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CN108531578A - A kind of Primer composition and kit for detecting hypertension medication related gene - Google Patents

A kind of Primer composition and kit for detecting hypertension medication related gene Download PDF

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CN108531578A
CN108531578A CN201810388078.3A CN201810388078A CN108531578A CN 108531578 A CN108531578 A CN 108531578A CN 201810388078 A CN201810388078 A CN 201810388078A CN 108531578 A CN108531578 A CN 108531578A
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primer
primer sets
cyp2c9
kit
cyp2d6
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田晓丽
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Ningbo Beautiful Life Medical Laboratory Ltd Co
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Abstract

The present invention relates to a kind of Primer compositions and kit for detecting hypertension medication related gene, and Primer composition includes NPPA primer sets, ADRB1 primer sets, CYP2D6 primer sets, AGTR1 primer sets, CYP2C9 primer sets, CYP3A5 primer sets and GNB3 primer sets；Kit includes above-mentioned Primer composition.Compared with prior art,The present invention uses the method that ARMS technologies are combined with SYBR dyestuffs,Obtaining one kind can be quick,Sensitivity and easily detection hypertension medication related gene (NPPA,ADRB1,CYP2D6,AGTR1,CYP2C9,CYP3A5 and GNB3) gene pleiomorphism kit,The kit includes specificity ARMS detection primers,Internal control primer and PCR reaction solution,By designing ARMS detection primers and Scorpions probes being replaced with SYBR dyestuffs,So that testing cost substantially reduces,It is more suitable for Chinese patients NPPA,ADRB1,CYP2D6,AGTR1,CYP2C9,The detection of CYP3A5 and GNB3 gene pleiomorphisms,It is quick with detection,High sensitivity,High specificity,Method is simple,As a result accurate advantage,It is suitable for popularization and application.

Description

A kind of Primer composition and kit for detecting hypertension medication related gene
Technical field
The invention belongs to technical field of gene detection, are related to a kind of primer sets for detecting hypertension medication related gene Close object and kit.
Background technology
Diuretics decompression starts from 1948, during coming out more than 30 years from nineteen fifty-seven chlorothiazide (chlorothiazide), with Thiazide diuretic based on Hydrochioro (hydrodiuril, hydrochlorothiazide) is always drug for hypertension One of main force have clear curative effect no matter being applied alone or being combined with other drugs for hypertension.Big rule international in decades Mould clinical test results further determined its status in Treatment of Hypertension.American-European several hypertension treatment principles committee All suggest uncomplicated hypertensive patient, using diuretics as choice drug.In recent years, novel diuretics indapamide (longevity ratio Mountain, indapmide) listing, so that diuretics is had new raising again in the treatment status of high blood pressure, its feature is common agent Amount is showed only as slight diuresis, is mainly shown as that vasorelaxation action, decompression effective percentage are not present 80% or so The metabolic disorder side effect of traditional diuretics, in wide clinical application.Atrial natriuretic peptide precursor A (natriuretic Peptide precursor A, NPPA) it is a variety of disease genetic susceptibilities " candidate gene " of research, it is located at human chromosome 1p36.22 is made of three exons and 2 intrones, and preceding ANP of the synthesis containing 151 amino acid first is former in vivo (prepro-ANP), obtain the peptide chain that carboxyl terminal 99-126 amino acids are formed by a series of processing, i.e. ANP, wherein The ring of 17 Amino acid profiles is that it plays the required structure of bioactivity.Atrial natriuretic peptide (ANP) is closed by atrial muscle cell At the object with natriuretic diuretic, expansion blood vessel and inhibition renin-angiotensin-aldosterone system (RAAS) effect with secretion Matter, encodes on the 3rd exon of its precursor substance (NPPA) gene that there are T2238C mutation, is altered by Plasma ANP Concentration and influence Treatment of Hypertension.The study found that there is the patient of C allele to take diuretics ratio on this site takes other The depressor better efficacy of type, and it is more advantageous to the generation for reducing cardiovascular and cerebrovascular disease.The study found that NPPA T2238C bases Because saltant type patient is good to the reaction more sensitivity treatment effect of diuretics, and not mutated patient is to using cerebrocrast to imitate More preferably, detection NPPA genotype is conducive to patient's selection and is suitable for the drug for hypertension of oneself fruit, prevents side reaction at a specified future date Occur.For TT genotype patients, Amlodipine is selected to be better than other diuresis classes and angiotensin converting enzyme inhibitors.
