SNP (Single Nucleotide Polymorphism) site combination, primer group and kit related to drug action of cardiovascular diseases and application of SNP site combination, primer group and kit

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site combination, a primer group and a kit for detecting drug effects related to cardiovascular diseases, and application of the SNP site combination, the primer group and the kit. The SNP site combination comprises rs9934438, rs1799853, rs1057910, rs4244285, rs498893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504. By detecting the SNP site combination related to the drug effect, individualized drug use suggestions and guidance can be given for specific drugs related to cardiovascular diseases, so that the drug curative effect is improved, side effects are avoided, and accurate drug use is achieved.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to a combination of SNP sites related to the action of cardiovascular disease drugs, a primer set, a kit and applications thereof. Background Art

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide, and its treatment drugs include anti-angina drugs, anti-arrhythmic drugs, anti-heart failure drugs, anti-hypertensive drugs, etc. Related studies have found that due to the influence of genetic factors, different patients have individual differences in their response to the same drug, so personalized drug genetic testing is extremely important in the treatment of cardiovascular disease.

[0003] At present, the genetic testing for personalized medication of cardiovascular diseases mainly adopts methods such as first-generation sequencing, second-generation sequencing, and qPCR. Each of the above methods has certain advantages, but at the same time, there are also problems such as low sample or site throughput, high detection cost, complex operation or analysis methods, and long detection time.

[0004] Therefore, there is an urgent need to develop new genetic testing technologies for personalized medication for cardiovascular diseases. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a SNP site combination, a primer set, a kit and its application related to the action of cardiovascular disease drugs. By detecting the SNP site combination related to the action of the drug of the present invention, individualized medication recommendations and guidance can be given for specific drugs for related cardiovascular diseases, thereby improving the efficacy of the drug, avoiding side effects, and achieving precision medication.

[0006] It should be stated that the present invention is completed based on the following work of the inventor:

[0007] By consulting a large number of scientific research literature and research analysis, the inventors found and determined that only the following 11 related genes of cardiovascular disease drugs can achieve accurate medication guidance and suggestions for cardiovascular disease drugs such as anti-angina drugs, anti-arrhythmic drugs, anti-heart failure drugs, anti-hypertensive drugs, anti-shock drugs, lipid-regulating and anti-atherosclerotic drugs. These 11 related genes are VKORC1, CYP2C9, ALDH2, CYP2D6, ADRB1, AGTR1, ACE, MTHFR, APOE, SLCO1B1, and CYP2C19. Further, the inventors conducted in-depth research and analysis on the SNP sites of the above genes and screened out 20 SNP site combinations. Further research results show that through the detection of these 20 SNP site combinations, on the one hand, it can guide the reasonable selection of cardiovascular disease drug types, dosages, and medication regimens, thereby improving drug efficacy and avoiding side effects; on the other hand, a single experiment can achieve simultaneous detection of 20 sites, which reduces the complexity of detection and further reduces detection costs.

[0008] Furthermore, based on the above 20 SNP site combinations, the inventors, through in-depth research and analysis and experimental design, screened and obtained a multiplex PCR amplification primer set and a multiplex single-base extension primer set with high specificity, high accuracy and high sensitivity. These two sets of multiplex primer sets can be used to specifically and accurately amplify the target gene and target site in the same reaction system, thereby accurately detecting the above SNP site combinations, and then determining the SNP site typing of the test sample DNA from patients with cardiovascular diseases, and providing individualized medication guidance and recommendations for specific drugs.

[0009] In the first aspect of the present invention, the present invention proposes a drug action related SNP site combination. The SNP site combination includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504. According to an embodiment of the present invention, by detecting the above 20 SNP site combinations of the present invention, individualized medication guidance and suggestions can be given for cardiovascular disease medication, thereby improving drug efficacy, avoiding side effects, and achieving precision medication.

[0010] According to an embodiment of the present invention, the above SNP site combination may also have the following additional technical features:

[0011] According to an embodiment of the present invention, the drug is selected from one or more of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists (AIIA) drugs, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs.

[0012] According to an embodiment of the present invention, the drug is selected from one or more of rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril (benazepril, lotensin), enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin, and folic acid.

[0013] In the second aspect of the present invention, the present invention proposes a primer set for detecting the above-mentioned drug action-related SNP sites. The primer set has a nucleotide sequence as shown in SEQ ID NO: 1 to 40. According to an embodiment of the present invention, using the primer set of the present invention, all target genes can be specifically amplified in the same reaction system for SNP site typing detection, and the detection results are accurate and the application value is high.

[0014] According to an embodiment of the present invention, the above primer set may also have the following additional technical features:

[0015] According to an embodiment of the present invention, the primer set further includes a single base extension primer;

[0016] According to an embodiment of the present invention, the single-base extension primer has a nucleotide sequence as shown in SEQ ID NOs: 41-61.

[0017] In the third aspect of the present invention, the present invention proposes a kit. The kit includes the aforementioned primer set for detecting SNP sites related to the action of cardiovascular disease drugs. According to an embodiment of the present invention, based on the aforementioned primer set for detecting SNP sites, a kit for detecting SNP sites related to the action of cardiovascular disease drugs can be further developed. The kit can specifically amplify the target gene in the same reaction system, thereby accurately detecting the aforementioned SNP sites, and then determining the SNP site typing of the DNA of the sample to be tested from the patient with cardiovascular disease. By using the kit of the present invention to detect SNP sites related to the action of cardiovascular disease drugs, a single experiment can achieve simultaneous detection of 20 sites, reducing the cumbersomeness of the detection while further reducing the detection cost. It has the advantages of high detection site throughput, simple operation, low cost, and short time consumption, and provides a better reagent selection for the scientific research and clinical detection of the aforementioned SNP sites.

[0018] In the fourth aspect of the present invention, the present invention proposes the use of the aforementioned primer set for detecting SNP sites in the preparation of a kit for detecting the aforementioned drug action-related SNP site combination.

