CN110257506A - Polymorphism detection kit and method for hypertension accurate medication related gene - Google Patents
Polymorphism detection kit and method for hypertension accurate medication related gene Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, in particular to a kit and a method for detecting polymorphism of genes related to accurate administration of hypertension. The kit comprises a reaction solution I, a reaction solution II and a reaction solution III. The reaction solution I comprises a PCR buffer system and enzyme, the reaction solution II comprises ACE, CYP2D6 and ADRB1 gene detection primers and corresponding fluorescent probes, and the reaction solution III comprises NPPA, AGTR1, CYP3A5 and CYP2C9 gene detection primers and corresponding fluorescent probes. The kit and the method can detect the polymorphism of the gene related to clinical 5-class antihypertensive requirements quickly, sensitively and characteristically.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of polymorphism of the accurate medication related gene of hypertension
Detection kit and method.
Background technique
Hyperpiesia is one of most common chronic disease, is to lead to the cardiovascular diseases such as coronary heart disease, cerebral apoplexy
The significant risk factor, a large amount of research has shown that hyperpiesia is mainly by the common shadow of inherent cause and environmental factor
It rings.Current clinically used antihypertensive drugs includes diuretics, beta-2 adrenoceptor retarding agent, calcium antagonist, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, blood
Angiotensin II receptor antagonist and alpha adrenergic receptor retarding agent, however, the drug in clinical treatment hypertension is anti-in individual
It answers difference very universal, is primarily due to drug metabolic enzyme relevant to drug and hereditary variation has occurred in receptor.
CYP2D6 only accounts for 1-2%, but up to more than 80 kinds of drug through its catalysis metabolism, Different Individual in the total CYP of liver
The active maximum of CYP2D6 can differ 1000 times.The most common polymorphism is CYP2D6*10 in Chinese population, about 51.6%
Chinese carry this hereditary variation.The polymorphism causes the enzymatic activity of CYP2D6 to reduce, and extremely unstable, to influence drug
Metabolism.
Beta-2 adrenoceptor belongs to g protein coupled receptor superfamily, is encoded by ADRB1 gene, and there are polymorphic for the gene
Property.Research shows that ADRB1 gene G1165C polymorphism is related to condition of medicine treatment for hypertension, metoprolol, Carvedilol etc. are used
After treatment, C1165 homozygotic individual effective percentage is higher.
Angiotensin-Converting is the key enzyme of renin-angiotensin system, is encoded by ACE gene, and the gene is fixed
Positioned at No. 17 2nd area of chromosome long arm, 3 bands, the long 21kb of sequence.There are a variety of genetic polymorphisms for ACE gene, wherein the 16th introne
Present in 287bp Alu Insert Fragment (I) and deletion fragment (D) polymorphism it is related to condition of medicine treatment for hypertension, study table
The homozygous individual of bright DD is significant in efficacy using benazepil and fosinopril, and the homozygous individual of II is reached using enalapril and miaow
Puli's is significant in efficacy, and ID heterozygous individual uses hypertensin inhibitor class drug therapy, effectively.
The listing of novel diuretics indapamide (Indapamide, indapmide), makes diuretics in the treatment of high blood pressure
There is new raising in position again, and its feature is that common dose is showed only as slight diuresis, is mainly shown as that blood vessel dilatation is made
With the metabolic disorder side effect of traditional diuretics is not present 80% or so in decompression effective percentage, in wide clinical application.
Atrial natriuretic peptide precursor A (natriure ticpeptide precursor A, NPPA) is a variety of disease inheritance susceptibles of research
Property " candidate gene ", be located at human chromosome 1p36, encode the precursor of ANP.ANP controls extracellular fluid volume as a kind of diuretics
With the balance of electrolyte, and low blood pressure can be caused by improving ANP level.Research shows that ANP (and NPPA gene) and a variety of heart and brain
Vascular diseases are related, such as cerebral apoplexy, heart failure, left atrial hypertrophy and hypertension.NPPA G664A is to be located at 1 extra of gene
Single nucleotide mutation (G/A) on aobvious son, makes the 7th amino acids of ANP precursor protein become methionine, and ANP activity is caused to drop
It is low.
