Primer probe combination for detecting APOE haplotype, kit and application thereof

Abstract

The invention discloses a primer probe combination for detecting APOE haplotype, a kit and application thereof. The method, the kit, the primer pair and the probe provided by the invention can realize direct interpretation of each haplotype and typing combination of the APOE gene, the detection result is accurate, and the sensitivity is high. The method solves the problems that haploid typing interpretation cannot be directly realized or haploid type interpretation is incomplete, interpretation results are complex, detection reagents are many and the like in a traditional detection technology, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to gene locus detection, and in particular to a primer-probe combination for detecting APOE haplotype, a kit and applications thereof. Background Art

[0002] Human apolipoprotein E (APOE) is a 34kDa glycoprotein composed of 299 amino acids, encoded by the APOE gene located on chromosome 19. The function of APOE is to transport lipids between cells in different organs and within specific tissues as a component of plasma lipoproteins. In the central nervous system, APOE is expressed in large quantities in astrocytes, microglia, vascular wall cells, and choroid plexus cells, and in small quantities in stressed neurons. APOE can act as a ligand to bind to specific cell surface receptors, thereby regulating the clearance of lipoproteins from plasma. These receptors include members of the low-density lipoprotein receptor family and heparan sulfate proteoglycans.

[0003] The ApoE gene has two functional SNP sites, rs429358 (c.388T>C) and rs7412 (c.526C>T). According to the different bases at these two sites, four haplotypes are formed, ε2 (388T-526T), ε3 (388T-526C), ε4 (388C-526C) and ε3r (388C-526T). Among them, the ε4 mutation is the most common, forming 10 possible genotypes: 4 homozygous (ε2 / ε2, ε3 / ε3, ε4 / ε4 and ε3r / ε3r) and 6 heterozygous (ε2 / ε3, ε2 / ε4, ε3 / ε4, ε2 / ε3r, ε3 / ε3r and ε3r / ε4). The four haplotypes encode four ApoE protein isoforms: ApoE2, ApoE3, ApoE4 and ApoE3r. These four ApoE proteins differ in amino acids 112 and 158, mainly in the changes of arginine (Arginine, Arg) and cysteine ​​(Cysteine, Cys). Although the four ApoE proteins differ in only one amino acid residue in the primary structure, their spatial conformations are very different, resulting in different ApoE phenotypes having completely different effects in diseases.

[0004] Genome-wide association studies (GWAS) have confirmed that the ε4 allele of APOE is the strongest genetic risk factor for Alzheimer's disease (AD). Regardless of the presence of ε2 or ε3 alleles, carrying one or two ε4 alleles increases the risk of AD by 3-4 times or 9-15 times, respectively. If the AD incidence of the most widely distributed APOE3 homozygous population is taken as a benchmark, the AD incidence of the APOE4 population is higher than that of the APOE3 homozygous population. The probability of AD in the two heterozygous APOE4 populations is 2.6 and 3.6 times that of the APOE3 homozygous population, respectively, while the AD incidence of homozygous APOE4 can increase to 15.5 times. In contrast, the prevalence of AD in people with the APOE2 genotype is significantly lower than that of APOE3, indicating the protective effect of the ε2 allele.

[0005] Statins are affected by the absorption, distribution, metabolism, and excretion of drugs in the body. ApoE is an important transport protein for exogenous TC and endogenous TG in humans. It binds to low-density lipoprotein (LDL) receptors as a ligand, controls the uptake of chylomicrons and very low-density lipoproteins in the liver, and plays a role in lowering blood lipids. The ε3 type population accounts for the majority of the population, and the lipid-lowering effect of statins is normal, and there is no obvious tendency for the onset of cardiovascular and cerebrovascular diseases. The ε4 type population has a higher risk of early-onset cardiovascular disease and Alzheimer's disease. When dyslipidemia occurs, the effect of statins is worse than that of ε2 and ε3 types. Probucol can be used for treatment, and a low-fat diet has a significant lipid-lowering effect. Most people who take statins are patients with cardiovascular diseases such as coronary heart disease and myocardial infarction. By testing the APOE genotype, doctors can be guided to use drugs accurately within the effective treatment period, improve the effectiveness of drug use, and reduce toxic side effects.

