PCR primer sequences are shown in Table 1:

[0057] Table 1

[0058]

[0059] In the present invention, the significance of APOE genotyping clinical testing is shown in Table 2 below:

[0060] Table 2

[0061]

[0062] 2. Shrimp alkaline phosphatase (SAP) purification reaction

[0063] 2 μL of SAP mixed solution was added to the above amplification reaction wells, including: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), to obtain a SAP purification system: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL first step PCR amplification product, a total of 7 μL reaction system. The reaction conditions for the purification reaction were 37 degrees for 40 minutes and 80 degrees for 5 minutes.

[0064] 3 Single base extension reaction

[0065] After the purification reaction, 2 μL of iPLEX reaction system was added to the reaction well, and the extension reaction system for each well was: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 0.2 μL of iPLEX Termination mix (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme (iPLEX Pro kit, purchased from Agena Bioscience Catalog No.: 10212), 7 μL of the second step purified product, a total of 9 μL of reaction system. The extended UEP Mix sequence is SEQ NO5 and SEQ NO6 in Table 3, and the concentration of each primer is 0.7 μM. The reaction conditions are shown in Table 4:

[0066] Table 3

[0067]

[0068] Table 4

[0069]

[0070] 4. Nucleic acid mass spectrometry analysis

[0071] Add 35 μL of deionized water to the extension product. Place the sample in the MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer, and set the detection parameters: Wells to Process is set to Automatic, Start Disoense Condition is set to 1000, Resin Volume is set to 13, Shot is set to 30, Maximum Acquisitions is set to 9, Minimum Good Spectra and Maximum Good Spectra are both set to 5, and 5 items need to be checked, including Tum OffHy After Analysis, Transfer Resin to MTP, Dispense Samples onto Chips, Transferchips to Anayzer, and AnalyzeChips. Then the mass spectrometer spotter spots the extension product onto the SpectroCHIP (chip) for detection. After the detection, the detector converts the electrical signal into a visible peak graph. Each genotype has a unique molecular weight. The software automatically analyzes the corresponding base type to obtain the final genotyping result. The results of the three test samples are shown in Tables 5 to 7.

[0072] Tables 5 to 7 are the test results of 3 samples respectively:

[0073] Table 5

[0074]

[0075] Table 6

[0076]

[0077] Table 7

[0078]

[0079] It can be seen from the test results that the kit of the present invention can accurately detect the 2 SNP genotypes.

[0080] The same sample was subjected to Sanger sequencing, and the sequencing results and mass spectrometry results were exactly the same. The detection spectrum of the representative sample (No. 13) is compared as follows Figures 1 to 4 shown.

[0081] in, Figure 1 This is the mass spectrometry result of the rs7412 locus of sample 13, and the result is boxed; Figure 2 This is the Sanger sequencing result of the rs7412 locus of sample 13, and the result is boxed; Figure 3This is the mass spectrometry result of the rs429358 locus of sample 13, and the result is boxed; Figure 4 This is the Sanger sequencing result of the rs429358 site of sample 13, and the result is boxed.

[0082] Figure 1 to Figure 4 The results showed that the sequencing method of the present invention was completely consistent with the results of Sanger sequencing.

Claims

1. A primer set for detecting polymorphism of APOE gene loci, characterized in that: The primer set includes multiple PCR amplification primers, the sequences of which are shown in SEQ NO1 to SEQ NO4, and are used to amplify two APOE genotyping-related gene loci:

2. The primer set for detecting APOE gene locus polymorphism according to claim 1, characterized in that: The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ NO5 to SEQ NO6:

3. A kit for detecting APOE gene locus polymorphism, characterized in that: The kit comprises the multiplex PCR amplification primers as claimed in claim 1 and the UEP primers as claimed in claim 2.

4. The kit according to claim 3, characterized in that The kit comprises a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase purification reaction system and a single base extension reaction system.

5. The kit according to claim 4, characterized in that The multiplex PCR amplification reaction solution comprises multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture, DNA polymerase and MgCl2; The SAP purification reaction system comprises SAP enzyme and SAP buffer; The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme.

6. Use of the kit as described in any one of claims 3 to 5 in detecting APOE gene locus polymorphism; the application includes APOE genotyping for health management, lipid-lowering drug guidance, and cardiovascular disease and cognitive impairment risk assessment.

7. A method for detecting APOE gene locus polymorphism using the kit according to any one of claims 3 to 5, characterized in that: The method comprises the following steps: (1) Multiplex PCR amplification reaction: Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products; The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components: Ultrapure water 0.8μL, 10xPCR Buffer 0.5μL, 25mM MgCl2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCREnzyme 0.2μL, DNA template 2μL; The P mix is ​​the primer sequence of SEQ NO1 to SEQ NO4, and the concentration of each primer is 0.5 μM; (2) Shrimp alkaline phosphatase purification reaction: The multiplex PCR amplification products were purified with SAP enzyme. The SAP purification reaction system included the following components: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL amplification product, a total of 7 μL reaction system; after the reaction, the purified product was obtained; (3) Single base extension reaction The purified product of step (2) is subjected to a single base extension reaction, wherein the single base extension reaction system comprises the following components: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus, 0.2 μL of iPLEX Termination mix, 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme, and 7 μL of purified product, a total of 9 μL of reaction system; after the reaction, an extension product is obtained; UEP Mix contained the primer sequences of SEQ NO5 and SEQ NO6, and the concentration of each primer was 0.7 μM; (4) Nucleic acid mass spectrometry analysis 35 μL of deionized water was added to the extension product, and then detected by a nucleic acid mass spectrometer to obtain the APOE gene site polymorphism.

8. The method according to claim 7, characterized in that The reaction conditions of the multiplex PCR amplification reaction were 95° C. for 2 minutes; 95° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 60 seconds, 45 cycles; and then 72° C. for 5 minutes.

9. The method according to claim 7, characterized in that In the step (2), the reaction conditions of the purification reaction are 37 degrees for 40 minutes and 80 degrees for 5 minutes.

10. The method according to claim 7, characterized in that In the step (3), the reaction procedure of the single base extension reaction is 94°C for 30s, {94°C for 5s, (41°C for 5s, 80°C for 5s) for 5 internal cycles} 39 external cycles, and 72°C for 3min.

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