CN107557432A - A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism - Google Patents
A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism Download PDFInfo
- Publication number
- CN107557432A CN107557432A CN201710720710.5A CN201710720710A CN107557432A CN 107557432 A CN107557432 A CN 107557432A CN 201710720710 A CN201710720710 A CN 201710720710A CN 107557432 A CN107557432 A CN 107557432A
- Authority
- CN
- China
- Prior art keywords
- gene polymorphism
- primer sets
- related gene
- cyp2d6
- adrb1
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The primer sets of detection hypertension medication related gene polymorphism disclosed by the invention, it is characterised in that include following primer：CYP2D6*10‑F；CYP2D6*10‑R；ADRB1‑F；ADRB1‑R；AGTR1‑F；AGTR1‑R；ACE‑F；ACE‑R；CYP2C9*3‑F；CYP2C9*3‑R.CYP2C9*3, ADRB1 (1165G of the present invention>C)、AGTR1(1166A>C), CYP2D6*10, ACBL (I/D) gene is proved to be closely related with the effect of hypertension therapeutic common drug, can be according to the result for detecting these gene polymorphism sites, to instruct the reasonable safety uses the medicine of hyperpietic.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of primer for detecting hypertension medication related gene polymorphism
Group and detection kit.
Background technology：
Conservative estimation, China's hyperpietic's number have exceeded 1.6 hundred million.The incidence of disease of hypertension is in rising trend, high blood
Pressure complication-cerebral apoplexy is the second largest cause of the death in China.Hypertension has become the third-largest disease economy in the whole world with its complication
Burden.It is current and expected future reduces that hypertension complication occurs primary hand in for quite a long time to control blood pressure by drug therapy
Section.
In the whole world preceding 200 kinds of medicines salable in 2000, the medicine for treating hypertension accounts for 17 kinds.Then, high blood is treated
The individual reaction difference that the medicine of pressure clinically occurs is very universal, and the patient for receiving drug therapy there are about 20%-50% blood
Pressure is not well controlled.Its main cause is also due to the drug metabolic enzyme related to medicine and acceptor, and there occurs heredity change
It is different.Curative effect of medication and the individual difference of adverse reaction are the universal phenomena in current drug treatment.Pharmacogenetics and medicine
The progress of thing genomics shows that the hereditary variation of drug metabolic enzyme, transporter and acceptor (drug target) is to cause
The main reason for individual drugs response difference.As cytochrome oxidase CYP2D6 producers be mutated, in same dose condition
Under, the blood concentration of the beta-blocker of its mediated metabolic in saltant type homozygote is higher than wild-type homozygote 2-3 times,
If that serious toxicity may not occur according to genotype regulating dosage, saltant type homozygote patient；If on the contrary, beta receptor
Generating function is mutated, and Bextra is still used in saltant type homozygotic individual, frequently can lead to Endodontic failure.This
Not only delay treatment, and result in the waste of medical resource.Therefore, transported according to the individual metabolic enzyme related to drug therapy
Different therapeutic schemes is specified in the hereditary variation of body and acceptor, realize drug-treated individual be not only current pharmacogenetics and
The developing direction of clinical drug therapy, and there is highly important social effect and economic implications.
At this stage, clinically detection hypertension medication gene pleiomorphism mainly takes PCR sequencing PCR and gene chips to be examined
Survey, PCR sequencing PCR sensitivity is low and whole detection cycle needs 7 days, and complex operation is not suitable for clinical expansion；Gene chips are sensitive
Degree is only capable of reaching 104Copies/ml, and operating process easily causes cross pollution causes false positive.
Human gene polymorphism is illustrating human body to disease, the neurological susceptibility of poisonous substance and tolerance, to Disease Clinical performance
Diversity, and to all being played an important role in the reactivity of drug therapy.
Beta receptor blocking agent, angiotensin converting enzyme inhibitor (ACEI), Angiotensin Ⅱ receptor antagonist (ARB),
Diuretics, calcium antagonist (CCB) this five hypotensor, between Different Individual the activity difference of drug metabolic enzyme can cause it is different very
To opposite drug effect difference.CYP2D6, ADRB1, CYP2C9, AGTR1 and ACE are related to beta receptor blocking agent, angiotensins conversion
Enzyme inhibitor (ACEI), the hypotensor of Angiotensin Ⅱ receptor antagonist (ARB) three.
