CN107287340A - A kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms - Google Patents
A kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms Download PDFInfo
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Abstract
The invention discloses a kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms, the mononucleotide polymorphism site in CYP2C9 and VKORC1 genes is detected by FAM, HEX, ROX (modification) triple channel multi-PRC reaction.In order to improve the simplicity and specificity of detection, realize that a pipe parting is detected by the technological means of probe parting, so that whole operation and course of reaction are simplified, and on this basis, lock nucleic acid modification and secondary structure modification are carried out to probe, increase the Tm values of probe, further improve the specific of probe combination, finally by the proportioning of adjustment primer pair, the consumption of Taq enzyme, consumption of magnesium ion etc., improve the minimum detection limit of whole kit, accuracy and specificity, kit minimal detectable concentration can reach 1ng/ μ l, accuracy and specificity can reach 100%.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to one kind is used to detect CYP2C9 and VKORC1 gene polymorphics
The technology of property.
Background technology
Warfarin is a kind of Coumarins oral anticoagulation of indirectly-acting, by suppressing vitamin K in liver cell
Synthesize several clotting factor and play anticoagulation.Warfarin can change the viscous pasty state of blood, clinically for prevent and
Treat thrombus.The warfarin of normal dose can anti-freezing, underdosage do not reach the therapeutic effect of anti-tampon；It is excessive then again
It is easily caused various bleedings.Bleeding once occurs for patient, just necessary decrement or deactivation warfarin, and then caused consequence is exactly
Decrease or stopping due to anticoagulation, cause patient to form thrombus again.Because medicament metabolism ability is present between individual patients
Difference, warfarin is different to the effective dose of each patient.Scientific research shows, the polymorphism and warfarin of Individual genes
Internal metabolic adsorption has substantial connection.The prediction accuracy that warfarin dose is taken to patient can be improved by being detected by science of heredity,
And available for optimization maintenance dose, reduction triggers the risk of the complication such as bleeding.
Warfarin is clinical conventional prevention of thromboembolic disorders oral drugs, but there is obvious individual difference.Wherein,
Cytochrome P 450 enzymes 2C9 genes (CYP2C9) and the gene (VKORC1) of vitamin K epoxide reductase complex subunit 1
Polymorphism is two major influence factors of warfarin individual dose difference, and about 37% and 6% dose difference is explained respectively.It is beautiful
Food and medicine Surveillance Authority of state (FDA) points out genotype detection to help to adjust in the modification warfarin dispensing label of in August, 2007
Whole warfarin dosage.U.S. FDA in 2009 changes warfarin dispensing label again, and according to patient VKORC1 and CYP2C9
Genotype determines warfarin initial dose.International Warfarin drug gene group federation (IWPC) have collected 5700 big from 4
9, continent national 21 research institution reaches the patient information of stable clinical efficacy using warfarin, and sets up database.Pass through
Screening and checking to this database, establish IWPC models, are to be related to the largest model of case at present, the model can be solved
Release warfarin individual dose difference 47%.It was verified that according to Patient genotype and combining patient clinical information's progress warfarin
Individual administration, can significantly improve warfarin anti-freezing compliance rate, reduce anti-freezing complication, reduce patient's admission rate again.Therefore, build
View carries out CYP2C9*3 and VKORC1 genetic tests before using warfarin, and warfarin is instructed to medicament according to Patient genotype
Amount.
People's CYP2C9 genes have genetic polymorphism, and hypotype is divided into * 1, * 2 and * 3, wherein the closest with asian population
It is that the c.1075 position nucleotides of CYP2C9 genes has A/C polymorphisms.When pleomorphism site is C (CYP2C9*3 type), enzymatic activity
80% is reduced than wild type, and CYP2C9*2 type allele is seldom found in Chinese.Research shows, warfarin by
CYP2C9 is metabolized, and its mutant substantially reduces warfarin metabolic rate, and medication individual is just having higher to go out using initial stage
Blood is dangerous.Warfarin is vitamin K epoxide reductase VKORC1 specific inhibitor.Numerous studies discovery,
The gene pleiomorphism (- 1639G/A) of VKORC1 promoters is racial difference and individual difference in influence warfarin dosage requirements
Main factor.
