CN104962606A - Detection kit and detection method for individualized medication of warfarin - Google Patents
Detection kit and detection method for individualized medication of warfarin Download PDFInfo
- Publication number
- CN104962606A CN104962606A CN201510105467.7A CN201510105467A CN104962606A CN 104962606 A CN104962606 A CN 104962606A CN 201510105467 A CN201510105467 A CN 201510105467A CN 104962606 A CN104962606 A CN 104962606A
- Authority
- CN
- China
- Prior art keywords
- site
- cyp2c9
- seq
- primer
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a detection kit and detection method for individualized medication of warfarin, belonging to the technical fields of molecular biology and nucleic acid detection. According to the detection method provided by the invention, mutant types of CYP2C9 and VKORC1 are detected by using a bidirectional allele-specific polymerase chain reaction (Bi-ASPCR) method; two cycles of PCR are carried out on site CYP2C9*2 and site VKORC1-1639; one cycle of PCR is carried out on site CYP2C9*3; and obtained PCR products are subjected to electrophoresis and electrophoresis results are compared with standards. According to the invention, PCR primers directed at mutant sites included in CYP2C9 and VKORC1 genes and closely related to metabolism of warfarin are designed, and the genotypes of to-be-detected genes are determined by reading the lengths of multiplex PCR products with naked eyes via common gel electrophoresis, so the dosage of warfarin in medication is guided and individualized medication of patients is realized; and the detection kit and detection method have the advantages of rapidness, accuracy, low cost, etc.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field, particularly relate to warfarin personalized medicine detection kit and detection method thereof.
Background technology
Warfarin is widely used in clinical coumarins oral anticoagulant thing at present, can the Fashion and Evolution of anti-hemostasis suppository, such as treat thromboembolic states phlebitis, rheumatic heart disease, artificial replacement's heart valve surgery, arthrodesis of hip and reduce the M & M of pulmonary infarction, also can be used for the adjuvant drug of myocardial infarction patient.Though the anticoagulant effect of warfarin is better than other anticoagulants of approval at present, but because the mobility of warfarin individual dose is very large, as serious anti-freezing deficiency or drug overdose can not caused to cause lethal hemorrhage according to individual difference medication, this just brings great challenge for the Clinical practice of warfarin.Present stage, be first give patient the medicine of certain routine dose substantially, clinician constantly adjusted drug dose according to the INR value of patient again, until patient INR reaches re-set target value clinically when applying Anticoagulation of Warfarin.Whole Period Process is very long, and patient need carry out INR detection repeatedly, so again can the psychological burden of making patients.
The factor affecting warfarin effect comprises intake and the inherited genetic factors of vitamin K in sex, age, disease, diet, in recent years along with molecular biological further investigation finds, heritable variation causes the most important factor of warfarin individual applications difference, it mainly concentrates on warfarin pharmacokinetics metabolism link------Cytochrome P450 isozyme 2C9 (CYP2C9) and pharmacodynamics acceptor link------vitamin K epoxide reductase complex body subunit 1 (VKORC1).Warfarin mainly in liver by liver cytochrome 450 enzymes metabolism, this is comprising CYP2C9, CYP2C19, CYP2C8, CYP2C18, CYP1A2 and CYP3A4, wherein the most importantly CYP2C9, its metabolite S-7-hydroxyl warfarin provides the anticoagulating active of warfarin 70%.Research finds that CYP2C9 exists several genes polymorphism, and current discovery is more than 60 kinds, but the most commonly CYP2C9*1, CYP2C9*2 and CYP2C9*3, other polymorphism is then comparatively rare.CYP2C9*2 (Arg144Cys) homozygote is the 16%-20% of wild-type CYP2C9*1 homozygote activity, and in white people, CYP2C9*2 mutation frequency is up to 11%, then very rare in asian population; CYP2C9*3 (Ile359Leu) homozygote activity is only the 4%-6% of wild-type homozygous body, and the mutation frequency in Chinese can reach 3.3%.Therefore, compared with having the patient of wild-type homozygous body, the patient carrying this mutated-genotype is after taking warfarin, and the time reached needed for stable state is longer, and when equal dosage, INR value is higher, life-threatening bleeding risk occurs and increases.The protein of VKORC1 (VITAMIN epoxide reductase complex subunit 1) coding containing 163 amino-acid residues, it is a most important composition subunit of VKORC (VITAMIN epoxide reductase complex body), it is thrombogen, VII, Ⅸ, Ⅹ and PROTEIN C, the required factor of S and Z activation, wherein, VKORC1 not only controls the generation of vitamin K-dependent clotting factor, also be the target spot of warfarin effect simultaneously, VKORC1 promoter region-1639 district AA genotype carriers is responsive to Fa Hualin, belong to hypermetabolism type, only need the warfarin taking smaller dose can reach and maintain INR target zone,-1639GG and-1639AG carrier be method woods relative insensitivity to China then, need larger dose warfarin just can reach the INR scope of expection.The individualized treatment of CYP2C9 and VKORC1 two gene polynorphisms and warfarin is the most closely related, by carrying out gene test to target patient, determine its gene type, namely take warfarin by fixing formulae discovery or according to the dosage that FDA recommends, the INR value scope of clinical regulation in the short period of time, can be reached.