Beta-blocker can be selectively combined with beta-2 adrenoceptor, to antagonism neurotransmitter and catecholamine To a kind of drug type of the agonism of beta receptor.Adrenocepter is distributed in most of sympathetic nerve postganglionic fibers and is propped up On the effector cell film matched, receptor is divided into 3 types, can excitement causes heart rate and myocardial contractive power to increase, bronchus expands , vasodilation, visceral smooth muscle relaxation and lipolysis etc., these effects can be blocked by beta-blocker and antagonism. Mankind Beta1 adrenoreceptors (ADRB1) albumen is the action target spot of Beta1 adrenoreceptor blocking agents, and research finds ADRB1 Albumen participates in regulating blood pressure, is the major protein in cardiac sympathetic nerve adrenal function system signal passageway.Arg389Gly is more State property is located at intracellular c-terminus, can influence ligand-mediated adenyl cyclase activity and G-protein coupling function, to shadow Ring heart rate and blood pressure.The difference of ADRB1 genotype may be ADRB1 retarding agents efficacy of antihypertensive treatment have individual difference the reason of it One.The study found that ADRB1 genotype is the patient couple of heterozygous mutation (Gly389Arg) and no mutant homozygote (Arg389Arg) Beta1 receptor antagonists are more sensitive, and genotype is that wild homozygote (Gly389Gly) is insensitive to drug.
Cytochrome P450 (CYP) family is a kind of important enzyme system, endogenous in vivo in drug metabolic enzyme It plays a significant role in the metabolism of substance and exogenous material.CYP2D6 is important one of the member of CYP enzyme families, although it The 2%-9% of liver enzyme total amount is only accounted for, the metabolism of 20%-30% drugs is but participated in.The metabolic phenotype of CYP2D6 can be divided into ultrafast Metabolic pattern (UMs), fast metabolic pattern (EMs), medium metabolic type (IMs), slow inactivation (PMs).Asian (including Japan, South Korea, China and the pacific island state crowd) probability of happening containing CYP2D6*10 is 33%-43%, but occur in white people Probability is but very low, only 2%-5%.The substrate of CYP2D6 includes the beta receptors such as essential hypertension medicine such as Propranolol Blocking agent, the mutation of CYP2D6 is based on CYP2D6*10 (C100T, P34S) in population of China, mutation rate 56.41%, therefore Heterozygous mutation type is most of in population of China, and metabolic enzyme activity is normal, and wild homozygous enzymatic activity is higher, need to increase agent Amount, and mutant homozygous subtype is slow metabolism, needs to be reduced.
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 Act on.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 genes are located at 3q21-25, contain only 1 exon, without in Containing subregion.The study found that in AGTR1 gene pleiomorphisms, the frequency highest that T573C, A1166C and A1062G are replaced, 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 antagonists (such as Losartan, irbesartan, Telmisartan) is as follows：A1166A Patient drugs are 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.
Cytochrome pathways (CYP2C9) are a kind of important drug metabolic enzymes in human liver, account for about mankind liver The 20% of middle CYP enzymes total amount.CYP2C9 is located on human chromosomal 10q24.2, overall length about 50.71kb, including 9 exons and 8 intrones encode 490 amino acid residues.The distribution of CYP2C9 allele has apparent crowd, race and region poor Different, in Chinese population, Primary mutations type gene is CYP2C9*3, is in the 1075th generation A of the 7th exon>C is mutated, and is made At the 359th isoleucine mutation on CYP2C9 polypeptide chains at leucine (Ile359>Leu359), gene frequency is 1.0-3.5%.CYP2C9 genes have genetic polymorphism, have now been found that a allele more than 30.The mutation etc. more early found Position gene is CYP*2-*6, and wherein CYP*2 and * 3 is most commonly seen while being also most studied mutation.CYP2C9 loses in gene Polymorphism is passed, causes CYP2C9 enzymatic activitys to be had differences between Different Individual, so that individual difference occurs in curative effect of medication.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 cause Losartan to drop Press decreased effectiveness；CYP2C9*3 is mutated the blood concentration that can increase Valsartan and Irbesartan, leads to Valsartan and Irbesartan Antihypertensive effect enhances.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.