Therefore, CYP2C9 and VKORC1 genetic polymorphism detections are significant for warfarin medication.And detect at present
The nucleotide primer and method accuracy and specificity of CYP2C9 and VKORC1 gene pleiomorphisms are relatively low, it is difficult to extensively should clinical
With.So explore a kind of fast and reliable nucleotide primer group of detection CYP2C9 and VKORC1 gene pleiomorphisms and method into
For the hot issue of clinical and experimental study.
The content of the invention
It is an object of the invention to provide it is a kind of be used for detect CYP2C9 and VKORC1 gene pleiomorphisms composition and its should
With consumption by adjusting the proportioning of primer, the consumption of Taq enzyme and magnesium ion etc. passes through FAM, HEX, ROX (modification) triple channel
Multi-PRC reaction, and primer is placed into parting detection in a pipe, improve the simplicity and specificity of detection so that whole
Operation and course of reaction are simplified.And on this basis, lock nucleic acid modification is carried out to probe and secondary structure is modified, increase is visited
The Tm values of pin, further improve that probe combines is specific, finally by the adjustment of above-mentioned system so that accuracy and special
Property can reach 100%, minimal detectable concentration can reach 1ng/ μ L, solve in the past detection CYP2C9 and VKORC1 genes
During polymorphism accuracy and it is specific relatively low the problem of.
To achieve these goals, a kind of group for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms that the present invention is provided
Compound, including for for detect in measuring samples respectively with the specific target gene of CYP2C9 and VKORC1 gene-correlations
Primer pair and probe, including：
CYP2C9*2 primers：
CYP2C9*2 gene forward primers (CYP2C9*2-F):GACGCTGCGGAATTTTG(SEQ ID NO.1)；
CYP2C9*2 genes reverse primer (CYP2C9*2-R):GGTTTTTCTCAACTCCTCC(SEQ ID NO.2)；
Probe CYP2C9*2-CFP：FAM-CCTCATTGAGGACCGTGTTCAAGAGG-BHQ1(SEQ ID NO.3)；
Probe CYP2C9*2-TFP：ROX-TCTTGGAGGACTGTGTTCAAGA-BHQ2(SEQ ID NO.4)；
CYP2C9*3 primer pairs：
CYP2C9*3 gene forward primers (CYP2C9*3-F):ACACAGATGCTGTGGT(SEQ ID NO.5)；
CYP2C9*3 genes reverse primer (CYP2C9*3-R):TAATGTCACAGGTCACTG(SEQ ID NO.6)；
Probe CYP2C9*3-AF：FAM-AGAAGCAGAGATACATTGACCTTCT-BHQ1(SEQ ID NO.7)；
Probe CYP2C9*3-CFP：ROX-GGAGAGAGATACCTTGACCTTCTCC-BHQ2(SEQ ID NO.8)；
VKORC1 primer pairs：
VKORC1 gene forward primers (VKORC1-F)：CTCCTGACCTCAAGTGATCCA(SEQ ID NO.9)；
VKORC1 genes reverse primer (VKORC1-R)：CAACAGTAAGGGATCCCTC(SEQ ID NO.10)；
Probe VKORC1-GFP:FAM-CCATTCACCGCACCCGGCCAATGG-BHQ1(SEQ ID NO.11)；
Probe VKORC1-AFP:ROX-AACACGCACCTGGCCAATGGTTGTT-BHQ2(SEQ ID NO.12)；
Interior label primer pair：
HER2-F:TGCTTGGATCTGGCGCTTTTGGCA(SEQ ID NO.13)；
HER2-R:CCAAACACTGCCTCCAGCTCTTGCC(SEQ ID NO.14)；
Probe HER2-FP:HEX-AGGGCCAGGTCCTGGGGTGGGC-BHQ1(SEQ ID NO.15)；
Wherein to the 5 of CYP2C9*2-CFP, CYP2C9*3-AF and VKORC1-GFP, end carries out FAM modifications, 3, end progress
BHQ1 is modified；To the 5 of CYP2C9*2-TFP, CYP2C9*3-CFP and VKORC1-AFP, end carries out ROX modifications, 3, end progress
BHQ2 is modified；To the 5 of HER2-FP, end carries out HEX modifications, 3, end progress BHQ1 modifications；
Increase the modification of lock nucleic acid (LNA) on the 14th bit base C of CYP2C9*2-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 12nd bit base T of CYP2C9*2-TFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base A of CYP2C9*3-AF sequences；
Increase the modification of lock nucleic acid (LNA) on the 13rd bit base C of CYP2C9*3-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base C of VKORC1-GFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 11st bit base T of VKORC1-AFP sequences.
