CN107794298A - A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application - Google Patents
A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application Download PDFInfo
- Publication number
- CN107794298A CN107794298A CN201711101301.3A CN201711101301A CN107794298A CN 107794298 A CN107794298 A CN 107794298A CN 201711101301 A CN201711101301 A CN 201711101301A CN 107794298 A CN107794298 A CN 107794298A
- Authority
- CN
- China
- Prior art keywords
- cyp2c19
- detection
- detection probe
- genotypings
- type detection
- 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
- 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
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application, belong to field of medical examination.The Nucleic acid combinations of CYP2C19 Genotypings detection provided by the invention exactly, specifically can participate in reacting, it is simple to quickly finish typing assay detection, the Nucleic acid combinations that CYP2C19 Genotypings detect are applied in the detection kit for preparing the detection of CYP2C19 Genotypings, obtained inspection kit, detection reaction can more easily be carried out, Genotyping is rapidly performed by, there is larger use value and social value.
Description
Technical field
The present invention relates to field of medical examination, in particular to a kind of Nucleic acid combinations of CYP2C19 Genotypings detection
And detection kit and application.
Background technology
Clopidogrel (Plavix) is used as oral anti-diabetic agent thing, is widely used in preventing and treating myocardial infarction, ischemic brain blood
The diseases such as bolt, thromboembolism, atherosclerosis and obliterans.Itself it is adiaphorous pro-drug, after oral
About 85% clopidogrel is by esterase hydrolyzed, only 15% cytochrome P 450 Enzyme of the medicine through liver
(cytochromeP450, CYP) plays a role after transforming into active component.CYP2C19 is a kind of highly important medicine generation
Thank to enzyme, be primarily present in liver microsomal body, many Endogenous Substrates, environmental contaminants and clinically about 2% medicine
All it is metabolized by its catalysis.FDA has added black surround warning in the packaging of Plavix (clopidogrel), it is desirable to which clinic makes
With detection patient's CYP2C19 genotype before Plavix.
Method currently used for SNP mutation detection includes：Direct sequencing, ARMSPCR methods, digestion (PCR-RLFR) method,
DNA chip method, TaqMan-MGB probe typings etc..Such as reagent testing cost in popularization all more or less be present in these methods
Height, complex operation, pollution is also easy to produce, the defects of producing certain false negative and false positive.
The content of the invention
The first object of the present invention is to provide a kind of Nucleic acid combinations of CYP2C19 Genotypings detection, the Nucleic acid combinations
It sensitive, specific can be reacted, parting carried out to CYP2C19 genes.
The Nucleic acid combinations that the second object of the present invention is to provide above-mentioned CYP2C19 Genotypings detection are in CYP2C19
Application in Genotyping detection, using for the purpose of non-diagnostic or non-treatment.
It is prepared by the Nucleic acid combinations that the third object of the present invention is to provide above-mentioned CYP2C19 Genotypings detection
Application in the detection kit of CYP2C19 Genotypings detection.
The fourth object of the present invention is to provide a kind of detection kit of CYP2C19 Genotypings detection, detection examination
Agent box can conveniently and efficiently be used for CYP2C19 Genotypings.
The detection kit that the fifth object of the present invention is to provide above-mentioned CYP2C19 Genotypings detection exists
Application in the detection of CYP2C19 Genotypings, using for the purpose of non-diagnostic or non-treatment.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
A kind of Nucleic acid combinations of CYP2C19 Genotypings detection, the detection primer that Nucleic acid combinations includes combines and detection probe
Combination；
Detection primer combination includes the first primer pair, the second primer pair, three-primer pair and the 4th primer pair；First primer
To base sequence respectively as shown in SEQ ID No.1-2, the base sequence of the second primer pair is respectively such as SEQ ID No.3-4 institutes
Show, the base sequence of three-primer pair is respectively as shown in SEQ ID No.5-6, and the base sequence of the 4th primer pair is respectively such as SEQ
Shown in ID No.7-8；
Detection probe combination includes the first wild type detection probe, the first saltant type detection probe, the detection of the second wild type
Probe, the second saltant type detection probe, the 3rd wild type detection probe, the 3rd saltant type detection probe, the detection of the 4th wild type
Probe and the 4th saltant type detection probe；For the base sequence of first wild type detection probe as shown in SEQ ID No.9, first is prominent
The base sequence of modification detection probe is as shown in SEQ ID No.10；The base sequence of second wild type detection probe such as SEQ ID
Shown in No.11, the base sequence of the second saltant type detection probe is as shown in SEQ ID No.12；3rd wild type detection probe
Base sequence as shown in SEQ ID No.13, the base sequence of the 3rd saltant type detection probe is as shown in SEQ ID No.14；
The base sequence of 4th wild type detection probe is as shown in SEQ ID No.15, and the base sequence of the 4th saltant type detection probe is such as
Shown in SEQ ID No.16；
First wild type detection probe, the second wild type detection probe, the 3rd wild type detection probe and the 4th wild type
5 ' ends of detection probe are marked with the first fluorophor, and 3 ' ends are marked with the second fluorescer group；First saltant type detection probe,
5 ' ends of the second saltant type detection probe, the 3rd saltant type detection probe and the 4th saltant type detection probe are marked with the 3rd fluorescence
Group, 3 ' ends are marked with the second fluorescer group.
