CN101928757A - Detection of curative effect of antihypertensive drug - Google Patents

Abstract

The invention discloses a kit for detecting a curative effect of an individual antihypertensive drug. The kit comprises specific primer pairs of targeted locus CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene polymorphic locus for detecting the antihypertensive drug, specific fluorescent probe pairs, a fluorescent quantitive PCR conventional assembly and the like. The kit of the invention evaluates the curative effect of the individual antihypertensive drug by using the targeted locus CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene polymorphic locus for detecting the antihypertensive drug.

Description

Curative effect of antihypertensive drug detects

Technical field

The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects individual curative effect of antihypertensive drug, assess individual curative effect of antihypertensive drug by detecting antihypertensive drugs medication target site CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene polymorphism sites.

Background technology

Hypertension can be divided into primary and Secondary cases two big classes.In most patients, hypertensive etiology unknown is referred to as essential hypertension, account for that total hypertension more than 95%; In less than 5% patient, elevation of blood pressure is a kind of clinical manifestation of some disease, and the clearly and independently cause of disease is arranged itself, is called secondary hypertension.

Essential hypertension (primary hypertension) is to be the syndrome of main clinical manifestation with the elevation of blood pressure, abbreviates hypertension as.Hypertension is the important cause of disease and the Hazard Factor of the multiple heart, cerebrovascular disease, influences the important organ for example structure and the function of the heart, brain, kidney, finally causes the nonfunction of these organs, is still one of cardiovascular disorder main causes of death so far.

Treat hypertensive major objective and be and reduce neovascularity to greatest extent and die of illness and die and invalid total danger.Principle of reatment comprises: first Primary Care, back pharmacological agent; Primary Care comprises the mode of making the life better, and eliminates to be unfavorable for psychology and healthy behavior and custom, reduces the initiation potential of hypertension and other cardiovascular diseasess.The current medicine that is used for step-down is mainly following 6 classes, i.e. hydragog(ue), beta-blockers, angiotensin converting enzyme inhibitor (ACEI), Angiotensin II body antagonist, calcium antagonist and alpha blocker.

CYP3A4, the 5th, important I phase metabolic enzyme mainly takes off reactions such as hydrocarbon, C-hydroxylation and comes metabolic drug by C-or N-.Participate in about 50% drug metabolism.Hypertension therapeutic common drug calcium channel blocker (amlodipine, Odizem, felodipine, lercanidipine, nifedipine, nicardipine, nisoldipine, nitrendipine, verapamil), beta-blockers (carvedilol), renin-angiotensin system Depressant (losartan, irbesartan, eplerenone) CYP3A4,5 have all participated in metabolic process, and what have is in main metabolic pathway, and this moment, the height of enzymic activity was very big to medicine effect and Influence on security.Therefore, CYP3A4, the enzyme activity change that 5 gene pleiomorphisms cause will reflect the medication effect of antihypertensive drug to a great extent, CYP3A4, the somatotype of 5 genes detect the important realistic meaning that the medication guide for clinical antihypertensive drug has.

CYP2D6 is important I phase metabolic enzyme, participates in the metabolism of about 25% clinical commonly used drug, comprises beta-blockers, antidepressant drug, antipsychotics and chemotherapeutics etc.Because the CYP2D6 gene exists genetic polymorphism to cause between the individuality CYP2D6 enzymic activity variant, the polymorphism that medicament metabolism ability occurs, be difficult to obtain optimum blood medicine concentration when causing patient's medication, cross as Plasma Concentration and lowly can't effectively bring into play curative effect of medication or toxic side effect appears in excessive concentration.Hypertension therapeutic common drug calcium channel blocker (verapamil), beta-blockers (H-56/28, Proprasylyte, carvedilol metoprolol, bufuralol, nebivolol), CYP2D6 has participated in metabolic process, and for some drugs 2D6 is the main metabolic enzyme, and this moment, the height of enzymic activity was very big to medicine effect and Influence on security.Therefore, the enzyme activity change that the CYP2D6 gene pleiomorphism causes will reflect the medication effect of antihypertensive drug to a great extent, and the somatotype of CYP2D6 gene detects the important realistic meaning that the medication guide for clinical antihypertensive drug also has.

CYP2C9 is important I phase metabolic enzyme, hypertension therapeutic common drug calcium channel blocker (Odizem), beta-blockers (carvedilol), and renin-angiotensin system Depressant (losartan, irbesartan) CYP2C9 has participated in metabolic process, and for some drugs 2C9 is the main metabolic enzyme, and this moment, the height of enzymic activity was very big to medicine effect and Influence on security.Therefore, the enzyme activity change that the CYP2C9 gene pleiomorphism causes will reflect the medication effect of antihypertensive drug to a great extent, and the somatotype of CYP2C9 gene detects the important realistic meaning that the medication guide for clinical antihypertensive drug also has.

CYP2C19 is important I phase metabolic enzyme, hypertension therapeutic common drug beta-blockers (Proprasylyte), and CYP2C19 participates in metabolic process, and the height of this enzymic activity is very big to the drug effect and the Influence on security of Proprasylyte.Therefore, the enzyme activity change that the CYP2C19 gene pleiomorphism causes will react the medication effect of this medicine to a great extent, and the somatotype of CYP2C19 gene detects the important realistic meaning that the medication guide for clinical antihypertensive drug also has.

Summary of the invention

Can be used to assess on the basis of individual curative effect of antihypertensive drug based on antihypertensive drugs medication target site CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene pleiomorphism, the invention provides a kind of test kit that detects individual curative effect of antihypertensive drug.

This test kit comprises:

The Auele Specific Primer that detects antihypertensive drugs medication target site CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene polymorphism sites is right to reaching the specificity fluorescent probe;

The quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, reaction buffer, deionized water etc.).

Specificity fluorescent probe described in this test kit is to finishing design easily, and the specificity fluorescent probe synthesizes the probe synthetic technology of available routine.

The component and the content of test kit of the present invention comprise:

10X quantitative fluorescent PCR reaction buffer 4 μ l,

25mM dNTP mixed solution 0.4 μ l,

25mM MgCl2 solution 2.4 μ l,

5units/ μ l Taq archaeal dna polymerase 0.1 μ l,

20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,

10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,

Deionized water 21.2 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

Use the quantitative fluorescent PCR suit in the detection kit, carry out 4 independently quantitative fluorescent PCR reactions, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l ₂The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.

React on the pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on quantitative real time PCR Instrument.

Step 4: gene type assay

According to the gene type diagram that the test kit working instructions indicate gene type assay is carried out in the SNP site.

The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence fluorescence probe signals.

Claims (2)

1. test kit that detects individual curative effect of antihypertensive drug is characterized in that: comprise the Auele Specific Primer that detects antihypertensive drugs medication target site CYP3A4 gene, CYP2D6 gene, CYP2C9 gene and CYP2C19 gene polymorphism sites to and the specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. test kit according to claim 1 is characterized in that: the component and the content of test kit comprise:

10X quantitative fluorescent PCR reaction buffer 4 μ l,

25mM dNTP mixed solution 0.4 μ l,

25mM MgCl2 solution 2.4 μ l,

5units/ μ l Taq archaeal dna polymerase 0.1 μ l,

20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,

10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,

Deionized water 21.2 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.