CN109082464B

CN109082464B - Primer group and kit for detecting hypertension drug metabolism related genes

Info

Publication number: CN109082464B
Application number: CN201710447185.4A
Authority: CN
Inventors: 刘娟; 周玲燕; 张定芳
Original assignee: Precedo Pharmaceuticals Co Ltd
Current assignee: Hefei Zhongke Purusheng Medical Laboratory Co ltd
Priority date: 2017-06-14
Filing date: 2017-06-14
Publication date: 2021-12-07
Anticipated expiration: 2037-06-14
Also published as: CN109082464A

Abstract

The invention relates to a primer group for detecting hypertension drug metabolism related genes based on a Sanger sequencing method and a kit containing the primer group. The primer set of the present invention is characterized by comprising a primer for detecting at least one gene selected from the following genes: BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene and NR3C2 gene.

Description

Primer group and kit for detecting hypertension drug metabolism related genes

Technical Field

The invention relates to the field of medicine and biotechnology, in particular to a primer group for detecting hypertension drug metabolism related genes based on a Sanger sequencing method, a kit using the primer group and application of the primer group in preparation of the kit.

Background

Hypertension is a worldwide problem and cannot be cured at present. Statistically, about 1/3 adults worldwide suffer from hypertension, which is easily complicated by heart disease, stroke, chronic kidney disease and premature death, and poses serious threats to human health and life. At present, the number of people suffering from hypertension in China is up to 3.3 hundred million, and the incidence of diseases rises year by year; drugs are the primary means of treating hypertension and its complications currently and for considerable time in the future. Five major classes of hypotensive drugs currently in use are calcium channel blockers, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, diuretics, and beta-blockers. Among the etiologies that can be determined, hypertension caused by a single genetic mutation occupies an important position. The five major drugs relate to polymorphic sites of a plurality of genes, and the different types of different genes have different influences on the metabolism or absorption of the antihypertensive drugs. 95 percent of patients with hypertension are essential hypertension, and the causes of the hypertension are difficult to find, so the treatment is not easy to be cured, and the best method at present is to control the blood pressure rise and avoid the occurrence of cardiovascular and cerebrovascular malignant events. However, due to the differences in the therapeutic efficacy of drug therapy and adverse drug reactions among different individuals, the blood pressure of about 20% to 50% of patients receiving drug therapy is not well controlled. At present, clinical hypotensor has a plurality of error areas, which can cause the failure of hypertension treatment, even cause hypertension emergency and threaten the life of patients.

The individual differences in the therapeutic effects and adverse reactions of antihypertensive drugs are common phenomena in the current treatment process. Patients receiving hypertension medication may experience various degrees of adverse reactions. A large number of researches show that the genotype of an individual determines the toxic reaction of the drug, and the drug-related gene polymorphism can cause the antihypertensive drug to generate toxic and side effects on a human body by changing the functions of enzymes, channels, receptors and the like in the traditional pharmacokinetic and pharmacokinetic pathways. Through long-term research, many gene mutations are related to hypertension drug response, and the typing of hypertension drug metabolism related genes on patients has important significance in the aspect of hypertension individual medication guidance.

With the successive proposals of the human genome project, the thousand human genome project and the Single Nucleotide Polymorphism (SNP) haplotype project, the research on the correlation between hypertension drugs and genomics has been greatly advanced in recent years, but at present, the studies are rarely really applied to clinical treatment. The relation between the related gene mutation of the hypertension drug and the drug safety can provide sufficient basis for the reasonable application of the drug and the individualized drug treatment, avoid adverse reaction, reduce the drug treatment cost and risk, have important clinical application value, can also relieve the pain and economic burden of patients, and have very important social and economic meanings.

At present, there are many methods for detecting gene mutation, and the most commonly used methods include polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP method), sequence-specific PCR, manual or automatic sequencing, gene chip (also called DNA chip) and the like. Sanger sequencing, also known as dideoxy chain-termination method (Sanger method), is a commonly used nucleic acid sequencing technique for DNA analysis, invented in 1977 by Fredrich Sanger, England Biochemist. The dideoxy chain termination method and chemical degradation method and their derivatives are collectively called first generation DNA sequencing technology, and are the main sequencing methods used by the human genome project. The sang-Ge method is simple and convenient to operate, and has the advantages of accurate qualification, high sensitivity and strong specificity; in addition, the method also has the advantages of simple sample treatment, simple sequencing step and intuitive result analysis.

Disclosure of Invention

The invention aims to provide a primer group for detecting hypertension drug metabolism related genes based on a Sanger sequencing method and a kit using the primer group. More specifically, the present invention aims to provide a primer set for comprehensively detecting gene variation of metabolic enzymes and receptors related to hypertension patients, and a kit using the same.

The present inventors have conducted extensive and intensive studies and have found that genotyping of BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene, and NR3C2 gene of hypertension patients is of great significance for individualized medication guidance of hypertension, thereby completing the present invention.

Specifically, the present invention relates to the following inventions.

1. A primer group for detecting hypertension drug metabolism related genes based on a Sanger sequencing method is characterized by comprising primers for detecting at least one gene selected from the following genes:

BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene and NR3C2 gene.

2. The primer set according to item 1 above, wherein the primer set comprises a primer that detects at least one of the following gene mutation sites:

BDKRB2(rs1799722), CYP11B2(rs1799998), OAT1/OAT3(rs10792367), AGT (rs699), KCNH2(rs1137617), LDLR (rs1799898), APOB (rs693), CACNA1C (rs2238032), CACNA1C (rs2239050), GRK C (R65C), GRK C (a 142C), GRK C (a 486C), ACE (rs1799752), CYP2D C (C188C), CYP2C C (C430C), CYP2C C (a1075C), CYP2C C (rs4986893), CYP2C C (rs4244285), ACE C (G3670C), AGTR C (a 1166), rb 1165572 (R365572), and NR 36363672 (adnr 365572).