[0019] Those skilled in the art will appreciate that the features and advantages described above for the SNP site combination, primer set, and kit are also applicable to this application and will not be described in detail here.

[0020] According to an embodiment of the present invention, the above application may also have the following additional technical features:

[0021] According to an embodiment of the present invention, the detection method of the kit includes: using the aforementioned primer set to amplify the DNA sample to be tested to obtain an amplification product; and determining the SNP site information of the amplification product.

[0022] According to an embodiment of the present invention, the method further comprises the following steps: (1) using the primer pair in the primer set described in claim 3 to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product; (2) using the single-base extension primer in the primer set described in claim 4 to perform a single-base extension reaction on the first amplification product to obtain a second amplification product; and (3) determining the SNP site information of the second amplification product.

[0023] According to an embodiment of the present invention, the SNP site information of the DNA sample to be tested is determined based on the SNP site information of the second amplification product.

[0024] According to an embodiment of the present invention, step (3) uses sequencing or mass spectrometry analysis to determine the SNP site information of the second amplification product.

[0025] According to an embodiment of the present invention, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase.

[0026] According to an embodiment of the present invention, the mass spectrometry analysis includes: purifying the second amplification product to obtain a purified product; and performing mass spectrometry analysis on the purified product.

[0027] In the fifth aspect of the present invention, the present invention proposes a method for detecting the aforementioned drug action-related SNP site combination for non-disease diagnosis purposes. The method comprises: using the aforementioned primer set to amplify the DNA sample to be tested to obtain an amplification product; and determining the SNP site information of the amplification product.

[0028] Those skilled in the art will appreciate that the features and advantages described above for SNP site combinations, primer sets, kits, and uses are also applicable to this application and will not be described in detail here.

[0029] According to an embodiment of the present invention, the above method may also have the following additional technical features:

[0030] According to an embodiment of the present invention, the method further comprises the following steps: (1) using the primer pair in the primer set described in claim 3 to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product; (2) using the single-base extension primer in the primer set described in claim 4 to perform a single-base extension reaction on the first amplification product to obtain a second amplification product; and (3) determining the SNP site information of the second amplification product.

[0031] According to an embodiment of the present invention, the SNP site information of the DNA sample to be tested is determined based on the SNP site information of the second amplification product.

[0032] According to an embodiment of the present invention, step (3) uses sequencing or mass spectrometry analysis to determine the SNP site information of the second amplification product.

[0033] According to an embodiment of the present invention, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase.

[0034] According to an embodiment of the present invention, the mass spectrometry analysis includes: purifying the second amplification product to obtain a purified product; and performing mass spectrometry analysis on the purified product.

[0035] In the sixth aspect of the present invention, the present invention proposes the use of a combination of drug-related SNP sites in predicting the efficacy, side effects or dosage of a drug for cardiovascular disease in a subject. The combination of SNP sites includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504.

[0036] Those skilled in the art will appreciate that the features and advantages described above for the SNP site combination, primer set, kit, and use are also applicable to this use and will not be described in detail here.

[0037] According to an embodiment of the present invention, the above-mentioned use may also have the following additional technical features:

[0038] According to an embodiment of the present invention, based on the genotyping results of each SNP site in the SNP site combination, the efficacy, side effects or dosage of the cardiovascular disease drug for the subject is predicted.

[0039] According to an embodiment of the present invention, the drug is selected from at least one of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists (AIIA) drugs, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs.

[0040] According to an embodiment of the present invention, the drug is selected from at least one of rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril (benazepril, lotensin), enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin and folic acid.

[0041] According to an embodiment of the present invention, the SNP site genotyping result is determined based on the aforementioned kit or the aforementioned method.

[0042] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0044] Figure 1 It is a first generation sequencing result diagram of rs429358 in Example 2 of the present invention;

[0045] Figure 2 The first generation sequencing result diagram of rs7412 in Example 2 of the present invention;

[0046] Figure 3 This is a diagram showing the nucleic acid mass spectrometry detection results of 20 SNP sites of the human genome standard NA12156 according to Example 2 of the present invention. DETAILED DESCRIPTION

[0047] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0048] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0049] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0050] Terms and Definitions

[0051] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0052] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0053] Herein, the term "single base extension enzyme" refers to a DNA polymerase used for a single base extension reaction, which can be obtained commercially.

[0054] In this article, the term "PCR premix" refers to a premixed solution of DNA polymerase and its reaction buffer before adding it to the PCR amplification system, which usually also contains Mg, which is beneficial to amplification. 2+ Metal ions such as chlorinated bicarbonate and chlorinated bicarbonate can be obtained commercially.

[0055] As used herein, the term "QuanNUA nucleic acid typing mass spectrometry system" refers to a time-of-flight mass spectrometry system.

[0056] In this article, the term "QuanSNP Genotyping System" refers to a software system applied to time-of-flight mass spectrometry for gene locus analysis.

[0057] The present invention provides a SNP site combination, a primer set, a kit and applications thereof, which will be described in detail below.

[0058] SNP site combination

[0059] The present invention proposes a drug action related SNP site combination. The SNP site combination includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504. According to an embodiment of the present invention, by detecting the above 20 SNP site combinations of the present invention, individualized medication guidance and suggestions can be given for cardiovascular disease medication, thereby improving drug efficacy, avoiding side effects, and achieving precision medication.

[0060] According to an embodiment of the present invention, the drug is selected from at least one of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists (AIIA) drugs, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs. Thus, by detecting the above 20 SNP site combinations, rational medication and personalized guidance and suggestions for the treatment of cardiovascular diseases can be achieved, thereby improving drug efficacy, avoiding side effects, and achieving individualized treatment.

