阅读说明：本技术 一种个体化用药相关基因多态性检测试剂盒及其应用 (Detection kit for polymorphism of gene related to individualized medication and application thereof ) 是由 廖南清 梁浩 袁志兵 张林峰 于 2020-07-08 设计创作，主要内容包括：本发明涉及一种用于指导个体化用药相关基因SNP的特异性引物探针组合,所述引物探针组合包括针对rs16960228、rs1801253、rs12750834、rs776746基因SNP的特异性引物探针组合。本发明还提供了包括了所述引物组合的试剂盒及其应用。本发明的有益效果在于：本发明提供了一种同时检测rs16960228、rs1801253、rs12750834、rs776746四个基因多态性,并能准确区分杂合子与纯合子的方法,并提供了一种用于同时指导美托洛尔、氢氯噻嗪、缬沙坦、氨氯地平的个体化用药相关基因SNP的分型试剂盒及应用,本发明检测耗时短,操作简便,结果精确可靠,适合临床推广应用。(The invention relates to a specific primer probe combination for guiding SNP (Single nucleotide polymorphism) of related genes for personalized medicine, which comprises specific primer probe combinations aiming at SNP of rs16960228, rs1801253, rs12750834 and rs776746 genes. The invention also provides a kit comprising the primer combination and application thereof. The invention has the beneficial effects that: the invention provides a method for simultaneously detecting four gene polymorphisms of rs16960228, rs1801253, rs12750834 and rs776746, and accurately distinguishing heterozygotes and homozygotes, and provides a typing kit for simultaneously guiding related gene SNPs of individualized medication of metoprolol, hydrochlorothiazide, valsartan and amlodipine and application thereof.)

1. A specific primer probe combination for the individual medication related gene polymorphism is characterized by comprising specific oligonucleotide primers aiming at the SNP of rs16960228, rs1801253, rs12750834 and rs776746 genes and a specific fluorescence labeling MGB probe, and the specific primer probe combination comprises the following components:

the rs16960228 primer probe sequence is as follows:

upstream primer 5 '-3': AGCTGTGGGCTGGATTCTGT

Downstream primer 5 '-3': ATGCTGGTCACTCTCCTCTTTCTG

Probe one 5 '-3': TAAGGTCACACATAAAG

Probe two 5 '-3': TAAGGTCACACGTAAAG

The rs1801253 primer probe sequence is as follows:

upstream primer 5 '-3': AGCCCCGACTTCCGCA

Downstream primer 5 '-3': GTCTCCGTGGGTCGCGT

Probe one 5 '-3': CCTTCCAGGGACTG

Probe two 5 '-3': CCTTCCAGCGACTG

The probe sequence of the rs12750834 primer is as follows:

upstream primer 5 '-3': GAATGCCTCCCAAGATTGATG

Downstream primer 5 '-3': CCTTAGAACACCAAAGCAGGCTTA

Probe one 5 '-3': CTGTAAGTGCCCTCAG

Probe two 5 '-3': CTGTAAGTGCCCCCAGA

The probe sequence of the rs776746 primer is:

upstream primer 5 '-3': GATGAAGGGTAATGTGGTCCAAA

Downstream primer 5 '-3': TGCTCTACTGTCATTTCTAACCATAATCT

Probe one 5 '-3': AGGGAAGAGATACTG

Probe two 5 '-3': AAGAGATATTGAAAGACA are provided.

2. Individual medicine application phaseThe kit for detecting gene polymorphism, characterized in that it comprises PCR mixed liquor of the specific primer probe combination of claim 1, wherein the PCR mixed liquor is rs16960228, rs1801253, rs12750834 and rs776746 PCR mixed liquor, and the PCR mixed liquor further comprises Tris-HCl, KCl and MgCl₂、dNTPs。

3. The kit for detecting polymorphism of gene related to personalized medicine according to claim 2, wherein the concentration of the primer pair in the PCR mixture is 0.20. mu.M; the concentration of the probe pair was 0.12. mu.M.