Cytochrome pathways (CYP2C9) are a kind of important drug metabolic enzymes in human liver, account for about mankind liver
The 20% of middle CYP enzyme total amount.The distribution of CYP2C9 allele has apparent crowd, race and areal variation, Chinese population
In, Primary mutations type gene is CYP2C9*3, is to cause CYP2C9 polypeptide in the 1075th generation A > C mutation of the 7th exon
The 359th isoleucine mutation on chain is at leucine (Ile359 > Leu359), gene frequency 1.0-3.5%.Lip river
The husky smooth class such as Sha Tan, Irbesartan is common blood-pressure drug, is a kind of effective angiotensin receptor antagonist, it leads
It to be metabolized by CYP2C9, studies have shown that sartans are mainly metabolized by CYP2C9 in vivo, the mutation of polymorphism CYP2C9*3
Enzymatic activity is caused to be remarkably decreased, toxicity increases, and curative effect reduces.Clinical research shows that CYP2C9*3 mutation can lead to Losartan drop
Press decreased effectiveness；CYP2C9*3 is mutated the blood concentration that can increase Valsartan and Irbesartan, leads to Valsartan and Irbesartan
Antihypertensive effect enhancing.Therefore, when hypertensive patient selects sartans, it should first carry out genetic test, root to CYP2C9
Sartans are selected according to the genotype of CYP2C9*3.
Angiotensin II receptor antagonist (Angiotensin II receptor antagonists) is a kind of effect
In the drug of renin-angiotensin system, it is mainly used in treatment hypertension, diabetic nephropathy and congestive heart failure.I
Type angiotensin-ii receptor (Angiotensin II receptor type 1, AGTR1) is Angiotensin II
The receptor of (angiotensin, Ang II), is distributed mainly in vascular smooth muscle cells, mediates the most biologies of Ang II
Effect.It is the component part of renin-angiotensin system (rennin-angiotensin system, RAS), to height
The generation of blood pressure and drug therapy are of great significance.Human AGT R1 gene is located at 3q21-25, contains only 1 exon, without in
Containing subregion.The study found that in AGTR1 gene pleiomorphism, the frequency highest of T573C, A1166C and A1062G replacement, A1166C
More significant blood pressure response is presented when using Losartan and Candesartan in the patient of genotype.Hypertensive patient is to vasotonia
The drug susceptibility of plain II receptor antagonist (such as Losartan, irbesartan, Telmisartan) is as follows: A1166A Patient drug is quick
It is perceptual normal, it is proposed that use routine dose；A1166C Patient drug's sensibility increases, it is proposed that reduces dosage；C1166C patient's medicine
Object sensibility increases, it is proposed that reduces dosage.
Calcium ion antagonist is the chemicals to be reduced blood pressure by retardance calcium channel.Its property of can choose inhibits Ca2+
Enter into the cell through the calcium channel on cell membrane, by expansion blood vessel and negative inotropic action, relaxation vascular smooth muscle reduces tip
Vascular resistence, to play antihypertensive effect.Cytochrome P450 (Cytochrome P450, CYP) is a major class drug metabolism
Enzyme participates in the metabolism of many drugs, xenobiotic substance and endogenous material.CYP3A5 is mainly in adult liver and small intestine
Expression, expression have apparent polymorphism.The gene mutation of CYP3A5 is the main reason of generation enzyme activity sex differernce, wherein
Mutation of the CYP3A5*3 at intron3 (6959A > G) causes variable sheer, produces unstable protein, so that prominent
It is denaturalized homozygotic individual, i.e., its people for carrying gene C YP3A5*3/*3 does not express CYP3A5.Studies have shown that calcium antagonist class medicine
Object is affected by CYP3A5*3 gene pleiomorphism, and containing mutated-genotype patient, poisonous side effect of medicine increases.
There are many gene pleiomorphism detecting method, it is most common include PCR sequencing PCR, DHPLC, PCR-SSCP/RFLP, ARMS,
Taqman PCR, ME-PCR, genetic chip etc..These methods are not only complicated for operation, and detection cycle is long, and influence testing result
There are many factor, are difficult to control, it is difficult to meet the requirement of clinical reagent detection, only carry out at present in scientific research field.
Summary of the invention
The purpose of the present invention is be in view of the deficiencies of the prior art, provide it is a kind of quickly, precisely systematically detect high blood
Press the kit and method of medication related gene polymorphism.
To solve the problem above-mentioned, the invention adopts the following technical scheme:
A kind of polymorphic detection kit of the accurate medication related gene of hypertension, kit includes reaction solution I, reaction solution
II, reaction solution III, positive reference substance, negative controls.The reaction solution I includes PCR buffer system and enzyme, reaction solution II
Including ACE, CYP2D6, ADRB1 genetic test primer and corresponding fluorescence probe, reaction solution III include NPPA, AGTR1,
CYP3A5, CYP2C9 genetic test primer and corresponding fluorescence probe, positive reference substance ACE, CYP2D6, ADRB1, NPPA,
The heterozygous plasmid of seven genes of AGTR1, CYP3A5, CYP2C9, negative controls are the Tris salt without target target gene.