3. The primer set according to item 1 above, wherein the primer set is at least one selected from the following primer sets 1 to 22:

primer set 1: a primer for detecting BDKRB2(rs1799722), which comprises nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3;

primer set 2: primers for detecting CYP11B2(rs1799998) comprising nucleotide sequences shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively;

primer set 3: primers for detecting OAT1/OAT3(rs10792367) comprising nucleotide sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively;

primer set 4: for detection and AGT (rs699) comprising primers having the nucleotide sequences shown in SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12, respectively;

primer set 5: (ii) primers for detecting KCNH2(rs1137617) comprising nucleotide sequences shown in SEQ ID nos. 13, 14 and 15, respectively;

primer set 6: primers for detecting LDLR (rs1799898) comprising nucleotide sequences shown in SEQ ID No.16, SEQ ID No.17, and SEQ ID No.18, respectively;

primer set 7: primers for detecting APOB (rs693) comprising nucleotide sequences shown in SEQ ID No.19, SEQ ID No.20, and SEQ ID No.21, respectively;

a primer set 8: for detecting CACNA1C (rs2238032), including primers having nucleotide sequences shown in SEQ ID No.22, SEQ ID No.23, and SEQ ID No.24, respectively;

primer set 9: the primers are used for detecting CACNA1C (rs2239050) and respectively comprise nucleotide sequences shown in sequence numbers SEQ ID No.25, SEQ ID No.26 and SEQ ID No. 27;

primer set 10: a primer for detecting GRK4(R65L), which comprises nucleotide sequences shown in SEQ ID No.28, SEQ ID No.29 and SEQ ID No. 30;

primer set 11: a primer for detecting GRK4(A142V), comprising nucleotide sequences shown in SEQ ID No.31, SEQ ID No.32, and SEQ ID No.33, respectively;

primer set 12: a primer for detecting GRK4(A486V), comprising nucleotide sequences shown in SEQ ID No.34, SEQ ID No.35, and SEQ ID No.36, respectively;

primer set 13: primers for detecting ACE (rs1799752) comprising nucleotide sequences shown in SEQ ID No.37, SEQ ID No.38, and SEQ ID No.39, respectively;

primer set 14: for CYP2D6(C188T), including primers having nucleotide sequences shown in SEQ ID No.40, SEQ ID No.41, and SEQ ID No.42, respectively;

primer set 15: primers for detecting CYP2C9(C430T) comprising nucleotide sequences shown in SEQ ID No.43, SEQ ID No.44, and SEQ ID No.45, respectively;

primer set 16: primers for detecting CYP2C9(A1075C) comprising nucleotide sequences shown in SEQ ID No.46, SEQ ID No.47, and SEQ ID No.48, respectively;

primer set 17: for detecting CYP2C19(rs4986893), including primers having nucleotide sequences shown in SEQ ID No.49, SEQ ID No.50, and SEQ ID No.51, respectively;

a primer set 18: primers for detecting CYP2C19(rs4244285) comprising nucleotide sequences shown as SEQ ID No.52, SEQ ID No.53 and SEQ ID No.54, respectively;

primer set 19: a primer for detecting ACE2(G9570A), which comprises nucleotide sequences shown in SEQ ID No.55, SEQ ID No.56 and SEQ ID No.57, respectively;

a primer set 20: for detecting AGTR1(A1166C), including primers having nucleotide sequences shown in SEQ ID No.58, SEQ ID No.59, and SEQ ID No.60, respectively;

primer set 21: primers for detecting ADRB2(R16G) and ADRB2(Q27E) comprising nucleotide sequences shown in SEQ ID No.61, SEQ ID No.62, and SEQ ID No.63, respectively;

primer set 22: for detecting NR3C2(rs5522), comprising primers having the nucleotide sequences shown in SEQ ID No.64, SEQ ID No.65 and SEQ ID No.66, respectively.

4. The primer set according to the above item 3, wherein the primer set comprises all of the primer sets 1 to 22.

5. The primer set according to any one of the above items 1 to 4, wherein the hypertensive agent is at least one selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium antagonist, an angiotensin II receptor antagonist, a beta-blocker, and a diuretic antihypertensive agent.

6. A kit for detecting hypertension drug metabolism-related genes based on a Sanger sequencing method is characterized by comprising the primer group of any one of the items 1-5.

7. The kit according to the item 6, characterized in that the kit further comprises a negative control substance and a quality control substance, wherein the negative control substance is sterilized distilled water, and the quality control substance is human whole genome DNA.

8. The kit according to item 6 or 7 above, wherein the hypertensive agent is at least one selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium antagonist, an angiotensin II receptor antagonist, a β -receptor blocker and a diuretic hypotensive agent.

9. Use of the primer set according to any one of items 1 to 5 in preparation of a kit for detecting genes related to hypertension drug metabolism based on a Sanger sequencing method.

10. The use according to the above item 9, wherein the hypertensive agent is at least one selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium antagonist, an angiotensin II receptor antagonist, a β -receptor blocker and a diuretic hypotensive agent.

According to the invention, different schemes can be formulated for hypertension patients according to individual differences of genes, and proper antihypertensive drugs are selected to realize accurate typing and accurate medication, so that the antihypertensive effect is improved, and adverse drug reactions are reduced.

In addition, the primer group has higher sensitivity and specificity, and has the advantages of accurate qualification, high sensitivity and strong specificity when the primer group is used for genotyping the genes related to the hypertension drug metabolism. In addition, the kit provided by the invention also has the advantages of simple sample treatment, simple sequencing steps and intuitive result analysis.

Drawings

FIG. 1 is a Sanger sequencing chart of BDKRB2 gene, wherein FIGS. 1-1, 1-2 and 1-3 are the Sanger sequencing charts of three types of wild, heterozygous and mutated BDKRB2(rs1799722) gene of clinical samples.

FIG. 2 is a sequence diagram of Sanger sequencing of CYP11B2 gene, wherein FIGS. 2-1, 2-2, and 2-3 are sequence diagrams of Sanger sequencing of three types of wild, heterozygous, and homozygous mutation of CYP11B2(rs 17998) gene in clinical samples, respectively.

FIG. 3 is a sequence diagram of Sanger's sequencing of OAT1/OAT3 gene, wherein FIGS. 3-1, 3-2, and 3-3 are sequence diagrams of Sanger's sequencing of wild, heterozygous, and homozygous mutant genes of OAT1/OAT3(rs10792367) clinical samples, respectively.