Preferably, a person-portion addition is：CYP2C9*2-F addition 0.1-0.3 μ L, CYP2C9*3-F additions 0.1-
0.3 μ L, VKORC1-F addition 0.1-0.3 μ L, CYP2C9*2-R addition 0.1-0.3 μ L, CYP2C9*3-R additions 0.1-
0.3 μ L, VKORC1-R addition 0.1-0.3 μ L, CYP2C9*2-CFP addition 0.01-0.2 μ L, CYP2C9*3-AFP additions
0.01-0.2 μ L, VKORC1-GFP addition 0.01-0.2 μ L, CYP2C9*2-TFP addition 0.01-0.2 μ L, CYP2C9*3-
CFP addition 0.01-0.2 μ L, VKORC1-AFP addition 0.01-0.2 μ L, HER2-F addition 0.01-0.2 μ L, HER2-R
Addition 0.01-0.2 μ L, HER2-FP addition 0.01-0.2 μ L.
A kind of kit for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms that the present invention is provided, the reagent
Box includes the above-described composition for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms.
Preferably, the kit includes archaeal dna polymerase (Taq enzyme), dNTPs, 10 × archaeal dna polymerase buffer, UDG enzyme
And Mg2+, a person-portion addition is：Archaeal dna polymerase addition 0.1-0.4 μ L, dNTPs addition 1-3 μ L, 10 × archaeal dna polymerase
Buffer addition 1-4 μ L, UDG enzyme addition 0.01-0.2 μ L, Mg2+Addition 2-5 μ L.
A kind of sample process side for being used to detect the kit of CYP2C9 and VKORC1 gene pleiomorphisms that the present invention is provided
Method, including：
(1) genomic DNA in sample is extracted, wherein sample can select whole blood sample；
(2) genomic DNA after extraction is subjected to concentration mensuration, and by after concentration dilution to 10-20ng/ μ L, carried out
Quantitative fluorescent PCR reacts；
(3) interpretation of result after being reacted according to PCR：The result expanded according to PCR carries out interpretation, draw CYP2C9 and
The specific type in each site of VKORC1.
Preferably, the site in step (3) includes CYP2C9*2 (C ＞ T), CYP2C9*3 (A ＞ C) and VKORC1 (G ＞
A)。
Preferably, PCR courses of reaction are：UDG enzyme reactions 2min at 37 DEG C, 95 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 15s,
60 DEG C of annealing extension 35s, 40 circulations.
A kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms that the present invention is provided, has
Following beneficial effect：
The present invention carries out related gene detection using this technology, and easy to operate, be easy to interpretation, the requirement to instrument is not
Height, and whole PCR processes use totally-enclosed form, it is to avoid the possibility of cross pollution, makes result more accurate.
The present invention is realized by the technological means of probe parting and SNP (SNP) site is carried out in a pipe
Detect simultaneously, improve the simplicity of detection, and then reach and precisely effectively detect in purpose site.