Application of the Nucleic acid combinations of above-mentioned CYP2C19 Genotypings detection in the detection of CYP2C19 Genotypings, application
For the purpose of non-diagnostic or non-treatment.
The Nucleic acid combinations of above-mentioned CYP2C19 Genotypings detection are preparing the detection reagent of CYP2C19 Genotypings detection
Application in box.
A kind of detection kit of CYP2C19 Genotypings detection, CYP2C19 gene parting detecting reagents include above-mentioned
CYP2C19 Genotypings detection Nucleic acid combinations.
Application of the detection kit of above-mentioned CYP2C19 Genotypings detection in the detection of CYP2C19 Genotypings, should
For the purpose of non-diagnostic or non-treatment.
Compared with prior art, beneficial effects of the present invention are：CYP2C19 Genotypings detection provided by the invention
Nucleic acid combinations exactly, specifically can participate in reacting, and typing assay detection simply be quickly finished, by CYP2C19 Genotypings
The Nucleic acid combinations of detection are applied in the detection kit for preparing the detection of CYP2C19 Genotypings, obtained inspection kit,
Detection reaction can be more easily carried out, is rapidly performed by Genotyping, there is larger use value and social value.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below by embodiment it is required use it is attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the wild type CYP2C19*2 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 2 is the saltant type CYP2C19*2 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 3 is the mixed type CYP2C19*2 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 4 is the wild type CYP2C19*3 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 5 is the saltant type CYP2C19*3 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 6 is the mixed type CYP2C19*3 amplified reaction curve maps that experimental example 1 of the present invention provides；
Fig. 7 is wild type CYP2C19*17 (C-806T) amplified reaction curve map that experimental example 1 of the present invention provides；
Fig. 8 is saltant type CYP2C19*17 (C-806T) amplified reaction curve map that experimental example 1 of the present invention provides；
Fig. 9 is mixed type CYP2C19*17 (C-806T) amplified reaction curve map that experimental example 1 of the present invention provides；
Figure 10 is wild type CYP2C19*17 (C-3402T) amplified reaction curve map that experimental example 1 of the present invention provides；
Figure 11 is saltant type CYP2C19*17 (C-3402T) amplified reaction curve map that experimental example 1 of the present invention provides；
Figure 12 is mixed type CYP2C19*17 (C-3402T) amplified reaction curve map that experimental example 1 of the present invention provides.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
The Nucleic acid combinations and detection kit that are detected below to a kind of CYP2C19 Genotypings of the embodiment of the present invention and should
With being specifically described.
At least 18 kinds of gene pleiomorphisms, wherein CYP2C19*2 types (681G be present in CYP2C19 genes>) and CYP2C19*3 A
Type (636G>A) cause the enzymatic activity of CYP2C19 gene codes to be lost, the reduced capability of metabolism substrate, receive recommended dose ripple
During vertical dimension treatment, cardiovascular event incidence rises compared with the normal patient of CYP2C19 genes.CYP2C19*17 (-806C>T；-
3402C>T) mutation can cause the abnormal enhancing of the activity of CYP2C19 enzymes so that the patient for taking clopidogrel medicine increases medication
The risk of bleeding afterwards.