Calcium ion antagonist is the chemicals to be reduced blood pressure by blocking calcium channel.It can be with selective depression Ca2+ Enter into the cell through the calcium channel on cell membrane, pass through and expand 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 the liver of adult and small intestine Expression, expression have apparent polymorphism.The gene mutation of CYP3A5 is the main reason of generation enzyme activity sex differernce, wherein CYP3A5*3 is in intron3 (6959A>G mutation) causes variable sheer, produces unstable protein, so that prominent It is denaturalized homozygotic individual, i.e., it carries the people of gene C YP3A5*3/*3 and does not express CYP3A5.Studies have shown that calcium antagonist class medicine Object is affected by CYP3A5*3 gene pleiomorphisms, and containing mutated-genotype patient, poisonous side effect of medicine increases.
1 receptors of α of 1 receptor blocker energy selective exclusion vascular smooth muscle postsynaptic membranes of α, distend the blood vessels, and cause periphery Vascular resistence decline and returned blood volume are reduced, to reduce systolic pressure and diastolic pressure.G-protein be one group can be with guanosine triphosphate (GTP) it combines, the protein with hydrolysing activity, in being risen in the information exchanging process between cell-membrane receptor and effect protein Jie acts on.G-protein is the downstream albumen of alpha adrenergic receptor, is autonomic nerves system, renin-angiotensin system, endothelium The coupling albumen of the regulating blood pressures system specific receptors such as prime system system, the sodium hydrogen that it influences cell exchange, and determine plasma renin activity With serum N a+、K+Concentration participates in regulating blood pressure.G-protein serves " molecular switch " in signal transduction.When G-protein structure, contain When amount, dysfunction, neurotransmitter, the effect of hormone can be zoomed in or out.Research is found outside GNB3 subunit genes the 10th There are one C825T polymorphisms, 825T carrier's GNB3 genetic transcription mRNA nucleic acid sequence 498-620 bit bases to lack for tool in aobvious son It loses, thus generates protein variants, referred to as GNB3-S.GNB3-S repeats sequence by 7 WD (being made of tryptophan and L-aminobutanedioic acid) Row, but rear 4 amino acid in the 3rd WD structure and preceding 37 amino acid deletions in the 4th WD structure, finally formed G Protein complexes than the active higher of wild-type G proteins complex, accelerate intracellular messengers and transmit, increase cell by this polymorphism Na+-H+Exchange activity, make hypertension cause danger and hypertension caused by related organ impairment's danger increase.There is research table Bright, GNB3C825T genotype exists with the antihypertensive effect of 1 receptor blocker of α to be associated with, wherein CC types Amplitude of Hypotensive is compared with TC/TT types Bigger.
Detection method of gene mutation has very much, and scholars have carried out this numerous studies, it has been reported that method packet Include direct sequencing, dhplc analysis (DHPLC), PCR-SSCP/RFLP, Scorpions ARMS, TaqMan PCR, ME-PCR etc..These methods respectively have advantage and disadvantage, in clinical and scientific research more common method be direct sequencing and ARMS (Amplification refractory mutation system, amplification refractory mutation system) method.
Wherein, direct sequencing is it can be found that some new unknown mutations, but detectability is limited, and detection sensitivity is about 20% or so, and step is complicated, and entire detection process is related to a series of steps such as the deciphering of PCR- electrophoresis-sequencing-sequencing result Suddenly, time-consuming and laborious.
ARMS methods are to be combined to create with the ARMS primers of specificity by molecular beacon (probe), ARMS primers 3 ' end designs are in mutational site, the last one base is matched with mutating alkali yl, using the Taq DNA without 3 ' → 5 ' 5 prime excision enzyme activities Polymerase specifically identifies that 3 ' ends of primer, only 3 ' end of primer match clock synchronization, could normally expand, work as primer completely When mispairing occurs for 3 ' ends, cannot effectively it expand.After primer is combined with mutagenesis template and extends corresponding product, probe The fluorophor and quenching group at both ends detach and generate fluorescence.However, similar reagents box currently on the market is expensive, answer With being restricted.