The present invention, can be by each site by designing lock nucleic acid and specific probe and primer containing secondary structure
Three gene types are accurately distinguished, and detect that least concentration can reach 1ng/ μ L.
The consumption of the present invention by adjusting the proportioning of primer, the consumption of Taq enzyme and magnesium ion etc., improves whole kit
Accuracy and specificity, accuracy and specificity can reach 100%.
The invention mainly comprises using specific multiplex PCR-fluorescence probe method detection CYP2C9 and VKORC1 genes
SNP C430T, A1075C and G1639A, point for judging pleomorphism site in CYP2C9 and VKORC1 genes
Cloth, is further used for aiding in the clinical diagnosis of warfarin dosage.
Brief description of the drawings
Fig. 1 is the gene peak of each detection site genotype in the present embodiment 1.
Embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to embodiment to this hair
It is bright to be described in further detail.
The present invention provide it is a kind of be used to detecting the compositions of CYP2C9 and VKORC1 gene pleiomorphisms, including for for
Detect primer pair and probe respectively with the specific target gene of CYP2C9 and VKORC1 gene-correlations in measuring samples, bag
Include：
CYP2C9*2 primers：
CYP2C9*2 gene forward primers (CYP2C9*2-F):GACGCTGCGGAATTTTG；
CYP2C9*2 genes reverse primer (CYP2C9*2-R):GGTTTTTCTCAACTCCTCC；
Probe CYP2C9*2-CFP：FAM-CCTCATTGAGGACCGTGTTCAAGAGG-BHQ1；
Probe CYP2C9*2-TFP：ROX-TCTTGGAGGACTGTGTTCAAGA-BHQ2；
CYP2C9*3 primer pairs：
CYP2C9*3 gene forward primers (CYP2C9*3-F):ACACAGATGCTGTGGT；
CYP2C9*3 genes reverse primer (CYP2C9*3-R):TAATGTCACAGGTCACTG；
Probe CYP2C9*3-AF：FAM-AGAAGCAGAGATACATTGACCTTCT-BHQ1；
Probe CYP2C9*3-CFP：ROX-GGAGAGAGATACCTTGACCTTCTCC-BHQ2；
VKORC1 primer pairs：
VKORC1 gene forward primers (VKORC1-F)：CTCCTGACCTCAAGTGATCCA；
VKORC1 genes reverse primer (VKORC1-R)：CAACAGTAAGGGATCCCTC；
Probe VKORC1-GFP:FAM-CCATTCACCGCACCCGGCCAATGG-BHQ1；
Probe VKORC1-AFP:ROX-AACACGCACCTGGCCAATGGTTGTT-BHQ2；
Interior label primer pair：
HER2-F:TGCTTGGATCTGGCGCTTTTGGCA；
HER2-R:CCAAACACTGCCTCCAGCTCTTGCC；
Probe HER2-FP:HEX-AGGGCCAGGTCCTGGGGTGGGC-BHQ1；
Increase the modification of lock nucleic acid (LNA) on the 14th bit base C of CYP2C9*2-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 12nd bit base T of CYP2C9*2-TFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base A of CYP2C9*3-AF sequences；
Increase the modification of lock nucleic acid (LNA) on the 13rd bit base C of CYP2C9*3-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base C of VKORC1-GFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 11st bit base T of VKORC1-AFP sequences.
When primer is synthesized, base to be finished is marked according to the regulation of synthesis unit, synthesis unit will be known
This site is modified in road by the way of LNA.
Embodiment 1：The present invention is configured to the primer of pre- mixed detection site on the basis of multiple fluorescence PCR
Composition.It can directly be detected to extracting the sample completed, so make detecting step more easy.
Comprise the following steps that：
1) genomic DNA (using commercialized peripheral blood genome DNA extracting reagent kit) in whole blood sample is extracted.