A kind of Nucleic acid combinations of CYP2C19 Genotypings detection, the detection primer that Nucleic acid combinations includes combines and detection probe
Combination；
Detection primer combination includes the first primer pair, the second primer pair, three-primer pair and the 4th primer pair；First primer
To base sequence respectively as shown in SEQ ID No.1-2, the base sequence of the second primer pair is respectively such as SEQ ID No.3-4 institutes
Show, the base sequence of three-primer pair is respectively as shown in SEQ ID No.5-6, and the base sequence of the 4th primer pair is respectively such as SEQ
Shown in ID No.7-8；
Detection probe combination includes the first wild type detection probe, the first saltant type detection probe, the detection of the second wild type
Probe, the second saltant type detection probe, the 3rd wild type detection probe, the 3rd saltant type detection probe, the detection of the 4th wild type
Probe and the 4th saltant type detection probe；For the base sequence of first wild type detection probe as shown in SEQ ID No.9, first is prominent
The base sequence of modification detection probe is as shown in SEQ ID No.10；The base sequence of second wild type detection probe such as SEQ ID
Shown in No.11, the base sequence of the second saltant type detection probe is as shown in SEQ ID No.12；3rd wild type detection probe
Base sequence as shown in SEQ ID No.13, the base sequence of the 3rd saltant type detection probe is as shown in SEQ ID No.14；
The base sequence of 4th wild type detection probe is as shown in SEQ ID No.15, and the base sequence of the 4th saltant type detection probe is such as
Shown in SEQ ID No.16；
First wild type detection probe, the second wild type detection probe, the 3rd wild type detection probe and the first wild type
5 ' ends of detection probe are marked with the first fluorophor, and 3 ' ends are marked with the second fluorescer group；First saltant type detection probe,
5 ' ends of the second saltant type detection probe, the 3rd saltant type detection probe and the 4th saltant type detection probe are marked with the 3rd fluorescence
Group, 3 ' ends are marked with the second fluorescer group.
When being reacted, the first wild type detection probe, the first saltant type detection probe and the first primer pair are same
When use；Second wild type detection probe, the second saltant type detection probe use simultaneously with the second primer pair；3rd wild type is examined
Probing pin, the 3rd saltant type detection probe use simultaneously with three-primer pair；4th wild type detection probe, the inspection of the 4th saltant type
Probing pin uses simultaneously with the 4th primer pair；Four pairs of different primer pairs, for different sites, and different probes, then may be used
To reflect different genotype；Due to the wild type detection probe and the fluorescence of saltant type detection probe mark of different genotype
Group is different, therefore produces different fluorescence；By different fluorescence signals it may determine that going out different genotype, carry out quick
Genotyping.First primer pair corresponds to CYP2C19*2 genes；Second primer pair corresponds to CYP2C19*3 genes；Three-primer pair
CYP2C19*17 genes are corresponded to the 4th primer pair.
Further, in presently preferred embodiments of the present invention, the first fluorophor be FAM, MGB, VIC, TET, JOE, HEX,
One kind in CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED；Second fluorophor be FAM, MGB, VIC,
One kind in TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED；3rd fluorophor is
One kind in FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED；The
One fluorophor, the second fluorophor and the 3rd fluorophor differ.
A kind of fluorophor is selected when the first fluorophor, the second fluorophor and the 3rd fluorophor difference, is conveniently entered
Row fluorescence is distinguished, and is more beneficial for the progress of detection.
Application of the Nucleic acid combinations of above-mentioned CYP2C19 Genotypings detection in the detection of CYP2C19 Genotypings.
Further, in presently preferred embodiments of the present invention, enter in performing PCR reaction, the first wild type detection probe and first
The ratio between concentration of saltant type detection probe is 1-3:1；The concentration of second wild type detection probe and the second saltant type detection probe
The ratio between be 1:1-3；The ratio between concentration of 3rd wild type detection probe and the 3rd saltant type detection probe is 1-2:1；4th is wild
The ratio between concentration of type detection probe and the 4th saltant type detection probe is 1:1-3.
By the rational concentration proportion for setting saltant type probe and wild-type probe, it is possible to increase the accuracy of detection.
The Nucleic acid combinations of above-mentioned CYP2C19 Genotypings detection are preparing the detection reagent of CYP2C19 Genotypings detection
Application in box.
A kind of detection kit of CYP2C19 Genotypings detection, CYP2C19 gene parting detecting reagents include above-mentioned
CYP2C19 Genotypings detection Nucleic acid combinations.
Further, in presently preferred embodiments of the present invention, in addition to PCR reaction buffers, Taq archaeal dna polymerases,
At least one of dNTPs.
In kit, including enter the reagent of performing PCR, sample can be carried out；It can disposably meet to detect needs
Some reagents, experiment flow is saved, shorten experimental period, improve the efficiency of detection.
Further, in presently preferred embodiments of the present invention, PCR reaction buffers are mainly by KCl, MgCl2, TrisHCl
It is made with water.
Further, in presently preferred embodiments of the present invention, detection kit also includes genome extracts reagent.
In kit directly provide extraction sample genome, the template that the genome of extraction is reacted as PCR,
Reacted；Detection cycle can equally be shortened, improve efficiency.
Application of the detection kit of above-mentioned CYP2C19 Genotypings detection in the detection of CYP2C19 Genotypings.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of Nucleic acid combinations of CYP2C19 Genotypings detection, and Nucleic acid combinations include detection primer group
Close and detection probe combines.