The genotypic method of current detection CYP2C9 and VKORC1 mainly direct sequencing, also has high temperature conjunction enzyme process etc. in addition.But these two kinds of methods all need DNA sequencer with the use of, DNA sequencer is expensive, mostly is foreign enterprise and monopolizes, and is unfavorable for the outfit of domestic fundamental research and vast medical institutions.In addition, DNA sequencer is very high to the requirement of operator, needs regular maintenance, and relating operation personnel must carry out strict training could be on duty, and test procedure is loaded down with trivial details.Direct sequencing needs a series of loaded down with trivial details step of easily makeing mistakes again of purifying, the selection of sequencing reaction chemical type etc. of the designs of PCR primer purifying, sequencing primer, sequencing reaction PCR, not easily promotes the use of.And above-mentioned two kinds of method test durations are longer, the fastest eight hours of direct sequencing, high temperature conjunction enzyme process soon five hours.
Two-way ApoE gene (Bidirectional Allele-specific PCR, Bi-ASPCR) be a kind of development and application of ApoE gene (Allele-specific PCR), Bi-ASPCR adopts four primers, two Outside primer and two allele specific inner primer respectively, when detecting, each allele specific primer coordinates an Outside primer to increase, same site can be detected from both direction simultaneously, meanwhile, article two, Outside primer can also increase template to be measured, as positive control.Therefore, detect CYP2C9 and VKORC1 by Bi-ASPCR method, to instructing, warfarin medication is significant.
Summary of the invention
The problem to be solved in the present invention is that existing detection CYP2C9 and VKORC1 genotypic method cost is higher, and complicated operation, the test duration of needs is long.
First object of the present invention is to provide warfarin personalized medicine to detect primer, and it comprises following primer pair:
(1) CYP2C9*2 site first round PCR
Upstream primer: 5 '-CTGTCTACTTTCCTAGCTCTCAAAGG-3 ' (SEQ ID No.1),
Downstream primer: 5 '-GATAGGGCCATTCCCACCATGTTG-3 ' (SEQ ID No.2);
(2) PCR is taken turns in CYP2C9*2 site second
Upstream primer: 5 '-TTTTCCCACTGGCTGAAAGAGCTAA-3 ' (SEQ ID No.3),
Downstream primer: 5 '-TGGGAAAAACACTGCTCTTTAACTC-3 ' (SEQ ID No.4),
Saltant type special primer: 5 '-AAGAGGAGCATTGAGGCCT-3 ' (SEQ ID No.5),
Wild-type special primer: 5 '-CGGGCTTCCTCTTGAACCCG-3 ' (SEQ ID No.6);
(3) CYP2C9*3 site PCR
Upstream primer: 5 '-TCTGGTTTATGGCAGTTACACA-3 ' (SEQ ID No.7),
Downstream primer: 5 '-GGGACTTCGAAAACATGGAGT-3 ' (SEQ ID No.8),
Wild-type special primer: 5 '-GCACGAGGTCCAGAGATGCA-3 ' (SEQ ID No.9),
Saltant type special primer: 5 '-GGTGGGGAGAAGSTCCAG-3 ' (SEQ ID No.10);
(4) VKORC1-1639 site first round PCR
Upstream primer: 5 '-ATTCATGCAGGGACATCTTTGGTG-3 ' (SEQ ID No.11),
Downstream primer: 5 '-TCCTCTCCCGGCATTATCCCATC-3 ' (SEQ ID No.12);
(5) PCR is taken turns in VKORC1-1639 site second
Upstream primer: 5 '-CCAGCAGGAGAGGGAAATATCAC-3 ' (SEQ ID No.13),
Downstream primer: 5 '-ACCAAGAYGCTAGACCCAA-3 ' (SEQ ID No.14), VKORC1-1639 site A second takes turns PCR special primer:
5 '-GACCTGAAAAACAACCATTGGACA-3 ' (SEQ ID No.15), VKORC1-1639 site G second takes turns PCR special primer:
5′-CGTGAGCCACCGCAACC-3′(SEQ ID No.16)。
Second object of the present invention is to provide a kind of warfarin personalized medicine detection kit.Described test kit comprises above-mentioned primer.
Further, also comprise PCR damping fluid in described test kit, Taq enzyme, dNTP, DNA ladderMarker.
Further, during use, the concentration of described each primer is 10uM; DNTP is the mixture of dATP, dGTP, dCTP, dTTP, and the concentration of described dATP, dGTP, dCTP, dTTP is 2.5mM.