FIG. 4 is a Sanger sequencing graph of AGT gene, wherein FIGS. 4-1, 4-2, 4-3 are the Sanger sequencing graphs of three types of wild, heterozygous and homozygous mutation of AGT (rs699) gene of clinical samples respectively.

FIG. 5 is a sequence diagram of Sanger's method for clinical sample KCNH2(rs1137617) gene wild type.

FIG. 6 is a sequence chart of Sanger's sequencing of LDLR (rs1799898) gene homozygous mutant in clinical samples.

FIG. 7 is a Sanger sequencing graph of APOB gene, wherein FIGS. 7-1 and 7-2 are the Sanger sequencing graphs of wild and heterozygous APOB (rs693) gene of clinical samples, respectively.

FIG. 8 is a Sanger sequencing graph of CACNA1C gene, wherein FIGS. 8-1 and 8-2 are the sequencing graphs of wild and heterozygous Sanger sequencing graphs of CACNA1C (rs2238032) gene of clinical samples respectively; FIGS. 8-3 and 8-4 are Sanger's sequencing charts of wild and heterozygous clinical samples of CACNA1C (rs2239050) gene.

FIG. 9 is a Sanger sequencing graph of GRK4 gene, wherein FIGS. 9-1 and 9-2 are the Sanger sequencing graphs of clinical samples GRK4(R65L) gene heterozygous and homozygous mutant types, respectively; FIGS. 9-3 and 9-4 are sequencing charts of Sanger's method for clinical samples GRK4(A142V) gene heterozygous and homozygous mutant; FIGS. 9-5 and 9-6 are Sanger's sequencing charts of wild type and heterozygous type of the GRK4(A486V) gene in clinical samples.

FIG. 10 is a sequence diagram of the Sanger method for sequencing ACE genes, wherein FIGS. 10-1, 10-2 and 10-3 are sequence diagrams of Sanger method for three types of insertion, hybridization and deletion mutation of ACE (rs1799752) genes of clinical samples respectively;

FIG. 11 is a sequence diagram of the CYP2D6 gene by Sanger's method, wherein FIGS. 11-1 and 11-2 are sequence diagrams of clinical samples CYP2D6(C188T) gene heterozygous and homozygous mutant by Sanger's method, respectively.

FIG. 12 is a Sanger sequencing chart of the CYP2C9 gene, wherein FIG. 12-1 is a Sanger sequencing chart of a wild type of the CYP2C9(C430T) gene of a clinical sample; FIGS. 12-2 and 12-3 are sequencing charts of wild-type and heterozygous-type Sanger method of the CYP2C9(A1075C) gene in clinical samples, respectively.

FIG. 13 is a Sanger sequencing map of the CYP2C19 gene, wherein FIG. 13-1 is a Sanger sequencing map of a clinical sample CYP2C19(rs4986893) gene wild type; FIG. 13-2 is a Sanger sequencing chart of clinical samples CYP2C19(rs4244285) gene heterozygous.

FIG. 14 is a sequence diagram of the ACE2 gene by Sanger's method, wherein FIGS. 14-1 and 14-2 are sequence diagrams of wild type and homozygous mutant type ACE2(G9570A) gene in clinical samples by Sanger's method, respectively.

FIG. 15 is a Sanger sequencing graph of AGTR1 gene, wherein FIGS. 15-1 and 15-2 are the Sanger sequencing graphs of wild and heterozygous clinical samples AGTR1(A1166C) gene, respectively.

FIG. 16 is a Sanger sequencing chart of ADRB2 gene, wherein, FIGS. 16-1, 16-2 and 16-3 are the Sanger sequencing charts of the wild, heterozygous and homozygous mutation three types of ADRB2(R16G) gene of clinical samples respectively; FIGS. 16-4 are sequencing charts of Sanger's method for ADRB2(Q27E) gene homozygous mutant type in clinical samples.

FIG. 17 is a Sanger sequencing graph of NR3C2 gene, wherein FIGS. 17-1, 17-2 and 17-3 are the Sanger sequencing graphs of wild, heterozygous and homozygous mutant genes of NR3C2(rs5522) in clinical samples.

Detailed Description

The gene and/or gene mutation site related to the present invention is obtained by the inventors of the present invention after conducting detailed investigation and scientific screening of a large number of genes and/or gene mutation sites that may be related to the reactivity of antihypertensive drugs. On the basis of a great deal of work, the invention finally determines 17 hypertension drug metabolism related genes: BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene and NR3C2 gene.

Moreover, the invention further determines 23 gene mutation sites related to hypertension drug metabolism in the genes: BDKRB2(rs1799722), CYP11B2(rs1799998), OAT1/OAT3(rs10792367), AGT (rs699), KCNH2(rs1137617), LDLR (rs1799898), APOB (rs693), CACNA1C (rs2238032), CACNA1C (rs2239050), GRK4(R65L), GRK4(a142 4), GRK4(a486 4), ACE (rs1799752), CYP2D 4 (C4), CYP2C 4 (C430 4), CYP2C 4(a 1075 4), CYP2C 4 (rs 494993), CYP2C 4 (rs4244285), 4 (G9570 4), AGTR 4(a 1166 4), ad3672 (R553616), rb 553672 (NR 4), and NR 4 (NR 4).

The specific relationship between the above gene and/or gene mutation site and the hypertension drug response is as follows:

BDKRB2 gene: the kallikrein kinase system has an important relationship with blood pressure regulation, wherein bradykinin is one of the strongest vasodilating substances in vivo, wherein the vasodilating effect is mainly generated by directly expanding blood vessels, resisting the vasoconstriction effect of angiotensin II and norepinephrine, promoting the synthesis of endogenous vasodilating substances such as nitric oxide and the like, and the kallikrein has a strong natriuretic effect. The physiological action of bradykinin is mediated mainly through a bradykinin beta 2 receptor (BDKRB2), and point mutation of T mutation to C exists in a BDKRB2 gene promoter region-58, and researches show that the reduction range of TT and TC is obvious compared with that of CC group blood pressure, which shows that T allele is related to the blood pressure reduction curative effect of Angiotensin Converting Enzyme Inhibitor (ACEI).