Detection primer combination includes the first primer pair, the second primer pair, three-primer pair and the 4th primer pair；First primer
To base sequence respectively as shown in SEQ ID No.1-2, the base sequence of the second primer pair is respectively such as SEQ ID No.3-4 institutes
Show, the base sequence of three-primer pair is respectively as shown in SEQ ID No.5-6, and the base sequence of the 4th primer pair is respectively such as SEQ
Shown in ID No.7-8.
Detection probe combination includes the first wild type detection probe, the first saltant type detection probe, the detection of the second wild type
Probe, the second saltant type detection probe, the 3rd wild type detection probe, the 3rd saltant type detection probe, the detection of the 4th wild type
Probe and the 4th saltant type detection probe；For the base sequence of first wild type detection probe as shown in SEQ ID No.9, first is prominent
The base sequence of modification detection probe is as shown in SEQ ID No.10；The base sequence of second wild type detection probe such as SEQ ID
Shown in No.11, the base sequence of the second saltant type detection probe is as shown in SEQ ID No.12；3rd wild type detection probe
Base sequence is as shown in SEQ ID No.13, and the base sequence of the 3rd saltant type detection probe is as shown in SEQ ID No.14；The
The base sequence of four wild type detection probes is as shown in SEQ ID No.15, and the base sequence of the 4th saltant type detection probe is such as
Shown in SEQ ID No.16.
First wild type detection probe, the second wild type detection probe, the 3rd wild type detection probe and the 4th wild type
5 ' ends of detection probe are marked with FAM fluorophors, and 3 ' ends are marked with MGB fluorophors；First saltant type detection probe, second
5 ' ends of saltant type detection probe, the 3rd saltant type detection probe and the 4th saltant type detection probe are marked with VIC fluorophors,
3 ' ends are marked with MGB fluorophors.
Certainly, in other embodiments of the invention, the first wild type detection probe, the second wild type detection probe,
The fluorophor of 5 ' end marks of three wild type detection probes and the 4th wild type detection probe can be used as the first fluorophor, also
Can select other kinds of fluorophor, for example, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610,
One kind in TEXASRED, RED670 and NED.
First wild type detection probe, the first saltant type detection probe, the second wild type detection probe, the inspection of the second saltant type
Probing pin, the 3rd wild type detection probe, the 3rd saltant type detection probe, the 4th wild type detection probe and the inspection of the 4th saltant type
3 ' ends of probing pin marked fluorophor MGB as the second fluorophor, can also similarly select other kinds of fluorescence
Group is marked, in FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED
One kind.
First saltant type detection probe, the second saltant type detection probe, the 3rd saltant type detection probe and the 4th saltant type
5 ' ends of detection probe are marked with VIC as the 3rd fluorophor, can also similarly select other kinds of fluorophor to carry out
Mark, such as one kind in FAM, MGB, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED.
In experimentation, from one kind when the first fluorophor, the second fluorophor and the 3rd fluorophor difference, and
The fluorophor selected between any two also differs, and ensures the accuracy of detection, while is also avoided that and interferes, and avoids the occurrence of
Phenomena such as false positive.
Embodiment 2
The present embodiment provides a kind of detection kit of CYP2C19 Genotypings detection, and kit contains embodiment 1 and provided
Nucleic acid combinations, the detection primer includes primer pair, and the base sequence of primer pair is respectively as shown in SEQ ID No.1-2.
CYP2C19 Genotypings detection detection kit also include PCR reaction buffers, Taq archaeal dna polymerases,
At least one of dNTPs.
PCR reaction buffers be 10 × reaction buffer, by 500mM KCl, 25mM MgCl2, 100mM Tris and
Double distilled deionized water is according to 1:1:1:Configuration forms after 2 autoclavings.
The detection kit of CYP2C19 Genotypings detection also includes genome extracts reagent.
The specifically used method of the detection kit for the CYP2C19 Genotypings detection that the present embodiment provides is as follows：
Sample genome extracts, and comprises the following steps that：
Sample preparation：Take the blood sample of person to be checked, preservation is standby.
Reagent preparation, including EDTA solution and SDS solution are made to the buffer solution LB of cell lysis, aqueous isopropanol
It is made and combines liquid I, TE solution is made with reference to liquid II, rinsing liquid W1 is made in Tris saturated phenols, chloroform isoamyl alcohol solution is made
Rinsing liquid W2.