[0006] There are relatively few studies and reports on the haplotype ε3r, on the one hand because it accounts for a relatively low proportion in the population, and on the other hand, it is not easy to detect its haplotype using current conventional detection methods. At the same time, since the sequences near these two SNP sites have a high GC content and the two SNP sites are close to each other, the design of primers and probes is greatly limited, which in turn affects the specificity and sensitivity of the test results.

[0007] Most of the kits reported so far can only interpret the results of two SNP sites of the APOE gene, and cannot detect its haploid type, which may lead to misjudgment. Some kits can interpret haploid types, but this method is to detect them by separately designing primers for the haploid types of ε2, ε3, and ε4. They can only interpret ε2 (388T-526T), ε3 (388T-526C), and ε4 (388C-526C), but cannot interpret the haploid type of ε3r (388C-526T). In addition, they are generally composed of three or more reaction solutions, which are complicated to operate and interpret. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a combination of primers and probes for detecting APOE haplotypes, a kit and their applications, which can quickly identify all haplotypes of the APOE gene and make the results easy to interpret.

[0009] In order to solve the above technical problems, the technical solution of the present invention is:

[0010] The present invention provides a combination of primers and probes for detecting haploid types of the ApoE gene, wherein the combination of primers and probes is divided into two groups:

[0011] The first set of primer probes:

[0012] The wild-type specific upstream primer of the rs429358 site is shown in SEQ ID NO: 1, the wild-type and mutant specific probes with different fluorescent labels at the rs7412 site are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the downstream primer is shown in SEQ ID NO: 5;

[0013] The second set of primer probes:

[0014] The mutant-specific upstream primer of the rs429358 site is shown in SEQ ID NO:2, the wild-type and mutant-specific probes with different fluorescent labels at the rs7412 site are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, and the downstream primer is shown in SEQ ID NO:5.

[0015] In some specific embodiments, the 3' end of the probe for the rs7412 site further includes an MGB probe.

[0016] In some embodiments, the fluorescent label is located at the 5' end of the probe of the rs7412 site and is selected from any one of VIC, CY5, FAM, ROX and HEX.

[0017] In some specific embodiments, the fluorescent label is selected from any one of FAM, ROX and CY5.

[0018] In some specific embodiments, the probe for the rs7412 site is shown as SEQ ID NO:3 and SEQ ID NO:4.

[0019] The present invention also provides a kit for detecting the haplotype of the APOE gene, wherein the kit comprises the combination of the primers and probes.

[0020] In some embodiments, the wild-type specific upstream primer of the rs429358 site and the mutant-type specific upstream primer of the rs429358 site are not mixed.

[0021] In some embodiments, the wild-type and mutant-specific probes labeled with different fluorescence and the downstream primer are formulated as a primer-probe mixture.

[0022] In some specific embodiments, the first set of primer probes is formulated as primer probe mixture A, and the second set of primer probes is formulated as primer probe mixture B.

[0023] In some embodiments, the kit further comprises primers and probes for an internal reference gene.

[0024] In some specific embodiments, the internal reference gene is GAPDH, the upstream primer is shown in SEQ ID NO:6, the probe is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8.

[0025] In some specific embodiments, the 3' end of the probe of the internal reference gene further includes a BHQ1 quenching group.

[0026] In some specific embodiments, the kit further comprises a PCR amplification reaction solution.

[0027] In some specific embodiments, the PCR amplification reaction solution includes Taq enzyme, buffer, dNTP, UDG enzyme, betaine and 10 nm nanogold particles.

[0028] In some embodiments, the final concentration of the primer is 200-600 nM.

[0029] In some specific embodiments, the final concentration of the primer is 400 nM.

[0030] In some embodiments, the final concentration of the probe is 100-500 nM.

[0031] In some specific embodiments, the final concentration of the probe is 200 nM.

[0032] In some embodiments, the final concentration of betaine is 0.2-0.4M.

