Kit and detection method for detecting rs429358 and rs7412 site mutation of human APOE gene

Abstract

The invention provides a kit for detecting human APOE gene rs429358 and rs7412 site mutation and a detection method, and relates to the technical field of gene detection.By optimizing components of a sample releasing agent, cells can be efficiently split, nucleic acid can be released, meanwhile, stable reaction is ensured through enzyme and a protective agent in a freeze-dried ball, and the detection sensitivity is high. High-specificity detection is achieved through specific primers and probes in the chip, the possibility of non-specific amplification and cross reaction is greatly reduced, the accuracy of APOE gene rs429358 and rs7412 site mutation detection is remarkably improved, cells are rapidly split through a sample releasing agent, efficient polymerase chain reaction can be achieved through rapid Taq enzyme in freeze-drying balls, and the detection sensitivity is high. The whole detection process is short in time consumption, a detection result can be rapidly obtained, the clinical requirements for rapid diagnosis and timely treatment are met, especially in some emergency situations, a timely diagnosis basis can be provided for a patient, and the kit adopts simple steps, so that the operation difficulty and the dependence on the technical level of operators are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and more specifically, particularly relates to a kit and a detection method for detecting mutations at sites rs429358 and rs7412 of the human APOE gene. Background Art

[0002] Apolipoprotein E gene (APOE) is currently the only recognized risk gene for late-onset Alzheimer's disease (AD). APOE gene polymorphism (rs429358 site and rs7412 site) is closely related to late-onset Alzheimer's disease (AD, Alzheimer's disease), coronary heart disease, hyperlipidemia, cerebral infarction and other diseases. In the normal metabolic process, APOE produces β-amyloid peptide to maintain nerve function. However, under appropriate conditions, β-amyloid peptide will form fibrillar polypeptides, which will deposit in nerve cells and produce toxic effects, leading to the death of neuronal cells, causing Alzheimer's patients to have dementia symptoms such as cognitive impairment and memory dysfunction. When APOE mutates, blood lipids cannot be transported. Over time, blood lipids in plasma gradually increase and deposit in the blood vessel wall, gradually forming small plaques, which will cause atherosclerosis, resulting in blood flow obstruction, blood supply obstruction in the corresponding parts, and functional defects. If blood flow obstruction occurs in the coronary artery, it will cause coronary heart disease; when it occurs in the cerebral blood vessels, cerebral infarction is prone to occur. By testing the ApoE gene type, the incidence of Alzheimer's disease, cardiovascular and cerebrovascular diseases, etc. can be predicted early, so as to achieve early detection and early intervention and maximize the patient's survival.

[0003] ApoE includes three subtypes: ApoE2, ApoE3 and ApoE4, among which ApoE3 is the most common, accounting for 78%, and is related to the metabolism of chylomicrons (CM) and low-density lipoprotein (LDL); ApoE2 accounts for 8%, which is related to the metabolism of high-density lipoprotein (HDL); and ApoE4 accounts for 14%, which is related to Alzheimer's disease (AD). The gene structure of the three subtypes has caused corresponding changes in the amino acid sequence of ApoE due to differences in base sequences. There are three ApoE alleles, ε2, ε3 and ε4. These three alleles can be combined into three homozygous types (ε2 / ε2, ε3 / ε3, ε4 / ε4, accounting for 1%, 60% and 2% respectively) and three heterozygous types (ε2 / ε3, ε2 / ε4, ε3 / ε4, accounting for 13%, 2% and 22% respectively). ApoE4 is widely distributed in patients with late-onset AD and sporadic AD. It is a risk factor in the development of AD, a susceptibility gene for AD, and may play an important role in the inheritance of AD. Homozygotes with the E4 allele develop AD at an earlier age than those with the E4 heterozygote. The incidence of AD increases threefold with each additional E4 allele. The ApoE4 genotype can significantly increase the occurrence of late-onset Alzheimer's disease (AD), the ApoE2 genotype can effectively prevent the occurrence of late-onset Alzheimer's disease (AD), and is a protective factor for AD. The ApoE3 genotype is not likely to cause the occurrence of Alzheimer's disease (AD) and is a normal factor in the human body.