1.1 take sample to be checked to centrifuge, and are then dissolved with PBS, and composition vibration mixes, and cell suspension is made, avoids
There is tissue agglomerate；
1.2 draw 1.8mL cell suspension, are added in EP pipes, 12000rpm centrifugations 2min；
1.3 abandon supernatant, collect the buffer solution LB that cell adds 200 μ L, and add 2 μ L Proteinase K Solution, and vibration is mixed
It is even；
1.4 digest 10min in 70 DEG C, brief centrifugation, purification column are placed in stand-by in collecting pipe；
1.5 add 200 μ L combination liquid I, add 200 μ L combination liquid II, overturn and mix, and solution is transferred to purification column
It is interior；
1.6 stand 2min, 12000rpm centrifugation 30s, the waste liquid abandoned in collecting pipe, and purification column is put back into collecting pipe；
1.7 add 600 μ L rinsing liquid W1,12000rpm centrifugation 30s into purification column, abandon waste liquid；
1.8 add 600 μ L rinsing liquid W2,12000rpm centrifugation 30s into purification column, abandon waste liquid；
1.9 add 600 μ L rinsing liquid W2,12000rpm centrifugation 2min into purification column, abandon waste liquid；
1.10 are transferred to purification column in other RNase-free EP pipes, and room temperature places 3-5min；
1.11 into purification column it is hanging add 50 eluent TE, stand 2min, 12000rpm centrifugation 2min, collect from
The Genomic DNA solution that gains in depth of comprehension arrive is stand-by.
The genomic DNA extracted in step 1.11 is taken as template, with the first primer pair, the second primer pair, three-primer
Pair and the 4th primer pair enter respectively performing PCR reaction.The present embodiment selects 25 μ L reaction system, the wherein μ of genomic DNA template 2
The μ L of L, PCR reaction buffer 2.5, the μ L of forward primer 0.75, reverse primer 0.75 μ L, MgCl2The μ L of solution 1.5, probe 2 μ L, Taq
The μ L of archaeal dna polymerase 0.5, then plus water complements to 25 μ L.By the first wild type detection probe and the first saltant type detection probe with
2:1 concentration ratio is added in the reaction system of the first primer pair；Second wild type detection probe and the detection of the second saltant type are visited
Pin is with 1:2 concentration ratio is added in the reaction system of the second primer pair；3rd wild type detection probe and the inspection of the 3rd saltant type
Probing pin is with 1:1 concentration ratio is added in the reaction system of three-primer pair；4th wild type detection probe and the 4th mutation
Type detection probe is with 1:2 concentration ratio is added in the reaction system of the 4th primer pair.
PCR response procedures are 95 DEG C of pre-degeneration 4min, and 95 DEG C of denaturation 15s, 60 DEG C are reacted 35s, 40 circulating collection fluorescence
Signal, cooling down.
The evaluation method of Genotyping is shown in Table 1.
The Analysis of test results of table 1
Experimental example 1
The specificity that this experimental example provides the Nucleic acid combinations in the kit provided embodiment 2 detects.
First primer pair corresponds to CYP2C19*2 genes, mutational site G681A；Second primer pair corresponds to CYP2C19*3 bases
Cause, mutational site G636A；Three-primer is corresponding to CYP2C19*3 genes, mutational site C-806T and the 4th primer pair
CYP2C19*17 gene mutation sites C-3402T.
Respectively using the corresponding wild plasmid in 4 sites, mutant plasmids and mixing plasmid as template, embodiment is used
2 detection kits provided are detected.PCR reaction systems and response procedures are with reference to embodiment 2.
CYP2C19*2 genes, mutational site are G681A experimental result as shown in Fig. 1 to Fig. 3；Fig. 1 is wild type matter
The experimental result of grain, the experimental result of Fig. 2 positions mutant plasmids, Fig. 3 are the experimental result of mixing plasmid.
CYP2C19*3 genes, mutational site are G636A experimental result as shown in Fig. 4 to Fig. 6；Fig. 4 is wild type matter
The experimental result of grain, Fig. 5 are the experimental result of mutant plasmids, and Fig. 6 is the experimental result of mixing plasmid.
CYP2C19*17 genes, mutational site are C-806T experimental result as shown in Fig. 7 to Fig. 9；Fig. 7 is wild type
The experimental result of plasmid, Fig. 8 are the experimental result of mutant plasmids, and Fig. 9 is the experimental result of mixing plasmid.
CYP2C19*17 genes, mutational site are C-3402T experimental result as shown in Figure 10 to Figure 12；Figure 10 is wild
The experimental result of type plasmid, Figure 11 are the experimental result of mutant plasmids, and Figure 12 is the experimental result of mixing plasmid.
From experimental result as can be seen that using this method, corresponding amplification curve can be preferably obtained, it is bent by expanding
The Ct values analysis of line, it is wild type, saltant type and heterozygous to obtain corresponding template, has preferable difference degree.