[0061] After in-depth research and analysis, the inventor creatively introduced SNP site primers numbered rs4149056, rs2306283, rs11045819, and rs11045852, so that the site combination can provide medication guidance and suggestions for rosuvastatin cardiovascular drugs; the gene SLCO1B1 (site rs4149056, rs2306283, rs11045819, rs11045852), gene AGTR1 rs5186, gene ACErs1799752SNP site primers, so that the site combination can provide medication guidance and suggestions for drugs such as atorvastatin; gene VKORC1 (rs9923231, rs9934438), CYP2C9rs1057910, APOErs429358 site primers are introduced, so that the site combination can provide medication guidance and suggestions for drugs such as acenocoumarol; gene CYP2C19 (rs4244285, rs4986893, rs12248560) and VKORC1rs9923231 are introduced, so that the site combination can provide medication guidance and suggestions for drugs such as warfarin.

[0062] In this article, the term "medication guidance" includes but is not limited to: guidance on drug efficacy, toxicity, metabolism, dosage, etc. Specific medication guidance methods include, for example, providing scientific basis for clinical individualized medication, predicting drug dosage, drug safety assessment, and research and development of new drugs.

[0063] According to the embodiments of the present invention, the drugs specifically involved include but are not limited to: rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril (benazepril, lotensin), enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin, folic acid. Therefore, it is widely applicable to the medication guidance and suggestions of cardiovascular disease drugs.

[0064] Those skilled in the art will appreciate that, in this article, the term "detection" refers to genetic testing for specific SNP sites or combinations thereof, which is also referred to as "gene polymorphism testing" in some literature reports and in some cases, "genetic testing for drug guidance."

[0065] Detailed information on the SNP sites related to the effects of the above cardiovascular disease drugs screened and determined by the present invention is shown in Table 1.

[0066] Table 1 Information on SNP sites related to the effects of cardiovascular disease drugs

[0067]

[0068] Primer set

[0069] The present invention proposes a primer set for detecting the above-mentioned drug-related SNP sites. The primer set has a nucleotide sequence as shown in SEQ ID NO: 1 to 40. According to an embodiment of the present invention, the primer set of the present invention can be used to specifically amplify the target gene in the same reaction system for SNP site typing detection, and the detection result is accurate and has high application value.

[0070] In some specific embodiments, a primer set having a nucleotide sequence as shown in SEQ ID NO: 1 to 40 is used to specifically amplify the SNP site, wherein:

[0071] The primer pair for specifically amplifying rsrs9934438 has the nucleotide sequences shown in SEQ ID NOs: 1 and 2;

[0072] The primer pair for specifically amplifying rsrs1799853 has the nucleotide sequences shown in SEQ ID NOs: 3 and 4;

[0073] The primer pair for specifically amplifying rs1057910 has the nucleotide sequences shown in SEQ ID NOs: 5 and 6;

[0074] The primer pair for specifically amplifying rs4244285 has the nucleotide sequences shown in SEQ ID NOs: 7 and 8;

[0075] The primer pair for specifically amplifying rs4986893 has the nucleotide sequences shown in SEQ ID NOs: 9 and 10;

[0076] The primer pair for specifically amplifying rs12248560 has the nucleotide sequences shown in SEQ ID NOs: 11 and 12;

[0077] The primer pair for specifically amplifying rs671 has the nucleotide sequences shown in SEQ ID NOs: 13 and 14;

[0078] The primer pair for specifically amplifying rs5186 has the nucleotide sequences shown in SEQ ID NOs: 15 and 16;

[0079] The primer pair for specifically amplifying rs1801133 has the nucleotide sequences shown in SEQ ID NOs: 17 and 18;

[0080] The primer pair for specifically amplifying rs4149056 has the nucleotide sequences shown in SEQ ID NOs: 19 and 20;

[0081] The primer pair for specifically amplifying rs1065852 has the nucleotide sequences shown in SEQ ID NOs: 21 and 22;

[0082] The primer pair for specifically amplifying rs1801253 has the nucleotide sequences shown in SEQ ID NOs: 23 and 24;

[0083] The primer pair for specifically amplifying rs1799752 has the nucleotide sequences shown in SEQ ID NOs: 25 and 26;

[0084] The primer pair for specifically amplifying rs429358 has the nucleotide sequences shown in SEQ ID NOs: 27 and 28;

[0085] The primer pair for specifically amplifying rs7412 has the nucleotide sequences shown in SEQ ID NOs: 29 and 30;

[0086] The primer pair for specifically amplifying rs9923231 has the nucleotide sequences shown in SEQ ID NOs: 31 and 32;

[0087] The primer pair for specifically amplifying rs11045819 has the nucleotide sequences shown in SEQ ID NOs: 33 and 34;

[0088] The primer pair for specifically amplifying rs2306283 has the nucleotide sequences shown in SEQ ID NOs: 35 and 36;

[0089] The primer pair for specifically amplifying rs11045852 has the nucleotide sequences shown in SEQ ID NOs: 37 and 38;

[0090] The primer pair for specifically amplifying rs28399504 has the nucleotide sequences shown in SEQ ID NOs: 39 and 40;

[0091] The mutual interference between the multiple PCR amplification systems of the present invention is complicated, especially for the same sample, in the same reaction system, to achieve accurate typing detection of dozens of SNP sites at the same time, the technical difficulty is very large, there are technical obstacles such as primer-specific amplification interference, multiple gene amplification efficiency interference, and multiple sites cannot be detected in a single hole. After a lot of design and experimental optimization, the inventor obtained a primer set including the above-mentioned primer pair. According to an embodiment of the present invention, the aforementioned SNP sites are subjected to multiple PCR amplification using a primer set including the above-mentioned primer pair, which can eliminate the interference between the multiple amplification systems and achieve accurate typing detection of 20 SNP sites for the same sample at the same time. Therefore, the primer set of the present invention is used to detect and analyze the aforementioned SNP sites, the detection results are accurate, the site detection flux is high, the operation is simple, the cost is low, the time consumption is short, and the application value is high.

[0092] According to an embodiment of the present invention, the primer set further includes a single base extension primer, thereby achieving a single base extension reaction of multiple PCR amplification products in the same reaction system, and obtaining an amplification product that can be further used to detect and analyze the aforementioned SNP site information.