The reaction solution I includes 10*taq buffer I, 5*taq buffer II, Hotstart Taq enzyme, UDG
Enzyme, dNTP, dUTP, reinforcing agent；The 10*taq buffer I includes 200mM Tris-HCl (pH8.4), 200mM KCl,
100mM(NH4)2SO4, 15mM MgCl2；The 5*taq buffer II includes 20mM Mg SO4, the reinforcing agent is packet
Glycine betaine containing 0.5M, 1% (V/V) formamide, 3% (V/V) glycerol and 0.2mg/mlBSA mixture.
Reaction solution II includes that ACE, CYP2D6, ADRB1 detection primer and correspondent probe are as follows:
ACE F1:TTTCTCTAGACCTGCTGCCTATAC
ACE R1:AGCTCAGAGAATTTCAGAGCTG
ACE P:CTGCTGCCTATACAGTCACTTTTATGTGG, 5 ends mark ROX, and 3 ends mark BHQ2.
D6 F1:CCTCCCTCACCTGGTCGAAGC
D6 R1:TTCCTGCTCCTGGTGGACCTG
D6 P:CTGCACGCTACTCACCAGGCCC, 5 end flag F AM, 3 ends mark BHQ1.
B1 F1:GCCTTCAACCCCATCATCT
B1 R1:GTCGTCGTCGTCGTCCGA
B1 P:TTCCAGCGACTGCTCTGCT, 5 ends mark HEX, and 3 ends mark BHQ1.
Reaction solution II includes following detection primer ratio are as follows: ACE F1:ACE R1=2pmol:20pmol；D6 F1:D6
R1=30pmol:3pmol；B1 F1:B1 R1=2pmol:20pmol；The additive amount of above-mentioned three detection probes is 5pmol.
Reaction solution III includes that NPPA, AGTR1, CYP3A5, CYP2C9 detection primer and correspondent probe are as follows:
R1 F1:TGAGTGACATGTTCGAAACC
R1 R1:CAGCCGTCATCTGTCTAATGC
R1 P:CAAATGAGCATTAGCTACTTTTCAGA, 5 ends mark ROX, and 3 ends mark BHQ2.
PA F1:GACACAAATGCAGCAGAGAC
PA R1:AGGATGGGCACACTCATAC
PA P:CTGTTATCTTCAGTACTGCAAAGAGAAC, 5 end flag F AM, 3 ends mark BHQ1.
A5 F1:GAGAGTGGCATAGGAGATAC
A5 R1:CACAGCAAGAGTCTCACACAG
A5 P:TGTCTTTCAATATCTCTTCCCTGTTTG, 5 ends mark CY5, and 3 ends mark BHQ3.
C9 F1:ATCAGCTAAAGTCCAGGAA
C9 R1:GAAACAAACTTACCTTGGGAAT
C9 P:CGAGGTCCAGAGATACATTGACC, 5 ends mark HEX, and 3 ends mark BHQ1.
Reaction solution III includes following detection primer ratio are as follows: R1 F1:R1 R1=4pmol:40pmol；PA F1:PA R1
=3pmol:30pmol；A5 F1:A5 R1=2pmol:20pmol；C9 F1:C9 R1=40pmol:4pmol, above-mentioned four inspections
The additive amount of probing needle is 5pmol.
A kind of pleiomorphism detecting method of the accurate medication related gene of hypertension, detecting step are as follows:
1) augmentation detection reagent I is prepared according to 19ul reaction solution I:2ul reaction solution II；It is anti-according to 19ul reaction solution I:2ul
Liquid III is answered to prepare augmentation detection reagent II.
2) each 4ul of sample to be examined DNA is drawn, is added separately in augmentation detection reagent I and augmentation detection reagent II, respectively
It mixes.
3) by added with the augmentation detection reagent I of DNA and augmentation detection reagent II reaction tube be placed in real-time fluorescence PCR instrument into
The detection of row PCR amplification.
4) real-time fluorescent PCR amplification program is provided that
A kind of pleiomorphism detecting method of the accurate medication related gene of hypertension, the result interpretation of 7 kinds of genotype:
Compared with prior art, the present invention has following features:
1) present invention is provided with kit and the side of a kind of detection five major class hypertension drug related gene polymorphisms detection
Method can effectively instruct the clinical application of mainstream drug for hypertension.Existing product more on the market covers more complete.
2) present invention carries out genetic polymorphism detection using single probe combination melting curve method, while multiple using two pipes
PCR reaction to related 7 genetic polymorphism detections of hypertension drug, have quickly, high sensitivity, high specificity, method it is simple,
The advantages such as testing result is accurate.
3) kit can carry out polymorphic detection to 10pg genomic DNA.
Detailed description of the invention
Fig. 1 is CYP2D6*10 loci polymorphism testing result.
Fig. 2 is ADRB1 (1165G > C) loci polymorphism testing result.
Fig. 3 is ACE (I/D) loci polymorphism testing result.
Fig. 4 is NPPA (664G > A) loci polymorphism testing result.
Fig. 5 is CYP2C9*3 loci polymorphism testing result.
Fig. 6 is AGTR1 (1166A > C) loci polymorphism testing result.
Fig. 7 is CYP3A5*3 loci polymorphism testing result.
Specific embodiment
The present invention will be further described with reference to the accompanying drawing.
Case study on implementation 1: design of the hyperpiesis individual medicine because of polymorphic detection kit primer and probe
According to ACE (rs4646994), CYP2D6 (rs1065852), ADRB1 (rs1801253), NPPA (rs5065),
Seven AGTR1 (rs5186), CYP3A5 (rs776746), CYP2C9 (rs56165452) gene-correlation detection site design detections
Probe and primer, correlated series are as follows:
ACE F1:TTTCTCTAGACCTGCTGCCTATAC
ACE R1:AGCTCAGAGAATTTCAGAGCTG
ACE P:CTGCTGCCTATACAGTCACTTTTATGTGG, 5 ends mark ROX, and 3 ends mark BHQ2.
D6 F1:CCTCCCTCACCTGGTCGAAGC
D6 R1:TTCCTGCTCCTGGTGGACCTG
D6 P:CTGCACGCTACTCACCAGGCCC, 5 end flag F AM, 3 ends mark BHQ1.
B1 F1:GCCTTCAACCCCATCATCT
B1 R1:GTCGTCGTCGTCGTCCGA
B1 P:TTCCAGCGACTGCTCTGCT, 5 ends mark HEX, and 3 ends mark BHQ1.
R1 F1:TGAGTGACATGTTCGAAACC
R1 R1:CAGCCGTCATCTGTCTAATGC
R1 P:CAAATGAGCATTAGCTACTTTTCAGA, 5 ends mark ROX, and 3 ends mark BHQ2.
PA F1:GACACAAATGCAGCAGAGAC
PA R1:AGGATGGGCACACTCATAC
PA P:CTGTTATCTTCAGTACTGCAAAGAGAAC, 5 end flag F AM, 3 ends mark BHQ1.
A5 F1:GAGAGTGGCATAGGAGATAC
A5 R1:CACAGCAAGAGTCTCACACAG
A5 P:TGTCTTTCAATATCTCTTCCCTGTTTG, 5 ends mark CY5, and 3 ends mark BHQ3.
C9 F1:ATCAGCTAAAGTCCAGGAA
C9 R1:GAAACAAACTTACCTTGGGAAT
C9 P:CGAGGTCCAGAGATACATTGACC, 5 ends mark HEX, and 3 ends mark BHQ1.Case study on implementation 2: hypertension
The preparation of personalized medicine related gene polymorphism detection kit
Reaction solution I is the UDG enzyme comprising 10*taq buffer I, 5*taq buffer II, Hotstart Taq enzyme,
DNTP, dUTP, the mixture of reinforcing agent provide the buffer system of an efficient amplification for real-time fluorescence PCR.Wherein, described
10*taq buffer I includes 200mM Tris-HCl (pH8.4), 200mM KCl, 100mM (NH4)2SO4, 15mM MgCl2；
The 5*taq buffer II includes 20mM Mg SO4, the reinforcing agent is to include 0.5M glycine betaine, 1% (V/V) first
The mixture of amide, 3% (V/V) glycerol and 0.2mg/mlBSA.
Reaction solution II contains ACE, CYP2D6, ADRB1 genetic test primer and corresponding fluorescence probe, wherein CE F1:ACE R1
=2pmol:20pmol；D6 F1:D6 R1=30pmol:3pmol；B1 F1:B1 R1=2pmol:20pmol；Three detections are visited
The additive amount of needle is 5pmol.
Reaction solution III contains NPPA, AGTR1, CYP3A5, CYP2C9 genetic test primer and fluorescence associated probe, wherein R1
F1:R1 R1=4pmol:40pmol；PA F1:PA R1=3pmol:30pmol；A5 F1:A5 R1=2pmol:20pmol；C9
F1:C9 R1=40pmol:4pmol, the additive amount of four detection probes are 5pmol.
Positive reference substance is the heterozygous of seven genes of ACE, CYP2D6, ADRB1, NPPA, AGTR1, CYP3A5, CYP2C9
Plasmid.
Negative controls are the Tris salt without target target gene.
Case study on implementation 3: hyperpiesis individual medicine is because of polymorphic detection kit application method
1) genomic DNA in sample to be tested is extracted.
2) augmentation detection reagent I is prepared according to 19ul reaction solution I:2ul reaction solution II；It is anti-according to 19ul reaction solution I:2ul
Liquid III is answered to prepare augmentation detection reagent II.
3) sample to be examined DNA, positive reference substance and each 4ul of negative controls are drawn, augmentation detection reagent I is added separately to
In augmentation detection reagent II, mix.
4) by added with the augmentation detection reagent I of DNA and augmentation detection reagent II reaction tube be placed in real-time fluorescence PCR instrument into
The detection of row PCR amplification.
Wherein amplification program is as follows:
As a result interpretation standard:
100 clinical blood samples are collected, genomic DNA is extracted using the blood extracts kit of commercialization, then adopts
Related gene polymorphism detection is carried out with kit of the invention, while being compared using sanger PCR sequencing PCR, it is all consistent.
Sequence table
<110>thousand base Biotechnology Co., Ltd of Hangzhou
Hangzhou Bo Xin Bioisystech Co., Ltd
<120>the polymorphic detection kit and method of a kind of accurate medication related gene of hypertension
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<400> 18
Thr Gly Thr Cys Thr Thr Thr Cys Ala Ala Thr Ala Thr Cys Thr Cys
1 5 10 15
Thr Thr Cys Cys Cys Thr Gly Thr Thr Thr Gly
20 25
<210> 19
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 19
Ala Thr Cys Ala Gly Cys Thr Ala Ala Ala Gly Thr Cys Cys Ala Gly
1 5 10 15
Gly Ala Ala
<210> 20
<211> 22
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 20
Gly Ala Ala Ala Cys Ala Ala Ala Cys Thr Thr Ala Cys Cys Thr Thr
1 5 10 15
Gly Gly Gly Ala Ala Thr
20
<210> 21
<211> 23
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 21
Cys Gly Ala Gly Gly Thr Cys Cys Ala Gly Ala Gly Ala Thr Ala Cys
1 5 10 15
Ala Thr Thr Gly Ala Cys Cys
20
Claims (8)
1. a kind of polymorphic detection kit of the accurate medication related gene of hypertension, which is characterized in that including reaction solution I, instead
Answer liquid II, reaction solution III, positive reference substance, negative controls；Wherein, the reaction solution I includes PCR buffer system, enhancing
Agent and enzyme, the reaction solution II include ACE, CYP2D6, ADRB1 genetic test primer and corresponding fluorescence probe, and described is anti-
Answering liquid III includes NPPA, AGTR1, CYP3A5, CYP2C9 genetic test primer and corresponding fluorescence probe, the positive control
Product are the heterozygous plasmid of seven genes of ACE, CYP2D6, ADRB1, NPPA, AGTR1, CYP3A5, CYP2C9, the feminine gender
Reference substance is the Tris salt without target target gene.
2. a kind of polymorphic detection kit of the accurate medication related gene of hypertension as described in claim 1, feature exist
In, the reaction solution I include 10*taq buffer I, 5*taq buffer II, Hotstart Taq enzyme, UDG enzyme,
DNTP, dUTP, reinforcing agent；The 10*taq buffer I includes 200mM Tris-HCl (pH8.4), 200mM KCl,
100mM(NH4)2SO4, 15mM MgCl2；The 5*taq buffer II includes 20mM Mg SO4, the reinforcing agent is packet
Glycine betaine containing 0.5M, 1% (V/V) formamide, 3% (V/V) glycerol and 0.2mg/mlBSA mixture.