3rd object of the present invention is to provide warfarin personalized medicine detection method, and it uses above-mentioned test kit, comprises the following steps:
(1) first round PCR
With nucleic acid to be detected for template, carry out first round PCR process to CYP2C9*2 site, CYP2C9*3 site and VKORC1-1639 site respectively, the product obtained is designated as M, B and N respectively;
(2) second take turns PCR
As template after M being diluted 100 times, carry out second to CYP2C9*2 site and take turns PCR process, its product is designated as A;
As template after diluting 100 times using N, carry out second to VKORC1-1639 site and take turns PCR process, its product is designated as C;
(3) electrophoresis
A and C obtained in the B obtained in step (1), step (2) is carried out gel electrophoresis, the electrophorogram obtained is contrasted with corresponding standard.
Further, in described step (1) to the condition that first round PCR process is carried out in CYP2C9*2 site, CYP2C9*3 site and VKORC1-1639 site be:
4min at (a) 95 DEG C;
B 30s at () 95 DEG C, 15s at 60 DEG C, 30s at 72 DEG C, circulate 35 times.
Further, in described step (2) to CYP2C9*2 site and VKORC1-1639 site the second condition of taking turns PCR process of carrying out be:
4min at (a) 95 DEG C;
B 30s at () 95 DEG C, 15s at 60 DEG C, 30s at 72 DEG C, circulate 30 times.
Further, the condition of electrophoresis is in step (3): get the B obtained in step (1), each 5ul of A and C obtained in step (2), carry out gel electrophoresis.
Further, the condition of electrophoresis is in step (3): get the B obtained in step (1), each 5ul of A and C obtained in step (2), and the sepharose with 2%, GOODVIEW to dye under 6v/cm voltage electrophoresis 40 minutes.
The advantage that the present invention has and positively effect are: warfarin personalized medicine provided by the invention detects and uses Bi-ASPCR method to detect CYP2C9 and VKORC1 saltant type, testing method is simple to operation, and design PCR primer in CYP2C9 and VKORC1 gene with the closely-related mutational site of warfarin metabolism, the genotype of testing gene is judged by the length of common gel electrophoresis naked eyes reading multiple PCR products, and then instruct warfarin dosage, realize the personalized medicine of patient, there is quick, accurate, low cost and other advantages.
(1) primer provided in the present invention, introduce the base of strong or weak or moderate mispairing accordingly in 3 ' end antepenulatimate, the very big specificity that must improve reaction, avoids the generation of false positive results.
(2) test kit testing cost provided by the invention is low: without the need to DNA sequencer, only need the Common Thermal Cycle instrument and common agarose gel electrophoresis equipment to carry out detection, be conducive to extending to civilian hospital and detect or common lab carries out scientific effort.
(3) this test kit is easy and simple to handle: without the need to the step such as purifying, the selection of sequencing reaction chemical type of PCR primer purifying, sequencing primer design, sequencing reaction PCR, only need these two operation stepss of PCR and electrophoresis, simple and easy to do.
(4) detection time is short: the present invention adopts Bi-ASPCR method, completes from DNA extraction to gene type, only needs four hours.And gene sequencing method wants eight hours at the soonest, high temperature conjunction enzyme process soon five hours, the present invention shortens detection time preferably.
(5) detection method provided by the invention, utilize Bi-ASPCR method, compared with other detection meanss based on PCR, do not need extra positive control, the two kinds of genotypic detections of a sample can be completed in same pipe simultaneously, both can avoid the pollution brought due to repeatable operation, and the operating time of at least half can have been saved again, the reagent consumption of half can also be saved.
Accompanying drawing explanation
Fig. 1 is the product (A that the CYP2C9*2 site detecting sample 1-11 obtains through two-wheeled PCR
1-A
11) electrophorogram.
Fig. 2 is the product (B that the CYP2C9*3 site one detecting sample 1-11 is taken turns PCR and obtained
1-B
11) electrophorogram.
Fig. 3 is the product (C that the VKORC1-1639 site detecting sample 1-11 obtains through two-wheeled PCR
1-C
11) electrophorogram.
Fig. 4 is the product (A that the CYP2C9*2 site detecting sample 39 obtains through two-wheeled PCR
39) electrophorogram.
Fig. 5 detects the CYP2C9*3 site one of sample 39 to take turns the product (B that PCR obtains
39) electrophorogram.
Fig. 6 is the product (C that the VKORC1-1639 site detecting sample 39 obtains through two-wheeled PCR
39) electrophorogram.
Fig. 7 is the product (A that the CYP2C9*2 site of special sample S obtains through two-wheeled PCR
s) electrophorogram.
Fig. 8-Figure 19 be respectively detect sample 1-11,39 the DNA sequencing collection of illustrative plates in CYP2C9*2 site.
Figure 20-Figure 31 be respectively detect sample 1-11,39 the Sequencing chromatogram of DNA in CYP2C9*3 site.
Figure 32-Figure 43 be respectively detect sample 1-11,39 the DNA sequencing collection of illustrative plates in VKORC1 site.
Embodiment
For a better understanding of the present invention, below in conjunction with specific embodiments and the drawings, the present invention is conducted further description.
Warfarin personalized medicine detection kit and method thereof, test kit comprises PCR damping fluid, Taq enzyme, dNTP, DNA ladder Marker.Wherein, following primer is also comprised in test kit provided by the invention:
(1) CYP2C9*2 site first round PCR
Upstream primer: 5 '-CTGTCTACTTTCCTAGCTCTCAAAGG-3 ' (SEQ IDNo.1, hereinafter referred to as 2C9-F),
Downstream primer: 5 '-GATAGGGCCATTCCCACCATGTTG-3 ' (SEQ IDNo.2, hereinafter referred to as 2C9-R);
(2) PCR is taken turns in CYP2C9*2 site second
Upstream primer: 5 '-TTTTCCCACTGGCTGAAAGAGCTAA-3 ' (SEQ IDNo.3, hereinafter referred to as P2),
Downstream primer: 5 '-TGGGAAAAACACTGCTCTTTAACTC-3 ' (SEQ IDNo.4, hereinafter referred to as Q2),
Saltant type special primer: 5 '-AAGAGGAGCATTGAGGCCT-3 ' (SEQ IDNo.5, hereinafter referred to as F2),
Wild-type special primer: 5 '-CGGGCTTCCTCTTGAACCCG-3 ' (SEQ IDNo.6, hereinafter referred to as R2);
(3) CYP2C9*3 site first round PCR
Upstream primer: 5 '-TCTGGTTTATGGCAGTTACACA-3 ' (SEQ ID No.7, hereinafter referred to as P3),
Downstream primer: 5 '-GGGACTTCGAAAACATGGAGT-3 ' (SEQ ID No.8 is hereinafter referred to as Q3),
Wild-type special primer: 5 '-GCACGAGGTCCAGAGATGCA-3 ' (SEQ IDNo.9, hereinafter referred to as F3),
Saltant type special primer: 5 '-GGTGGGGAGAAGSTCCAG-3 ' (SEQ IDNo.10, hereinafter referred to as R3);
(4) VKORC1-1639 site first round PCR
Upstream primer: 5 '-ATTCATGCAGGGACATCTTTGGTG-3 ' (SEQ IDNo.11, hereinafter referred to as VK-F),
Downstream primer: 5 '-TCCTCTCCCGGCATTATCCCATC-3 ' (SEQ IDNo.12, hereinafter referred to as VK-R);
(5) PCR is taken turns in VKORC1-1639 site second
Upstream primer: 5 '-CCAGCAGGAGAGGGAAATATCAC-3 ' (SEQ IDNo.13, hereinafter referred to as PV),
Downstream primer: 5 '-ACCAAGAYGCTAGACCCAA-3 ' (SEQ ID No.14, hereinafter referred to as QV),
VKORC1-1639 site A second takes turns PCR special primer:
5 '-GACCTGAAAAACAACCATTGGACA-3 ' (SEQ ID No.15, hereinafter referred to as FV),
VKORC1-1639 site G second takes turns PCR special primer:
5 '-CGTGAGCCACCGCAACC-3 ' (SEQ ID No.16, hereinafter referred to as RV).
The concentration of above primer in test kit is 100uM.During storage, each primer is the storage liquid of 100uM, during use, it is diluted to 10uM with ultrapure water.
Use this test kit, when using warfarin personalized medicine detection method provided by the invention to detect CYP2C9 and VKORC1 saltant type, comprise the following steps:
1 nucleic acid extraction:
After extracting 98 people 1ml venous blood, preserve with EDTA anticoagulant tube, often 200ul blood got by pipe, and by the DNA extraction kit of full formula gold or other company, reference reagent box specification sheets extracts complete genome DNA as the template detected, and be designated as T, number consecutively is T
1, T
2, T
3by that analogy to T
98.
2. first round PCR
First round PCR reaction conditions is:
(1) denaturation: temperature 95 DEG C of time 4min.
(2) amplification cycles: (2-1) sex change: temperature 95 DEG C, time 30s;
(2-2) anneal: temperature 60 C, time 15s;
(2-3) extend: temperature 72 DEG C, time 30s;
Circulate 35 times.
2.1 CYP2C9*2 site first round PCR
Reaction system amount to 25ul, composition and content as shown in table 1:
Table 1
| Composition | Specification | Content |
| Template (T n) | 0.5ul(20-100ng) | |
| 2C9-F | 10uM | 0.5ul |
| 2C9-R | 10uM | 0.5ul |
| dNTP | 2.5mM each | 2ul |
| Taq enzyme | 5U/ul | 0.25ul |
| PCR damping fluid | 5X | 5ul |
| Water | Ultrapure water | 16.25ul |
The product obtained is the DNA fragmentation of 896bp, is designated as M, and number consecutively is M
1, M
2, M
3, by that analogy to M
98.
2.2 CYP2C9*3 site PCR
Reaction system 25ul, composition and content as shown in table 2:
Table 2
| Composition | Specification | Content |
| Template (T n) | 0.5ul(20-100ng) | |
| P3 | 10uM | 0.5ul |
| Q3 | 10uM | 0.5ul |
| F3 | 10uM | 0.5ul |
| R3 | 10uM | 0.5ul |
| dNTP | 2.5mM each | 1ul |
| Taq enzyme | 5U/ul | 0.25ul |
| PCR damping fluid | 5X | 5ul |
| Water | Ultrapure water | 16.25ul |
The product obtained is designated as B, and number consecutively is B
1, B
2, B
3, by that analogy to B
98.
2.3 VKORC1-1639 site first round PCR
Reaction system amount to 25ul, composition and content as shown in table 3:
Table 3
| Composition | Specification | Content |
| Template (T n) | 0.5ul(20-100ng) | |
| VK-F | 10uM | 0.5ul |
| VK-R | 10uM | 0.5ul |
| dNTP | 2.5mM each | 2ul |
| Taq enzyme | 5U/ul | 0.25ul |
| PCR damping fluid | 5X | 5ul |
| Water | Ultrapure water | 16.25ul |
The product obtained is the DNA fragmentation of 697bp, is designated as N, and number consecutively is N
1, N
2, N
3, by that analogy to N
98.
3. second take turns PCR
Second takes turns PCR reaction conditions is:
(1) denaturation: temperature 95 DEG C, time 4min.
(2) amplification cycles: (2-1) sex change: temperature 95 DEG C, time 30s;
(2-2) anneal: temperature 60 C, time 15s;
(2-3) extend: temperature 72 DEG C, time 30s;
Circulate 30 times.
M to be diluted the template as epicycle PCR after 100 times, reaction system 25ul by 3.1, composition and content as shown in table 4:
Table 4
| Composition | Specification | Content |
| Template | M nDilute 100 times | 0.5ul |
| P2 | 10uM | 0.5ul |
| Q2 | 10uM | 0.5ul |
| F2 | 10uM | 0.5ul |
| R2 | 10uM | 0.5ul |
| dNTP | 2.5mM each | 1ul |
| Taq enzyme | 5U/ul | 0.25ul |
| PCR damping fluid | 5X | 5ul |
| Water | Ultrapure water | 16.25ul |
The product obtained is designated as the product obtained and is designated as A, and number consecutively is A
1, A
2, A
3, by that analogy to A
98.
N to be diluted the template as epicycle PCR after 100 times, reaction system 25ul by 3.2, composition and content as shown in table 5:
Table 5
| Composition | Specification | Content |
| Template | N nDilute 100 times | 0.5ul |
| PV | 10uM | 0.5ul |
| QV | 10uM | 0.5ul |
| FV | 10uM | 0.5ul |
| RV | 10uM | 0.5ul |
| dNTP | 2.5mM each | 1ul |
| Taq enzyme | 5U/ul | 0.25ul |
| PCR damping fluid | 5X | 5ul |
| Water | Ultrapure water | 16.25ul |
The product obtained is designated as the product obtained and is designated as C, and number consecutively is C
1, C
2, C
3, by that analogy to C
98.
4. gel electrophoresis
B step 2.2 obtained, the A that step 3.1 obtains, the C that step 3.2 obtains respectively gets 5ul, the electrophoresis 40 minutes under 6v/cm voltage of the sepharose (GOODVIEW dyeing) with 2%, observations.Fig. 1-Fig. 6 is the electrophorogram detecting 1-11 and 39 in sample, and in figure, Marker is respectively 1200bp, 900bp, 750bp, 600bp, 450bp, 300bp and 150bp from top to bottom.
Known standard electrophoresis interpretation of result is as shown in table 6-table 8:
The analysis of PCR primer electrophoresis result is taken turns in two of table 6 standard C YP2C9*2 site
The analysis of PCR primer electrophoresis result is taken turns in one of table 7 standard C YP2C9*3 site
Note: haplotype is mutually exclusive, namely on same karyomit(e), CYP2C9 can only be * 2 or * 3, if therefore the detected result existing * 2 of a certain sample has * 3 again, so the CYP2C9 of this sample is the heterozygote of * 2/*3.
The analysis of PCR primer electrophoresis result is taken turns in two of table 8 standard VKORC1-1639 site
The electrophorogram that Fig. 1-Fig. 6 is obtained and the Comparison of standards in table 6-table 8, the genotypic result of CYP2C9 and VKORC1-1639 of 12 samples of detection is as shown in table 9:
Table 9
In order to verify the accuracy of this test kit and detection method, detection sample 1-11,39 is carried out gene sequencing, and its result is as shown in Fig. 8-Figure 43.Fig. 8-Figure 19 be respectively detect sample 1-11,39 CYP2C9*2DNA Sequencing chromatogram.Figure 20-Figure 31 be respectively detect sample 1-11,39 the Sequencing chromatogram of CYP2C9*3DNA.Figure 32-Figure 43 be respectively detect sample 1-11,39 VKORC1DNA Sequencing chromatogram.
Contrast known by the DAN sequencing result of the result obtained in table 9 and Fig. 8-Figure 19, result and the DNA sequencing result of the test of this test kit are coincide rate 100%.