[0093] In some specific embodiments, in the single-base extension primer:

[0094] The single base extension primer for rs9934438 has the nucleotide sequence shown in SEQ ID NO: 41;

[0095] The single base extension primer for rs1799853 has the nucleotide sequence shown in SEQ ID NO: 42;

[0096] The single base extension primer for rs1057910 has the nucleotide sequence shown in SEQ ID NO: 43;

[0097] The single base extension primer for rs4244285 has the nucleotide sequence shown in SEQ ID NO: 44;

[0098] The single base extension primer for rs4986893 has the nucleotide sequence shown in SEQ ID NO: 45;

[0099] The single base extension primer for rs12248560 has the nucleotide sequence shown in SEQ ID NO: 46;

[0100] The single base extension primer for rs671 has the nucleotide sequence shown in SEQ ID NO: 47;

[0101] The single base extension primer for rs5186 has the nucleotide sequence shown in SEQ ID NO: 48;

[0102] The single base extension primer for rs1801133 has the nucleotide sequence shown in SEQ ID NO: 49;

[0103] The single base extension primer for rs4149056 has the nucleotide sequence shown in SEQ ID NO: 50;

[0104] The single base extension primer for rs1065852 has the nucleotide sequence shown in SEQ ID NO: 51;

[0105] The single base extension primer for rs1801253 has the nucleotide sequence shown in SEQ ID NO: 52;

[0106] The single base extension primer for rs1799752 has a nucleotide sequence as shown in SEQ ID NO: 53;

[0107] The single base extension primer for rs429358 has the nucleotide sequence shown in SEQ ID NO: 54;

[0108] The single base extension primer for rs7412 has the nucleotide sequence shown in SEQ ID NO: 55;

[0109] The single base extension primer for rs9923231 has the nucleotide sequence shown in SEQ ID NO: 56;

[0110] The single base extension primer for rs11045819 has the nucleotide sequence shown in SEQ ID NO: 57;

[0111] The single base extension primer for rs2306283 has the nucleotide sequence shown in SEQ ID NO: 58;

[0112] The single base extension primer for rs11045852 has the nucleotide sequence shown in SEQ ID NO: 59;

[0113] The single base extension primer for rs28399504 has the nucleotide sequence shown in SEQ ID NO: 60;

[0114] After a large number of experimental optimizations, the inventors obtained a primer set further including the above-mentioned single-base extension primer. According to an embodiment of the present invention, using the above-mentioned primer set, a single-base extension reaction of multiple PCR amplification products can be realized in the same reaction system to obtain amplification products that can be used for subsequent SNP site detection and analysis, further realizing accurate and simultaneous detection of the aforementioned SNP sites, and then determining the SNP site typing of the DNA of the sample to be tested from patients with cardiovascular diseases, thereby providing specific and personalized medication guidance and suggestions, improving drug efficacy, avoiding side effects, and realizing precision medication.

[0115] It can be understood by those skilled in the art that the primer pairs and extension primers in the present invention correspond one to one, and the corresponding primer pairs and extension primer pairs are used to detect the nucleotides of the same SNP site. For example, the primer pair for detecting the rs1799853 site includes an upstream primer of the nucleotide sequence shown in SEQ ID NO: 3, a downstream primer of the nucleotide sequence shown in SEQ ID NO: 4, and a single base extension primer of the nucleotide sequence shown in SEQ ID NO: 42.

[0116] The primer nucleotide sequence information involved in the above primer set is specifically shown in Table 2 and Table 3.

[0117] Table 2 Nucleotide sequence information of multiplex PCR primers for 20 SNP loci

[0118]

[0119]

[0120] Table 3 Nucleotide sequence information of single base extension primers at 20 SNP sites

[0121]

[0122]

[0123] Reagent test kit

[0124] The present invention proposes a kit. The kit includes the aforementioned primer set for detecting SNP sites related to the action of cardiovascular disease drugs. According to an embodiment of the present invention, based on the aforementioned primer set for SNP site detection, a kit for detecting SNP sites related to the action of cardiovascular disease drugs can be further developed. The kit can specifically amplify the target gene in the same reaction system, thereby accurately detecting the aforementioned SNP sites, and then determining the SNP site typing of the DNA of the sample to be tested from the patient with cardiovascular disease. By using the kit of the present invention to detect SNP sites related to the action of cardiovascular disease drugs, a single experiment can achieve simultaneous detection of 20 sites, which reduces the cumbersomeness of the detection and further reduces the detection cost. It has the advantages of high detection site throughput, simple operation, low cost, short time consumption, etc., and provides a better reagent selection for the scientific research and clinical detection of the aforementioned SNP sites.

[0125] application

[0126] The present invention proposes the use of the aforementioned primer set for detecting SNP sites in preparing a kit, and the kit is used to detect the aforementioned SNP site combination.

[0127] Those skilled in the art will appreciate that the features and advantages described above for the SNP site combination, primer set, and kit are also applicable to this application and will not be described in detail here.

[0128] According to an embodiment of the present invention, the detection method of the kit comprises:

[0129] Amplifying the DNA sample to be tested using the aforementioned primer set to obtain an amplified product;

[0130] Determine the SNP site information of the amplified product.

[0131] According to an embodiment of the present invention, the method further comprises the following steps:

[0132] (1) using the primer pair in the aforementioned primer set to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product;

[0133] (2) using the single-base extension primer in the aforementioned primer set to perform a single-base extension reaction on the first amplification product to obtain a second amplification product;

[0134] (3) Determine the SNP site information of the second amplification product.

[0135] According to an embodiment of the present invention, in step (3), the SNP site information of the DNA sample to be tested is determined based on the SNP site information of the second amplification product.

[0136] According to an embodiment of the present invention, step (3) determines the SNP site information of the second amplification product by sequencing or mass spectrometry analysis to determine the SNP site information of the DNA sample to be tested.

[0137] According to an embodiment of the present invention, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase.