[0004] ApoE genotype characteristics:

[0005] ApoE2: mutant type, accounting for 7% of the natural population; strong ability to metabolize fat; protective effect on vascular integrity, protective effect on atherosclerosis, reduced risk of Alzheimer's disease, coronary heart disease, cerebral infarction and other diseases; ApoE2 has also been proven to be a longevity gene.

[0006] ApoE3: Wild type, accounting for about 78% of the natural population; plays a key role in the normal physiological functions of the body.

[0007] ApoE4: mutant type, accounting for 15% of the natural population; the body's ability to metabolize and utilize lipids is poor, a large amount of lipids accumulate in the body, leading to the occurrence of hyperlipidemia; it is a potential risk factor for atherosclerosis, and is prone to Alzheimer's disease, coronary heart disease, cerebral infarction, retinitis pigmentosa and other diseases; it is advisable to eat unsaturated fatty acid foods, eat less fatty meat, and exercise more.

[0008] Clinical significance of APOE typing test:

[0009] 1. Used for risk prediction of senile dementia, namely Alzheimer's disease (AD): The detection rate of apoE4 in the serum of AD patients is significantly increased. Homozygotes of the ε4 allele develop AD at an earlier age than heterozygotes with ε4. The incidence of AD increases threefold with each additional ε4 allele. ApoE4 genotype can significantly increase the occurrence of late-onset Alzheimer's disease (AD), ApoE2 genotype can effectively prevent the occurrence of late-onset Alzheimer's disease (AD) and is a protective factor for (AD). ApoE3 genotype is not likely to cause the occurrence of Alzheimer's disease (AD) and is a normal factor in the human body.

[0010] 2. The relationship between ApoE and dementia treatment drugs: The first-class cholinesterase inhibitor drugs (such as tacrine, etc.) are more effective in alleviating the symptoms of non-ε4 AD patients than ε4 AD patients. The second-class cholinesterase inhibitors (such as donepezil and trimefoxim) can play a good therapeutic role in ε4 AD patients;

[0011] 3. The relationship between ApoE and lipoproteins: ApoE can bind to VLDL and HDL through its lipid binding domain. VLDL is responsible for transporting triglycerides from the liver to all tissues except the brain. HDL distributes cholesterol in a balanced manner within cells. The binding activity of ApoE2 to LDL receptors is very low, less than 1% of the binding activity of ApoE3, while low doses of ApoE2 can increase triglyceride levels by 7 times, and high-dose injections of ApoE2 can increase triglyceride levels by 50 times. It has been found that type III HLP patients are almost without exception E2 / 2 type. However, ApoE2 type can significantly increase HDL, and ε4 is closely related to high total cholesterol (TC), high low-density lipoprotein cholesterol (LDL-C) and high apolipoprotein B (ApoB) levels, and is a genetic susceptibility factor for hypercholesterolemia and coronary heart disease;

[0012] 4. Drug selection for hyperlipidemia: Treatment plans for different genotypes: E2 type uses: Atorvastatin calcium, pravastatin, lovastatin, rosuvastatin, fluvastatin; insensitive: methylhydroxyglutaryl coenzyme A, probucol, simvastatin. E4 (opposite to E2) uses probucol and simvastatin, and others are insensitive;

[0013] 5. The relationship between apolipoprotein E and health: E2: Longevity gene: The phenotype is E2, and the corresponding genotypes are ε2 / ε2, ε2 / ε3, accounting for 7% of the human population. Individuals with this genotype are not susceptible to Alzheimer's disease, coronary heart disease, cerebral infarction and other diseases, but are susceptible to macular degeneration. Their blood lipid profile is as follows: high triglycerides, low LDH-C, and uncertain HDL; E3: Wild-type gene: The phenotype is E3, and the corresponding genotypes are ε3 / ε3, ε2 / ε4, accounting for 78% of the human population. This genotype is a common gene; E4: Dangerous gene: The phenotype is E4, and the corresponding genotypes are ε3 / ε4; ε4 / ε4, accounting for 15% of the human population. Individuals with this genotype are susceptible to Alzheimer's disease, coronary heart disease, cerebral infarction, retinitis pigmentosa and other diseases. Their blood lipid profile is as follows: low triglycerides, high LDH-C, and low HDL.