[0033] In some specific embodiments, the final concentration of betaine is 0.3M.

[0034] In some embodiments, the final concentration of the 10 nm gold nanoparticles is 0.2-0.6 nM.

[0035] In some specific embodiments, the final concentration of the 10 nm gold nanoparticles is 0.4 nM.

[0036] In some embodiments, the Taq enzyme is selected from Champagne Taq DNA Polymerase, Hieff HotStart Taq DNA Polymerase, Golden Taq DNA Polymerase and TaqPath TM ProAmp TM Master Mixes.

[0037] In some specific embodiments, the Taq enzyme is Golden Taq DNA Polymerase.

[0038] The present invention also provides a method for detecting the haploid type of the APOE gene, the method comprising using the combination or the kit, the method comprising the following steps:

[0039] a) taking two samples to be tested, respectively adding ① the wild-type specific upstream primer of the rs429358 site, the wild-type and mutant-specific probes of the rs7412 site with different fluorescent labels and the downstream primer; and ② the mutant-specific upstream primer of the rs429358 site, the wild-type and mutant-specific probes of the rs7412 site with different fluorescent labels and the downstream primer as shown in SEQ ID NO:5, adding PCR amplification reaction solution, placing in a fluorescent quantitative PCR instrument and performing PCR amplification reaction simultaneously;

[0040] b) Performing fluorescence detection on the amplified product, and interpreting the APOE gene haploid type of the sample to be tested directly based on the fluorescence curve;

[0041] Wherein, the PCR amplification reaction solution is as defined in any of the preceding items; and the method is for non-diagnostic purposes.

[0042] In this article, application scenarios for non-diagnostic purposes include, for example, detecting the APOE gene in an in vitro sample for research and development purposes.

[0043] In some specific embodiments, the conditions for performing the PCR amplification reaction in step a) are:

[0044] 60℃ for 30 seconds, pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 15 seconds, annealing at 60℃ for 30 seconds, 40 cycles.

[0045] The present invention also provides the use of the primer-probe combination or the kit in detecting the haploid type of the ApoE gene.

[0046] In some specific embodiments, the ApoE gene haplotypes include 4 homozygous (ε2 / ε2, ε3 / ε3, ε4 / ε4, ε3r / ε3r) and 6 heterozygous (ε2 / ε3, ε2 / ε4, ε2 / ε3r, ε3 / ε4, ε3 / ε3r, ε3r / ε4).

[0047] In some specific embodiments, the present invention designs a specific primer-probe combination based on the polymorphism of two SNP sites of the APOE gene, which is used to amplify and identify all haploid types of the APOE gene. More precisely, different polymorphisms are detected by designing allele-specific amplification primers for the rs429358 site, and different polymorphisms are detected by designing specific MGB probes for the rs7412 site. Further explanation: an allele-specific amplification primer refers to a polymorphic base detected at the last base at the 3' end of the amplification primer, and amplification is performed only when it matches the base at that position of the amplification template. In order to further improve the specificity, it is usually necessary to introduce additional non-fixed positions of the amplification primer, and the position and base type of the additionally introduced non-matching base are difficult to accurately predict. Only by designing multiple different amplification primers and conducting experimental screening can allele-specific amplification primers with both good specificity and high amplification efficiency be obtained. It is further explained that when MGB probes are used to detect SNP polymorphisms, the length and sequence selection of the MGB probes will affect both the efficiency and specificity of amplification. However, it is difficult to design a probe that is both efficient in amplification and highly specific through theoretical design. Different probes need to be designed and combined with different amplification systems to obtain the best detection solution.

[0048] Through repeated exploration and optimization of experiments, the primer and probe combination of the present invention was obtained: the first group of primer-probe combinations: the wild-type specific upstream primer of the rs429358 site is shown in SEQ ID NO:1, the wild-type and mutant-specific probes of the rs7412 site using different fluorescent labels are shown in SEQ ID NO:3 and SEQ ID NO:4, and the downstream primer of the rs7412 site is shown in SEQ ID NO:5; the second group of primer-probe combinations: the wild-type specific upstream primer of the rs429358 site is shown in SEQ ID NO:2, the wild-type and mutant-specific probes of the rs7412 site using different fluorescent labels are shown in SEQ ID NO:3 and SEQ ID NO:4, and the downstream primer of the rs7412 site is shown in SEQ ID NO:5.