Invention content
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one kind for detecting high blood Press the Primer composition and kit of medication related gene.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of Primer composition for detecting hypertension medication related gene, which includes NPPA primers Group, ADRB1 primer sets, CYP2D6 primer sets, AGTR1 primer sets, CYP2C9 primer sets, CYP3A5 primer sets and GNB3 primers Group；
The NPPA primer sets include following primer：
NPPA-FW：5’-CTGTGTTCTCTTTGCAGTAGT-3’；
NPPA-FS1：5’-TGTGTTCTCTTTGCAGTATC-3’；
NPPA-R：5’-GTGAGAAGTGTTGACAGGAA-3’；
The ADRB1 primer sets include following primer：
ADRB1-FW1：5’-CTTCCGCAAGGCCTTCCAAC-3’；
ADRB1-FS1：5’-ACTTCCGCAAGGCCTTCCATG-3’；
ADRB1-R：5’-TGGCCCCGACGACATCGTCG-3’；
The CYP2D6 primer sets include following primer：
CYP2D6-FW1：5’-ACGCTGGGCTGCACGCTAAC-3’；
CYP2D6-FS1：5’-AACGCTGGGCTGCACGCTAGT-3’；
CYP2D6-R：5’-ACCCACCACCCATGTTTG-3’；
The AGTR1 primer sets include following primer：
AGTR1-FW1：5’-CACTTCACTACCAAATGACCA-3’；
AGTR1-FS1：5’-CACTTCACTACCAAATGATCC-3’；
AGTR1-R：5’-TCTCAAATCAACACATTCATCG-3’；
The CYP2C9 primer sets include following primer：
CYP2C9-FW1：5’-GCACGAGGTCCAGAGATAGA-3’；
CYP2C9-FS1：5’-GCACGAGGTCCAGAGATAGC-3’；
CYP2C9-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
The CYP3A5 primer sets include following primer：
CYP3A5-FW1：5’-AGAGCTCTTTTGTCTTTCTA-3’；
CYP3A5-FS1：5’-AAGAGCTCTTTTGTCTTTCGG-3’；
CYP3A5-R：5’-ATGTAATCCATACCCCTA-3’；
The GNB3 primer sets include following primer：
P1-FW1：5’-CATCTGCGGCATCACGTAC-3’；
P1-FS1：5’-CATCTGCGGCATCACGTAT-3’；
P1-R：5’-ACCCAGTGACAAGGGACAGC-3’.
Further, which further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
Primer composition described in a kind of is in the genetic polymorphism detection reagent for preparing hypertension medication related gene Using.
It is a kind of for detecting the kit of hypertension medication related gene, which includes the Primer composition.
Further, which further includes PCR reaction solution, the PCR reaction solution include PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I dyestuffs.The PCR buffer solutions are Tris-HCl buffer solutions, Tris- acetic acid (TAE) Buffer solution or Tris- boric acid (TBE) buffer solution.
The PCR reaction solution includes 3 ' → 5 ' 5 prime excision enzyme activity high-fidelity Taq enzyme (enzyme activities as a preferred technical solution, For 2.5U), 1.0-5.0mM MgCl2, 4.0-20.0mM dNTPs (including each 1.0-5.0mM of dATP, dUTP, dGTP, dCTP) And SYBR Green I dyestuffs.
Further, which further includes positive control solution and negative controls.
The positive control solution is to be corresponded to containing detectable 7 sites in kit as a preferred technical solution, Plasmid mixed liquor, in plasmid mixed liquor containing 7 kinds of different NPPA, ADRB1 with mutational site to be detected, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3 allele, the plasmid concentration in plasmid mixed liquor are 1000copies/ μ l；It is described Negative controls be Tris-HCL buffer solutions, a concentration of 7-13mM of the Tris-HCL buffer solutions, pH 7.5-8.5.