Experimental example 2
This experimental example provides to be detected to the sample to be checked of acquisition, the extraction of genomic DNA, PCR reaction systems and anti-
Program is answered with reference to embodiment 2.
14 kinds of mutators and 18 kinds of allelic mutations at least be present in CYP2C19.Encoding the gene of normal enzymatic activity is
CYP2C19*1, CYP2C19 allele are mainly * 1-*17；Wherein CYP2C19*2 types (681G>) and * 3 type (636G A>A) draw
The enzymatic activity for playing CYP2C19 gene codes is lost, the reduced capability of metabolism substrate, when receiving the treatment of recommended dose Plavix,
Cardiovascular event incidence rises compared with the normal patient of CYP2C19 genes.CYP2C19*17(-806C>T；-3402C>T) it is mutated
The abnormal enhancing of activity of CYP2C19 enzymes can be caused so that the patient for taking clopidogrel medicine increases the risk of bleeding after medication.
Therefore, the genotype information for detecting CYP2C19 may determine that metabolic rate of the patient to such medicine, instruct clopidogrel to use
Medicine.Specific genotype see the table below 2 with accretion rate correlation：
The correlation that the CYP2C19 different genotypes of table 2 are metabolized with clopidogrel
| Gene type | Accretion rate |
| CYP2C19*1/*1 | It hurry up |
| CYP2C19*1/*17 | It hurry up |
| CYP2C19*17/*17 | It hurry up |
| CYP2C19*1/*2 | It is medium |
| CYP2C19*1/*3 | It is medium |
| CYP2C19*2/*2 | Slowly |
| CYP2C19*3/*3 | Slowly |
Genotype is calculated in the Ct values that amplification curve is reacted by reacting PCR；Remaining 9 sample is similarly obtained experiment knot
Fruit.As a result it is as shown in table 3.
The analysis result of 3 10 parts of samples to be checked of table
From table 2 it can be seen that sample 1,2,3,5,7,9,10 belongs to clopidogrel medium metabolic type, sample 4,8 belongs to chlorine
The fast metabolic pattern of pyrrole Gray, sample 6 belong to clopidogrel slow inactivation.
In summary, the Nucleic acid combinations of CYP2C19 Genotypings detection provided in an embodiment of the present invention have preferably special
Different in nature and higher sensitivity, it is applied to detection using the detection primer, and is applied to prepare the detection of CYP2C19 Genotypings
The detection kit of Nucleic acid combinations, all there is preferably specific and higher sensitivity；With higher practicality and higher
Application value.
Embodiments described above is part of the embodiment of the present invention, rather than whole embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Surexam Biotechnology Co., Ltd.
<120>A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
cttagatatg caataatttt cccac 25
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
cactttccat aaaagcaagg ttttt 25
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
attgaatgaa aacatcagga ttg 23
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
caatatagaa ttttggattt cccag 25
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
gtttggaagt tgttttgttt tgc 23
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
acacagctca tagctggcag aact 24
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
caccttgatt aaaaaaatgg gc 22
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
tagcttgcag ttccctaatg ac 22
<210> 9
<211> 17
<212> DNA
<213>Artificial sequence
<400> 9
actatttccc gggaacc 17
<210> 10
<211> 14
<212> DNA
<213>Artificial sequence
<400> 10
tttcccggga accc 14
<210> 11
<211> 14
<212> DNA
<213>Artificial sequence
<400> 11
ctccccctgg atcc 14
<210> 12
<211> 14
<212> DNA
<213>Artificial sequence
<400> 12
ccccctgaat ccag 14
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<400> 13
catctgttct caaagcatc 19
<210> 14
<211> 18
<212> DNA
<213>Artificial sequence
<400> 14
ttctgttctc aaagtatc 18
<210> 15
<211> 15
<212> DNA
<213>Artificial sequence
<400> 15
cagaacagac acctc 15
<210> 16
<211> 17
<212> DNA
<213>Artificial sequence
<400> 16
acagacatct caccaag 17
Claims (10)
1. a kind of Nucleic acid combinations of CYP2C19 Genotypings detection, it is characterised in that the Nucleic acid combinations include detection primer group
Close and detection probe combines；
The detection primer combination includes the first primer pair, the second primer pair, three-primer pair and the 4th primer pair；Described first
The base sequence of primer pair is respectively as shown in SEQ ID No.1-2, and the base sequence of second primer pair is respectively such as SEQ ID
Shown in No.3-4, the base sequence of the three-primer pair is respectively as shown in SEQ ID No.5-6, the alkali of the 4th primer pair
Basic sequence is respectively as shown in SEQ ID No.7-8；
The detection probe combination includes the first wild type detection probe, the first saltant type detection probe, the detection of the second wild type
Probe, the second saltant type detection probe, the 3rd wild type detection probe, the 3rd saltant type detection probe, the detection of the 4th wild type
Probe and the 4th saltant type detection probe；The base sequence of the first wild type detection probe is as shown in SEQ ID No.9, institute
The base sequence of the first saltant type detection probe is stated as shown in SEQ ID No.10；The base of the second wild type detection probe
Sequence is as shown in SEQ ID No.11, and the base sequence of the second saltant type detection probe is as shown in SEQ ID No.12；Institute
The base sequence of the 3rd wild type detection probe is stated as shown in SEQ ID No.13, the base of the 3rd saltant type detection probe
Sequence is as shown in SEQ ID No.14；The base sequence of the 4th wild type detection probe is as shown in SEQ ID No.15, institute
The base sequence of the 4th saltant type detection probe is stated as shown in SEQ ID No.16；
The first wild type detection probe, the second wild type detection probe, the 3rd wild type detection probe and institute
5 ' the ends for stating the 4th wild type detection probe are marked with the first fluorophor, and 3 ' ends are marked with the second fluorescer group；Described
One saltant type detection probe, the second saltant type detection probe, the 3rd saltant type detection probe and the 4th mutation
5 ' ends of type detection probe are marked with the 3rd fluorophor, and 3 ' ends are marked with the second fluorescer group.