The content of the invention：
The purpose of the present invention one of is to provide a kind of primer sets for detecting hypertension medication related gene polymorphism.
Two a kind of primers using above-mentioned detection hypertension medication related gene polymorphism of offer that the purpose of the present invention is
Detection kit prepared by group.
As the primer sets of the detection hypertension medication related gene polymorphism of first aspect present invention, comprising drawing as follows
Thing：
CYP2D6*10-F:aggtgtgtccagaggagcccat；
CYP2D6*10-R:CCACCATCCATGTTTGCTTCTGG；
ADRB1-F:CGCCTCTTCGTCTTCTTCAA；
ADRB1-R:AGTTCACCTGCTATCGGTCTTA；
AGTR1-F:ctcagataatgtaagctcatccacc；
AGTR1-R:AGCAGCCGTCATCTGTCTAATGC；
ACE-F:gactctgtaagccactgctggag；
ACE-R:GACGTGGCCATCACATTCGTCA；
CYP2C9*3-F:GTCCAGGAAGAGATTGAACGTGTGA；
CYP2C9*3-R:TATCACCCGGTGATGGTAGAGGTT；
Detection prepared by the primer sets using above-mentioned detection hypertension medication related gene polymorphism as the present invention tries
Agent box, include above-mentioned primer sets.
CYP2C9*3、ADRB1(1165G>C)、AGTR1(1166A>C), CYP2D6*10, ACBL (I/D) gene is proved to
It is closely related with the effect of hypertension therapeutic common drug, can be according to the result for detecting these gene polymorphism sites, to refer to
Lead the reasonable safety uses the medicine of hyperpietic.
Brief description of the drawings
Fig. 1 is ACE electrophoretograms of the present invention, if only 500bp or so band in figure, illustrates it is II types；If have 200/
The band of 500bp or so two, then explanation is ID types；If only 200 or so band, illustrate it is DD types.
Fig. 2 a to Fig. 2 c are CYP2D6*10 of the present invention (C100T) testing result schematic diagram.
Fig. 3 is CYP2C9*3 of the present invention (A1075C) testing result schematic diagram.
Fig. 4 is ADRB1 of the present invention (G1165C) testing result schematic diagram.
Fig. 5 is AGTR1 of the present invention (A1166C) testing result schematic diagram.
Embodiment
The primer sets of the detection hypertension medication related gene polymorphism of the present invention, include following primer：
CYP2D6*10-F:aggtgtgtccagaggagcccat；
CYP2D6*10-R:CCACCATCCATGTTTGCTTCTGG；
ADRB1-F:CGCCTCTTCGTCTTCTTCAA；
ADRB1-R:AGTTCACCTGCTATCGGTCTTA；
AGTR1-F:ctcagataatgtaagctcatccacc；
AGTR1-R:AGCAGCCGTCATCTGTCTAATGC；
ACE-F:gactctgtaagccactgctggag；
ACE-R:GACGTGGCCATCACATTCGTCA；
CYP2C9*3-F:GTCCAGGAAGAGATTGAACGTGTGA；
CYP2C9*3-R:TATCACCCGGTGATGGTAGAGGTT；
Detection prepared by the primer sets using above-mentioned detection hypertension medication related gene polymorphism as the present invention tries
Agent box, include above-mentioned primer sets.
The preparation method and testing result of the primer sets of above-mentioned detection hypertension medication related gene polymorphism are as follows：
First, genome extracts：Extract human gene group DNA (exemplified by saliva sample)
1. the SOP is applied to useReagent extraction is collected by Oragene and ORAcollect series of products
Saliva sample genomic DNA.
1) sample in OrageneDNA (OG-500) saliva collection pipe is gently overturned and mixes 10s.
2) by saliva collection pipe in 50 DEG C of water-bath at least 1h (air bath at least 2h).
3) the 500 mixed saliva samples of μ l are taken in 1.5ml centrifuge tubes, remaining sample preserves in normal temperature.