A kind of hypertension medication related gene (NPPA, ADRB1, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3) Gene pleiomorphism detecting method includes the following steps：
1) extraction of the processing of sample to be tested and template；
2) quantitative fluorescent PCR reaction system is prepared using the Primer composition；
3) ARMS methods are used, divide wild type and mutated genes sequence using each guiding region, are contaminated by SYBR Green I The hybridization of material and amplified production, detects the SYBR fluorescence intensities of reaction system to judge testing result.SYBR is detection signal, with SYBR signals reach the cycle-index Ct values needed for the threshold value of setting as criterion, and Ct values are less than 32 for the positive, and Ct values are big It is feminine gender in 35, Ct values are weakly positive between 32 to 35.
The sample to be tested described in step 1) includes whole blood, blood plasma, serum or pleural effusion as a preferred technical solution,.
As a preferred technical solution, in the PCR reaction systems described in step 2), the content of each component is as follows：
Wherein, the concentration of each primer is 300nM in Primer composition.
The genetic polymorphism detection kit of hypertension medication related gene of the present invention uses SYBR dye methods, establishes needle To gene pleiomorphism (such as table 1 of mankind's related gene (NPPA, ADRB1, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3) It is shown) Multiplex real-time PCR detection method.The present invention passes through a large number of experiments, and research and analysis, which successfully filter out, can be used in soon Primer fast, sensitive, that NPPA, ADRB1, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3 gene pleiomorphism is effectively detected Composition, and go out the method and kit for detecting hypertension medication related gene polymorphism using these primer developments.
The gene pleiomorphism form of 1 hypertension medication related gene of table
The specific primer of hypertension medication related gene see the table below 2-8, and table 9 is the detection primer of internal reference.
The detection primer of table 2 NPPA (2238) gene pleiomorphism
The detection primer of table 3 ADRB1 (389) gene pleiomorphism
The detection primer of table 4 CYP2D6 (100) gene pleiomorphism
The detection primer of table 5 AGTR1 (1166) gene pleiomorphism
The detection primer of table 6 CYP2C9 (1075) gene pleiomorphism
The detection primer of table 7 CYP3A5 (6959) gene pleiomorphism
The detection primer of table 8 GNB3 (825) gene pleiomorphism
The detection primer of 9 internal reference of table
In the present invention, according to the base mutation form of each gene, using ARMS-PCR methods, design and wild type and prominent The compatible primer of modification genetic fragment, and these primers are synthesized using custom primer synthetic method, for related gene Gene pleiomorphism be detected.The basic principle of ARMS-PCR methods is：If 3 ' the end bases and template base of primer are not Complementation can not then be extended with general hot resistant DNA polymerase, therefore design corresponding primer, 3 ' end alkali according to known point mutation Base respectively with mutation and normal template base complementrity, so that the template for having certain point mutation and normal template be distinguished.
SYBR Green I are a kind of dyes with green excitation wavelength being incorporated into all dsDNA minor grooves region Material.Under free state, SYBR Green I send out faint fluorescence, but once combined with double-stranded DNA, fluorescence increases greatly By force.Therefore, the fluorescence signal intensity of SYBR Green I and the quantity of double-stranded DNA are related, can be detected according to fluorescence signal Double-stranded DNA quantity existing for PCR system.The maximum absorption wavelength of SYBR Green I is about 497nm, and launch wavelength is about 520nm。
Compared with prior art, the invention has the characteristics that：
1) use the method that is combined with SYBR dyestuffs of ARMS technologies, obtain one kind can quickly, sensitivity and easily Detect hypertension medication related gene (NPPA, ADRB1, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3) gene pleiomorphism Kit, the kit include specificity ARMS detection primers, internal control primer and PCR reaction solution, pass through design ARMS detect Scorpions probes are simultaneously replaced with SYBR dyestuffs by primer so that testing cost substantially reduces, be more suitable for Chinese patients NPPA, The detection of ADRB1, CYP2D6, AGTR1, CYP2C9, CYP3A5 and GNB3 gene pleiomorphism, have detection quickly, high sensitivity, High specificity, method are simple, the accurate advantage of result, are suitable for popularization and application；
2) kit can accurately detect 1% mutant DNA under 50ng wild type gene group DNA backgrounds, and specific aim is set The specific primer for counting different mutational sites, is detected using quantitative fluorescent PCR, and detection process is stopped pipe reaction, significantly Reduce pollution.