4. The kit for detecting polymorphism of genes related to individualized drugs according to claim 2, further comprising DNA polymerase, a positive quality control product A, a positive quality control product B, a positive quality control product C and a positive quality control product D, wherein the positive quality control product A is a plasmid mixed solution containing 2 rs16960228 allelic loci, and the positive quality control product B is a plasmid mixed solution containing 2 rs1801253 allelic loci; the positive quality control substance C is plasmid mixed liquor containing 2 rs12750834 allelic loci, and the positive quality control substance D is plasmid mixed liquor containing 2 rs776746 allelic loci.

5. The kit for detecting polymorphism of gene related to personalized medicine according to claim 4, wherein the kit is used in PCR amplification reaction system as follows: 36.5. mu.l of the PCR mixture, 0.5. mu.l of DNA polymerase, and 3. mu.l of the template DNA in a total volume of 40. mu.l.

6. The kit for detecting polymorphism of gene related to personalized medicine according to claim 5, wherein the amplification program of the kit for PCR amplification reaction system is: 95 ℃, 5 minutes, 1 cycle; at 95 deg.C, 45 seconds, 58 deg.C, 1 minute, 40 cycles; 38 ℃ for 5 seconds, 1 cycle.

7. The use of the kit for detecting polymorphism of gene related to individualized medication according to any one of claims 2 to 6, wherein the kit is used for simultaneously instructing the individualized medication of metoprolol, hydrochlorothiazide, valsartan and amlodipine.

Technical Field

The invention relates to the technical field of gene diagnosis, in particular to a primer and probe combination for gene polymorphism detection for blood pressure lowering drug personalized medication guidance, a kit and application thereof.

Background

Hypertension is taken as the most important risk factor of cardiovascular and cerebrovascular diseases, the prevalence situation is serious, the disability fatality rate of main complications such as stroke, myocardial infarction, heart failure, chronic kidney diseases and the like is high, medical and social resources are seriously consumed, heavy burden is caused to families and society, and the hypertension becomes an important public health problem in China.

Genetic background is an important factor that varies greatly between individuals in response to drugs. The drug enters the human body and plays a role through the processes of absorption, transportation, metabolism, effect and elimination, and patients carrying different genotypes may show different degrees of reaction in the drug action process, which is expressed as individual difference of drug reactivity.

The hypertension rational medication guide (2 nd edition) indicates that pharmacogenomics has become an important tool for guiding clinical personalized medication and evaluating the risk of serious adverse drug reactions. Through detecting drug metabolizing enzyme and drug target gene, the clinician can be guided to select proper antihypertensive drug and administration dosage for specific patients, and the effectiveness and safety of treatment of the antihypertensive drug are improved.

At present, the types of the blood pressure lowering drugs are various, and most of the clinicians make treatment plans to experimentally select the types and the dosages of the drugs according to the ages, the weights, the hypertension degrees, the complications and the like of patients. The application of pharmacogenomics will provide an effective reference for drug administration at the molecular level and at the microscopic level. The accurate medication guide is to take the individual genotype into consideration, customize the medication scheme, avoid improper medication and really realize accurate personalized medication.

The protein kinase C-alpha (PRKCA) gene is a member of serine-threonine specific protein kinase C family, participates in various biological events such as cell growth, differentiation and apoptosis, relates to different cell signal pathways, such as vascular smooth muscle contraction and vascular endothelial growth factor pathways, and is also a regulatory factor of myocardial contraction. The rs16960228 SNP is an important prediction factor of the antihypertensive effect of hydrochlorothiazide, and the antihypertensive effect of hydrochlorothiazide of an AA type hypertension patient is better than that of GG type genotype.