4) 20 μ l (1/25 saliva volume) PT-L2P, vortex oscillation 10 seconds, of short duration centrifugation are added in 500 μ l salivas.
5) 10min is incubated on ice.
6) 15,000 × g of room temperature centrifuges 5min (centrifugation 15min effects are more preferable if the time allows).
7) pipettor range is adjusted to 200 μ l, is carefully transferred to clean supernatant in new 1.5ml centrifuge tubes,
Abandon the centrifuge tube containing contamination precipitation.
Note：Pipette tips not encounter contamination precipitation necessarily during transfer supernatant.If imprudence encounters precipitation, must incite somebody to action
Microcentrifugal tube centrifuges and retransfers supernatant again.
8) absolute ethyl alcohol that 600ul room temperatures are placed is added in 500ul supernatants, gently overturns and mixes 10 times.
9) said mixture is incubated at room temperature 10min, DNA is fully precipitated.
10) centrifuge tube is put into centrifuge by the direction of lid opening towards rotor center, room temperature 15,000 × g centrifugations
2min。
11) carefully remove supernatant with pipettor and abandoned.
Note：Pipettor is adjusted to 500 μ l first when absorbing supernatant, removes supernatant once.Pipettor is adjusted to again
200 μ l, thoroughly remove remaining supernatant as far as possible.DNA precipitations are not encountered during this.
12) 250 μ l 70% ethanol is carefully added into the centrifuge tube precipitated containing DNA, room temperature places 1min.
Ethanol is thoroughly removed in the case of being precipitated without impinging on DNA.
Note：Residual ethanol can influence DNA mass, must thoroughly remove ethanol.If precipitation separation, can by sample in
15,000 × g centrifuges 5min at room temperature.
13) 50-100ul TE dissolving DNAs precipitation, vortex oscillation at least 5s are added.DNA after dissolving places 1h at 50 DEG C
To be completely dissolved DNA, during which vibration is several times.
2.DNA quality testings
1) DNA quality testings：Take 3 μ l DNA samples to add 1 μ l 6x loading buffer, inhaled with rifle and play mixing.Adjust
Rifle is drawn whole samples and added in 1% Ago-Gel hole, put 3 μ l DL15000Marker, 140V electrophoresis 15 minutes to 4 μ l.
After electrophoresis terminates, blob of viscose is put into gel imaging system and taken pictures, picture is stored in corresponding file.
2) 1 μ L DNA samples (10 times can be diluted) are taken, 30 times is diluted with TE, adds 30 μ L PicoGreen fluorescent dyes
(TE dilutes 200 times), the μ L mixed solutions of transferase 45 0 are into Minicell after of short duration concussion centrifugation.If the liquid in Minicell has
Bubble is not all added to bottom, can drive bubble away by way of hand gets rid of several times and get rid of liquid into Minicell bottoms
Portion.Minicell is put into TBS380, Read keys is pressed, takes reading.Using standard curve, sample concentration is calculated, and record.
2nd, design of primers and synthesis
It is corresponding using primer-design software Primer5 designs in 400bp or so sequence above and below corresponding SNP site
Primer, synthesized by upper Shanghai's style Sen Nuo bio tech ltd, prepared using PAGE method of purification.PAGE method of purification is to use
Denaturing polyacrylamide gel electrophoresis, DNA fragmentation is separated, the method that target DNA is then reclaimed from gel.PAGE is pure
Change method is also a kind of very effective DNA purification process, and DNA purity after purification is more than 95%.
3rd, PCR is expanded：STb gene is expanded, obtains corresponding genetic fragment.
PCR is expanded
1. prepare PCR reaction solutions (50 μ L)：
2 x PCR master mix:25μL
Primer mix(10μM each):1.5μL
template(DNA):20ng
dH2O:up to 50μL
Wherein, 2 × PCR master (1mL) are：
2. PCR programs (touchdown)
15cycles (annealing temperature is each to circulate 0.5 DEG C since circulating second)
Target fragment detects
Take 3 μ l DNA samples to add 0.5 μ l 10x loading buffer, inhaled with rifle and play mixing.Rifle is adjusted to be drawn to 5 μ l
Whole samples are added in 2% Ago-Gel hole, put 2 μ l DL15000Marker, 140V electrophoresis 15 minutes.After electrophoresis terminates,
Blob of viscose is put into gel imaging system and taken pictures, picture is stored in corresponding file.