Description of the drawings
Fig. 1 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects NPPA (2238) sample；
Fig. 2 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects ADRB1 (389) sample；
Fig. 3 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP2D6 (100) sample；
Fig. 4 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects AGTR1 (1166) sample；
Fig. 5 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP2C9 (1075) sample；
Fig. 6 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP3A5 (6959) sample；
Fig. 7 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects GNB3 (825) sample.
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.The present embodiment is with technical solution of the present invention Premised on implemented, give detailed embodiment and specific operating process.Those skilled in the art can be by this explanation Content disclosed by book understands other advantages and effect of the present invention easily.The present invention can also be by addition different specific Embodiment is embodied or practiced, and the various details in this specification can also be based on different viewpoints and application, not carry on the back Various modifications or alterations are carried out under spirit from the present invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Embodiment 1：
1, sample process and nucleic acid extraction：
Sample is handled using the DNA extraction kit of commercialization, concrete operations are needed referring to kit specification, carried DNA With ultraviolet specrophotometer measured concentration and purity, DNA OD260/OD280Value should 1.8~2.0, concentration should 5~ Between 50ng/ μ L, sample DNA person off quality must not be used to detect, and re-start nucleic acid extraction less than 5ng/ μ L persons, be higher than 50ng/ μ L persons are suitably diluted to defined concentration range, and the DNA extracted should be detected immediately, otherwise in -20 DEG C with Lower preservation, the holding time was no more than 6 months.Kit quality-control product is melted using preceding room temperature, vortex oscillation 10 seconds, 2000rpm Centrifuge 15 seconds for use.
2, preparation of reagents：
2.1 prepare explanation：Detection reaction setting internal control detection, negative quality-control product detection and positive quality control product detection.
2.2 process for preparation：
Reagent was taken out in 30 minutes in advance, room temperature is melted, vortex oscillation 10 seconds, and 2000rpm centrifuges 15 seconds for use.It determines every A detection site stoichiometric number N and internal control testing number M, N=sample numbers (n) to be checked+quality-control product number (2)+1；M=sample numbers to be checked (n)+1.The amount for being added to each reagent in reaction mixture is calculated, such as the following table 10 is calculated：
10 PCR reaction systems of table are with tabulation
15 sterile centrifugation tubes are taken to prepare above-mentioned 15 reaction systems, vortex oscillation 10 seconds after reagent is all added, 2000rpm is centrifuged 15 seconds.Then 18 μ L/ pipes of above-mentioned mixed liquor are dispensed into PCR reaction tubes (sterile and RNase-Free).
3, it is loaded：
11 sample of table and quality-control product loading table
Title Ingredient Sample-adding amount
Sample to be checked Sample DNA 2μL
Internal control Quality-control product 2μL
According to loading ratio shown in table 11, processed sample, quality-control product are added in reaction tube, pipe lid is covered tightly and (avoids Bubble generates), 2000rpm is centrifuged 15 seconds and is all got rid of the liquid on tube wall to tube bottom, carries out pcr amplification reaction immediately after.
4, PCR amplification program is arranged：
The setting of 12 PCR response procedures of table
5, the deciphering of inspection result：
Divide wild type and mutated genes sequence using the guiding regions ARMS, passes through SYBR Green dyestuffs and amplified production Hybridization, detects the SYBR fluorescence intensities of reaction system to judge testing result.Detect amplification curve diagram such as Fig. 1, the figure of each sample 2, shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7.SYBR is detection signal, reaches the cycle needed for the threshold value of setting time with SYBR signals Number Ct values are as criterion, and Ct values are less than 32 for the positive, and Ct values are more than 35 for feminine gender, and Ct values are weak sun between 32 to 35 Property.