[0138] According to an embodiment of the present invention, the mass spectrometry analysis comprises:

[0139] Purifying the second amplification product to obtain a purified product;

[0140] The purified product was subjected to mass spectrometry analysis.

[0141] In some embodiments of the present invention, the mass spectrometry analysis is performed using a time-of-flight mass spectrometer, which detects the molecular weight of the second amplification product, determines the information of the 20 SNP sites in the sample to be tested based on the molecular weight markers, and then types the 20 SNP sites.

[0142] According to an embodiment of the present invention, the instrument and software used in the mass spectrometry analysis are selected from the QuanNUA nucleic acid typing mass spectrometry system and the QuanSNP genotyping system. According to an embodiment of the present invention, using the above-mentioned mass spectrometry analysis instrument, the test results can be directly output without the need for a complex bioinformatics analysis process. The test operation is simple, low-cost, and short in time, and is widely applicable to the rapid detection needs of batch samples.

[0143] According to an embodiment of the present invention, the reaction system of PCR amplification is as follows:

[0144] 2.0 μL PCR premix,

[0145] 1.0 μL PCR primer pair,

[0146] 2.0 μL DNA template.

[0147] The inventors obtained the above-mentioned optimal PCR amplification reaction system after a large number of experiments, thereby further eliminating the interference between multiple amplification systems, achieving specific amplification of dozens of SNP sites in the same amplification system, and further improving the detection accuracy.

[0148] According to an embodiment of the present invention, the concentration of the DNA template is greater than 10 ng / μL; and the purity of the DNA template is 1.7 to 2.0 according to the ratio of the absorbance values ​​of OD260 and OD280. The inventors obtained the concentration and purity of the above-mentioned preferred DNA template through a large number of experiments, thereby further improving the amplification efficiency and eliminating interference between multiple amplification systems.

[0149] According to an embodiment of the present invention, the reaction system of the single base extension reaction is as follows:

[0150] 0.6μL single base extension enzyme,

[0151] 1.4 μL single base extension buffer,

[0152] 1.0 μL ddNTPmix,

[0153] 1.0 μL single base extension primer.

[0154] According to the embodiments of the present invention, the inventors obtained the above-mentioned optimal single-base extension reaction system after a large number of experiments, thereby further eliminating the interference between different single-base extension primers, realizing the single-base extension reaction of multiple PCR amplification products in the same reaction system, thereby further improving the detection accuracy.

[0155] Therefore, the kit developed based on the primer set for detecting the aforementioned SNP sites can detect the combination of SNP sites related to the action of cardiovascular disease drugs, and can specifically detect the specific typing of 20 SNP sites on the action of cardiovascular disease drugs. The test results are accurate, the detection site throughput is high, the operation difficulty is low, the detection cost is low, the detection time is short, and the application value is high.

[0156] Detection Methods

[0157] The present invention proposes a method for detecting the combination of SNP sites related to the aforementioned drug effects for non-disease diagnosis purposes. The method comprises:

[0158] Amplifying the DNA sample to be tested using the aforementioned primer set to obtain an amplified product;

[0159] Determine the SNP site information of the amplified product.

[0160] It should be particularly noted that the method provided by the present invention for detecting SNP site combinations related to the effects of cardiovascular disease drugs is for non-disease diagnosis and treatment purposes.

[0161] This method is specifically used for the detection and confirmation of genotyping of 20 SNP sites for cardiovascular disease drugs.

[0162] According to an embodiment of the present invention, the method further comprises the following steps:

[0163] (1) using the primer pair in the aforementioned primer set to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product;

[0164] (2) using the single-base extension primer in the aforementioned primer set to perform a single-base extension reaction on the first amplification product to obtain a second amplification product;

[0165] (3) Determine the SNP site information of the second amplification product.

[0166] According to an embodiment of the present invention, in step (3), the SNP site information of the DNA sample to be tested is determined based on the SNP site information of the second amplification product.

[0167] According to an embodiment of the present invention, step (3) uses sequencing or mass spectrometry to analyze the SNP site information of the second amplification product to determine the SNP site information of the DNA sample to be tested.

[0168] According to an embodiment of the present invention, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase.

[0169] According to an embodiment of the present invention, the mass spectrometry analysis comprises:

[0170] Purifying the second amplification product to obtain a purified product;

[0171] The purified product was subjected to mass spectrometry analysis.

[0172] In some embodiments of the present invention, the mass spectrometry analysis is performed using a time-of-flight mass spectrometry analysis instrument. The molecular weight of the second amplification product is detected using a time-of-flight mass spectrometry, and the information of the SNP site in the sample to be tested is determined based on the molecular weight marker, thereby determining the typing of the relevant SNP site.

[0173] According to an embodiment of the present invention, the instrument and software used in the mass spectrometry analysis are selected from the QuanNUA nucleic acid typing mass spectrometry system and the QuanSNP genotyping system.

[0174] According to an embodiment of the present invention, the reaction system of PCR amplification is as follows:

[0175] 2.0 μL PCR premix,

[0176] 1.0 μL PCR primer pair,

[0177] 2.0 μL DNA template.

[0178] According to an embodiment of the present invention, the concentration of the DNA template is greater than 10 ng / μL; and the purity of the DNA template is 1.7-2.0 according to the ratio of the absorbance values ​​of OD260 and OD280.

[0179] According to an embodiment of the present invention, the reaction system of the single base extension reaction is as follows:

[0180] 0.6μL single base extension enzyme,

[0181] 1.4 μL single base extension buffer,

[0182] 1.0 μL ddNTPmix,

[0183] 1.0 μL single base extension primer.

[0184] Those skilled in the art will appreciate that the features and advantages described above for SNP site combinations, primer sets, kits, and uses are also applicable to this application and will not be described in detail here.

[0185] Therefore, the method of the present invention for detecting the combination of SNP sites related to the effects of the aforementioned cardiovascular disease drugs for non-disease diagnosis purposes can achieve simultaneous detection of 20 sites in a single experiment, and has the advantages of accurate detection results, high detection site throughput, simple operation, low cost, short time consumption, and high application value.