2. the Nucleic acid combinations of CYP2C19 Genotypings detection according to claim 1, it is characterised in that first fluorescence
Group is one in FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED
Kind；Second fluorophor be FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED,
One kind in RED670 and NED；3rd fluorophor be FAM, MGB, VIC, TET, JOE, HEX, CY3, CY5, ROX,
One kind in RED610, TEXASRED, RED670 and NED；First fluorophor, second fluorophor and described
Three fluorophors differ.
3. the Nucleic acid combinations of CYP2C19 Genotypings detection as claimed in claim 1 or 2 detect in CYP2C19 Genotypings
In application, it is described application for the purpose of non-diagnostic or non-treatment.
4. the Nucleic acid combinations of CYP2C19 Genotypings detection according to claim 3 are in the detection of CYP2C19 Genotypings
Application, it is characterised in that enter performing PCR reaction in, the first wild type detection probe and first saltant type detection visit
The ratio between concentration of pin is 2:1；The ratio between concentration of the second wild type detection probe and the second saltant type detection probe is
1:2；The ratio between concentration of the 3rd wild type detection probe and the 3rd saltant type detection probe is 1:1；The vast expanse of open ground
The ratio between concentration of raw type detection probe and the 4th saltant type detection probe is 1:2.
5. the Nucleic acid combinations of CYP2C19 Genotypings detection as claimed in claim 1 or 2 are preparing the inspection of CYP2C19 Genotypings
Application in the detection kit of survey.
A kind of 6. detection kit of CYP2C19 Genotypings detection, it is characterised in that the CYP2C19 Genotypings detection
Kit includes the Nucleic acid combinations of CYP2C19 Genotypings as claimed in claim 1 or 2 detection.
7. the detection kit of CYP2C19 Genotypings detection according to claim 6, it is characterised in that also including PCR
At least one of reaction buffer, Taq archaeal dna polymerases, dNTPs.
8. the detection kit of CYP2C19 Genotypings detection according to claim 7, it is characterised in that the PCR is anti-
Answer buffer solution mainly by KCl, MgCl2, TrisHCl and water is made.
9. the detection kit of CYP2C19 Genotypings detection according to claim 6, it is characterised in that the detection
Kit also includes genome extracts reagent.