Embodiment 2：
A kind of Primer composition for detecting hypertension medication related gene include NPPA primer sets, ADRB1 primer sets, CYP2D6 primer sets, AGTR1 primer sets, CYP2C9 primer sets, CYP3A5 primer sets and GNB3 primer sets；
NPPA primer sets include following primer：
NPPA-FW：5’-CTGTGTTCTCTTTGCAGTAGT-3’；
NPPA-FS1：5’-TGTGTTCTCTTTGCAGTATC-3’；
NPPA-R：5’-GTGAGAAGTGTTGACAGGAA-3’；
ADRB1 primer sets include following primer：
ADRB1-FW1：5’-CTTCCGCAAGGCCTTCCAAC-3’；
ADRB1-FS1：5’-ACTTCCGCAAGGCCTTCCATG-3’；
ADRB1-R：5’-TGGCCCCGACGACATCGTCG-3’；
CYP2D6 primer sets include following primer：
CYP2D6-FW1：5’-ACGCTGGGCTGCACGCTAAC-3’；
CYP2D6-FS1：5’-AACGCTGGGCTGCACGCTAGT-3’；
CYP2D6-R：5’-ACCCACCACCCATGTTTG-3’；
AGTR1 primer sets include following primer：
AGTR1-FW1：5’-CACTTCACTACCAAATGACCA-3’；
AGTR1-FS1：5’-CACTTCACTACCAAATGATCC-3’；
AGTR1-R：5’-TCTCAAATCAACACATTCATCG-3’；
CYP2C9 primer sets include following primer：
CYP2C9-FW1：5’-GCACGAGGTCCAGAGATAGA-3’；
CYP2C9-FS1：5’-GCACGAGGTCCAGAGATAGC-3’；
CYP2C9-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
CYP3A5 primer sets include following primer：
CYP3A5-FW1：5’-AGAGCTCTTTTGTCTTTCTA-3’；
CYP3A5-FS1：5’-AAGAGCTCTTTTGTCTTTCGG-3’；
CYP3A5-R：5’-ATGTAATCCATACCCCTA-3’；
GNB3 primer sets include following primer：
P1-FW1：5’-CATCTGCGGCATCACGTAC-3’；
P1-FS1：5’-CATCTGCGGCATCACGTAT-3’；
P1-R：5’-ACCCAGTGACAAGGGACAGC-3’.
Above-mentioned Primer composition is applied in the genetic polymorphism detection reagent for preparing hypertension medication related gene.
A kind of kit for detecting hypertension medication related gene includes above-mentioned Primer composition and PCR reaction solution, The PCR reaction solution includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I dyestuffs.
Embodiment 3：
A kind of Primer composition for detecting hypertension medication related gene include NPPA primer sets, ADRB1 primer sets, CYP2D6 primer sets, AGTR1 primer sets, CYP2C9 primer sets, CYP3A5 primer sets and GNB3 primer sets；
NPPA primer sets include following primer：
NPPA-FW：5’-CTGTGTTCTCTTTGCAGTAGT-3’；
NPPA-FS1：5’-TGTGTTCTCTTTGCAGTATC-3’；
NPPA-R：5’-GTGAGAAGTGTTGACAGGAA-3’；
ADRB1 primer sets include following primer：
ADRB1-FW1：5’-CTTCCGCAAGGCCTTCCAAC-3’；
ADRB1-FS1：5’-ACTTCCGCAAGGCCTTCCATG-3’；
ADRB1-R：5’-TGGCCCCGACGACATCGTCG-3’；
CYP2D6 primer sets include following primer：
CYP2D6-FW1：5’-ACGCTGGGCTGCACGCTAAC-3’；
CYP2D6-FS1：5’-AACGCTGGGCTGCACGCTAGT-3’；
CYP2D6-R：5’-ACCCACCACCCATGTTTG-3’；
AGTR1 primer sets include following primer：
AGTR1-FW1：5’-CACTTCACTACCAAATGACCA-3’；
AGTR1-FS1：5’-CACTTCACTACCAAATGATCC-3’；
AGTR1-R：5’-TCTCAAATCAACACATTCATCG-3’；
CYP2C9 primer sets include following primer：
CYP2C9-FW1：5’-GCACGAGGTCCAGAGATAGA-3’；
CYP2C9-FS1：5’-GCACGAGGTCCAGAGATAGC-3’；
CYP2C9-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
CYP3A5 primer sets include following primer：
CYP3A5-FW1：5’-AGAGCTCTTTTGTCTTTCTA-3’；
CYP3A5-FS1：5’-AAGAGCTCTTTTGTCTTTCGG-3’；
CYP3A5-R：5’-ATGTAATCCATACCCCTA-3’；
GNB3 primer sets include following primer：
P1-FW1：5’-CATCTGCGGCATCACGTAC-3’；
P1-FS1：5’-CATCTGCGGCATCACGTAT-3’；
P1-R：5’-ACCCAGTGACAAGGGACAGC-3’.
The Primer composition further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
Above-mentioned Primer composition is applied in the genetic polymorphism detection reagent for preparing hypertension medication related gene.
A kind of kit for detecting hypertension medication related gene includes above-mentioned Primer composition and PCR reaction solution, The PCR reaction solution includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I dyestuffs.
Kit further includes positive control solution and negative controls.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention's Within protection domain.

Claims (6)

1. a kind of for detecting the Primer composition of hypertension medication related gene, which is characterized in that the Primer composition includes NPPA primer sets, ADRB1 primer sets, CYP2D6 primer sets, AGTR1 primer sets, CYP2C9 primer sets, CYP3A5 primer sets and GNB3 primer sets；
The NPPA primer sets include following primer：
NPPA-FW：5’-CTGTGTTCTCTTTGCAGTAGT-3’；
NPPA-FS1：5’-TGTGTTCTCTTTGCAGTATC-3’；
NPPA-R：5’-GTGAGAAGTGTTGACAGGAA-3’；
The ADRB1 primer sets include following primer：
ADRB1-FW1：5’-CTTCCGCAAGGCCTTCCAAC-3’；
ADRB1-FS1：5’-ACTTCCGCAAGGCCTTCCATG-3’；
ADRB1-R：5’-TGGCCCCGACGACATCGTCG-3’；
The CYP2D6 primer sets include following primer：
CYP2D6-FW1：5’-ACGCTGGGCTGCACGCTAAC-3’；
CYP2D6-FS1：5’-AACGCTGGGCTGCACGCTAGT-3’；
CYP2D6-R：5’-ACCCACCACCCATGTTTG-3’；
The AGTR1 primer sets include following primer：
AGTR1-FW1：5’-CACTTCACTACCAAATGACCA-3’；
AGTR1-FS1：5’-CACTTCACTACCAAATGATCC-3’；
AGTR1-R：5’-TCTCAAATCAACACATTCATCG-3’；
The CYP2C9 primer sets include following primer：
CYP2C9-FW1：5’-GCACGAGGTCCAGAGATAGA-3’；
CYP2C9-FS1：5’-GCACGAGGTCCAGAGATAGC-3’；
CYP2C9-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
The CYP3A5 primer sets include following primer：
CYP3A5-FW1：5’-AGAGCTCTTTTGTCTTTCTA-3’；
CYP3A5-FS1：5’-AAGAGCTCTTTTGTCTTTCGG-3’；
CYP3A5-R：5’-ATGTAATCCATACCCCTA-3’；
The GNB3 primer sets include following primer：
P1-FW1：5’-CATCTGCGGCATCACGTAC-3’；
P1-FS1：5’-CATCTGCGGCATCACGTAT-3’；
P1-R：5’-ACCCAGTGACAAGGGACAGC-3’.
2. a kind of Primer composition for detecting hypertension medication related gene according to claim 1, feature exist In the Primer composition further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
3. a kind of Primer composition as claimed in claim 1 or 2 is in the gene pleiomorphism for preparing hypertension medication related gene Application in detection reagent.
4. a kind of kit for detecting hypertension medication related gene, which is characterized in that the kit includes that right such as is wanted Seek the Primer composition described in 1 or 2.
5. a kind of kit for detecting hypertension medication related gene according to claim 4, which is characterized in that should Kit further includes PCR reaction solution, which includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I dyestuffs.
6. a kind of kit for detecting hypertension medication related gene according to claim 4, which is characterized in that should Kit further includes positive control solution and negative controls.
CN201810388078.3A 2018-04-26 2018-04-26 A kind of Primer composition and kit for detecting hypertension medication related gene Pending CN108531578A (en)

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Application publication date: 20180914