The beta 1-adrenergic receptor (ADRB 1) is a tissue receptor mediating the action of catecholamine, is a G-protein coupled receptor, participates in the regulation of human body on blood pressure, and the polymorphism of the gene is closely related to the curative effect and safety of hypertension and antihypertensive drugs. Arg389Gly (rs 1801253) is located at the carboxyl terminal in the cell, and influences the activity of ligand-mediated adenylate cyclase and the coupling function of G protein, thereby influencing the heart rate and the blood pressure. Research shows that Arg389 is a susceptible gene of hypertension, and people with the genotype of Arg389 homozygote are more likely to develop essential hypertension. Arg389 has 3-4 times stronger signal transduction ability than Gly389, longer activity and more sensitive response to agonists, so Arg389Arg genotype hypertensive patients may require higher doses of beta-blockers to achieve the same therapeutic effect as Arg389Gly or Gly389Gly genotype patients. Patients with the Arg389Arg genotype received twice the increased risk of death for low doses of beta-blockers compared to high doses, while Gly389 carriers had no significant difference in risk of death when receiving high or low doses of beta-blockers.

The renin gene (REN) G1051A may affect the enzymatic activity of renin, thus being associated with essential hypertension. The plasma renin activity of the G/G genotype is significantly higher than that of the A/A and G/A genotypes, so that the G/G genotype may cause relatively high renin activity to increase blood pressure, while the A/A genotype may cause relatively low renin activity to decrease blood pressure.

CYP3A5 gene (rs 776746) belongs to human cytochrome P450 enzyme system, and its polymorphism can make the medicine have different blood concentration in human body, thus affecting the therapeutic effect of lowering blood pressure. At present, research has proved that the mutant genotype CYP3A5 x 3 of CYP3A5 can generate shearing mutation, generate unstable protein, form mutant homozygote GG type, and the gene reduces the expression of CYP3A5 in liver and intestine, can cause the metabolism of calcium antagonist to slow down, leads to the increase of blood concentration in vivo, and further correspondingly increases the toxicity of the medicine. Amlodipine is one of the drugs that are mainly metabolized via CYP3a 5.

The gene detection technology is utilized to interpret the gene polymorphism of the hypertension patient, different treatment schemes are made according to the genetic diversity of individuals and genes related to drug treatment, and the realization of individuation of drug treatment has important clinical significance. The appropriate antihypertensive drug is selected according to the detection result, and the dosage of the patient is adjusted, so that the drug can exert the optimal curative effect, the toxic and side effects are prevented to the maximum extent, and the waste of drug resources can be avoided.

At present, the conventional gene polymorphism detection methods such as Restriction Fragment Length Polymorphism (RFLP), Denaturing High Performance Liquid Chromatography (DHPLC), direct sequencing and the like are mostly adopted for gene detection in the market. These conventional methods are either not qualitative, or time and labor consuming, expensive in equipment and consumables, and more importantly, they are difficult to detect multiple mutation sites of different genes simultaneously. So far, no kit for rapidly detecting the gene of the individual administration of the antihypertensive drug exists in the market, and in conclusion, a rapid, efficient and accurate gene detection kit for guiding the safe administration of the antihypertensive drug is developed, so that the kit has great clinical significance and social significance.

Disclosure of Invention

The invention provides a primer pair and a probe pair for simultaneously detecting PRKCA (rs 16960228), ADRB1 (rs 1801253), REN (rs 12750834) and CYP3A5 (rs 776746) locus gene polymorphisms, and the specific sequences are as follows:

rs16960228 site of PRKCA gene:

PRKCA-F 5’-3’：AGCTGTGGGCTGGATTCTGT

PRKCA-R 5’-3’：ATGCTGGTCACTCTCCTCTTTCTG

PRKCA-A 5’-3’：TAAGGTCACACATAAAG

PRKCA-G 5’-3’：TAAGGTCACACGTAAAG

rs1801253 site of ADRB1 gene:

ADRB1-F 5’-3’：AGCCCCGACTTCCGCA

ADRB1-R 5’-3’：GTCTCCGTGGGTCGCGT

ADRB1-G 5’-3’：CCTTCCAGGGACTG

ADRB1-C 5’-3’：CCTTCCAGCGACTG

REN gene rs12750834 site:

REN-F 5’-3’： GAATGCCTCCCAAGATTGATG

REN-R 5’-3’： CCTTAGAACACCAAAGCAGGCTTA

REN-T 5’-3’： CTGTAAGTGCCCTCAG

REN-C 5’-3’： CTGTAAGTGCCCCCAGA

site rs776746 of CYP3A5 gene:

CYP3A5-F 5’-3’： GATGAAGGGTAATGTGGTCCAAA

CYP3A5-R 5’-3’： TGCTCTACTGTCATTTCTAACCATAATCT

CYP3A5-C 5’-3’： AGGGAAGAGATACTG

CYP3A5-T 5’-3’： AAGAGATATTGAAAGACA。

the invention provides a kit for simultaneously detecting rs16960228, rs1801253, rs12750834 and rs776746 gene polymorphisms, which comprises the following components:

the concentration of the PCR mixed solution primer pair in the kit is 0.20 mu M; the concentration of the probe pair was 0.12. mu.M.

The kit adopts a multiple PCR-fluorescent probe method, and is used for a PCR amplification reaction system as follows: 36.5. mu.l of the PCR mixture, 0.5. mu.l of DNA polymerase, and 3. mu.l of template DNA in a total volume of 40. mu.l. The amplification procedure was: 95 ℃, 5 minutes, 1 cycle; at 95 deg.C, 45 seconds, 58 deg.C, 1 minute, 40 cycles; 38 ℃ for 5 seconds, 1 cycle.

The invention has the beneficial effects that: the kit adopts a multiple PCR-fluorescence probe method to realize nucleic acid amplification and detection in a totally closed reaction system, greatly reduces the detection time compared with a gold-labeled sequencing method and a molecular hybridization method, and has the advantages of simple and convenient operation, high sensitivity and high specificity. The kit simultaneously detects four gene polymorphisms of rs16960228 (G > A), rs1801253 (G > C), rs12750834 (G > A) and rs776746 (G > A), designs a wild allele as a control point, can detect definite heterozygous or homozygous genotype at one time, guides individualized medication of related genes, particularly the individualized medication of metoprolol, hydrochlorothiazide, valsartan and amlodipine, and ensures medication safety.

Drawings

FIG. 1 is a graph of the results of the positive control in the detection of locus rs16960228 of PRKCA gene in example 1.

FIG. 2 is a result chart of the negative control product of the rs16960228 locus detection of the PRKCA gene in example 1.

FIG. 3 is a result chart of the test sample for detecting locus rs16960228 of PRKCA gene in example 1.

FIG. 4 is a graph showing the result of detecting the positive control at site rs1801253 of ADRB1 gene in example 1.

FIG. 5 is a graph showing the result of detecting the negative control at the rs1801253 site of ADRB1 gene in example 1.

FIG. 6 is a diagram showing the result of detecting a sample to be tested at site rs1801253 of ADRB1 gene in example 1.

FIG. 7 is a graph showing the result of the positive control in the detection of the locus rs12750834 of the REN gene in example 1.

FIG. 8 is a graph showing the result of the negative control in the detection of the locus rs12750834 of the REN gene in example 1.

FIG. 9 is a graph showing the results of the test samples for the detection of the locus rs12750834 of the REN gene in example 1.

FIG. 10 is the result chart of the positive control product detected at site rs662799 of ApoA5 gene in example 1.

FIG. 11 is the result chart of the negative control product of the apoA5 gene rs662799 site detection in example 1.

FIG. 12 is a result chart of the detection of the locus rs662799 of the ApoA5 gene in example 1.

Detailed Description

The following examples are intended only to further illustrate the invention. It should be understood that these examples are only for illustrating the detection method and procedure of the present invention, and are not intended to limit the scope of the present invention. Any changes or modifications of the present invention by those skilled in the art are within the scope of the present invention.