[0186] use

[0187] The present invention proposes the use of a drug action-related SNP site combination in predicting the efficacy, side effects or dosage of a drug for cardiovascular disease in a subject. The SNP site combination includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504.

[0188] Those skilled in the art will appreciate that the features and advantages described above for the SNP site combination, primer set, kit, and use are also applicable to this use and will not be described in detail here.

[0189] According to an embodiment of the present invention, based on the genotyping results of each SNP site in the SNP site combination, the efficacy, side effects or dosage of the drug for cardiovascular diseases of the subject are predicted.

[0190] According to an embodiment of the present invention, the drug is selected from at least one of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists (AIIA) drugs, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs.

[0191] According to an embodiment of the present invention, the drug is selected from at least one of rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril (benazepril, lotensin), enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin, and folic acid. Thus, it is widely applicable to the medication guidance and suggestions of cardiovascular disease drugs.

[0192] According to an embodiment of the present invention, the SNP site genotyping result is determined based on the aforementioned kit or the aforementioned method.

[0193] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0194] Example 1: Method for detecting SNP sites of cardiovascular disease medication genes using the primer set of the present invention

[0195] (1) Extraction and quality control of genomic DNA

[0196] Extraction of genomic DNA from human peripheral blood samples or EDTA whole blood samples: Use the blood / cell / tissue genomic DNA extraction kit (DP304) of Tiangen Biochemical Technology (Beijing) Co., Ltd. and perform extraction according to the instructions.

[0197] Extraction of genomic DNA from human saliva samples: The extraction was performed using the high-efficiency oral swab genomic DNA extraction kit (DP362) from Tiangen Biochemical Technology (Beijing) Co., Ltd. according to the instructions.

[0198] Quality detection of extracted DNA: The genomic DNA samples extracted above were measured by UV spectrophotometer (OD 260 / OD 280 ) and Qubit fluorescence spectrophotometer to measure the purity and concentration. The purity of the extracted genomic DNA sample (OD 260 / OD 280 ) between 1.7 and 2.0 and the concentration must be above 10ng / μL before it can be used in subsequent experiments.

[0199] (2) Multiplex PCR amplification

[0200] All the reaction systems in this example were carried out in 96-well plates or PCR-specific EP tubes.

[0201] The PCR premix reagent for multiplex PCR amplification uses Thermo Fisher's Platinum multiplex PCR mastermix, the main components of which are PCR amplification enzyme, buffer (PCR amplification buffer), Mg 2+ , dNTPmix; the primers involved in multiplex PCR were synthesized by Shenggong Biotechnology, and the concentration of each primer was 0.1uM.

[0202] The preparation of the multiplex PCR amplification reaction system is shown in Table 4.

[0203] Table 4 PCR amplification reaction system

[0204]

[0205]

[0206] Note: "1x" means that the corresponding component is its working solution and no further dilution is required when preparing the reaction system.

[0207] Place the prepared PCR amplification reaction system tube on an oscillator, shake, mix, and centrifuge instantly. According to the number of test samples (including quality control products), add the PCR amplification reaction system to the corresponding PCR reaction tube (0.2mL PCR eight-row reaction tube, or 96-well plate) at 3μL per well, and add each DNA sample to be tested to the corresponding PCR reaction tube at 2μL per well, and cover the tube cap or stick the sealing film. After shaking and mixing, centrifuge thoroughly to centrifuge all the liquid on the tube wall to the bottom of the tube. Put the PCR reaction tube with the sample into the PCR instrument for amplification.

[0208] The PCR amplification reaction conditions are shown in Table 5.

[0209] Table 5 PCR amplification reaction conditions

[0210]

[0211] (3) Enzymatic hydrolysis

[0212] First, prepare the enzymatic reaction system, then add 2 μL of the enzymatic reaction system to each well where the PCR amplification reaction has been completed, and cover the tube or apply a sealing film. After shaking and mixing, centrifuge thoroughly to centrifuge all the liquid on the tube wall to the bottom of the tube to obtain a PCR reaction tube with the sample added.

[0213] The configuration of the enzymatic hydrolysis reaction system is shown in Table 6.

[0214] Table 6 Enzymatic reaction system preparation

[0215] Reaction components Reaction volume 1x (μL) SAP enzyme 0.3 SAP Buffer 0.17 Nuclease-free water 1.53 Total amount of enzymatic reaction system 2.0

[0216] Note: "1x" means that the corresponding component is its working solution and no further dilution is required when preparing the reaction system.

[0217] Place the PCR reaction tube with the sample into the PCR instrument, set the enzymatic reaction conditions, and perform the enzymatic reaction.

[0218] The enzymatic reaction conditions are shown in Table 7.

[0219] Table 7 Enzymatic reaction conditions settings

[0220] temperature time 37℃ 40min 85℃ 5min 12℃ Save and prepare for the next experiment

[0221] (4) Preparation of single base extension reaction system

[0222] The single-base extension reaction body single-base extension enzyme uses UltraHiFidelity PCR Mix of Tiangen Biochemical Technology (Beijing) Co., Ltd., and its main component is PCR amplification enzyme; the single-base extension buffer uses UltraHiFidelity PCR Mix of Tiangen Biochemical Technology (Beijing) Co., Ltd., and its main components are water, ammonium sulfate, potassium chloride, etc.; the single-base extension primers are all synthesized by Shenggong Biotechnology, and the concentration of each primer is 0.1μM; ddNTPmix uses a mixture of ddATP, ddTTP, ddCTP, and ddGTP customized and synthesized by Rongzhi Biotechnology (Qingdao) Co., Ltd., and the concentration is 0.2mM.

[0223] First, prepare the single base extension reaction system, then add 4 μL of the single base extension reaction system to each well where the PCR amplification reaction has been completed, and cover the tube cap or affix a sealing film. After shaking and mixing, centrifuge thoroughly to centrifuge all the liquid on the tube wall to the bottom of the tube to obtain a PCR reaction tube with the sample added.