2) genomic DNA after extraction is subjected to concentration mensuration, and concentration dilution to 10-20ng/ μ L is carried out subsequently
PCR courses of reaction.
3) the composition composition of reaction system：dNTPs、Mg2+, archaeal dna polymerase buffer, reverse transcription buffer, gene draw
Thing is to, archaeal dna polymerase etc..
4) PCR reaction conditions：UDG enzyme reactions 2min at 37 DEG C；95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 15s, are moved back at 60 DEG C
Fire extension 35s, 40 circulations.
5) preparation of PCR reaction systems：
PCR amplification MIX (12.5 μ L, remaining uses purified water polishing)
PCR primer mixture system (6.5 μ L, remaining uses purified water polishing)
PCR amplification system
PCR amplification programs are as follows：
First amplification stage：37 DEG C of UDG enzyme reactions 2min；
Second amplification stage：95 DEG C of pre-degeneration 3min；
3rd amplification stage：94 DEG C of denaturation 15s, 60 DEG C of annealing extension 35s, 40 circulations.
6) interpretation of result
1. each sample internal standard passage (HEX) Ct values are answered in≤35, and two amplified reactions of target gene passage at least within
One Ct ＜ 38, (target gene passage is calculated without amplification curve, Ct values by 40) is met after this condition by the interpretation of table 1：
If 2. sample internal standard passage (HEX) Ct values ＞ 35, it is proposed that extract sample again, then detect；
If 3. sample internal standard channel C t value≤35, and equal Ct >=38 of target gene channel C t values, then it is invalid to detect.
The result interpretation of table 1
| Detect title | Ct values | As a result | Judge |
| CYP2C9*2(C) | Ct=A | B-A≥5 | CC genotype |
| CYP2C9*2(T) | Ct=B | | B-A | ＜ 5 | CT genotype |
| A-B≥5 | TT genotype | ||
| CYP2C9*3(A) | Ct=C | D-C≥5 | AA genotype |
| CYP2C9*3(C) | Ct=D | | D-C | ＜ 5 | AC mixed types |
| C-D≥5 | CC genotype | ||
| VKORC1(G) | Ct=E | F-E≥5 | GG genotype |
| VKORC1(A) | Ct=F | | F-E | ＜ 5 | GA genotype |
| E-F≥5 | AA genotype |
7) performance verification of finished product kit
The finished product kit completed using configuration carries out product specificity, minimum detection limit detection, the spy of product kit
The opposite sex can reach 100%, and minimal detectable concentration can reach 1ng/ μ L.
Whole blood sample 40 is collected, specific detection is carried out using above-mentioned finished product kit, results of comparison is surveyed using a generation
Sequence is made comparisons, as a result as follows：
It can draw from the above, the detection accuracy of this kit reaches 100%, and specificity also reaches 100%.
50ng/ μ L, 25ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.5ng/ μ L various concentrations sample is entered using this kit
Row detection, this kit minimal detectable concentration can reach 1ng/ μ L, therefore, and the detection minimal detectable concentration of this kit is 1ng/
μL。
Embodiment 2：
The 200 Patients with Peripheral blood check samples from Wuhan hospital of Tongji University are collected, are examined using above-mentioned finished product kit
Survey.Concrete operation step is as described in case study on implementation 1.
Specific case used herein is elaborated to inventive concept, and the explanation of above example is only intended to
Help to understand core concept of the invention.It should be pointed out that for those skilled in the art, not departing from this
On the premise of inventive concept, any obvious modification, equivalent substitution or other improvement made should be included in the present invention
Protection domain within.
SEQUENCE LISTING
<110>Beijing Xinnuo Meidi Gene Inspection Technology Co., Ltd.