[0014] So far, NMPA has approved 15 APOE genotyping test kits, and most of them are fluorescent PCR technology methods. Fluorescent PCR technology is currently the most widely used detection technology in the field of molecular diagnosis. It is widely used in pathogen detection, early tumor screening, food detection and many other fields due to its high detection sensitivity and strong specificity. Its core principle is PCR amplification and fluorescent signal detection. The PCR amplification process simulates the semi-conservative replication process of DNA in the body. In the presence of biological enzymes, dNTPs, metal ions and other substances, the primers complementary to the target gene nucleic acid sequence are used as guides to synthesize new nucleic acid fragments. The reaction conditions include denaturation, annealing and extension. The fluorescent signal is detected by a probe that is complementary to the target sequence and labeled with a reporter group and a quencher group at the 5' end and 3' end respectively. Common probes include the most commonly used Taqman probes, molecular beacon probes, scorpion probes, MGB probes, etc. Different types of probes are used in different specific detection fields.

[0015] The present invention optimizes the composition and concentration of the sample release agent, and uses a certain concentration of surfactant and other chemical components to lyse and break human cells, so that nucleic acids are fully released. At the same time, components such as EDTA in the sample release agent have a certain protective effect on nucleic acids, so that the exposed nucleic acids are not degraded by nucleases in the environment, thereby ensuring the stability of nucleic acids.

[0016] The LNA+MGB probe in the present invention is a novel specific fluorescent PCR probe, the 5' end of which is labeled with a conventional fluorescent reporter group, such as FAM / VI C / HEX / ROX / CY5, etc., and the 3' end is not only labeled with a non-luminescent quenching group (NFQ), but also connected with a minor groove binder (MGB). The modification of MGB can significantly increase the Tm value of the probe (10-20° C.), thereby increasing the specificity of binding to the nucleic acid template. Locked nucleic acid (LNA) is a class of modified high-affinity RNA analogs. The addition of LNA nucleotides can improve the thermal stability of the probe binding to the nucleic acid template (2-8° C.), and has higher recognition and affinity for the target, thereby improving the specificity of detection. The sample does not need to be subjected to nucleic acid extraction, and can be directly used for detection after a one-step nucleic acid release. Summary of the invention

[0017] In order to solve the above technical problems, the present invention provides a kit and a detection method for detecting mutations at the rs429358 and rs7412 sites of the human APOE gene to solve the above problems.

[0018] A kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene, comprising three components: a sample releaser, a freeze-dried ball and a chip;

[0019] Among them, the chip contains primer probes.

[0020] Preferably, the sample release agent is a reagent for rapidly lysing cells in human oral exfoliated samples and releasing nucleic acids, and its main components include: 10-20 mM TrisCl, 10-50 mM KCl, 0.25-2.5 mM EDTA, 0.001-0.1% SDS (w / v), 0.05-0.5% TritonX-100 (v / v), 0.05-0.5% Tween-20 (v / v), 0.05-0.5% NP-40 (v / v), and 0.01%-0.1% Procline-300, wherein TrisCl can provide a suitable buffer environment to maintain the pH stability of the system; KCl helps to maintain the ionic strength and promote the reaction; EDTA can chelate metal ions to prevent nucleases from degrading nucleic acids; SDS can destroy cell membranes and nuclear membranes, promote cell lysis and nucleic acid release; Non-ionic surfactants such as itonX-100, Tween-20 and NP-40 can enhance the cell lysis effect and improve the efficiency of nucleic acid extraction; Proc lin-300 acts as a preservative to extend the shelf life of the reagent. Through the synergistic effect of these ingredients, cells can be lysed quickly, efficiently and stably to release nucleic acids, thereby ensuring the accuracy and reliability of the detection of APOE gene rs429358 and rs7412 site mutations.