[0049] To further illustrate, the fluorescent label of the MGB probe in the present invention can be any combination of two of the following fluorescent groups: FAM, VIC or HEX or JOE, TAMRA, ROX, CY5.

[0050] The reaction system and detection method included in the present invention are as follows:

[0051] The wild-type specific upstream primer SEQ ID NO: 1 of the rs429358 (T) site, two specific probes SEQ ID NO: 3 and SEQ ID NO: 4 with different fluorescent labels of the rs7412 site, and a downstream primer are placed in reaction system A, and the mutant-specific upstream primer SEQ ID NO: 2 of the rs429358 (C) site, two specific probes SEQ ID NO: 3 and SEQ ID NO: 4 with different fluorescent labels of the rs7412 site, and a downstream primer are placed in reaction system B, thereby realizing the detection of APOE gene haplotype typing. It is further explained that haplotypes ε2 (388T-526T) and ε3 (388T-526C) can be detected by reaction system A, haplotypes ε4 (388C-526C) and ε3r (388C-526T) can be detected by reaction system B, and all haplotype combinations can be detected by A and B reactions.

[0052] The rs429358 and rs7412 sites of the APOE gene have high GC content, which is about 75%. Professionals and technicians in this field are aware that high GC content will bring great difficulties to amplification efficiency and specificity. In order to overcome the amplification efficiency and specificity problems caused by high GC in the APOE gene detection area, by screening the combination of adding different concentrations of betaine and different concentrations of 10nm nanogold particles, and screening different commercial taq DNA polymerases, the concentrations of betaine and 10nm nanogold particles with the highest amplification efficiency and the best specificity were finally screened out, and the concentrations were 0.3M and 0.4nM respectively, and the DNA polymerase was Golden Taq DNA Polymerase from Suzhou Yuanqi Biotechnology Co., Ltd.

[0053] Another object of the present invention is to provide a human APOE haplotype detection kit, which comprises the two sets of specific primer-probe combinations described above, wherein the specific primers are specific wild-type and mutant primers designed for the rs429358 site of the APOE gene; and the specific probes are specific wild-type and mutant probes designed for the rs7412 site of the APOE gene.

[0054] The components of the PCR amplification reaction solution include Taq enzyme, buffer, dNTP, UDG enzyme, and sterile water.

[0055] Further explanation, this reaction system uses the above two sets of primer probe combinations A and B and contains 0.3M betaine and different concentrations of 0.4nM 10nm nanogold particles to amplify each type of APOE haploid. The primer concentration in the reaction system is 400nM, and the probe concentration is 200nM.

[0056] The human APOE haplotype detection kit also includes a PCR amplification reaction solution, two sets of specific primer probe mixtures, a reference positive control solution, and a negative control solution.

[0057] The reaction program of the kit is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 15 seconds, extension at 60°C for 30 seconds, for a total of 40 cycles.

[0058] The result interpretation of the kit is based on the amplification of the two fluorescent channels in the reaction solutions of tubes A and B and the size of the amplification ct values ​​to perform typing interpretation. Further explanation, when an amplification curve of a corresponding color (depending on the specific fluorescent labeling group) appears and the Ct value is less than 38, it means that the haploid type is contained in the test sample.

[0059] The reagents and raw materials used in the present invention are commercially available.

[0060] The positive and progressive effects of the present invention are:

[0061] The present invention is aimed at the 139bp interval between the sites rs429358 and rs7412 of the APOE gene, and uses the base mutation types of the rs429358 site and the rs7412 site to respectively design specific amplification primers and specific MGB probes to achieve direct interpretation of each haploid type of the APOE gene and its typing combination. The typing interpretation can be performed using an ordinary fluorescent PCR instrument, and the detection result is accurate and highly sensitive. This solves the problems that traditional detection technology fails to directly realize haploid typing interpretation or haploid type interpretation is incomplete, the interpretation result is complex, and there are many detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a graph showing the results of APOE genotyping ε2 / ε2 homozygous type detection in an embodiment of the present invention.