CYP11B2 gene: the occurrence and development of hypertension are closely related to a renin-angiotensin-aldosterone system (RAAS), which controls secretion of aldosterone, and aldosterone has an extremely important role in controlling arterial blood pressure due to its functions of sodium retention, potassium excretion and catecholamine activation. Aldosterone synthesis is affected by aldosterone synthase (CYP11B2) in adrenal cortex mitochondria. Many researches show that the polymorphism of the CYP11B2 gene is related to human essential hypertension, at present, the most researched polymorphism site is the CYP11B2 gene 344C/T polymorphism site, the site can influence the combination of a CYP11B2 gene promoter and a sFI factor, so that the transcription of the mRNA of the aldo-keto synthetase and the synthesis and secretion of the aldo-keto synthetase are influenced, and the polymorphism of the site can influence the curative effects of various antihypertensive drugs such as hydrochlorothiazide, atenolol, losartan and the like.

OAT1/OAT3 gene: the genes OAT1 (organic anion transporter 1) and OAT3 (organic anion transporter 3) encode organic anion transporters, which may be involved in the transport of kidney urate, and a SNP site exists between the two genes and is related to hydrochlorothiazide antihypertensive drugs.

AGT gene: the AGT (angiotensinogen) gene is the most important component of the renin-angiotensin-aldosterone system (RAAS), and its concentration fundamentally affects the production of angiotensin I and angiotensin II. The gene has 2 gene mutation sites in the No.2 exon area, which can result in the mutation of the 235 th amino acid from methionine to threonine (M235T) and the mutation of the 174 th amino acid from threonine to methionine (T174M). The literature reports that the gene mutation of AGT may be the pathogenesis of partial essential hypertension patients and influence the curative effect of antihypertensive drugs.

KCNH2 gene: KCNH2 is a potassium channel gene and plays an important role in excitation regulation of smooth muscle cells, and certain signal pathways mediated by angiotensin, nitric oxide and adrenoceptor blockers are all involved in the function regulation of KCNH2 potassium channels, and are closely related to the regulation pathways of blood pressure. The KCNH2(1956, C > T) gene polymorphism is obviously related to the curative effect of antihypertensive drugs on Chinese essential hypertension patients, and the polymorphism can be a useful biomarker for individualized treatment of specific antihypertensive drugs, so that the detection of the site has important significance.

LDLR gene: the LDLR (low density lipoprotein receptor) gene plays an important role in balancing cholesterol in the body, and its physiological role is to transport cholesterol-containing lipoprotein particles into cells through receptor-mediated endocytosis, with the primary ligand being Low Density Lipoprotein (LDL). The document reports that the polymorphism of the LDLR gene T1773C site has certain relation with human Essential Hypertension (EH) and influences the curative effect of partial hypotensor of beta receptor blocker.

APOB gene: the APOB gene is a major apolipoprotein in Low Density Lipoproteins (LDL) and Very Low Density Lipoproteins (VLDL), and plays an important role in maintaining the blood lipid level in vivo constant. Since there are multiple genetic mutation sites in the APOB gene, among which the site located in exon 26 of the APOB gene is closely related to blood lipid levels, literature studies indicate that this site gene is related to the increase of LDL-C and TG levels.

CACNA1C gene: amlodipine (Amlodipine) is a common drug widely used in the treatment of hypertension as dihydropyridine Calcium Channel Blockers (CCBs). Recent researches suggest that a single nucleotide polymorphism site of an L-type calcium channel Q1C subunit gene (CACNALC) is possibly associated with the antihypertensive effect of a dihydropyridine calcium channel blocker, the antihypertensive effect of an antihypertensive drug is possibly influenced, the fact that the GG genotype at the site carries a patient with the antihypertensive amplitude far lower than that of a T allele (TT + TC genotype) carrier is proved, and the systolic pressure and diastolic pressure drop amplitude of the CC genotype at the other site are lower than those of the GG and CG genotypes.

GRK4 gene: g protein related receptor kinase (GRK4) belongs to serine/threonine protein kinase family, and regulates sodium ion transport by influencing dopamine receptor, angiotensin II receptor and the like, so as to regulate the level of blood pressure, wherein the GRK4 gene has R65L, A486V and A142V variant polymorphism, the correlation between the GRK4 gene polymorphism and hypertension is reported in the current research, and different gene types of three sites have different influences on the curative effect of antihypertensive drugs.

The ACE gene: angiotensin Converting Enzyme (ACE) is a key enzyme of renin-angiotensin system (RAS), when the ACE gene contains D allele, the concentration of ACE in serum is increased, leading to the rapid conversion of angiotensin i (Ang i) to angiotensin II (Ang II), Ang II acts on angiotensin II type 1 receptor, leading to the blood vessel being in tension state, leading to the rise of blood pressure, producing a series of related pathological effects. The ACE gene is located on chromosome long arm 17q23, has a total length of 21kb, and comprises 26 exons and 25 introns. At present, a plurality of ACE gene variations are found, wherein intron 16 has insertion and deletion polymorphism with a length of 288bp sequence, the gene has three genotypes of I/I, I/D and D/D, and each type has different curative effects on antihypertensive drugs.

CYP2D6 gene: cytochrome oxidase P450 is an enzyme participating in the metabolism of endogenous and exogenous compounds, and at present, 35P 450 genes are known in human bodies, and CYP2D6 is one of the genes. CYP2D6 has been found to be involved in a variety of antiarrhythmic agents, beta blockers, antihypertensive agents, and the like. These drugs, when used in combination, are susceptible to substrate competitive inhibition, which affects CYP2D6 activity. Meanwhile, the activity of CYP2D6 can be inhibited by a plurality of specific inhibitors such as quinidine, fluoxetine, paroxetine and the like, and the CYP has gene polymorphism, which is one of the most important factors causing individual difference of drug action.