Because CYP2C9*2 mutatant ratio is less, this detection does not find CYP2C9*2 saltant type, for the feasibility of confirmatory experiment scheme, we people is point mutation CYP2C9*2 base, it is mixed with wild-type DNA equal proportion and is designated as special sample S (the CYP2C9 genotype of sample S is * 1/*2).Using S as template, detect its CYP2C9*2 site as stated above, the electrophoresis result obtained as shown in Figure 7.Standard in the result obtained by Fig. 7 and table 6 contrasts, and known CYP2C9 genotype of refering in particular to sample S is * 1/*2.This experimental result and expection fit like a glove, and primer designed in illustrative experiment and PCR system are applicable to the discrimination analysis in CYP2C9*2 site, and experimental program is feasible.
Above embodiments of the invention have been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the scope of the invention change and improve, and all should still belong within this patent covering scope.
SEQUENCE LISTING
Jin Qi bio tech ltd, <110> Beijing
<120> warfarin personalized medicine detection kit and detection method thereof
<130> 2014
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213> artificial sequence
<400> 1
ctgtctactt tcctagctct caaagg 26
<210> 2
<211> 24
<212> DNA
<213> artificial sequence
<400> 2
gatagggcca ttcccaccat gttg 24
<210> 3
<211> 25
<212> DNA
<213> artificial sequence
<400> 3
ttttcccact ggctgaaaga gctaa 25
<210> 4
<211> 25
<212> DNA
<213> artificial sequence
<400> 4
tgggaaaaac actgctcttt aactc 25
<210> 5
<211> 19
<212> DNA
<213> artificial sequence
<400> 5
aagaggagca ttgaggcct 19
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
cgggcttcct cttgaacccg 20
<210> 7
<211> 22
<212> DNA
<213> artificial sequence
<400> 7
tctggtttat ggcagttaca ca 22
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
gggacttcga aaacatggag t 21
<210> 9
<211> 20
<212> DNA
<213> artificial sequence
<400> 9
gcacgaggtc cagagatgca 20
<210> 10
<211> 18
<212> DNA
<213> artificial sequence
<400> 10
ggtggggaga agstccag 18
<210> 11
<211> 24
<212> DNA
<213> artificial sequence
<400> 11
attcatgcag ggacatcttt ggtg 24
<210> 12
<211> 23
<212> DNA
<213> artificial sequence
<400> 12
tcctctcccg gcattatccc atc 23
<210> 13
<211> 23
<212> DNA
<213> artificial sequence
<400> 13
ccagcaggag agggaaatat cac 23
<210> 14
<211> 19
<212> DNA
<213> artificial sequence
<400> 14
accaagaygc tagacccaa 19
<210> 15
<211> 24
<212> DNA
<213> artificial sequence
<400> 15
gacctgaaaa acaaccattg gaca 24
<210> 16
<211> 17
<212> DNA
<213> artificial sequence
<400> 16
cgtgagccac cgcaacc 17
Claims (9)
1. warfarin personalized medicine detects primer, it is characterized in that: comprise following primer:
(1) CYP2C9*2 site first round PCR
Upstream primer: 5 '-CTGTCTACTTTCCTAGCTCTCAAAGG-3 ' (SEQ ID No.1),
Downstream primer: 5 '-GATAGGGCCATTCCCACCATGTTG-3 ' (SEQ ID No.2);
(2) PCR is taken turns in CYP2C9*2 site second
Upstream primer: 5 '-TTTTCCCACTGGCTGAAAGAGCTAA-3 ' (SEQ ID No.3),
Downstream primer: 5 '-TGGGAAAAACACTGCTCTTTAACTC-3 ' (SEQ ID No.4),
Saltant type special primer: 5 '-AAGAGGAGCATTGAGGCCT-3 ' (SEQ ID No.5),
Wild-type special primer: 5 '-CGGGCTTCCTCTTGAACCCG-3 ' (SEQ ID No.6);
(3) CYP2C9*3 site PCR
Upstream primer: 5 '-TCTGGTTTATGGCAGTTACACA-3 ' (SEQ ID No.7),
Downstream primer: 5 '-GGGACTTCGAAAACATGGAGT-3 ' (SEQ ID No.8),
Wild-type special primer: 5 '-GCACGAGGTCCAGAGATGCA-3 ' (SEQ ID No.9),
Saltant type special primer: 5 '-GGTGGGGAGAAGSTCCAG-3 ' (SEQ ID No.10);
(4) VKORC1-1639 site first round PCR
Upstream primer: 5 '-ATTCATGCAGGGACATCTTTGGTG-3 ' (SEQ ID No.11),
Downstream primer: 5 '-TCCTCTCCCGGCATTATCCCATC-3 ' (SEQ ID No.12);
(5) PCR is taken turns in VKORC1-1639 site second
Upstream primer: 5 '-CCAGCAGGAGAGGGAAATATCAC-3 ' (SEQ ID No.13),
Downstream primer: 5 '-ACCAAGAYGCTAGACCCAA-3 ' (SEQ ID No.14), VKORC1-1639 site A second takes turns PCR special primer:
5 '-GACCTGAAAAACAACCATTGGACA-3 ' (SEQ ID No.15), VKORC1-1639 site G second takes turns PCR special primer:
5′-CGTGAGCCACCGCAACC-3′(SEQ ID No.16)。
2. warfarin personalized medicine detection kit, is characterized in that: comprise primer according to claim 1.
