CN105861739A - CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit - Google Patents
CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit Download PDFInfo
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Abstract
The invention discloses a CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit. The amplification system comprises two forward primers and a reverse fluorescent primer of six SNPs (single nucleotide polymorphisms) respectively and can simultaneously amplify the six SNPs. The system is characterized by achieving one-tube amplification of three SNPs on the gene CYP2C19, two SNPs on the gene CYP2C9 and one SNP on the gene VKORC1 through multiple PCR (polymerase chain reaction). In particular, the amplification system can achieve direct amplification of blood and blood spot samples and dispenses with the step of extracting DNA (deoxyribonucleic acid). The amplification system can integrate the UDG-dUTP antipollution measure and can effectively prevent product pollution. The detection system is comprehensive in site detection, is simple and convenient to operate, has high specificity, high sensitivity and strong reliability, is low in cost and has the capacity of mass detection.
Description
Technical field
The present invention relates to one and detect clopidogrel and warfarin medicine based on multiple fluorescence PCR technology and capillary electrophoresis technique simultaneously
The gene pleiomorphism of CYP2C19, CYP2C9 and VKORC1 that thing metabolic rate is relevant, for clinic in diseases such as treatment cardiovascular
Sick personalized medicine provides reference, belongs to the clinical molecular detection technique in biomedical sector.
Background technology
Cytochrome P450 superfamily (Cytochrome P450proteins, CYP) is one group of structurally and functionally related superfamily base
Because of the isozyme of coding, it is primarily present in the monooxygenase in liver, intestinal.Prokaryote is free in kytoplasm, is one
Plant soluble protein；In eukaryote, as a kind of embrane-associated protein, it is mainly distributed on endoplasmic reticulum, microsome and mitochondrion
On inner membrance, major function is to play an important role in terms of the metabolism and removing toxic substances of medicine (such as clopidogrel, warfarin etc.).Grind
Study carefully the medicine generation finding that CYP2C9, CYP2C19, CYP2C17, CYP2D6, CYP1A2 and CYP3A4 are responsible for about 90%
Thank.Vitamin K epoxide reductase (VKOR) is main by VKOR complex 1 (VKORC1) gene code, its effect
Being to make inactive epoxidation type vitamin K be changed into activated hydroquinone type vitamin K, hydroquinone type vitamin K is γ-paddy ammonia
The necessary cofactor of acyl group carboxylase (GGCX), prothrombin, VII, IX, X, protein C and protein S are passed through
Activate after the carboxylation of GGCX, and then occur a series of cascade reaction to cause blood coagulation.
Clopidogrel trade name Plavix (Plavix), is a kind of anticoagulant, depends on CYP2C19 enzyme generation
Thank to the activation producing active metabolite, play antiplatelet curative effect, for currently used widest thiophene pyridines antiplatelet
Medicine, is applied to prevent and treat what myocardial infarction, ischemic cerebral thrombosis, thromboangiitis obliterans, atherosclerosis and thromboembolism caused
Complication, can reduce the generation of cardiovascular death and myocardial infarction etc. after treatment.Clinical research shows that CYP2C19 enzymatic activity is deposited
In obvious individual variation, the metabolism of the many clinical medicines of impact, this with this gene on there is multiple pleomorphism site and have very high point
System.Wherein slow inactivation (poor metabolizer, PM) sports master, fast metabolism with CYP2C19*2, CYP2C19*3
Type (extensive metabolizer, EM) sports master with CYP2C19*17.CYP2C19*2 sudden change can cause exon 5 '
The front 40bp base deletion of end, changes mRNA reading frame, makes albumen synthesize premature end, produces one by disconnected cut scarce
The nonfunctional pheron of weary heme binding sites.CYP2C19*3 suddenlys change, and makes terminator codon in advance, produces metabolic function pole
Low CYP2C19 protease.Both sudden changes can be explained > the Asians poor metabolizer of 99% and about 88% white people weak
The phenotype of metabolizer.And the sudden change of CYP2C19*17 makes it easier to combine liver cell nuclear protein transcription enzyme, accelerate transcription rate,
Improve the CYP2C19 enzyme metabolic efficiency to medicine, thus accelerate clopidogrel and be converted into its active metabolite.According to investigations
CYP2C19*17 gene frequency in Chinese Han Population is 2%, and the ratio in Uygur nationality crowd is but up to 18%,
CYP2C19*17 carrier has significant clopidogrel strong reactivity and bleeding risk substantially to increase.FDA and american heart
The suggestion of sick association needs increase clopidogrel dosage or consider to change therapeutic scheme for slow inactivation patient；Fast metabolism is suffered from
Person need to reduce clopidogrel dosage.The most most of CYP2C19 detection kit only detect CYP2C19*2 and
CYP2C19*3 site, such typing is incomplete.This research adds CYP2C19*17 site so that typing is more complete
Face, diagnoses the reference providing more specific to clinician.