[0021] Preferably, the freeze-dried ball mainly includes: 0.05-0.4U / μl commercial fast Taq enzyme, commercial PCR Buffer matched with Taq enzyme, 0.01-0.02U / μl commercial UDG enzyme, and 1× lyophilized protective agent. It contains a certain amount of fast Taq enzyme, which can ensure efficient polymerase chain reaction and achieve rapid amplification of target gene fragments. The matching commercial PCR Buffer can provide a suitable reaction environment to ensure the activity of Taq enzyme and the smooth progress of the reaction. The presence of UDG enzyme can effectively degrade possible DNA fragments containing uracil, reduce interference, and improve the accuracy of detection. The lyophilized protective agent can protect the enzyme and other biologically active ingredients during the freeze-drying process, maintain its stability and activity, and facilitate the long-term storage and transportation of the test kit. At the same time, it can be quickly re-dissolved and play a role when used, ensuring the reliability of the test results.

[0022] Preferably, the chip is respectively embedded with specific primers and probes for human APOE gene rs429358 and rs7412. By embedding specific primers and probes in the chip, highly specific detection of target gene sites (rs429358 and rs7412) can be achieved, and specific gene fragments can be accurately identified and amplified, greatly reducing the possibility of nonspecific amplification and cross-reaction, thereby improving the accuracy and reliability of detection. At the same time, this integrated design facilitates operation and use, reduces the risk of human operation errors and reagent contamination, and makes the detection process more convenient, efficient and stable.

[0023] Another technical problem to be solved by the present invention is to provide a detection method for detecting mutations at sites rs429358 and rs7412 of the human APOE gene, comprising the following steps:

[0024] S1: Use a sterile swab to carefully swab oral exfoliated cells. Select a suitable oral site, such as the buccal mucosa, and gently wipe to obtain sufficient cell samples. Carefully dip the swab head with the cell sample into the pre-prepared sample release agent, then place it in a centrifuge tube, turn it upside down several times, and mix it thoroughly to promote cell lysis and nucleic acid release. This step can effectively destroy the cell structure and expose the nucleic acid in it;

[0025] S2: Use a pipette to directly draw 125μl of the sample in S1 and add it to the centrifuge tube containing the freeze-dried balls. Gently blow several times to ensure that the freeze-dried balls are completely dissolved. During this process, be careful to avoid bubbles to avoid affecting subsequent reactions. The nucleic acid in the sample is fully in contact and reacts with the fast Taq enzyme, PCR Buffer, UDG enzyme, and freeze-dried protective agent in the freeze-dried balls. The fast Taq enzyme begins to work and prepares for subsequent nucleic acid amplification; the UDG enzyme can degrade possible DNA fragments containing uracil to reduce interference;

[0026] S3: Accurately pipette 110μl of the sample treated with S2 and carefully add it to the chip. Ensure that the sample is evenly distributed in each reaction area of ​​the chip, especially the area containing the specific primers and probes for the human APOE gene rs429358 and rs7412. Insert the chip smoothly into the nucleic acid amplification analyzer to ensure a good connection for subsequent testing. At this point, the specific primers and probes in the chip will specifically bind to the target gene fragment, laying the foundation for subsequent amplification and detection;

[0027] S4: Start the nucleic acid amplification analyzer and begin testing. The instrument will automatically run according to the preset program and parameters, including heating, cooling, amplification and other steps. During the entire testing process, the nucleic acid amplification analyzer will monitor the progress of the reaction and signal changes in real time. As the amplification proceeds, the target gene fragments are continuously replicated and amplified, and the generated signal gradually increases. When the test is completed, the nucleic acid amplification analyzer will automatically issue detailed test results and reports. The report may include information such as the mutation status of the gene locus and the credibility of the test. By analyzing and interpreting this information, the human APOE gene can be accurately determined.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. Improve detection accuracy: By optimizing the composition of the sample releaser, cells can be efficiently lysed and nucleic acids can be released. At the same time, the enzymes and protective agents in the freeze-dried balls ensure that the reaction proceeds stably. The specific primers and probes in the chip achieve highly specific detection, which greatly reduces the possibility of nonspecific amplification and cross-reaction, and significantly improves the accuracy of detection of mutations at the rs429358 and rs7412 sites of the APOE gene.