3. a kind of polymorphic detection kit of the accurate medication related gene of hypertension as described in claim 1, feature exist
In the reaction solution II includes following detection primer and fluorescence probe:
ACE F1:TTTCTCTAGACCTGCTGCCTATAC
ACE R1:AGCTCAGAGAATTTCAGAGCTG
ACE P:CTGCTGCCTATACAGTCACTTTTATGTGG, 5 ends mark ROX, and 3 ends mark BHQ2；
D6 F1:CCTCCCTCACCTGGTCGAAGC
D6 R1:TTCCTGCTCCTGGTGGACCTG
D6 P:CTGCACGCTACTCACCAGGCCC, 5 end flag F AM, 3 ends mark BHQ1；
B1 F1:GCCTTCAACCCCATCATCT
B1 R1:GTCGTCGTCGTCGTCCGA
B1 P:TTCCAGCGACTGCTCTGCT, 5 ends mark HEX, and 3 ends mark BHQ1.
4. a kind of polymorphic detection kit of the accurate medication related gene of hypertension as claimed in claim 3, feature exist
In the ratio of detection primer in the reaction solution II are as follows: ACE F1:ACE R1=2pmol:20pmol；D6 F1:D6 R1=
30pmol:3pmol；B1 F1:B1 R1=2pmol:20pmol；The additive amount of three detection probes is 5pmol.
5. a kind of polymorphic detection kit of the accurate medication related gene of hypertension as described in claim 1, feature exist
In the reaction solution III includes following detection primer and fluorescence probe:
R1 F1:TGAGTGACATGTTCGAAACC
R1 R1:CAGCCGTCATCTGTCTAATGC
R1 P:CAAATGAGCATTAGCTACTTTTCAGA, 5 ends mark ROX, and 3 ends mark BHQ2；
PA F1:GACACAAATGCAGCAGAGAC
PA R1:AGGATGGGCACACTCATAC
PA P:CTGTTATCTTCAGTACTGCAAAGAGAAC, 5 end flag F AM, 3 ends mark BHQ1；
A5 F1:GAGAGTGGCATAGGAGATAC
A5 R1:CACAGCAAGAGTCTCACACAG
A5 P:TGTCTTTCAATATCTCTTCCCTGTTTG, 5 ends mark CY5, and 3 ends mark BHQ3；
C9 F1:ATCAGCTAAAGTCCAGGAA
C9 R1:GAAACAAACTTACCTTGGGAAT
C9 P:CGAGGTCCAGAGATACATTGACC, 5 ends mark HEX, and 3 ends mark BHQ1.
6. a kind of polymorphic detection kit of the accurate medication related gene of hypertension as claimed in claim 5, feature exist
In the ratio of detection primer in the reaction solution III are as follows: R1 F1:R1 R1=4pmol:40pmol；PA F1:PA R1=
3pmol:30pmol；A5 F1:A5 R1=2pmol:20pmol；C9 F1:C9 R1=40pmol:4pmol, four detection probes
Additive amount be 5pmol.
7. a kind of pleiomorphism detecting method of the accurate medication related gene of hypertension, which is characterized in that each gene pleiomorphism is adopted
It carries out Taqman melting curve method with single probe to be detected, specific detecting step is as follows:
1) augmentation detection reagent I is prepared according to 19ul reaction solution I:2ul reaction solution II；According to 19ul reaction solution I:2ul reaction solution
III prepares augmentation detection reagent II；
2) each 4ul of sample to be examined DNA is drawn, is added separately in augmentation detection reagent I and augmentation detection reagent II, mixes respectively
It is even；
3) it will be placed in real-time fluorescence PCR instrument and carry out with augmentation detection reagent II reaction tube added with the augmentation detection reagent I of DNA
PCR amplification detection；
4) real-time fluorescent PCR amplification program is provided that
1:50 DEG C of processing 2min of stage first,
2:95 DEG C of processing 3min of stage,
Stage 3: use landing procedure, it is every reduce by 1 degree carry out 2 circulation, condition be 95 DEG C of 10s, 57-60 DEG C of 30s of annealing temperature,
72 DEG C of 30s of elongating temperature,
Stage 4: successively carrying out 52 circulations in 95 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 30s,
5:95 DEG C of processing 2min of stage, then 40 DEG C of processing 2min, finally rise to 80 DEG C from 40 DEG C with 0.06 DEG C/s.
8. a kind of pleiomorphism detecting method of the accurate medication related gene of hypertension as claimed in claim 7, which is characterized in that
The result interpretation method of 7 kinds of genotype is as follows:
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