Wherein ACE (I/D) this be to primer detection one section of sequence missing, can directly from electrophoretogram it may determine that
Go out the polymorphism of the gene loci.
4th, generation sequencing carries out generation sequencing, sequenator used using the technology based on Sanger dideoxy chain terminations
For ABI3730xl.
Purpose fragment size is correct in glue figure and band is clear, can directly send to PCR primer and be sequenced and correctly fill in survey
Sequence list.Any primer for choosing one end is as sequencing primer, 10 μM of primer concentration.
Control sequence is compared with sequencing sequence with BioEdit or AlignX softwares, or directly by sequence inputting
NCBI websites nucleic acid compares, and finds pleomorphism site, observe the site whether undergo mutation or peak figure in there is heterozygosis peak.
Testing result is referring to Fig. 1 to Fig. 5.
Claims (2)
1. detect the primer sets of hypertension medication related gene polymorphism, it is characterised in that include following primer：
CYP2D6*10-F:aggtgtgtccagaggagcccat；
CYP2D6*10-R:CCACCATCCATGTTTGCTTCTGG；
ADRB1-F:CGCCTCTTCGTCTTCTTCAA；
ADRB1-R:AGTTCACCTGCTATCGGTCTTA；
AGTR1-F:ctcagataatgtaagctcatccacc；
AGTR1-R:AGCAGCCGTCATCTGTCTAATGC；
ACE-F:gactctgtaagccactgctggag；
ACE-R:GACGTGGCCATCACATTCGTCA；
CYP2C9*3-F:GTCCAGGAAGAGATTGAACGTGTGA；
CYP2C9*3-R:TATCACCCGGTGATGGTAGAGGTT。
2. detection kit prepared by the primer sets for detecting hypertension medication related gene polymorphism, it is characterised in that include power
Profit requires the primer sets of the detection hypertension medication related gene polymorphism described in 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710720710.5A CN107557432A (en) | 2017-08-21 | 2017-08-21 | A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710720710.5A CN107557432A (en) | 2017-08-21 | 2017-08-21 | A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107557432A true CN107557432A (en) | 2018-01-09 |
Family
ID=60976532
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710720710.5A Pending CN107557432A (en) | 2017-08-21 | 2017-08-21 | A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107557432A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977531A (en) * | 2018-09-05 | 2018-12-11 | 武汉康录生物技术股份有限公司 | A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application |
| CN109182509A (en) * | 2018-10-29 | 2019-01-11 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes |
| CN109355368A (en) * | 2018-10-22 | 2019-02-19 | 江苏美因康生物科技有限公司 | A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism |
| CN112608989A (en) * | 2020-12-18 | 2021-04-06 | 成都和合医学检验所有限公司 | Primer group, kit and method for detecting gene polymorphism |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101067152A (en) * | 2007-02-06 | 2007-11-07 | 上海生物芯片有限公司 | Pharmacogenetic detection chip for cardiac and cerebral vascular diseases and its application |
| CN101886129A (en) * | 2010-06-30 | 2010-11-17 | 首都医科大学附属北京安贞医院 | Method for detecting mononucleotide polymorphism locus rs388915 of hypertension susceptibility genes AGTR 1 and detection kit |
| CN101892298A (en) * | 2010-02-01 | 2010-11-24 | 首都医科大学附属北京安贞医院 | Method for detecting mononucleotide polymorphism rs2236055 locus of hypertension susceptibility gene and kit thereof |
| CN106755560A (en) * | 2017-03-30 | 2017-05-31 | 德必碁生物科技（厦门）有限公司 | A kind of multiple fluorescence PCR method detects the kit of hypertension medication gene pleiomorphism |
-
2017
- 2017-08-21 CN CN201710720710.5A patent/CN107557432A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101067152A (en) * | 2007-02-06 | 2007-11-07 | 上海生物芯片有限公司 | Pharmacogenetic detection chip for cardiac and cerebral vascular diseases and its application |
| CN101892298A (en) * | 2010-02-01 | 2010-11-24 | 首都医科大学附属北京安贞医院 | Method for detecting mononucleotide polymorphism rs2236055 locus of hypertension susceptibility gene and kit thereof |