CYP2C9 gene: CYP2C9 is an isozyme in CYP2C subfamily, is mainly distributed in liver tissues, and approximately 10% of clinical common drugs are subjected to oxidative metabolism through CYP2C9, wherein the drugs comprise some hypertension-related drugs such as glipizide, glibenclamide, tolazamide, losartan, irbesartan and the like. The CYP2C9 gene has genetic polymorphism, so that the CYP2C9 enzyme activity is different, and the gene mutation with functional significance can reduce the curative effect of CYP2C9 enzyme substrate drugs or generate more adverse reactions, which is one of the reasons for the difference between drug metabolism ethnicity and individuals.

CYP2C19 gene: CYP2C19, one of the most important drug metabolizing enzymes in the CYP450 family, is involved in the metabolism of endogenous substances and exogenous substances including drug environmental compounds, by which about 2% of drugs are clinically catalyzed. The research finds that CYP2C19 can affect the metabolism of clinical drugs omeprazole, diazepam, phenytoin sodium, propranolol and the like.

ACE2 gene: ACE2 is a carboxyl monopeptidase, belongs to zinc-dependent metalloprotease family member, and angiotensin converting enzyme 2 competes with angiotensin converting enzyme to reversely regulate renin-angiotensin-aldosterone system, and catalyzes angiotensinogen to generate metabolite with vasodilation function, thereby influencing the curative effect of hypertension and hypertension lowering drugs.

AGTR1 gene: angiotensin II (Ang II) is the most important bioactive substance in RAS, one of the most potent hormones known as endogenous pressor substances, whereas Ang II must act through receptors. Ang II receptors have two subtypes, type I (AT1R) and type II (AT 2R). AT1R is mainly distributed on vascular smooth muscle cells to mediate most biological effects of Ang II, and the type I angiotensin receptor gene AGTR1 naturally becomes an important candidate gene for hypertension research, and researches find that the genotype can influence the curative effect of antihypertensive drugs.

ADRB2 gene: the beta 2-adrenoceptor (ADRB 2) is a member of a G protein coupled receptor family, and researches show that the expression and function of the ADRB2 gene can influence the pharmaceutical effects of bronchodilators taking the beta 2-adrenoceptor as an action target, including beta 2 receptor agonists, theophyllines, anticholinergic drugs and the like, and play a certain role in the aspect of hypertension drug metabolism.

NR3C2 gene: the NR3C2 gene encodes the Mineralocorticoid Receptor (MR). The NR3C2 gene is located in the q31.1 region of human chromosome 4, has a length of over 400kb, and comprises 9 exons, and the amino acid number of the encoded protein is 984 and is encoded by the exons from the second to the ninth. It is found that the NR3C2rs5522G allele carrier has reduced diastolic blood pressure lowering reaction after taking enalapril. Therefore, the NR3C2 gene polymorphism can be used for predicting the curative effect of the ACEi antihypertensive drug taken by a patient.

In order to detect the screened target gene and/or the mutation site thereof, the invention provides a PCR primer group for detecting hypertension drug related genes based on a Sanger sequencing method and a kit comprising the primer group.

In one embodiment, the primer set for detecting a gene associated with hypertension drug metabolism based on Sanger sequencing according to the present invention includes a primer for detecting at least one gene selected from the above 17 genes. In another embodiment, the primer set for detecting genes related to hypertension drug metabolism based on the Sanger sequencing method comprises a primer for detecting at least one of the 23 gene mutation sites.

The primer sequence contained in the primer set of the present invention can be designed based on the DNA sequence of the gene and/or gene mutation site related to hypertension drug metabolism of the present invention by using a conventional technique in the art, and is not particularly limited. Specific examples of the primer sequences include primer sequences included in the primer sets 1 to 22 shown in Table 1, and specifically, primer sequences having nucleotide sequences shown in SEQ ID Nos. 1 to 66.

TABLE 1 primer sequences for detecting hypertension drug metabolism-related genes of the present invention based on Sanger sequencing method

(continued) Table 1. detection of primer sequences of genes related to hypertension drug metabolism according to the present invention based on Sanger sequencing method

The primer sets 1-22 have high sensitivity and specificity, and when the Sanger sequencing method is used for genotyping the genes related to the metabolism of the hypertension drug, the primer sets 1-22 have the advantages of accurate qualification, high sensitivity and strong specificity. Therefore, the primer set for detecting a gene associated with hypertension drug metabolism by Sanger sequencing according to the present invention preferably includes at least one of the primer sets 1 to 22, and more preferably includes all of the primer sets 1 to 22.

In another embodiment, the invention also provides a kit for detecting genes related to hypertension drug metabolism based on the Sanger sequencing method. The kit of the present invention is characterized by comprising the primer set for detecting the gene related to the metabolism of the hypertensive drug by Sanger sequencing according to the present invention.

According to needs, the kit of the invention can also comprise a negative control substance and a positive quality control substance. The negative control includes sterilized distilled water, and the positive control includes commercially available human whole genome DNA. By arranging the negative control substance and the positive quality control substance in the kit, the accuracy of the detection result can be better ensured when the kit is used for detecting the genotyping.

In addition, the kit of the present invention may further comprise other reagents required for Sanger sequencing, as necessary. As the other reagent, a commonly used reagent known in the art may be used, and examples thereof include DNA polymerase, buffer, dNTP, and the like. The kit of the present invention may further comprise instructions describing the protocol for use of the kit.

By using the primer group or the kit, the BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene and NR3C2 gene of a hypertensive can be subjected to genotyping based on a Sanger sequencing method, so that the individual medication of the hypertensive can be guided. Examples of the hypertension drugs to which the primer set and kit of the present invention can be applied to guide medication include angiotensin converting enzyme inhibitors, calcium antagonists, angiotensin II receptor antagonists, β -receptor blockers, and diuretic hypotensive drugs.

In another embodiment, the invention also provides application of the primer group in preparation of a kit for detecting hypertension drug metabolism-related genes based on a Sanger sequencing method. Examples of the hypertension drugs include angiotensin converting enzyme inhibitors, calcium antagonists, angiotensin II receptor antagonists, β -receptor blockers, and diuretic hypotensives.