[0063] Figure 2 This is a graph showing the results of APOE genotyping ε2 / ε3 homozygous type detection in an embodiment of the present invention.

[0064] Figure 3 This is a graph showing the results of APOE genotyping ε2 / ε4 homozygous type detection in an embodiment of the present invention.

[0065] Figure 4 This is a diagram of the APOE genotyping ε2 / ε3r homozygous detection results in an embodiment of the present invention.

[0066] Figure 5 This is a graph showing the results of APOE genotyping ε3 / ε3 homozygous type detection in an embodiment of the present invention.

[0067] Figure 6 This is a graph showing the results of APOE genotyping ε3 / ε4 homozygous type detection in an embodiment of the present invention.

[0068] Figure 7 This is a graph showing the results of APOE genotyping ε3 / ε3r homozygous type detection in an embodiment of the present invention.

[0069] Figure 8 This is a graph showing the results of APOE genotyping ε4 / ε4 homozygous type detection in an embodiment of the present invention.

[0070] Fig. 9 This is a graph showing the results of APOE genotyping ε3r / ε4 homozygous type detection in an embodiment of the present invention.

[0071] Fig.10 This is a graph showing the results of APOE genotyping ε3r / ε3r homozygous type detection in an embodiment of the present invention.

[0072] Fig.11 Schematic diagram of the detection method of the present invention.

[0073] Figures 12A-12D The results are shown in Table 1 for the screening of primer-probe combinations 1-4 in Example 1.

[0074] Figures 13A-13D This is the screening result of combination 1-4 in Example 2.

[0075] Figures 14A-14D is the Taq enzyme screening result in Example 2, wherein Fig.14A TaqPath TM ProAmp TM Master Mixes, Fig. 14B Golden taq DNA polymerase, Fig. 14C for Champagne Taq DNA Polymerase, Fig.14D Hieff HotStart Taq DNA Polymerase. DETAILED DESCRIPTION

[0076] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0077] Example 1

[0078] 1. Primer and probe design

[0079] The human APOE genotype is mainly determined by the base changes of two SNP sites, namely rs429358 (chromosome position 44.908,684) and rs7412 (chromosome position 44,908,822). Different genotypes are formed according to the different bases of the two sites. The APOE gene has four allele types, namely ε2 (rs429358-T and rs7412-T), ε3 (rs429358-T and rs7412-C), ε4 (rs429358-C and rs7412-C), and ε3r (388C-526T). The present invention is directed to the rs429358 (T>C) site and the rs7412 (C>T) site of the human APOE gene, and uses the site base mutation type to differentially design amplification primers and probes for specific amplification of all APOE gene haplotypes. Primers and probes were analyzed using Primer Premier5 software and custom synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0080] The specific wild-type and mutant upstream primer sequences designed for the rs429358 (T>C) site are shown in SEQ ID NO: 1 and SEQ ID NO: 2; the specific wild-type and mutant probe sequences designed for the rs7412 (C>T) site are shown in SEQ ID NO: 3 and SEQ ID NO: 4, the downstream primer sequence designed for the rs7412 (C>T) site is shown in SEQ ID NO: 5, and the upstream primer, probe and downstream primer designed for the internal reference gene GAPDH are shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0081] The above primer probe sequences are shown in Table 1 below:

[0082] Table 1 Primer probe sequences used in the examples

[0083] Primer / probe number Primer / probe name Primer / probe sequences SEQ ID NO:1 APOE-388-WF 5'-GCGGACATGGAGGACGTAT-3' SEQ ID NO:2 APOE-388-MF 5'-CGGACATGGAGGACGCGC-3' SEQ ID NO:3 APOE-526-WP FAM-CCAGGCGCTTCT-MGB SEQ ID NO:4 APOE-526-MP ROX-CCAGGCACTTCTG-MGB SEQ ID NO:5 APOE-526-R 5'-CGGATGGCGCTGAGGC-3' SEQ ID NO:6 GAPDH-F 5'-GACCTGACCTGCCGTCTA-3' SEQ ID NO:7 GAPDH-P CY5-TCCGAGCCTGCTTCACCACCTTCTT-BHQ1 SEQ ID NO:8 GAPDH-R 5'-CAGTGTAGCCCAGGATGC-3'

[0084] Table 2 shows the screening of different primer-probe combinations. The screening results of primer-probe combinations 1-4 are as follows: Figures 12A-12D shown.