| CN101886129A (en) * | 2010-06-30 | 2010-11-17 | 首都医科大学附属北京安贞医院 | Method for detecting mononucleotide polymorphism locus rs388915 of hypertension susceptibility genes AGTR 1 and detection kit |
| CN106755560A (en) * | 2017-03-30 | 2017-05-31 | 德必碁生物科技（厦门）有限公司 | A kind of multiple fluorescence PCR method detects the kit of hypertension medication gene pleiomorphism |
Non-Patent Citations (2)
| Title |
|---|
| YAN-HONG PAN等: "ACE Gene I/D Polymorphism and Obesity in 1,574 Patients with Type 2 Diabetes Mellitus", 《DIS MARKERS》 * |
| 薛探等: "ACE基因(I/D)多态性与家族性原发性高血压的关系", 《心血管康复医学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977531A (en) * | 2018-09-05 | 2018-12-11 | 武汉康录生物技术股份有限公司 | A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application |
| CN109355368A (en) * | 2018-10-22 | 2019-02-19 | 江苏美因康生物科技有限公司 | A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism |
| CN109182509A (en) * | 2018-10-29 | 2019-01-11 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes |
| CN109182509B (en) * | 2018-10-29 | 2022-05-06 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting polymorphic sites of hypertension-related drug genes |
| CN112608989A (en) * | 2020-12-18 | 2021-04-06 | 成都和合医学检验所有限公司 | Primer group, kit and method for detecting gene polymorphism |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112080563B (en) | Kit for detecting accurate medication genes of chronic diseases | |
| CN107557432A (en) | A kind of primer sets and detection kit for detecting hypertension medication related gene polymorphism | |
| CN107034273B (en) | CYP2C19 and ABCB1 gene detecting kits | |
| JP2010521156A (en) | System and method for detection of HIV drug resistant variants | |
| CN106755560A (en) | A kind of multiple fluorescence PCR method detects the kit of hypertension medication gene pleiomorphism | |
| CN106148498B (en) | KRAS gene mutation detection kit and application thereof | |
| CN105506118A (en) | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method | |
| CN109777871B (en) | Primer group and kit for detecting SNP (single nucleotide polymorphism) of susceptibility gene related to deafness | |
| CN107447030A (en) | The kit of Alzheimer's disease APOE genetic tests | |
| CN107513578A (en) | A kind of nucleic acid Mass Spectrometry detection method early sieved for lung cancer driving gene and tumor susceptibility gene | |
| CN111808944A (en) | Gene detection method for children personalized medicine | |
| CN113736878A (en) | Gene panel for detecting nervous system tumor, kit and application thereof | |
| CN108823301A (en) | It is a kind of for detecting the multiple PCR detection kit of people's drug gene polymorphism | |
| CN112458166A (en) | Method for optimizing rheumatoid disease gene SNP locus typing | |
| CN107022611B (en) | Method and special primer for detecting accurate medication of 4 common clinical cardiovascular and cerebrovascular disease medicines | |
| CN113528638B (en) | Primer group for amplifying personalized medicine gene locus of children, primer group for detection and application of primer group | |
| CN113584147A (en) | Gene polymorphism detection kit for guiding medication of psychosis | |
| Hsiao et al. | Detection and genetic characterization of the novel torque teno virus group 6 in Taiwanese general population | |
| CN111621553A (en) | Reagent for detecting NPC1L1 mutant genotyping and application thereof | |
| Veneri et al. | Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance | |
| CN106947815B (en) | Method and special primer for detecting accurate medication of aspirin and nitroglycerin | |
| CN113981069B (en) | Primer and kit for detecting ADRB1 gene G1165C polymorphism, and detection method and application thereof | |
| Nishida et al. | Genome-wide association study reveals host genetic factors for liver diseases | |
| CN107988356A (en) | A kind of primer sets and detection kit for detecting hypertension relative gene polymorphism | |
| CN111197080A (en) | Detection product for distinguishing nifedipine individualized medication type |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |