CN108531579A - Primer composition and kit for detecting sulfonylureas related gene - Google Patents
Primer composition and kit for detecting sulfonylureas related gene Download PDFInfo
- Publication number
- CN108531579A CN108531579A CN201810388083.4A CN201810388083A CN108531579A CN 108531579 A CN108531579 A CN 108531579A CN 201810388083 A CN201810388083 A CN 201810388083A CN 108531579 A CN108531579 A CN 108531579A
- Authority
- CN
- China
- Prior art keywords
- primer
- sulfonylureas
- kit
- related gene
- cyp2c9
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the Primer composition and kit for detecting sulfonylureas related gene, Primer composition includes CYP2C9 primer sets, KCNJ11 primer sets and ABCC8 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 sulfonylureas curative effect related gene (CYP2C9, KCNJ11 and ABCC8) gene pleiomorphism kit, the kit includes specificity ARMS detection primers, internal control primer and PCR reaction solution, by designing ARMS primers and Scorpions probes being changed to SYBR dyestuffs, so that testing cost substantially reduces, it is more suitable for Chinese patients CYP2C9, the detection of KCNJ11 and ABCC8 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
Technical field
The invention belongs to technical field of gene detection, are related to a kind of primer for detecting sulfonylureas related gene
Composition and kit.
Background technology
Sulfonylureas (sulfonylureas, SU) is one of the main oral drugs for treating diabetes B, is passed through
Beta Cell of islet potassium channel is set to close in conjunction with sulfonylurea receptor, to stimulate insulin secretion.The study found that sulfonylurea medicine
The drug effect individual difference of object is larger, and about 10%-20% patient's curative effect is higher than average level, but still has 10%-20% patient most
Just take treatment failure after sulfonylureas.Studies have pointed out that occur this phenomenon mostly be due to act on the channels KATP,
The CYP2C9 of the Kir6.2 and SUR1 receptors and metabolism sulfonylureas that are encoded respectively by KCNJ11 and ABCC8 morphs
It is caused.
CYP2C9 is drug enzyme metabolic gene common in CYP450 families, and common allele is with wild type
CYP2C9*1 and saltant type CYP2C9*2 and CYP2C9*3 are most commonly seen.The study found that the activity of CYP2C9*2 is only normal work(
The activity of 40% or so, the CYP2C9*3 of energy person is only normal function the 10% of person, and mutation patient can make sulfonylureas
Renal clearance significantly reduces, and drug half-life is obviously prolonged.
Sulfonylurea drugs act on the ATP sensitive potassium channels (KATP) on beta Cell of islet film, to close potassium channel,
Beta Cell of islet is stimulated to discharge insulin.ATP sensitive potassium channels form：Subunit sulfonylureas receptor (SUR)+subunit inward rectification
Potassium channel (Kir).The study found that the E23K polymorphisms of the gene KCNJ11 of encoded K ir6.2 may influence β cells to sulfonylureas
The reaction of class drug increases the risk that secondary failure occurs to reduce the secretion of insulin.
ABCC8 gene code SUR1 receptors, sulfonylureas with SUR1 receptors by being combined, to make potassium channel close
It closes, stimulates the secretion of insulin, and then generate hypoglycemic effect.The study found that ABCC8 gene S1369A loci polymorphisms with
The effect of sulfonylureas, is related, and site G allele carrier is more sensitive to sulfonylurea, and therapeutic effect is better than T
Allelotype carrier.
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 sulphonyl
The Primer composition and kit of carbamide type medicine related gene.
The purpose of the present invention can be achieved through the following technical solutions：
Primer composition for detecting sulfonylureas related gene, the Primer composition include CYP2C9 primers
Group, KCNJ11 primer sets and ABCC8 primer sets；
The CYP2C9 primer sets include following primer：
P1-FW：5’-AGAGGAGCATTGAGGAAC-3’；
P1-FS：5’-AAGAGGAGCATTGAGGACT-3’；
P1-R：5’-TAACAACCAGGACTCAT-3’；
P2-FW：5’-GCACGAGGTCCAGAGATAGA-3’；
P2-FS：5’-GCACGAGGTCCAGAGATAGC-3’；
P2-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
The KCNJ11 primer sets include following primer：
P3-F：5’-GCACGGTACCTGGGCAC-3’；
P3-RW：5’-GCACGGTACCTGGGCAT-3’；
P3-RS：5’-CACCGAGAGGACTCTGCA-3’；
The ABCC8 primer sets include following primer：
P4-FW：5’-CGTCAATGCCCTCATGT-3’；
P4-FS：5’-CGTCAATGCCCTCATGT-3’；
P4-R：5’-ACTGCGATGTCTGAATA-3’.
Further, which further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
The Primer composition answering in the genetic polymorphism detection reagent for preparing sulfonylureas related gene
With.
Kit for detecting sulfonylureas related gene, the kit include 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 4 sites in kit as a preferred technical solution,
Plasmid mixed liquor, in plasmid mixed liquor containing 4 kinds of different CYP2C9, KCNJ11 with mutational site to be detected and
ABCC8 allele, the plasmid concentration in plasmid mixed liquor are 1000copies/ μ l；The negative controls are Tris-
HCL buffer solutions, a concentration of 7-13mM of the Tris-HCL buffer solutions, pH 7.5-8.5.
The gene pleiomorphism detecting method of sulfonylureas related gene (CYP2C9, KCNJ11 and ABCC8), including with
Lower step：
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, divides wild type and mutated genes sequence using each guiding region, passes through SYBR GreenI dyestuffs
With the hybridization of amplified production, the SYBR fluorescence intensities of reaction system are detected 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 sulfonylureas related gene of the present invention uses SYBR dye methods, establishes
For the Multiplex real-time PCR inspection of the gene pleiomorphism (as shown in table 1) of mankind's related gene (CYP2C9, KCNJ11 and ABCC8)
Survey method.The present invention pass through a large number of experiments, research and analysis successfully filter out can be used in it is quick, sensitive, be effectively detected
The Primer composition of CYP2C9, KCNJ11 and ABCC8 gene pleiomorphism, and gone out for detecting sulfonylureas using these primer developments
The method and kit of class pharmaceutical relevant gene polymorphism.
The gene pleiomorphism form of 1 sulfonylureas related gene of table
The specific primer of sulfonylureas related gene see the table below 2-5, and table 6 is the detection primer of internal reference.
The detection primer of table 2 CYP2C9 (430) gene pleiomorphism
The detection primer of table 3 CYP2C9 (1075) gene pleiomorphism
The detection primer of table 4 KCNJ11 (23) gene pleiomorphism
The detection primer of table 5 ABCC8 (1369) gene pleiomorphism
The detection primer of 6 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 the kit of sulfonylureas curative effect related gene (CYP2C9, KCNJ11 and ABCC8) gene pleiomorphism, the kit
Including specific ARMS detection primers, internal control primer and PCR reaction solution, by designing ARMS primers and visiting Scorpions
Needle is changed to SYBR dyestuffs, so that testing cost substantially reduces, it is more to be more suitable for Chinese patients CYP2C9, KCNJ11 and ABCC8 gene
The detection of state property, have detection quickly, high sensitivity, high specificity, method be simple, the accurate advantage of result, answered suitable for promoting
With；
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 CYP2C9 (430) sample；
Fig. 2 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects CYP2C9 (1075) sample；
Fig. 3 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects KCNJ11 (23) sample；
Fig. 4 is the amplification curve diagram that genetic polymorphism detection kit of the present invention detects ABCC8 (1369) 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 7 is calculated：
7 PCR reaction systems of table are with tabulation
9 sterile centrifugation tubes are taken to prepare above-mentioned 9 reaction systems, vortex oscillation 10 seconds, 2000rpm after reagent is all added
Centrifugation 15 seconds.Then 23 μ L/ pipes of above-mentioned mixed liquor are dispensed into PCR reaction tubes (sterile and RNase-Free).
3, it is loaded：
8 sample of table and quality-control product loading table
| Title | Ingredient | Sample-adding amount |
| Sample to be checked | Sample DNA | 2μL |
| STD1 | Positive quality control product P1 | 2μL |
| STD2 | Positive quality control product P2 | 2μL |
| STD3 | Positive quality control product P3 | 2μL |
| STD4 | Positive quality control product P4 | 2μL |
| STD5 | Positive quality control product P5 | 2μL |
| STD6 | Positive quality control product P6 | 2μL |
| STD7 | Positive quality control product P7 | 2μL |
| STD8 | Positive quality control product P8 | 2μL |
| NTC | Negative quality-control product P9 | 2μL |
Note：STD represents positive quality control product；NTC represents negative quality-control product.
According to the loading ratio that table 8 prompts, processed sample, positive quality control product and negative quality-control product are separately added into reaction
Guan Zhong, internal control primer need to only detect sample DNA, cover tightly pipe lid (bubble is avoided to generate), and 2000rpm centrifuges 15 seconds by tube wall
On liquid all get rid of to tube bottom, carry out pcr amplification reaction immediately after.
4, PCR amplification program is arranged：
The setting of 9 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.SYBR is detection signal, reaches the cycle-index Ct values needed for the threshold value of setting as sentencing using SYBR signals
Disconnected standard, Ct values are less than 32 for the positive, and Ct values are more than 35 for feminine gender, and Ct values are weakly positive between 32 to 35.
Embodiment 2：
Primer composition for detecting sulfonylureas related gene includes CYP2C9 primer sets, KCNJ11 primer sets
And ABCC8 primer sets；
CYP2C9 primer sets include following primer：
P1-FW：5’-AGAGGAGCATTGAGGAAC-3’；
P1-FS：5’-AAGAGGAGCATTGAGGACT-3’；
P1-R：5’-TAACAACCAGGACTCAT-3’；
P2-FW：5’-GCACGAGGTCCAGAGATAGA-3’；
P2-FS：5’-GCACGAGGTCCAGAGATAGC-3’；
P2-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
KCNJ11 primer sets include following primer：
P3-F：5’-GCACGGTACCTGGGCAC-3’；
P3-RW：5’-GCACGGTACCTGGGCAT-3’；
P3-RS：5’-CACCGAGAGGACTCTGCA-3’；
ABCC8 primer sets include following primer：
P4-FW：5’-CGTCAATGCCCTCATGT-3’；
P4-FS：5’-CGTCAATGCCCTCATGT-3’；
P4-R：5’-ACTGCGATGTCTGAATA-3’.
Above-mentioned Primer composition is applied in the genetic polymorphism detection reagent for preparing sulfonylureas related gene.
Kit for detecting sulfonylureas related gene includes above-mentioned Primer composition and PCR reaction solution, is somebody's turn to do
PCR reaction solution includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I dyestuffs.
Embodiment 3：
Primer composition for detecting sulfonylureas related gene includes CYP2C9 primer sets, KCNJ11 primer sets
And ABCC8 primer sets；
CYP2C9 primer sets include following primer：
P1-FW：5’-AGAGGAGCATTGAGGAAC-3’；
P1-FS：5’-AAGAGGAGCATTGAGGACT-3’；
P1-R：5’-TAACAACCAGGACTCAT-3’；
P2-FW：5’-GCACGAGGTCCAGAGATAGA-3’；
P2-FS：5’-GCACGAGGTCCAGAGATAGC-3’；
P2-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
KCNJ11 primer sets include following primer：
P3-F：5’-GCACGGTACCTGGGCAC-3’；
P3-RW：5’-GCACGGTACCTGGGCAT-3’；
P3-RS：5’-CACCGAGAGGACTCTGCA-3’；
ABCC8 primer sets include following primer：
P4-FW：5’-CGTCAATGCCCTCATGT-3’；
P4-FS：5’-CGTCAATGCCCTCATGT-3’；
P4-R：5’-ACTGCGATGTCTGAATA-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 sulfonylureas related gene.
Kit for detecting sulfonylureas related gene includes above-mentioned Primer composition and PCR reaction solution, is somebody's turn to do
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. for detecting the Primer composition of sulfonylureas related gene, which is characterized in that the Primer composition includes
CYP2C9 primer sets, KCNJ11 primer sets and ABCC8 primer sets；
The CYP2C9 primer sets include following primer：
P1-FW：5’-AGAGGAGCATTGAGGAAC-3’；
P1-FS：5’-AAGAGGAGCATTGAGGACT-3’；
P1-R：5’-TAACAACCAGGACTCAT-3’；
P2-FW：5’-GCACGAGGTCCAGAGATAGA-3’；
P2-FS：5’-GCACGAGGTCCAGAGATAGC-3’；
P2-R：5’-CGGTGATGGTAGAGGTTTAA-3’；
The KCNJ11 primer sets include following primer：
P3-F：5’-GCACGGTACCTGGGCAC-3’；
P3-RW：5’-GCACGGTACCTGGGCAT-3’；
P3-RS：5’-CACCGAGAGGACTCTGCA-3’；
The ABCC8 primer sets include following primer：
P4-FW：5’-CGTCAATGCCCTCATGT-3’；
P4-FS：5’-CGTCAATGCCCTCATGT-3’；
P4-R：5’-ACTGCGATGTCTGAATA-3’.
2. the Primer composition according to claim 1 for detecting sulfonylureas related gene, which is characterized in that
The Primer composition further includes internal control primer sets, which includes following primer：
CF：5’-AGCAAGCAGGAGTATGACG-3’；
CR：5’-GAAAGGGTGTAACGCAACT-3’.
3. Primer composition as claimed in claim 1 or 2 is in the gene pleiomorphism inspection for preparing sulfonylureas related gene
Application in test agent.
4. the kit for detecting sulfonylureas related gene, which is characterized in that the kit includes such as claim 1
Or the Primer composition described in 2.
5. the kit according to claim 4 for detecting sulfonylureas related gene, which is characterized in that the examination
Agent box further includes PCR reaction solution, which includes PCR buffer solutions, Taq enzyme, MgCl2, dNTPs and SYBR Green I
Dyestuff.
6. the kit according to claim 4 for detecting sulfonylureas related gene, which is characterized in that the examination
Agent box further includes positive control solution and negative controls.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810388083.4A CN108531579A (en) | 2018-04-26 | 2018-04-26 | Primer composition and kit for detecting sulfonylureas related gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810388083.4A CN108531579A (en) | 2018-04-26 | 2018-04-26 | Primer composition and kit for detecting sulfonylureas related gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108531579A true CN108531579A (en) | 2018-09-14 |
Family
ID=63478177
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810388083.4A Pending CN108531579A (en) | 2018-04-26 | 2018-04-26 | Primer composition and kit for detecting sulfonylureas related gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108531579A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066868A (en) * | 2019-04-08 | 2019-07-30 | 大连美纳医学检验实验室有限公司 | A kind of primer sets, kit and the detection method of aspirin pharmaceutical relevant gene genetic polymorphism detection |
| CN110066869A (en) * | 2019-04-08 | 2019-07-30 | 大连美纳医学检验实验室有限公司 | A kind of primer sets, kit and the detection method of Warfarin drug related gene polymorphic detection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008032A (en) * | 2006-01-26 | 2007-08-01 | 北京华安佛医药研究中心有限公司 | Uses and method for polymorphic point genetype for predicting sulfaurea drugs effect |
| CN101445824A (en) * | 2007-11-28 | 2009-06-03 | 北京华安佛医药研究中心有限公司 | Primer for detecting sulfonylureas receptor 1 gene polymorphism sites, the application thereof and kits thereof |
| CN107868819A (en) * | 2016-09-20 | 2018-04-03 | 宁波美丽人生医药生物科技发展有限公司 | Fluorouracil curative effect related gene genetic polymorphism detection kit and detection method |
-
2018
- 2018-04-26 CN CN201810388083.4A patent/CN108531579A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008032A (en) * | 2006-01-26 | 2007-08-01 | 北京华安佛医药研究中心有限公司 | Uses and method for polymorphic point genetype for predicting sulfaurea drugs effect |
| CN101445824A (en) * | 2007-11-28 | 2009-06-03 | 北京华安佛医药研究中心有限公司 | Primer for detecting sulfonylureas receptor 1 gene polymorphism sites, the application thereof and kits thereof |
| CN107868819A (en) * | 2016-09-20 | 2018-04-03 | 宁波美丽人生医药生物科技发展有限公司 | Fluorouracil curative effect related gene genetic polymorphism detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| J. KLEN ET AL: "CYP2C9, KCNJ11 and ABCC8 polymorphisms and the response to sulphonylurea treatment in type 2 diabetes patients", 《EUR J CLIN PHARMACOL》 * |
| 孙冰等: "乳腺癌冰冻和石蜡包埋样本的RNA以及基因表达情况的比较研究", 《临床肿瘤学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066868A (en) * | 2019-04-08 | 2019-07-30 | 大连美纳医学检验实验室有限公司 | A kind of primer sets, kit and the detection method of aspirin pharmaceutical relevant gene genetic polymorphism detection |
| CN110066869A (en) * | 2019-04-08 | 2019-07-30 | 大连美纳医学检验实验室有限公司 | A kind of primer sets, kit and the detection method of Warfarin drug related gene polymorphic detection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102925562B (en) | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene | |
| CN102534031B (en) | High-specificity kit for detecting deafness predisposing genes and uses | |
| CN102010894B (en) | Nucleotide sequence, method and kit for detecting exons 12, 13 mutation of human K-ras gene | |
| CN103074434A (en) | CYP2C19 gene polymorphyism detection kit and detection method thereof | |
| CN104328164A (en) | Kit for detecting human EGFR gene mutation by using fluorescence probe hybridization method | |
| CN106755360B (en) | Nucleic acid, kit and method for detecting human CYP2D6 gene polymorphism | |
| CN114085903A (en) | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof | |
| CN110699440A (en) | Primer and method for detecting SNP (single nucleotide polymorphism) locus of gene related to metformin personalized medicine | |
| CN108531579A (en) | Primer composition and kit for detecting sulfonylureas related gene | |
| CN106119381A (en) | A kind of CYP2C19 genetic polymorphism detection test kit and detection method | |
| CN107653317A (en) | A kind of kit of molecular beacon probe detection mankind's CYP2C9 gene pleiomorphisms, method and its application | |
| CN110257496A (en) | Clopidogrel pharmaceutical relevant gene CYP2C19 genetic polymorphism detection primer sets, kit and detection method | |
| CN110066868A (en) | A kind of primer sets, kit and the detection method of aspirin pharmaceutical relevant gene genetic polymorphism detection | |
| CN108315398A (en) | A kind of Primer composition and kit for detecting biguanides related gene | |
| CN102534030B (en) | Kit for jointly detecting four deafness predisposing genes and application thereof | |
| CN108546753A (en) | Baclofen pharmaceutical relevant gene GABBR1 genetic polymorphism detection kits | |
| CN106636365B (en) | Nucleic acid, kit and method for detecting A1166C polymorphic site of AGTR1 gene | |
| CN107868819A (en) | Fluorouracil curative effect related gene genetic polymorphism detection kit and detection method | |
| CN112029851A (en) | Method and kit for detecting gene polymorphism of clopidogrel medication and application of kit | |
| CN111690736A (en) | Warfarin medication gene detection kit and use method thereof | |
| CN108315409A (en) | A kind of Primer composition and kit for detecting folic acid metabolism related gene | |
| CN108315408A (en) | Primer composition and kit for detecting nitroglycerin pharmaceutical relevant gene | |
| CN104046687A (en) | Leber's hereditary optic neuropathy gene diagnosis kit or reagent | |
| CN116970704B (en) | Primer probe combination for detecting mutation of BCR-ABL kinase region and application thereof | |
| CN107841557A (en) | Drug taxol curative effect related gene genetic polymorphism detection kit and detection method |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180914 |