[0030] 2. Ease of operation: The kit uses simple steps, such as obtaining oral exfoliated cells with a sterile swab, which can be completed by subsequent operations such as absorption, dissolution, and addition to the chip. It does not require complicated sample pre-treatment processes, reduces the difficulty of operation and the dependence on the operator's technical level, makes the detection process more convenient, and can be widely used in different scenarios.

[0031] 3. Fast and efficient: The sample release agent quickly lyses cells, and the fast Taq enzyme in the freeze-dried ball can achieve efficient polymerase chain reaction. The entire detection process takes a short time and can quickly obtain test results, meeting the clinical needs for rapid diagnosis and timely treatment, especially in some emergency situations, and can provide patients with timely diagnostic basis.

[0032] 4. Stability and reliability: The combination of ingredients in the sample release agent and the presence of freeze-drying protectants ensure the long-term stability of the reagent, allowing it to maintain good performance during storage and transportation. The precise design of each step in the detection method and the automated operation of the instrument ensure the reliability of the results and reduce the impact of human errors and uncertain factors.

[0033] 5. Cost-effectiveness: This detection method does not require expensive equipment and complex experimental environment. The cost of the test kit is relatively low, and batch testing can be achieved, which can effectively reduce the cost of testing in large-scale applications. At the same time, accurate test results can help avoid misdiagnosis and unnecessary treatment, and save medical costs in the long run.

[0034] 6. Clinical application value: Accurate detection of mutations at the rs429358 and rs7412 sites of the human APOE gene is of great significance for early diagnosis, risk assessment and individualized treatment of cardiovascular diseases and other related diseases. This helps clinicians develop more accurate treatment plans, improve patient prognosis, and enhance the quality of life of patients, and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the sequence diagram of the primer probe in the present invention. DETAILED DESCRIPTION

[0036] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0037] See also Figure 1 The present invention provides a kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene. The kit comprises three components: a sample release agent, a freeze-dried ball and a chip.

[0038] Among them, the chip contains primer probes.

[0039] The sample release agent is a reagent used to quickly lyse cells in human oral exfoliated samples and release nucleic acids, and its main components include: 10-20mM TrisCl, 10-50mM KCl, 0.25-2.5mM EDTA, 0.001-0.1% SDS (w / v), 0.05-0.5% TritonX-100 (v / v), 0.05-0.5% Tween-20 (v / v), 0.05-0.5% NP-40 (v / v), 0.01%-0.1% Procline-300;

[0040] TrisCl can provide a suitable buffer environment to maintain the pH stability of the system; KCl helps maintain the ionic strength and promotes the reaction; EDTA can chelate metal ions to prevent nucleases from degrading nucleic acids; SDS can destroy cell membranes and nuclear membranes, promote cell lysis and nucleic acid release; non-ionic surfactants such as TritonX-100, Tween-20 and NP-40 can enhance the cell lysis effect and improve the efficiency of nucleic acid extraction; Proclin-300 acts as a preservative to extend the shelf life of the reagent. Through the synergistic effect of these components, cells can be lysed quickly, efficiently and stably to release nucleic acids, thereby ensuring the accuracy and reliability of the detection of APOE gene rs429358 and rs7412 site mutations.