Examples

The present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: preparation of the kit

1. Design and synthesis of primer of hypertension drug metabolism related gene

PCR amplification primers and sequencing primers of 17 hypertension drug metabolism-related genes, namely BDKRB2 gene, CYP11B2 gene, OAT1/OAT3 gene, AGT gene, KCNH2 gene, LDLR gene, APOB gene, CACNA1C gene, GRK4 gene, ACE gene, CYP2D6 gene, CYP2C9 gene, CYP2C19 gene, ACE2 gene, AGTR1 gene, ADRB2 gene and NR3C2 gene, are designed by using oligo software (see Table 1). Primer design follows several principles: the length range of the primer is about 25-30 bp; the primer has no dimer; no hair pin structure; the difference between the Tm values of the upstream primer and the Tm value of the downstream primer is not more than 3 ℃; the GC content was around 50%. After the upstream and downstream primers are determined, performing comparative analysis on the upstream and downstream primers by using Blast in an NCBI database to ensure the specificity and amplification efficiency of the primers; designing a proper primer pair near the upstream and downstream of the SNP locus of each gene to ensure that the mutation locus is positioned in the middle of an amplified fragment as much as possible, and the average amplification length is about 500 bp.

2. Selection of control

Human whole genome DNA is taken as a quality control product; sterilized distilled water was used as a negative control.

Composition of PCR reaction solution

The composition of the PCR reaction solution is shown in Table 2.

TABLE 2 composition of PCR reaction solution

Example 2: use of the kit

1. Sample detection

Taking out related reagents, and preparing a corresponding system according to the amount of the template as follows: and (3) uniformly mixing the PCR reaction solution, subpackaging, and adding the sample DNA and the negative control or the positive control as a template to form a reaction system. PCR amplification was performed according to the PCR reaction program.

The major components of each gene testing system for hypertension are shown in table 3.

TABLE 3 essential components of the gene detection system for hypertension

The PCR reaction procedure for this system is shown in Table 4.

TABLE 4 PCR reaction procedure

After the amplification is finished, agarose gel electrophoresis is carried out to check the PCR amplification product, and the PCR amplification product is purified and then carried out to the next procedure.

Sanger sequencing

The method is carried out according to Sanger sequencing standard operating procedures and mainly comprises the following steps:

2.1 sequencing PCR was performed on the purified PCR amplification product, the sequencing PCR system was as follows:

TABLE 5 sequencing PCR System

Reagent	Dosage of
		PCR product	X μ L (about 10-50ng)
BigDye	2μL
		BigDye Buffer	3μL
Sequencing primer	1μL
		Deionized water	4-XμL
Total volume	20μL

The reaction parameters are set as follows: 1min at 96 ℃ (10S at 96 ℃, 5S at 50 ℃ and 4min at 60 ℃) multiplied by 25 cycles; keeping the temperature at 4 ℃.

2.2 sequencing product purification

The specific purification steps are as follows

Terminator v3.1cycle Sequencing Kit Sequencing reagents were performed with the instructions attached.

2.3 sequencing on machine

Data of ABI company was used when ABI gene analyzer was used

Data collection and Analysis with Sequencing Analysis software, detailed step referencing

And the Sequencing Analysis user manual.

3. Result judgment

The sequencing peak diagram of the quality control product is normal, the sequence is accurate, and the detection result of the quality control product is positive; negative controls were not amplified.

The results were judged according to the test standards described in table 6, and the test results of the samples were reported. The Sanger sequencing charts of the respective genes are shown in FIGS. 1 to 17.

TABLE 6

(continuation) Table 6

Example 3: the primer set and/or the kit of the invention have the effect of guiding the administration of a drug to a patient with hypertension

In order to verify the medication guidance effect of the primer set and/or the kit on the hypertension patients, the volunteers of the hypertension patients are selected in Purehei medical laboratory, genotype detection is carried out by using the primer set and/or the kit, the medication scheme is accurately formulated according to the detection result of each volunteer, and clinical tracking is carried out to observe the clinical medication effect.

Medication guide example 1

The effect of taking spironolactone before detection is poor for a person to be detected, the primer group and/or the kit provided by the invention are used for detecting hypertension-related genes of the person to be detected, and the curative effects of hydrochlorothiazide and propranolol are predicted to be better through pharmacodynamic analysis according to the gene detection result of the person to be detected. After taking propranolol for half a year, the blood pressure is stably controlled.

TABLE 7 hypertension-related Gene detection and drug efficacy analysis of drug administration guide example 1

Diuretic agents

Beta receptor blockers

Medication guide example 2

The kit is used for detecting hypertension related genes of the kuri, and the curative effects of felodipine, amlodipine and losartan are better predicted through pharmacodynamic analysis according to the gene detection result of the kuri. When picrorhiza scrophulariiflora Pennell is taken for 3 months, the blood pressure is effectively controlled.

TABLE 8 hypertension-related Gene detection and drug efficacy analysis of drug administration guide example 2

Calcium antagonists

Angiotensin receptor blockers

Medication guide example 3

Wu Chi, the effect of taking metoprolol before detection is poor, the primer group and/or the kit of the invention are used for detecting hypertension related genes of Tian Chi, and according to the gene detection result of Wu Chi, the curative effects of enalapril, imidapril and captopril are predicted to be better through pharmacodynamic analysis. Wu takes captopril for 5 months, and the blood pressure is effectively controlled.

TABLE 9 detection of hypertension-related genes and drug efficacy analysis in drug administration guide example 3

Angiotensin converting enzyme inhibitor

Beta receptor blockers

Medication guide example 4

The primer group and/or the kit are used for detecting genes related to hypertension of the Tianji, and the curative effects of imidapril and benazepril are predicted to be better through pharmacodynamic analysis according to the gene detection result of the Li. The blood pressure of the plum after taking benazepril is effectively controlled.

TABLE 10 hypertension-related Gene detection and drug efficacy analysis of drug administration guide example 4

Angiotensin converting enzyme inhibitor

Angiotensin receptor blockers

Medication guide example 5

The detection subject dandy is subjected to hydrochlorothiazide and atenolol alternately before detection, the blood pressure reduction is not obvious, the primer group and/or the kit provided by the invention are used for detecting hypertension-related genes of dandy, and according to the gene detection result of dandy, the spironolactone blood pressure reduction curative effect is predicted to be better through pharmacodynamic analysis. After taking the medicine, the blood pressure of Deng is reduced obviously and kept at a stable level.

TABLE 11 detection of hypertension-related genes and drug efficacy analysis in drug administration guide example 5

Diuretic medicine

Beta receptor blockers

Medication guide example 6

The blood pressure of a patient Wu-chi is not obviously reduced by taking captopril and nitrendipine before detection, the primer group and/or the kit provided by the invention are used for detecting the hypertension related genes of the Tian-chi, and the pharmacological analysis predicts that the blood pressure reduction curative effects of felodipine, amlodipine, enalapril and imidapril are better according to the gene detection result of the Wu-chi. Blood pressure is effectively controlled after Wu takes amlodipine, and adverse reaction is small.

TABLE 12 hypertension-related Gene detection and drug efficacy analysis of the administration guide example 6

Calcium antagonists

ACE inhibitors

In conclusion, the primer group and the kit containing the primer group can be used for rapidly, simply, efficiently and accurately detecting the parting of hypertension drug metabolism related genes BDKRB2, CYP11B2, OAT1/OAT3, AGT, KCNH2, LDLR, APOB, CACNA1C, GRK4, ACE, CYP2D6, CYP2C9, CYP2C19, ACE2, AGTR1, ADRB2 and NR3C2, so that different schemes can be formulated for hypertension patients according to the individual difference of the genes, appropriate antihypertensive drugs can be selected, accurate parting and accurate medication can be realized, the antihypertensive effect is increased, and adverse drug reactions are reduced.

After reading the above statements of the invention, various modifications and changes may be made by those skilled in the art to the present invention, including variations of the primers, which equivalents are also within the scope of the claims appended hereto.

Sequence listing

<110> science and technology of coppery and biomedical science and technology limited in fertilizer

<120> primer group and kit for detecting hypertension drug metabolism related genes

<160> 60

<170> Patent In version 3.3

<210> 1

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 1

cgggctacgc aaacatggaa atcttcc

<210> 2

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 2

ccacgaccac agggaaactt ctccccg

<210> 3

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 3

acagtttgtc ctcccagcag aggtgag

27

<210> 4

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 4

gagaccccat gtgactccag gacc

<210> 5

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 5

tggaggcccc cagccaaagg tagat

<210> 6

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 6

atgctgagaa aaggcgtggg gtct

<210> 7

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 7

agtgttgaag agatggtaga gcgag

<210> 8

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 8

aagagaataa ggcagacaca cagagaaa

<210> 9

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 9

ttaatgaatt ccaggtctcg gttcctca

<210> 10

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 10

gctgcaaaac ttgacaccga agacaagt

<210> 11

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 11

gatctcagct acacattgga tactaagtcc

<210> 12

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 12

acagtgagct atggggcgtg gtccat

<210> 13

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 13

caacgtctct cccaacacca actcag

<210> 14

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 14

tttcctcatg ggcaaaaagg ggcaacg

<210> 15

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 15

tgaggccctg gaacttagag actccag

<210> 16

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 16

ctgagtagct gagattacag acacgt

<210> 17

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 17

tagtcacaac cagttttctg cgttcatct

<210> 18

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 18

cttccaaact gctgggatta caggcgt

<210> 19

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 19

gctctggaac taccacaaaa agctgaag

<210> 20

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 20

gaacagtgaa cccttgctct accaatgct

<210> 21

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 21

caaagtcagt aaggttctta gcagcaag

<210> 22

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 22

aggtgggggt gataggttct ggaaagc

<210> 23

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 23

tctgtaaggt ttgccaagcg atcagct

<210> 24

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 24

ataagagttg gggaggtggc tgggaga

<210> 25

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 25

tttgcccttg gccaccttct ctgaa

<210> 26

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 26

accctatctc tcagacatca cccagt

<210> 27

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 27

tgacttcagg tttggtcttt gcctctggt

<210> 28

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 28

gtctgaaaca aacaacaaca acaacaacaa g

31

<210> 29

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 29

tccactccta catcattgcc caggtc

<210> 30

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 30

cctggttcaa gtgattcccc tgcct

<210> 31

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 31

agtacagttg acccttgaac aatgcgg

<210> 32

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 32

acaacagcag aactctggag agtgg

<210> 33

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 33

gctaatggta gttaagcttt tgggggagt

<210> 34

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 34

agtgagaggg gcctggcaaa agaatg

<210> 35

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 35

agcaaaccga gcatagaagt cttcatct

<210> 36

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 36

ttgggaactg agggagctga gaattgc

<210> 37

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 37

aggagaggag agagactcaa gcac

24

<210> 38

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 38

cttggggttt cacagcacct ctttg

25

<210> 39

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 39

tgttggcagc agggactcac cattg

25

<210> 40

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 40

gagcccattt ggtagtgagg cagg

24

<210> 41

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 41

ccatgtataa atgcccttct ccaggac

<210> 42

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 42

gggagcctca gcacctctgc c

21

<210> 43

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 43

ggatggggag gatggaaaac agagact

27

<210> 44

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 44

tctctggtga catgttctgg aataggtaa

29

<210> 45

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 45

tgcaaattcc cttggctctc agcttc

<210> 46

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 46

catccaggtc agtaacaggt cagtatgtct

<210> 47

<211> 32

<212> DNA

<213> Artificial Synthesis

<400> 47

ccatgacaac aaattcacaa cattcctggt ta

32

<210> 48

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 48

acacacactg ccagacacta ggacctg

<210> 49

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 49

gagcaaccat tatttaacca gctaggct

28

<210> 50

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 50

tctgtggttc caaatattct ctgtccttga

<210> 51

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 51

cactgatcag ggagctaatg ggctt

<210> 52

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 52

caaccagagc ttggcatatt gtatctatac c

31

<210> 53

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 53

gttcctcctg tgctgatctc actgga

<210> 54

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 54

atcacaaata cgcaagcagt cacataacta

30

<210> 55

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 55

catggctgcc tcactgtcct atga

<210> 56

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 56

agcccagagc ctctcattgt agtc

<210> 57

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 57

cactgaaatg acttacttac tgacttgatc

<210> 58

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 58

ctcagataat gtaagctcat ccaccaag

<210> 59

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 59

acaatctgga actctcatct cctgttgc

<210> 60

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 60

ctcatctcct gttgctcctc taacga

<210> 61

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 61

gagccccgcc gtgggtccg

19

<210> 62

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 62

tgggtggccc ggtaccagtg catc

<210> 63

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 63

atcacccggg ccttattctt ggtcag

<210> 64

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 64

gcaactgtag ctgagtccat gggtt

25

<210> 65

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 65

tacttaacgg acttgagaga ggagagc

27

<210> 66

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 66

tctgtaagag atgctgacta ttcctatg

Claims

1. A primer group for detecting hypertension drug metabolism related genes based on a Sanger sequencing method is characterized by comprising the following primer groups 1-22:

primer set 1: primers for detecting rs1799722 site of BDKRB2 gene, which are composed of nucleotide sequences respectively shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3;

primer set 2: a primer consisting of nucleotide sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively for detecting the rs1799998 site of the CYP11B2 gene;

primer set 3: primers consisting of nucleotide sequences shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 respectively for detecting rs10792367 sites of OAT1/OAT3 genes;

primer set 4: a primer consisting of nucleotide sequences shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12 respectively and used for detecting the rs699 locus of the AGT gene;

primer set 5: a primer consisting of nucleotide sequences shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15 respectively for detecting the rs1137617 site of the KCNH2 gene;

primer set 6: primers of nucleotide sequences shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively are used for detecting the site rs1799898 of the LDLR gene;

primer set 7: primers for detecting the rs693 site of the APOB gene and consisting of nucleotide sequences respectively shown as SEQ ID No.19, SEQ ID No.20 and SEQ ID No. 21;

a primer set 8: a primer consisting of nucleotide sequences shown as SEQ ID No.22, SEQ ID No.23 and SEQ ID No.24 respectively for detecting rs2238032 locus of CACNA1C gene;

primer set 9: primers for detecting rs2239050 site of CACNA1C gene, which are composed of nucleotide sequences respectively shown as SEQ ID No.25, SEQ ID No.26 and SEQ ID No. 27;

primer set 10: a primer consisting of nucleotide sequences shown as SEQ ID No.28, SEQ ID No.29 and SEQ ID No.30 respectively for detecting R65L site of GRK4 gene;

primer set 11: primers consisting of nucleotide sequences shown as SEQ ID No.31, SEQ ID No.32 and SEQ ID No.33 respectively and used for detecting the A142V site of the GRK4 gene;

primer set 12: primers for detecting the A486V locus of the GRK4 gene and consisting of nucleotide sequences shown as SEQ ID No.34, SEQ ID No.35 and SEQ ID No.36 respectively;

primer set 13: primers for detecting an rs1799752 locus of an ACE gene, which are respectively composed of nucleotide sequences shown as SEQ ID No.37, SEQ ID No.38 and SEQ ID No. 39;

primer set 14: primers used for CYP2D6 gene C188T locus and composed of nucleotide sequences shown as SEQ ID No.40, SEQ ID No.41 and SEQ ID No.42 respectively;

primer set 15: a primer consisting of nucleotide sequences shown as SEQ ID No.43, SEQ ID No.44 and SEQ ID No.45 respectively for detecting the C430T locus of the CYP2C9 gene;

primer set 16: a primer consisting of nucleotide sequences shown as SEQ ID No.46, SEQ ID No.47 and SEQ ID No.48 respectively for detecting the CYP2C9 gene A1075C site;

primer set 17: a primer consisting of nucleotide sequences shown as SEQ ID No.49, SEQ ID No.50 and SEQ ID No.51 respectively for detecting the rs4986893 site of the CYP2C19 gene;

a primer set 18: primers for detecting rs4244285 locus of CYP2C19 gene and consisting of nucleotide sequences respectively shown as SEQ ID No.52, SEQ ID No.53 and SEQ ID No. 54;

primer set 19: a primer consisting of nucleotide sequences shown as SEQ ID No.55, SEQ ID No.56 and SEQ ID No.57 respectively and used for detecting the G9570A locus of the ACE2 gene;

a primer set 20: a primer consisting of nucleotide sequences shown as SEQ ID No.58, SEQ ID No.59 and SEQ ID No.60 respectively for detecting the A1166C site of the AGTR1 gene;

primer set 21: primers consisting of nucleotide sequences shown as sequence numbers SEQ ID No.61, SEQ ID No.62 and SEQ ID No.63 respectively for detecting the R16G site of the ADRB2 gene and the Q27E site of the ADRB2 gene;

primer set 22: the primers are used for detecting the rs5522 site of the NR3C2 gene and consist of nucleotide sequences respectively shown as SEQ ID No.64, SEQ ID No.65 and SEQ ID No. 66.

2. The primer set as claimed in claim 1, wherein the hypertensive agent is at least one selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium antagonist, an angiotensin II receptor antagonist, a β -receptor blocker and a diuretic hypotensive agent.

3. A kit for detecting a gene related to hypertension drug metabolism based on a Sanger sequencing method, which is characterized by comprising the primer set according to claim 1 or 2.

4. The kit of claim 3, further comprising a negative control and a positive quality control, wherein the negative control is sterilized distilled water, and the positive quality control is human whole genome DNA.

5. The kit according to claim 3 or 4, wherein the hypertensive agent is at least one selected from the group consisting of an angiotensin converting enzyme inhibitor, a calcium antagonist, an angiotensin II receptor antagonist, a beta-blocker, and a diuretic hypotensive agent.

6. Use of the primer set according to claim 1 or 2 in the preparation of a kit for detecting genes related to hypertension drug metabolism based on Sanger sequencing.

7. The use as claimed in claim 6, wherein the hypertensive agent is selected from at least one of angiotensin converting enzyme inhibitors, calcium antagonists, angiotensin II receptor antagonists, beta-blockers, and diuretic hypotensives.