[0224] The configuration of the single base extension reaction system is shown in Table 8.

[0225] Table 8 Single base extension reaction system preparation

[0226] Reaction components Reaction volume 1x (μL) Single base extension enzyme 0.6 Single base extension buffer 1.4 ddNTPmix 1.0 20-plex single base extension primer mix 1.0 Total amount of reaction system 4.0

[0227] Note: "1x" means that the corresponding component is its working solution and no further dilution is required when preparing the reaction system.

[0228] Place the PCR reaction tube with the sample into the PCR instrument, set the single base extension reaction conditions, and perform amplification.

[0229] The single base extension reaction condition settings are shown in Table 9.

[0230] Table 9 Single base extension reaction condition settings

[0231]

[0232]

[0233] (5) Product purification and chip spotting

[0234] The product of the above-mentioned single base extension reaction is desalted and purified by using a cation exchange resin to obtain a sample solution that can be used for mass spectrometry analysis. The specific method is as follows:

[0235] Add 14 μL of enzyme-free water to each of the reaction wells that have completed the single-base extension reaction, then add 6 mg of cation exchange resin (BioRad) to each reaction well, and cover with a new tube cap or a new plate sealing film. After shaking and mixing, centrifuge thoroughly to centrifuge all the liquid on the tube wall to the bottom of the tube to obtain a product PCR tube with resin. Place the product PCR tube with resin on a flip mixer, mix at 20 rpm for 30 minutes, and perform desalting and purification to obtain a sample solution that can be used for mass spectrometry analysis.

[0236] According to the number of test samples, 1 μL per well was spotted on the chip pre-spotted with the matrix.

[0237] (6) Mass spectrometry test

[0238] Start the QuanNUA nucleic acid typing mass spectrometry system (Rongzhi Biotechnology (Qingdao) Co., Ltd.), load the sample-speckled and dried crystallized chip into the nucleic acid mass spectrometer, and perform sample editing and testing. After the test, use the QuanSNP genotyping software system (Rongzhi Biotechnology (Qingdao) Co., Ltd.) to analyze the sample results and obtain the typing results of each SNP site of each sample. If necessary, the typing results of all samples can be exported for backup.

[0239] The data acquisition conditions of the QuanNUA nucleic acid typing mass spectrometry system are:

[0240] Laser energy: 10uJ;

[0241] Collection mass range: 1k~10kDa.

[0242] Example 2: Verification of the accuracy and reproducibility of the reaction system

[0243] This example is carried out under the reaction system of Example 1, and the accuracy, specificity and repeatability of the reaction system of Example 1 are verified twice. The specific implementation plan is as follows:

[0244] A human genomic DNA standard (Coriell, catalog number: NA12156, hereinafter referred to as “human genomic standard NA12156”) was selected, and the reaction system and detection method in Example 1 were used to detect and analyze the 20 SNP sites related to cardiovascular disease as shown in Table 1, and the detection was repeated 20 times.

[0245] The human genome standard NA12156 was sequenced to determine the SNP site information, and the first-generation sequencing results were compared with the mass spectrometry test results described in Example 1 for consistency.

[0246] For example, the inventors selected two SNP sites, rs429358 and rs7412, for the first generation sequencing results to display the sequencing results. Figures 1-2 .

[0247] For example, the inventors selected the repeatability test results of 4 SNP sites of the human genome standard NA12156 for display, as shown in Table 9.

[0248] Table 9 Repeated test results of 4 SNP sites of human genome standard NA12156

[0249]

[0250]

[0251] The comparison of mass spectrometry detection results and first-generation sequencing results of 20 SNP sites of human genome standard NA12156 is shown in Table 10.

[0252] Table 10 Test results of 20 SNP sites of human genome standard NA12156

[0253]

[0254]

[0255] The results of nucleic acid mass spectrometry detection of 20 SNP sites of human genome standard NA12156 are shown in Figure 3 .

[0256] The above results show that the detection method for detecting SNP sites related to the action of cardiovascular diseases developed by using the primer set of the drug action-related SNP site combination of the present invention has the following characteristics: (1) the consistency between the detection result and the first-generation sequencing result is as high as 100%, and the detection result is accurate and reliable; (2) the detection method has good repeatability and high specificity; (3) a single experiment can realize the simultaneous detection of 20 SNP sites, with high throughput, simple operation, low detection cost and short time consumption.

[0257] It can be seen that by detecting the SNP site combination related to the action of the cardiovascular disease drugs of the present invention, it is possible to provide guidance on the use of drugs for related cardiovascular diseases, improve drug efficacy and avoid side effects; the detection method described in Example 1 of the present invention has the advantages of high throughput, simple operation, low detection cost, short time consumption, etc., and can be widely used in clinical drug gene detection, scientific research and development and other fields.

[0258] Example 3: A typical case of providing guidance and suggestions on the use of cardiovascular disease medication for the sample based on the test results of the clinical sample

[0259] This example is carried out under the reaction system of Example 1, and 20-fold SNP site typing detection is performed on 2 clinical samples, and medication guidance and suggestions for drugs such as rosuvastatin, acenocoumarol, warfarin and folic acid for treating cardiovascular diseases are given according to the typing results. The specific implementation plan is as follows:

[0260] Two clinical samples were selected, and the reaction system and detection method described in Example 1 were used to detect and analyze the 20 SNP sites related to cardiovascular disease medication as shown in Table 1, and the typing results of the 20 SNP sites of the two clinical samples were obtained.

[0261] The test results of 20 SNP sites in 2 clinical samples are shown in Table 11.