<120>A kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms
<130> P20170149
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
gacgctgcgg aattttg 17
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
ggtttttctc aactcctcc 19
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence
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cctcattgag gaccgtgttc aagagg 26
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
tcttggagga ctgtgttcaa ga 22
<210> 5
<211> 16
<212> DNA
<213>Artificial sequence
<400> 5
acacagatgc tgtggt 16
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> 6
taatgtcaca ggtcactg 18
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence
<400> 7
agaagcagag atacattgac cttct 25
<210> 8
<211> 25
<212> DNA
<213>Artificial sequence
<400> 8
ggagagagat accttgacct tctcc 25
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
ctcctgacct caagtgatcc a 21
<210> 10
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<212> DNA
<213>Artificial sequence
<400> 10
caacagtaag ggatccctc 19
<210> 11
<211> 24
<212> DNA
<213>Artificial sequence
<400> 11
ccattcaccg cacccggcca atgg 24
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence
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aacacgcacc tggccaatgg ttgtt 25
<210> 13
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<213>Artificial sequence
<400> 13
tgcttggatc tggcgctttt ggca 24
<210> 14
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<212> DNA
<213>Artificial sequence
<400> 14
ccaaacactg cctccagctc ttgcc 25
<210> 15
<211> 25
<212> DNA
<213>Artificial sequence
<400> 15
ccaaacactg cctccagctc ttgcc 25
Claims (8)
1. a kind of composition for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms, it is characterised in that the composition includes
For for detect in measuring samples respectively with the primer pair of the specific target gene of CYP2C9 and VKORC1 gene-correlations and
Probe, including：
CYP2C9*2 primers：
CYP2C9*2 gene forward primers (CYP2C9*2-F):SEQ ID NO.1；
CYP2C9*2 genes reverse primer (CYP2C9*2-R):SEQ ID NO.2；
Probe CYP2C9*2-CFP：SEQ ID NO.3；
Probe CYP2C9*2-TFP：SEQ ID NO.4；
CYP2C9*3 primer pairs：
CYP2C9*3 gene forward primers (CYP2C9*3-F):SEQ ID NO.5；
CYP2C9*3 genes reverse primer (CYP2C9*3-R):SEQ ID NO.6；
Probe CYP2C9*3-AF：SEQ ID NO.7；
Probe CYP2C9*3-CFP：SEQ ID NO.8；
VKORC1 primer pairs：
VKORC1 gene forward primers (VKORC1-F)：SEQ ID NO.9；
VKORC1 genes reverse primer (VKORC1-R)：SEQ ID NO.10；
Probe VKORC1-GFP:SEQ ID NO.11；
Probe VKORC1-AFP:SEQ ID NO.12；
Interior label primer pair：
HER2-F:SEQ ID NO.13；
HER2-R:SEQ ID NO.14；
Probe HER2-FP:SEQ ID NO.15；
FAM modifications wherein are carried out to CYP2C9*2-CFP, CYP2C9*3-AF and VKORC1-GFP 5 ' ends, 3 ' ends carry out BHQ1
Modification；ROX modifications are carried out to CYP2C9*2-TFP, CYP2C9*3-CFP and VKORC1-AFP 5 ' ends, 3 ' ends carry out BHQ2 and repaiied
Decorations；HEX modifications are carried out to HER2-FP 5 ' ends, 3 ' ends carry out BHQ1 modifications；
Increase the modification of lock nucleic acid (LNA) on the 14th bit base C of CYP2C9*2-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 12nd bit base T of CYP2C9*2-TFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base A of CYP2C9*3-AF sequences；
Increase the modification of lock nucleic acid (LNA) on the 13rd bit base C of CYP2C9*3-CFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 15th bit base C of VKORC1-GFP sequences；
Increase the modification of lock nucleic acid (LNA) on the 11st bit base T of VKORC1-AFP sequences.