[0085] Table 2 Primer probe sequence screening

[0086]

[0087] Example 2

[0088] 2. Composition of the kit

[0089] The present embodiment is a kit for detecting the haplotype of the human APOE gene, comprising a PCR amplification reaction solution, a primer-probe mixture A, and a primer-probe mixture B.

[0090] The components of the PCR amplification reaction solution include Taq enzyme, buffer, dNTP, UDG enzyme, sterile water, betaine and 10 nm gold nanoparticles.

[0091] Buffer, UDG enzyme, betaine were from Suzhou Yuanqi Biotechnology Co., Ltd., dNTP was from Shanghai Zhaowei Technology Development Co., Ltd., 10nm nanogold particles were from M erc k KGaA.

[0092] Table 3 Betaine, 10nm gold nanoparticle concentration screening

[0093] Combination 1 Combination 2 Combination 3 Combination 4 Betaine / 0.2M 0.3M 0.5M 10nm gold nanoparticles / 0.2nM 0.4nM 0.6nM

[0094] The screening results of combinations 1-4 are as follows Figures 13A-13D shown.

[0095] Table 4 Taq enzyme screening

[0096]

[0097] The screening results of the four enzymes are shown in Figures 14A-14D shown.

[0098] Example 3

[0099] 3. How to use the kit

[0100] The specific detection steps of the detection kit for detecting human APOE gene haplotype in this embodiment are as follows:

[0101] 1. DNA Extraction

[0102] For blood sample type and specific DNA extraction procedures, please refer to the DNA kit extraction product manual.

[0103] 2. DNA quality testing

[0104] After DNA extraction, the DNA quality was controlled by measuring the nucleic acid DNA concentration and the OD260 / 280 ratio. A ratio between 1.7 and 2.0 was appropriate and the best reaction results could be obtained.

[0105] 3. PCR reaction

[0106] (1) Prepare 25 μL PCR reaction system:

[0107] Calculate the number of PCR reaction tubes required for the number of samples (PCR reaction tubes = number of samples * 2 + positive control * 2 + negative control * 2), that is, 1 sample requires 6 tubes. Taking the preparation of reaction solution A as an example, the typing reaction system is 25μL: 12.5μL of PCR amplification reaction solution, 3μL of primer probe mixture A, 5μL of sample, and the rest of the water to make up the total system to 25μL. Prepare reaction solution B in the same way. Cover the PCR tube cap and mark it according to the known template.

[0108] (2) Perform PCR reaction. The PCR amplification program is shown in Table 5 below:

[0109] Table 5 PCR amplification program

[0110]

[0111] The above PCR reactions were performed on ABI7500, MA6000, and SLAN-96P fluorescence quantitative PCR instruments.

[0112] 4. Result analysis and interpretation

[0113] (1) The effectiveness of the kit is determined by referring to the positive control and negative control. The amplification curve of the positive control should show good amplification without abnormalities. The test results of the negative control should show no specific amplification, otherwise there may be contamination and the sample test results are unreliable.

[0114] (2) The validity of the experimental results is determined based on the internal reference CY5 channel. When the Ct value of the internal reference CY5 channel of the sample to be tested is ≤32, the result is valid and the result is interpreted according to the following principles:

[0115] (3) The determination of genotyping by the kit is shown in Table 6-7 below:

[0116] Table 6 Genotyping determination of the kit

[0117]

[0118] Table 7 Genotyping determination of the kit

[0119]

[0120] The sample test results can be found in Figure 1-10 .