[0262] Table 11 Test results of 20 SNP sites in 2 clinical samples

[0263] SNP loci Clinical sample-1 Clinical sample-2 rs1801253 C C rs9934438 G,A G,A rs1801133 G,A G,A rs5186 A A rs5186 G G,A rs11045819 C C rs1799853 C C rs11045852 A A rs9923231 C,T C,T rs1057910 A A rs4244285 G,A G rs671 G G,A rs4149056 T T rs12248560 C C rs7412 C C rs1799752 T / ins / I,A / del289 / D T / ins / I,A / del289 / D rs2306283 G A rs28399504 A A rs1065852 A A rs429358 T T

[0264] According to the typing results of these 20 SNP sites, cardiovascular disease drugs were analyzed and specific medication guidance and suggestions were obtained. Based on the analysis results of the efficacy, toxic side effects and dosage of cardiovascular disease drugs, the two patients were provided with medication recommendations and guidance for statins and anticoagulants commonly used for cardiovascular diseases.

[0265] The medication instructions and recommendations for 2 statins and anticoagulants are shown in Table 12 .

[0266] The above results show that the detection results of the SNP site combination related to the action of cardiovascular disease drugs according to the present invention can accurately analyze the response of different individuals to cardiovascular disease drugs, and thus provide targeted guidance for individual clinical medication to improve drug efficacy and avoid side effects, thereby achieving precision medication.

[0267] Table 12 Medication guidance and recommendations for statins and anticoagulants in two clinical samples

[0268]

[0269]

[0270]

[0271] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0272] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A drug action related SNP site combination, characterized in that: The SNP site combination includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504.

2. The SNP site combination according to claim 1, characterized in that: The drug is selected from drugs for cardiovascular diseases; Optionally, the drug is selected from one or more of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs; Optionally, the drug is selected from one or more of rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril, enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin and folic acid.

3. A primer set for detecting the SNP site associated with the drug action of claim 1 or 2, characterized in that: The primer set has the nucleotide sequences shown in SEQ ID NOs: 1-40.

4. The primer set according to claim 3, characterized in that: The primer set further includes a single base extension primer; Optionally, the single-base extension primer has a nucleotide sequence as shown in SEQ ID NOs: 41-61.

5. A kit, characterized in that: It comprises the primer set for detecting SNP sites related to the effects of cardiovascular disease drugs as described in claim 3 or 4.

6. Use of the primer set for detecting SNP sites according to claim 3 or 4 in preparing a kit for detecting the SNP site combination according to claim 1 or 2.

7. The use according to claim 6, characterized in that: The detection method of the kit comprises: Amplifying the DNA sample to be tested using the primer set described in claim 3 or 4 to obtain an amplified product; Determining the SNP site information of the amplified product; Optionally, further comprising the steps of: (1) using the primer pair in the primer set of claim 3 to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product; (2) using the single-base extension primer in the primer set of claim 4 to perform a single-base extension reaction on the first amplification product to obtain a second amplification product; (3) determining the SNP site information of the second amplification product; Optionally, based on the SNP site information of the second amplification product, determining the SNP site information of the DNA sample to be tested; Optionally, step (3) determines the SNP site information of the second amplification product by sequencing or mass spectrometry analysis; Optionally, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase; Optionally, the mass spectrometry analysis comprises: Purifying the second amplification product to obtain a purified product; The purified product was subjected to mass spectrometry analysis.

8. A method for detecting a combination of SNP sites associated with the drug effect of claim 1 or 2, characterized in that: include: Amplifying the DNA sample to be tested using the primer pair described in claim 3 or 4 to obtain an amplified product; Determine the SNP site information of the amplified product.

9. The method according to claim 8, characterized in that Further comprising the steps of: (1) using the primer pair in the primer set of claim 3 to perform PCR amplification on the DNA sample to be tested to obtain a first amplification product; (2) using the single-base extension primer in the primer set of claim 4 to perform a single-base extension reaction on the first amplification product to obtain a second amplification product; (3) determining the SNP site information of the second amplification product; Optionally, based on the SNP site information of the second amplification product, determining the SNP site information of the DNA sample to be tested; Optionally, step (3) determines the SNP site information of the second amplification product by sequencing or mass spectrometry analysis; Optionally, before performing a single base extension reaction on the first amplification product in step (2), the first amplification product is enzymatically treated with alkaline phosphatase, and the alkaline phosphatase is preferably shrimp alkaline phosphatase; Optionally, the mass spectrometry analysis comprises: Purifying the second amplification product to obtain a purified product; The purified product was subjected to mass spectrometry analysis.

10. Use of a drug-effect-related SNP site combination in predicting drug efficacy, side effects or dosage for cardiovascular diseases in a subject, wherein the use is for non-diagnostic purposes, characterized in that: The SNP site combination includes: rs9934438, rs1799853, rs1057910, rs4244285, rs4986893, rs12248560, rs671, rs5186, rs1801133, rs4149056, rs1065852, rs1801253, rs1799752, rs429358, rs7412, rs9923231, rs11045819, rs2306283, rs11045852 and rs28399504; Optionally, based on the genotyping results of each SNP site in the SNP site combination, predicting the efficacy, side effects or dosage of the cardiovascular disease drug for the subject; Optionally, the drug is selected from the group consisting of drugs for cardiovascular diseases; Optionally, the drug is selected from one or more of statins, beta lipid-lowering drugs, anticoagulants, beta adrenergic receptor blockers, diuretics, angiotensin II receptor antagonists, beta receptor agonists, angiotensin converting enzyme inhibitors, calcium ion antagonists, vasodilators and coronary heart disease drugs; Optionally, the drug is selected from one or more of rosuvastatin, atorvastatin, pitavastatin, simvastatin, fluvastatin, lovastatin, pravastatin, hydroxymethylglutaryl coenzyme A reductase inhibitors, fenofibrate, acenocoumarol, phenprocoumon, warfarin, clopidogrel, prasugrel, ticagrelor, metoprolol, carvedilol, atenolol, bucindolol, spironolactone, hydrochlorothiazide, losartan, irbesartan, candesartan, dobutamine, captopril, perindopril, benazepril, enalapril, nitrendipine, verapamil, diltiazem, sodium nitroprusside, catecholamines, digoxin, nitroglycerin and folic acid; Optionally, the SNP site genotyping result is determined based on the kit described in claim 5 or the method described in any one of claims 8 or 9.

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