[0041] The freeze-dried pellet mainly includes: 0.05-0.4U / μl commercial fast Taq enzyme, commercial PCR Buffer matching Taq enzyme, 0.01-0.02U / μl commercial UDG enzyme, and 1× freeze-dried protective agent;

[0042] It contains a certain amount of fast Taq enzyme, which can ensure efficient polymerase chain reaction and achieve rapid amplification of target gene fragments. The matching commercial PCRBuffer can provide a suitable reaction environment to ensure the activity of Taq enzyme and the smooth progress of the reaction. The presence of UDG enzyme can effectively degrade possible DNA fragments containing uracil, reduce interference, and improve the accuracy of detection. The lyophilization protectant can protect enzymes and other biologically active ingredients during the lyophilization process, maintain their stability and activity, and facilitate the long-term storage and transportation of the kit. At the same time, it can be quickly re-dissolved and play a role when used, ensuring the reliability of the test results.

[0043] The chip contains specific primers and probes for rs429358 and rs7412 of the human APOE gene, and their sequences are shown in Figure 1 , where the 5' end of the probe is labeled with a FAM luminescent group, the 3' end is connected to an MGB group, and the mutant bases are modified with LNA. By embedding specific primers and probes in the chip, highly specific detection of target gene sites (rs429358, rs7412) can be achieved, and specific gene fragments can be accurately identified and amplified, greatly reducing the possibility of nonspecific amplification and cross-reaction, thereby improving the accuracy and reliability of detection. At the same time, this integrated design facilitates operation and use, reduces the risk of human operation errors and reagent contamination, and makes the detection process more convenient, efficient and stable.

[0044] The components in the kit of the present invention, such as Tris, KCl, EDTA, SDS, Triton X-100, Tween-20, NP-40 in the sample release agent, Taq enzyme, PCR Buffer, UDG enzyme in the freeze-dried ball, and specific primer probes in the chip are all commercial reagents or raw materials, among which the specific primer probe sequences are designed by the company itself and commissioned to be synthesized by a synthesis company.

[0045] A method for detecting mutations at sites rs429358 and rs7412 of the human APOE gene comprises the following steps:

[0046] S1: Use a sterile swab to carefully swab oral exfoliated cells. Select a suitable oral site, such as the buccal mucosa, and gently wipe to obtain sufficient cell samples. Carefully dip the swab head with the cell sample into the pre-prepared sample release agent, then place it in a centrifuge tube, turn it upside down several times, and mix it thoroughly to promote cell lysis and nucleic acid release. This step can effectively destroy the cell structure and expose the nucleic acid in it;

[0047] S2: Use a pipette to directly draw 125μl of the sample in S1 and add it to the centrifuge tube containing the freeze-dried balls. Gently blow several times to ensure that the freeze-dried balls are completely dissolved. During this process, be careful to avoid bubbles to avoid affecting subsequent reactions. The nucleic acid in the sample is fully in contact and reacts with the fast Taq enzyme, PCR Buffer, UDG enzyme, and freeze-dried protective agent in the freeze-dried balls. The fast Taq enzyme begins to work and prepares for subsequent nucleic acid amplification; the UDG enzyme can degrade possible DNA fragments containing uracil to reduce interference;

[0048] S3: Accurately pipette 110μl of the sample treated with S2 and carefully add it to the chip. Ensure that the sample is evenly distributed in each reaction area of ​​the chip, especially the area containing the specific primers and probes for the human APOE gene rs429358 and rs7412. Insert the chip smoothly into the nucleic acid amplification analyzer to ensure a good connection for subsequent testing. At this point, the specific primers and probes in the chip will specifically bind to the target gene fragment, laying the foundation for subsequent amplification and detection;

[0049] S4: Start the nucleic acid amplification analyzer and begin testing. The instrument will automatically run according to the preset program and parameters, including heating, cooling, amplification and other steps. During the entire testing process, the nucleic acid amplification analyzer will monitor the progress of the reaction and signal changes in real time. As the amplification proceeds, the target gene fragments are continuously replicated and amplified, and the generated signal gradually increases. When the test is completed, the nucleic acid amplification analyzer will automatically issue detailed test results and reports. The report may include information such as the mutation status of the gene locus and the credibility of the test. By analyzing and interpreting this information, the human APOE gene can be accurately determined.

[0050] By optimizing the composition of the sample releaser, cells can be efficiently lysed and nucleic acids can be released. At the same time, the enzymes and protective agents in the freeze-dried balls ensure that the reaction proceeds stably. The specific primers and probes in the chip achieve highly specific detection, greatly reducing the possibility of nonspecific amplification and cross-reaction, and significantly improving the accuracy of detection of mutations at the rs429358 and rs7412 sites of the APOE gene.

[0051] The kit uses simple steps, such as obtaining oral exfoliated cells with a sterile swab, which can be completed through subsequent operations such as absorption, dissolution, and addition to a chip. It does not require complicated sample pre-treatment processes, reduces the difficulty of operation and dependence on the operator's technical level, makes the detection process more convenient, and can be widely used in different scenarios.

[0052] The sample releaser quickly lyses cells, and the fast Taq enzyme in the freeze-dried balls can achieve efficient polymerase chain reaction. The entire detection process is time-saving and can quickly obtain test results, meeting the clinical needs for rapid diagnosis and timely treatment, especially in some emergency situations, and can provide patients with timely diagnostic basis.

[0053] The combination of ingredients in the sample release agent and the presence of lyophilization protectants ensure the long-term stability of the reagent, allowing it to maintain good performance during storage and transportation. The precise design of each step in the detection method and the automated operation of the instrument ensure the reliability of the results and reduce the impact of human errors and uncertainties.

[0054] This detection method does not require expensive equipment and complex experimental environment. The cost of the test kit is relatively low, and batch testing can be achieved, which can effectively reduce the cost of testing in large-scale applications. At the same time, accurate test results can help avoid misdiagnosis and unnecessary treatment, and save medical costs in the long run.

[0055] Accurate detection of mutations at the rs429358 and rs7412 sites of the human APOE gene is of great significance for early diagnosis, risk assessment and individualized treatment of cardiovascular diseases and other related diseases. This helps clinicians develop more accurate treatment plans, improve patient prognosis, and enhance the quality of life of patients, and has significant clinical application value.

[0056] The nucleic acid amplification analyzer used in the detection method of the present invention is the LunaDxNano nucleic acid amplification detection analyzer produced by Jiangsu Huixian Pharmaceutical Technology Co., Ltd.

[0057] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene, characterized in that: The kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene includes three components: a sample release agent, a freeze-dried ball, and a chip; Among them, the chip contains primer probes.

2. A kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene as claimed in claim 1, characterized in that: The sample release agent is a reagent used to quickly lyse cells in human oral exfoliated samples and release nucleic acids. Its main components include: 10-20mM Tris-Cl, 10-50mM KCl, 0.25-2.5mM EDTA, 0.001%-0.1% SDS (w / v), 0.05%-0.5% TritonX-100 (v / v), 0.05%-0.5% Tween-20 (v / v), 0.05%-0.5% NP-40 (v / v), and 0.01%-0.1% Proclin-300.

3. A kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene as claimed in claim 1, characterized in that: The freeze-dried balls mainly include: 0.05-0.4U / μl commercial fast Taq enzyme, commercial PCR Buffer matching Taq enzyme, 0.01-0.02U / μl commercial UDG enzyme, and 1× freeze-dried protective agent.

4. A kit for detecting mutations at sites rs429358 and rs7412 of the human APOE gene as claimed in claim 1, characterized in that: The chip is embedded with specific primers and probes for human APOE gene rs429358 and rs7412 respectively.

5. A method for detecting mutations at sites rs429358 and rs7412 of the human APOE gene, characterized in that: The following steps are involved: S1: Use a sterile swab to collect oral exfoliated cells, immerse the swab head in the sample release agent, and turn it upside down several times; S2: Directly pipette 125 μl of the sample in S1 and add it to the freeze-dried pellets, pipetting several times to fully dissolve the freeze-dried pellets; S3: Take 110 μl of the sample treated in S2, add it to the chip, and insert the chip into the nucleic acid amplification analyzer for detection; S4: After the test is completed, the nucleic acid amplification analyzer automatically issues and prints the test results and report.

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