3. warfarin personalized medicine detection kit according to claim 2, is characterized in that: also comprise PCR damping fluid, Taq enzyme and dNTP.
4. warfarin personalized medicine detection kit according to claim 3, is characterized in that: during use, and the concentration of described each primer is 10uM; DNTP is the mixture of dATP, dGTP, dCTP, dTTP, and the concentration of described dATP, dGTP, dCTP, dTTP is 2.5mM.
5. warfarin personalized medicine detection method, is characterized in that: use the test kit described in claim 2-4, comprise the following steps:
(1) first round PCR
With nucleic acid to be detected for template, carry out first round PCR process to CYP2C9*2 site, CYP2C9*3 site and VKORC1-1639 site respectively, the product obtained is designated as M, B and N respectively;
(2) second take turns PCR
As template after M being diluted 100 times, carry out second to CYP2C9*2 site and take turns PCR process, its product is designated as A;
As template after diluting 100 times using N, carry out second to VKORC1-1639 site and take turns PCR process, its product is designated as C;
(3) electrophoresis
A and C obtained in the B obtained in step (1), step (2) is carried out gel electrophoresis, the electrophorogram obtained is contrasted with corresponding standard.
6. warfarin personalized medicine detection method according to claim 5, is characterized in that: in described step (1) to the condition that first round PCR process is carried out in CYP2C9*2 site, CYP2C9*3 site and VKORC1-1639 site be:
4min at (a) 95 DEG C;
B 30s at () 95 DEG C, 15s at 60 DEG C, 30s at 72 DEG C, circulate 35 times.
7. warfarin personalized medicine detection method according to claim 5, is characterized in that: in described step (2) to CYP2C9*2 site and VKORC1-1639 site the second condition of taking turns PCR process of carrying out be:
4min at (a) 95 DEG C;
B 30s at () 95 DEG C, 15s at 60 DEG C, 30s at 72 DEG C, circulate 30 times.
8. warfarin personalized medicine detection method according to claim 5, it is characterized in that: the condition of electrophoresis is in step (3): get the B obtained in step (1), each 5ul of A and C obtained in step (2), carry out gel electrophoresis.
9. warfarin personalized medicine detection method according to claim 8, it is characterized in that: the condition of electrophoresis is in step (3): get the B obtained in step (1), each 5ul of A and C obtained in step (2), after the sepharose with 2% is dyed under 6v/cm voltage electrophoresis 40 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510105467.7A CN104962606A (en) | 2015-03-11 | 2015-03-11 | Detection kit and detection method for individualized medication of warfarin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510105467.7A CN104962606A (en) | 2015-03-11 | 2015-03-11 | Detection kit and detection method for individualized medication of warfarin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104962606A true CN104962606A (en) | 2015-10-07 |
Family
ID=54216691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510105467.7A Pending CN104962606A (en) | 2015-03-11 | 2015-03-11 | Detection kit and detection method for individualized medication of warfarin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962606A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274097A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP2C19*2 and CYP2C19*3 detection primer and detection system thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824466A (en) * | 2009-12-29 | 2010-09-08 | 广州益善生物技术有限公司 | Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes |
| CN101899519A (en) * | 2010-07-23 | 2010-12-01 | 苏州大学 | Kit for detecting SNP locus of gene associated with individualized medication of warfarin and PCR amplification method thereof |
| CN102251043A (en) * | 2011-07-27 | 2011-11-23 | 协和干细胞基因工程有限公司 | Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same |
| CN102329885A (en) * | 2011-10-26 | 2012-01-25 | 李艳 | Kit for detecting polymorphism of VKORC1 and CYP2C9 genes |
| CN102690888A (en) * | 2012-06-15 | 2012-09-26 | 向华 | Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system |
| CN102719524A (en) * | 2010-07-23 | 2012-10-10 | 苏州大学 | Kit for warfarin personalized medication related gene SNP locus detection and PCR amplification method using same |
| CN103074438A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Multi-gene detection kit for guiding administration of warfarin and detection method of multi-gene detection kit |
| CN103146692A (en) * | 2013-02-21 | 2013-06-12 | 丁虎 | Kit for rapidly detecting warfarin individualized medication related gene SNP sites, and its detection method |
| CN103184271A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Warfarin drug gene VKORC1 and CYP2C9 mutation detection kit |
| CN103525911A (en) * | 2013-09-22 | 2014-01-22 | 上海中优医药高科技有限公司 | Method for detecting relative gene polymorphism of warfarin personalized medication by using high-resolution melting curve analysis technology |
| CN103820553A (en) * | 2014-02-27 | 2014-05-28 | 厦门大学附属中山医院 | Multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and detection method thereof |
| CN104031978A (en) * | 2013-03-06 | 2014-09-10 | 北京宏微特斯生物科技有限公司 | ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof |
-
2015