2. the composition according to claim 1 for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms, it is characterised in that
One person-portion addition is：CYP2C9*2-F addition 0.1-0.3 μ L, CYP2C9*3-F addition 0.1-0.3 μ L, VKORC1-F add
Enter amount 0.1-0.3 μ L, CYP2C9*2-R addition 0.1-0.3 μ L, CYP2C9*3-R addition 0.1-0.3 μ L, VKORC1-R adds
Enter amount 0.1-0.3 μ L, CYP2C9*2-CFP addition 0.01-0.2 μ L, CYP2C9*3-AFP addition 0.01-0.2 μ L,
VKORC1-GFP addition 0.01-0.2 μ L, CYP2C9*2-TFP addition 0.01-0.2 μ L, CYP2C9*3-CFP additions
0.01-0.2 μ L, VKORC1-AFP addition 0.01-0.2 μ L, HER2-F addition 0.01-0.2 μ L, HER2-R additions
0.01-0.2 μ L, HER2-FP addition 0.01-0.2 μ L.
3. the composition according to claim 1 or 2 for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms is preparing use
Application in the reagent of detection CYP2C9 and VKORC1 gene pleiomorphisms.
4. a kind of be used for the kit of detection CYP2C9 and VKORC1 gene pleiomorphisms, it is characterised in that the kit
The composition of CYP2C9 and VKORC1 gene pleiomorphisms is detected including being used for described in claim 1 or 2.
5. the kit according to claim 4 for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms, it is characterised in that
The kit includes archaeal dna polymerase (Taq enzyme), dNTPs, 10 × archaeal dna polymerase buffer, UDG enzyme and Mg2+, a person-portion adds
Entering amount is：Archaeal dna polymerase addition 0.1-0.4 μ L, dNTPs addition 1-3 μ L, 10 × archaeal dna polymerase buffer additions 1-4
μ L, UDG enzyme addition 0.01-0.2 μ L, Mg2+Addition 2-5 μ L.
6. it is a kind of according to claim 5 for detecting at the sample of kit of CYP2C9 and VKORC1 gene pleiomorphisms
Reason method, it is characterised in that the sample processing method includes：
(1) genomic DNA in sample is extracted, wherein sample can select whole blood sample；
(2) genomic DNA after extraction is subjected to concentration mensuration, and by after concentration dilution to 10-20ng/ μ L, carries out fluorescence
Quantitative PCR reacts；
(3) interpretation of result after being reacted according to PCR：The result expanded according to PCR carries out interpretation, draws CYP2C9 and VKORC1
The specific type in each site.
7. it is a kind of according to claim 6 for detecting at the sample of kit of CYP2C9 and VKORC1 gene pleiomorphisms
Reason method, it is characterised in that the site in step (3) includes CYP2C9*2 (C ＞ T), CYP2C9*3 (A ＞ C) and VKORC1 (G
＞ A).
8. the sample process side according to claim 6 for being used to detect the kit of CYP2C9 and VKORC1 gene pleiomorphisms
Method, it is characterised in that PCR courses of reaction are：UDG enzyme reactions 2min at 37 DEG C, 95 DEG C of pre-degeneration 3min, 94 DEG C are denatured 15s, 60
DEG C annealing extension 35s, 40 circulation.
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| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
| CN109457026A (en) * | 2018-10-22 | 2019-03-12 | 江苏美因康生物科技有限公司 | A kind of kit and method of quick detection antithrombotic personalized medicine gene pleiomorphism |
| CN110184343A (en) * | 2019-06-26 | 2019-08-30 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C9 and VKORC1 gene pleiomorphism |
| CN110938688A (en) * | 2019-12-30 | 2020-03-31 | 武汉光谷联合医学检验所股份有限公司 | CYP2C9 gene polymorphism detection kit and application thereof |
| CN112301121A (en) * | 2019-07-29 | 2021-02-02 | 上海利康精准医疗技术有限公司 | Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene |
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| CN107841540A (en) * | 2017-12-13 | 2018-03-27 | 益善生物技术股份有限公司 | A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit |
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| CN110184343A (en) * | 2019-06-26 | 2019-08-30 | 湖南健基生物技术有限公司 | Detect composition, kit, method and the application of CYP2C9 and VKORC1 gene pleiomorphism |
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