10. the detection kit of the CYP2C19 Genotypings detection as described in claim any one of 6-9 is in CYP2C19 genes
Application in parting detection, the application is for the purpose of non-diagnostic or non-treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711101301.3A CN107794298A (en) | 2017-11-09 | 2017-11-09 | A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711101301.3A CN107794298A (en) | 2017-11-09 | 2017-11-09 | A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107794298A true CN107794298A (en) | 2018-03-13 |
Family
ID=61534709
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711101301.3A Pending CN107794298A (en) | 2017-11-09 | 2017-11-09 | A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107794298A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592234A (en) * | 2019-09-26 | 2019-12-20 | 益善生物技术股份有限公司 | Nucleic acid combination, kit and method for detecting gene polymorphism |
| CN114410768A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting single nucleotide polymorphism of cyp2c19 x 2 gene and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399561A (en) * | 2016-11-22 | 2017-02-15 | 武汉海吉力生物科技有限公司 | Prime, probe and kit for detecting polymorphism of CYP2C19 gene |
| CN107217107A (en) * | 2017-08-03 | 2017-09-29 | 首都医科大学附属北京天坛医院 | The method and kit of a kind of quick detection CYP2C19 gene pleiomorphisms |
| CN107287339A (en) * | 2017-08-15 | 2017-10-24 | 北京鑫诺美迪基因检测技术有限公司 | A kind of composition and its application for being used to detect CYP2C19 gene pleiomorphisms |
-
2017
- 2017-11-09 CN CN201711101301.3A patent/CN107794298A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399561A (en) * | 2016-11-22 | 2017-02-15 | 武汉海吉力生物科技有限公司 | Prime, probe and kit for detecting polymorphism of CYP2C19 gene |
| CN107217107A (en) * | 2017-08-03 | 2017-09-29 | 首都医科大学附属北京天坛医院 | The method and kit of a kind of quick detection CYP2C19 gene pleiomorphisms |
| CN107287339A (en) * | 2017-08-15 | 2017-10-24 | 北京鑫诺美迪基因检测技术有限公司 | A kind of composition and its application for being used to detect CYP2C19 gene pleiomorphisms |
Non-Patent Citations (4)
| Title |
|---|
| DA SYCHEV等: ""The frequency of CYP2C19 genetic polymorphisms in Russian patients with peptic ulcers treated with proton pump inhibitors"", 《PHARMACOGENOMICS AND PERSONALIZED MEDICINE》 * |
| LINA ZHANG等: ""Comparison of High-Resolution Melting Analysis, TaqMan Allelic Discrimination Assay, and Sanger Sequencing for Clopidogrel Efficacy Genotyping in Routine Molecular Diagnostics"", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
| 刘秀卿等: ""CYP2C19 基因多态性荧光定量PCR检测方法的建立"", 《临床检验杂志》 * |
| 黄杰等: ""CYP2C19 基因多态性检测方法的建立"", 《药物分析杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592234A (en) * | 2019-09-26 | 2019-12-20 | 益善生物技术股份有限公司 | Nucleic acid combination, kit and method for detecting gene polymorphism |
| CN114410768A (en) * | 2021-12-22 | 2022-04-29 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting single nucleotide polymorphism of cyp2c19 x 2 gene and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104212904B (en) | A kind of kit and using method thereof for fast detecting mankind CYP2C19 gene pleiomorphism | |
| CN104846085A (en) | Human SLCO1B1 and ApoE (apolipoprotein E) gene polymorphism detection kit | |
| CN106929591A (en) | A kind of human HLA B*5801 genetic polymorphism detection kits | |
| CN104372093A (en) | SNP (single-nucleotide polymorphism) detection method based on high-flux sequencing | |
| CN105002275A (en) | Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection | |
| CN104988149A (en) | Human CYP2C19 gene polymorphism detection specific primer and kit | |
| CN109182501B (en) | Folate metabolism gene polymorphism detection primer and kit | |
| CN107034277A (en) | A kind of method for detecting low abundance gene mutation | |
| CN107841537A (en) | A kind of full premix MTHFR and MTRR multiplex PCR genetic polymorphism detection kits and method | |
| CN107217107A (en) | The method and kit of a kind of quick detection CYP2C19 gene pleiomorphisms | |
| CN104894256A (en) | Primers and kit for detecting acetaldehyde dehydrogenase 2 gene rs671 polymorphism site, and PCR (polymerase chain reaction) method thereof | |
| CN104975100A (en) | Specific primer and kit for human ALDH2 gene polymorphism detection | |
| CN104962609A (en) | Primer and kit for detecting CYP2C9 and VKORC1 gene polymorphism and PCR method of primer and kit | |
| CN110923314B (en) | Primer for detecting SNP locus rs9263726, crRNA sequence and application thereof | |
| CN107794298A (en) | A kind of Nucleic acid combinations of CYP2C19 Genotypings detection and detection kit and application | |
| CN101812537B (en) | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus | |
| CN105886606A (en) | Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit | |
| CN107164521A (en) | A kind of CYP2C19*2 genotype detections kit and its detection method | |
| CN104818340A (en) | Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof | |
| CN108060213A (en) | Isothermal duplication method detection SNP site probe and kit based on the recombinase-mediated that probe is oriented to | |
| EP1715049A1 (en) | Method of detecting and quantifying human P450 molecular species and probe and kit for this method | |
| EP2529034B1 (en) | Methods for the detection of fungus | |
| CN105969908A (en) | CTNNB1 third exon mutation detection primer probes and kit thereof | |
| CN108913774A (en) | C-KIT somatic mutation gene detection KIT and detection method thereof | |
| CN116004775A (en) | Primer probe composition, kit and method for quantifying copy number of human motor neurons |
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: 20180313 |