Kit for detecting cardiovascular disease medication gene and detection method
By providing a kit containing a variety of gene polymorphism detection kits, combining non-invasive oral mucosal sampling and multiple PCR amplification technology, the problem of low detection sensitivity of existing kits is solved, and medication for cardiovascular diseases is achieved The rapid and accurate detection of genes provides accurate guidance on medication.
Patent Information
- Application Number
- CN202411580106.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-02-11
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing kits are not sensitive when detecting drug genes for cardiovascular diseases, and it is difficult to provide accurate drug guidance.
Provide a kit for detecting drug genes for cardiovascular diseases, including CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism, MTHFR gene polymorphism detection kit, etc., through non-invasive oral mucosa detachment Cell sampling was performed using multiple PCR amplification reaction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
It realizes rapid and accurate detection of drug genes for cardiovascular diseases, and can provide accurate drug dosage and plan within 1 hour, improves the sensitivity and accuracy of the detection, and meets the clinical fast and immediate testing needs.
Smart Images
Figure CN119410766A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of kits, and in particular to a kit and a detection method for detecting cardiovascular disease medication genes. Background Art
[0002] The test kit is a box for containing chemical reagents for testing chemical components, drug residues, virus types, etc. It is generally used by hospitals and pharmaceutical companies. The test kit is used to qualitatively detect the gene polymorphism of drug metabolizing enzymes in human oral mucosal exfoliated cells in vitro. The results are used to determine the patient's drug metabolism type for drugs such as clopidogrel, statins, and folic acid, providing a reference for clinicians to select appropriate drugs or adjust drug dosages;
[0003] However, the detection sensitivity of ordinary test kits is not high, so we proposed a test kit and method for detecting cardiovascular disease drug genes. Summary of the invention
[0004] This application provides an intelligent operation and maintenance platform architecture for industrial robots and intelligent equipment to solve the above-mentioned problems.
[0005] The present application provides a kit for detecting cardiovascular disease medication genes, comprising:
[0006] CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism, MTHFR gene polymorphism detection kit, ALDH2 gene polymorphism detection kit, HLA gene polymorphism detection kit, VK0RC1&CYP2C9 gene polymorphism detection kit, SLCO1B1&ApoE gene polymorphism detection kit.
[0007] Preferably, the CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism detection sites are CYP2C19*1, CYP2C19*2 and CYP2C19*3, which are used for medication guidance of clopidogrel, proton pump inhibitors, voriconazole and (es)citalopram.
[0008] Preferably, the detection site of the MTHFR gene polymorphism detection kit is the C677T polymorphism site, which is used for guidance of folic acid supplementation during pregnancy.
[0009] Preferably, the detection location of the ALDH2 gene polymorphism detection kit is rs671, which is used for the evaluation of nitroglycerin efficacy, alcohol metabolism capacity, cardiovascular disease and tumor risk.
[0010] Preferably, the detection location of the HLA gene polymorphism detection kit is site B, which is used for guidance of allopurinol medication.
[0011] Preferably, the detection sites of the VK0RC1&CYP2C9 gene polymorphism detection kit are VK0RC1, CYP2C9*2, and CYP2C9*3, which are used for warfarin medication guidance.
[0012] Preferably, the SLCO1B1&ApoE gene polymorphism detection kit detects SLCO1B1 and ApoE, and is used for guiding statin medication.
[0013] A kit detection method for detecting cardiovascular disease drug genes, comprising the following steps:
[0014] Step 1: Get the basic test kit;
[0015] Step 2: Non-invasive DNA extraction;
[0016] Step 3, using multiplex PCR amplification reaction to obtain the target fragment;
[0017] Step 4: Designing complementary beacons for specific hybridization;
[0018] Step 5: Single base extension of gene fragment;
[0019] Step 6: Collect and measure the target DNA sequence to obtain genetic variations that can lead to individual differences in enzyme activity, including rapid metabolism, medium metabolism, and slow metabolism.
[0020] Preferably, the conditions of the PCR amplification reaction are: pre-denaturation: 95°C for 30s, cycle: 95°C denaturation for 30s, 55°C annealing for 20s, 75°C extension for 30s, a total of 30 cycles, 10 cycles of annealing and extension inserted in each cycle, final extension: 72°C for 10min.
[0021] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:
[0022] The overall structure provided by the embodiment of the present application avoids the risk of blood collection and secondary physical and mental trauma of patients by non-invasively scraping off cells from the oral mucosa. Non-invasive oral rapid sampling avoids the possibility of cross-contamination of samples to a certain extent and improves the accuracy of detection. The test results are obtained in 1 hour, and the accurate dosage and medication regimen are determined in a short time, providing a basis for clinicians to quickly and instantly formulate accurate medication regimens, meeting the clinical demand for accurate and rapid detection. The medical PCR analysis system is applied, and oral sampling is performed. The sample does not need a DNA purification process to be directly tested on the machine, and the one-button operation is performed. The collected probe fluorescent group signal is analyzed according to the software analysis system for genotyping. By performing multiple PCR amplification and combining matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the simultaneous detection of 12 polymorphic sites is achieved, which improves work efficiency, has high detection throughput, strong specificity, high sensitivity and high accuracy, and can provide medication guidance for patients with cardiovascular diseases in one test, which has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.
[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0025] Figure 1 It is a schematic diagram of the overall process flow of the present invention. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.
[0027] Various embodiments of the present application may exist in the form of a range. It should be understood that the description in the form of a range is only for convenience and simplicity and should not be understood as a hard limit to the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which are applicable regardless of the range. In addition, whenever a numerical range is indicated in the present application, it is meant to include any quoted numbers (fractions or integers) within the indicated range. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application, etc., can be purchased from the market or can be prepared by existing equipment.
[0028] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the present application, the terms "include", "comprise", etc. refer to "including but not limited to". In the present application, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In the present application, "and / or" describes the association relationship of the associated objects, indicating that three relationships may exist, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In the present application, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular items or plural items. For example, "at least one of a, b, or c" or "at least one of a, b and c" can both mean: a, b, c, ab, i.e. a and b, ac, bc or abc, where a, b, c can be single or plural, respectively.
[0029] like Figure 1 As shown: This embodiment of the application provides a kit for detecting cardiovascular disease medication genes, including:
[0030] CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism, MTHFR gene polymorphism detection kit, ALDH2 gene polymorphism detection kit, HLA gene polymorphism detection kit, VK0RC1&CYP2C9 gene polymorphism detection kit, SLCO1B1&ApoE gene polymorphism detection kit.
[0031] Specifically: a kit for detecting cardiovascular disease drug genes based on the establishment and optimization of a fully automated medical PCR analysis system platform, a rapid detection kit for drug metabolizing enzyme gene polymorphisms in human oral mucosal exfoliated cells;
[0032] According to the technical requirements for product testing and in accordance with the requirements of the "Appendix to the Medical Device Production Quality Management Standards - In Vitro Diagnostic Reagents", a complete production system should be established, a test kit should be established to detect genes for cardiovascular disease medications, and a traceability procedure should be established and process documents should be formed, including the scope, degree, identification and records of traceability.
[0033] The CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism detection sites are CYP2C19*1, CYP2C19*2 and CYP2C19*3, which are used for medication guidance of clopidogrel, proton pump inhibitors, voriconazole and (es)citalopram.
[0034] The detection site of the MTHFR gene polymorphism detection kit is the C677T polymorphism site, which is used for guiding folic acid supplementation during pregnancy.
[0035] The ALDH2 gene polymorphism detection kit has a detection location of rs671, and is used for evaluating the efficacy of nitroglycerin, evaluating alcohol metabolism capacity, and evaluating the risk of cardiovascular disease and tumors.
[0036] The detection location of the HLA gene polymorphism detection kit is site B, which is used for allopurinol medication guidance.
[0037] The VK0RC1&CYP2C9 gene polymorphism detection kit detects VK0RC1, CYP2C9*2, and CYP2C9*3, and is used for warfarin medication guidance.
[0038] The SLCO1B1&ApoE gene polymorphism detection kit detects SLCO1B1 and ApoE and is used for guiding statin medication.
[0039] A kit detection method for detecting cardiovascular disease drug genes, comprising the following steps:
[0040] Step 1: Get the basic test kit;
[0041] Step 2: Non-invasive DNA extraction;
[0042] Step 3, using multiplex PCR amplification reaction to obtain the target fragment;
[0043] Step 4: Designing complementary beacons for specific hybridization;
[0044] Step 5: Single base extension of gene fragment;
[0045] Step 6: Collect and measure the target DNA sequence to obtain genetic variations that can lead to individual differences in enzyme activity, including rapid metabolism, medium metabolism, and slow metabolism.
[0046] The conditions of the PCR amplification reaction are: pre-denaturation: 95°C for 30s, cycle: 95°C denaturation for 30s, 55°C annealing for 20s, 75°C extension for 30s, a total of 30 cycles, 10 cycles of annealing and extension inserted in each cycle, final extension: 72°C for 10min.
[0047] Specifically: prepare the genomic DNA of the sample to be tested; use the extracted genomic DNA as a template and use the prepared PCR reaction system of the detection kit to perform multiple PCR amplification reactions; PCR product purification and digestion: use exonuclease and alkaline phosphatase to digest the excess dNTP and primer amplification products in the PCR product; product mass spectrometry detection: after the reaction is completed, use matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze the genotype of the detection site according to the built-in Typer software.
[0048] Specifically: This detection technology is used to detect DNA sequences at specific sites. To ensure detection sensitivity, two pairs of specific primers are used to amplify the two target fragments respectively. Two molecular beacon probes labeled with different fluorescent groups and complementary to different bases at the site are specifically hybridized with the amplified fragments. The hybridization process causes the fluorescent group to move away from the quenching group, thereby releasing the fluorescent signal. As the number of amplified fragments accumulates, the fluorescent signal also increases accordingly and is collected by the instrument. By analyzing the collected fluorescent signals through the supporting software, it can be obtained that CYP2C19 genetic variations can lead to individual differences in enzyme activity: rapid metabolizers (extensive metabolizer, EM), intermediate metabolizers (intermediate
[0049] metabolizer, IM) and slow metabolizer (poor metabolizer, PM).
[0050] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.
Claims
1. A kit for detecting cardiovascular disease medication genes, characterized in that: include: CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism, MTHFR gene polymorphism detection kit, ALDH2 gene polymorphism detection kit, HLA gene polymorphism detection kit, VK0RC1&CYP2C9 gene polymorphism detection kit, SLCO1B1&ApoE gene polymorphism detection kit.
2. A kit for detecting cardiovascular disease medication genes according to claim 1, characterized in that: The CYP2C19*2 (c.G681A) and *3 (c.G636A) gene polymorphism detection sites are CYP2C19*1, CYP2C19*2 and CYP2C19*3, which are used for medication guidance of clopidogrel, proton pump inhibitors, voriconazole and (es)citalopram.
3. A kit for detecting cardiovascular disease medication genes according to claim 1, characterized in that: The detection site of the MTHFR gene polymorphism detection kit is the C677T polymorphism site, which is used for guiding folic acid supplementation during pregnancy.
4. A kit for detecting cardiovascular disease drug genes according to claim 3, characterized in that: The ALDH2 gene polymorphism detection kit has a detection location of rs671, and is used for evaluating the efficacy of nitroglycerin, evaluating alcohol metabolism capacity, and evaluating the risk of cardiovascular disease and tumors.
5. A kit for detecting cardiovascular disease medication genes according to claim 1, characterized in that: The detection location of the HLA gene polymorphism detection kit is site B, which is used for allopurinol medication guidance.
6. A kit for detecting cardiovascular disease medication genes according to claim 1, characterized in that: The VK0RC1&CYP2C9 gene polymorphism detection kit detects VK0RC1, CYP2C9*2, and CYP2C9*3, and is used for warfarin medication guidance.
7. A kit for detecting cardiovascular disease drug genes according to claim 1, characterized in that: The SLCO1B1&ApoE gene polymorphism detection kit detects SLCO1B1 and ApoE and is used for guiding statin medication.
8. A kit detection method for detecting cardiovascular disease drug genes, characterized in that: The steps include: Step 1: Get the basic test kit; Step 2: Non-invasive DNA extraction; Step 3, using multiplex PCR amplification reaction to obtain the target fragment; Step 4: Designing complementary beacons for specific hybridization; Step 5: Single base extension of gene fragment; Step 6: Collect and measure the target DNA sequence to obtain genetic variations that can lead to individual differences in enzyme activity, including rapid metabolism, medium metabolism, and slow metabolism.
9. The intelligent operation and maintenance platform architecture of industrial robots and intelligent equipment according to claim 8, characterized in that: The conditions of the PCR amplification reaction are: pre-denaturation: 95°C for 30s, cycle: 95°C denaturation for 30s, 55°C annealing for 20s, 75°C extension for 30s, a total of 30 cycles, 10 cycles of annealing and extension inserted in each cycle, final extension: 72°C for 10min.