- 2015-03-11 CN CN201510105467.7A patent/CN104962606A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824466A (en) * | 2009-12-29 | 2010-09-08 | 广州益善生物技术有限公司 | Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes |
| CN101899519A (en) * | 2010-07-23 | 2010-12-01 | 苏州大学 | Kit for detecting SNP locus of gene associated with individualized medication of warfarin and PCR amplification method thereof |
| CN102719524A (en) * | 2010-07-23 | 2012-10-10 | 苏州大学 | Kit for warfarin personalized medication related gene SNP locus detection and PCR amplification method using same |
| CN102251043A (en) * | 2011-07-27 | 2011-11-23 | 协和干细胞基因工程有限公司 | Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same |
| CN102329885A (en) * | 2011-10-26 | 2012-01-25 | 李艳 | Kit for detecting polymorphism of VKORC1 and CYP2C9 genes |
| CN103184271A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Warfarin drug gene VKORC1 and CYP2C9 mutation detection kit |
| CN102690888A (en) * | 2012-06-15 | 2012-09-26 | 向华 | Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system |
| CN103074438A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Multi-gene detection kit for guiding administration of warfarin and detection method of multi-gene detection kit |
| CN103146692A (en) * | 2013-02-21 | 2013-06-12 | 丁虎 | Kit for rapidly detecting warfarin individualized medication related gene SNP sites, and its detection method |
| CN104031978A (en) * | 2013-03-06 | 2014-09-10 | 北京宏微特斯生物科技有限公司 | ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof |
| CN103525911A (en) * | 2013-09-22 | 2014-01-22 | 上海中优医药高科技有限公司 | Method for detecting relative gene polymorphism of warfarin personalized medication by using high-resolution melting curve analysis technology |
| CN103820553A (en) * | 2014-02-27 | 2014-05-28 | 厦门大学附属中山医院 | Multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| NOUHA BEN GAIED ET AL: "End-capped HyBeacon probes for the analysis of human genetic polymorphisms related to warfarin metabolism", 《ORGANIC & BIOMOLECULAR CHEMISTRY》 * |
| 都丽萍等: "CYP2C9及VKORC1基因多态性对华法林剂量和抗凝效果的影响", 《中国药学杂志》 * |
| 高红等: "双向等位基因特异性PCR快速区分纯合子和杂合子SNP分型的新方法", 《医学分子生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274097A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP2C19*2 and CYP2C19*3 detection primer and detection system thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106434940A (en) | Kit used for SNP detection of personalized medicine relaed genes of statin lipid-lowering medicine and detecting method thereof | |
| CN107119107A (en) | A kind of method and kit for detecting mankind's CYP2C19 gene pleiomorphisms | |
| CN106498036A (en) | A kind of construction method in the Drug Discovery SNP variations library for high-flux sequence detection and its application | |
| CN103525925A (en) | Pair of specific primers and probe for detection of CYP2C19 gene chip | |
| CN104404163A (en) | Probes and kit for detecting human CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) gene polymorphism | |
| CN104328200B (en) | The detection kit of auxiliary diagnosis alzheimer's disease and detection method thereof | |
| CN105671190A (en) | High-throughput molecular marker for identifying neck rot and root rot resistance of tomatoes and marking method and application thereof | |
| CN110564861A (en) | Fluorescence labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN109371132A (en) | It is a kind of for detecting the kit of CYP2D6 gene copy number variation | |
| CN108531574A (en) | It detects alcohol dehydrogenase ADH1B and ethyl alcohol takes off the primer and method of acetaldehyde dehydrogenase ALDH2 gene pleiomorphisms | |
| CN110982895B (en) | Primer group and kit for detecting human erythrocyte ABO blood type genotyping and application | |
| CN105274220A (en) | CYP4F2*3 detection primer and detection system thereof | |
| CN104962606A (en) | Detection kit and detection method for individualized medication of warfarin | |
| CN107653317A (en) | A kind of kit of molecular beacon probe detection mankind's CYP2C9 gene pleiomorphisms, method and its application | |
| CN105274221A (en) | CYP3A5*3 detection kit | |
| CN102952858B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD | |
| CN106701947A (en) | Kit for detecting genotype of gene CYP2C19 at SNP site rs4986893 | |
| CN105861690A (en) | Primer combination, reagent kit and method for detecting CYP2C19*2 and CYP2C19*3 polymorphism | |
| CN103627789A (en) | Kit for detecting warfarin sensitivity gene by using electrochemical gene sensor method | |
| CN105274097A (en) | CYP2C19*2 and CYP2C19*3 detection primer and detection system thereof | |
| CN110578014A (en) | Kit and method for detecting candida parapsilosis nucleic acid | |
| CN105602947B (en) | The detection primer group of CYP3A5 gene, its reaction system and application for constituting | |
| Veeranagouda et al. | Next-generation sequencing to investigate urinary microRNAs from Macaca fascicularis (Cynomolgus monkey) | |
| CN104561302A (en) | Detection method for CYP4F2 gene polymorphism, as well as nucleic acid probe and kit for method | |
| CN109825572A (en) | A kind of kit and its detection method of detection and the susceptibility related gene polymorphism of propofol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into 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: 20151007 |