Claims

1. A combination of primers and probes for detecting haploid types of the ApoE gene, characterized in that: The combinations of primers and probes are divided into two groups: The first set of primer probes: The wild-type specific upstream primer of the rs429358 site is shown in SEQ ID NO: 1, the wild-type and mutant specific probes with different fluorescent labels at the rs7412 site are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the downstream primer is shown in SEQ ID NO: 5; The second set of primer probes: The mutant-specific upstream primer of the rs429358 site is shown in SEQ ID NO:2, the wild-type and mutant-specific probes with different fluorescent labels at the rs7412 site are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, and the downstream primer is shown in SEQ ID NO:

5.

2. The combination of primers and probes according to claim 1, characterized in that: The 3' end of the probe at the rs7412 site also includes an MGB probe; And / or, the fluorescent label is located at the 5' end of the probe at the rs7412 site and is selected from any one of VIC, CY5, FAM, ROX and HEX, preferably any one of FAM, ROX and CY5.

3. A kit for detecting the haplotype of the APOE gene, characterized in that: The kit comprises a combination of primers and probes as claimed in claim 1 or 2, wherein the wild-type specific upstream primer of the rs429358 site and the mutant-type specific upstream primer of the rs429358 site are not mixed; Preferably, the first set of primer probes is prepared as primer-probe mixed solution A, and the second set of primer probes is prepared as primer-probe mixed solution B.

4. The kit according to claim 3, characterized in that The kit further comprises primers and probes of an internal reference gene; preferably, the internal reference gene is GAPDH, the upstream primer thereof is shown in SEQ ID NO:6, the probe is shown in SEQ ID NO:7, and the downstream primer is shown in SEQ ID NO:8; more preferably, the 3' end of the probe of the internal reference gene further comprises a BHQ1 quenching group; The kit also includes a PCR amplification reaction solution; the PCR amplification reaction solution preferably includes Taq enzyme, buffer, dNTP, UDG enzyme, betaine and 10 nm nano-gold particles.

5. The kit according to claim 3 or 4, characterized in that The final concentration of the primer is 200-600 nM, preferably 400 nM, and the final concentration of the probe is 100-500 nM, preferably 200 nM.

6. The kit according to claim 4 or 5, characterized in that The final concentration of the betaine is 0.2-0.4M, preferably 0.3M, and the final concentration of the 10nm nano-gold particles is 0.2-0.6nM, preferably 0.4nM; And / or, the Taq enzyme is selected from Champagne Taq DNA Polymerase, Hieff HotStartTaq DNA Polymerase, Golden Taq DNA Polymerase and TaqPath TM ProAmp TM Master Mixes, preferably Golden Taq DNA Polymerase.

7. A method for detecting the haplotype of the APOE gene, characterized in that: The method comprises using the combination according to claim 1 or 2 or the kit according to any one of claims 3 to 6, and the method comprises the following steps: a) taking two samples to be tested, respectively adding ① the wild-type specific upstream primer of the rs429358 site, the wild-type and mutant-specific probes of the rs7412 site with different fluorescent labels and the downstream primer; and ② the mutant-specific upstream primer of the rs429358 site, the wild-type and mutant-specific probes of the rs7412 site with different fluorescent labels and the downstream primer as shown in SEQ ID NO:5, adding PCR amplification reaction solution, placing in a fluorescent quantitative PCR instrument and performing PCR amplification reaction simultaneously; b) Performing fluorescence detection on the amplified product, and interpreting the APOE gene haploid type of the sample to be tested directly based on the fluorescence curve; Wherein, the PCR amplification reaction solution is as defined in any one of claims 4-6; The method is not intended for diagnostic purposes.

8. The method according to claim 7, characterized in that The conditions for performing the PCR amplification reaction in step a) are: 60℃ for 30 seconds, pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 15 seconds, annealing at 60℃ for 30 seconds, 40 cycles.

9. Use of the combination of primers and probes as described in claim 1 or 2 or the kit as described in claims 3-6 in detecting haplotypes of the ApoE gene; the use is for non-diagnostic purposes.

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