Warfarin (Warfarin) is a kind of Coumarins oral anticoagulation, and its mechanism of action is the competitive work to antivitamin K
With, the synthesis of thrombin in suppression hepatocyte, reduces the platelet aggregation reaction of thrombin induction, is widely used in prevention and controls
Treat multiple thrombotic disease and the Cardiac valve prosthesis implant surgeries such as phlebothrombosis, myocardial infarction, ischemic shock.Clinic shows
Warfarin pharmacological action is the most affected by many factors, including vitamin K absorption in age, sex, body weight, disease, diet etc.
Environmental factors, and the factor such as hereditary variation, wherein VKORC1 (-1639G ＞ A) gene mutation is to affect clinical medicine demand
The topmost factor of dosage individual variation, and wild type G gene promoter activity is higher by 44% than A genic mutation type, variation can show
Write and increase patient's sensitivity to warfarin.In addition warfarin has 2 kinds of isomers, and S type is mainly through liver CYP2C9 medicament
Amount, R type is by CYP3A4 dosage, and the anticoagulating active of S type is stronger than R type 3-5 times, and causing CYP2C9 is to affect medication
One of dosage Major Enzymes.Allelotype CYP2C9*2 common for CYP2C9 in crowd and CYP2C9*3, sudden change can cause
The miopragia of this enzyme, causes the dosage ability to warfarin and Scavenging activity to be substantially reduced, so that patient's method to China
Woods is sensitive, just has higher hemorrhage risk at the use initial stage.If there is the variation of the two gene in U.S. FDA suggestion patient,
Need to use relatively low predose.
In sum, CYP2C19, CYP2C9 and VKORC1 gene pleiomorphism directly or indirectly affects clopidogrel and Hua Fa
Woods metabolism in vivo and anticoagulant effect, and both medicines are all the conventional anticoagulants in treating cardiovascular disease.Therefore originally
Test kit can be simultaneously for the personalized medicine offer reference of both medicines.In addition have been reported that find about 12% clinical medicine by
CYP2C19 metabolism, the clinical medicine of about 16% is responsible for metabolism by CYP2C9, therefore this research to CYP2C19, CYP2C9
The other drug consumption relevant with VKORC1 gene pleiomorphism instructs also certain reference value.
But, the method being currently used for detecting CYP2C19, CYP2C9 and VKORC1 gene pleiomorphism is a lot, as directly surveyed
Sequence method, fluorescence quantitative PCR method, gene chips and restricted enzyme enzymatic fragment length polymorphism (RFLP) analytic process
Deng.1, direct sequencing: the detection method detecting CYP2C19, CYP2C9 and VKORC1 gene mutation at present is typically
Traditional direct sequencing: design sequencing primer, extracts DNA and carries out PCR amplification and product order-checking.The main of the method lacks
Point is: needs to carry out sample extracting DNA, detection quality and concentration, operation complexity before (1) PCR reaction, exists and obscure
The risk of sample.(2) when detecting multiple site, one sample needs to carry out multiple reaction, and testing cost is high, the need to template amount
Asking high, manipulation strength is big, and easily causes operation mistake and pollution.(3) need product to be purified and surveys after PCR reaction
The steps such as sequence chemical reaction.Being exactly complex steps on the whole, the test period is long, and cost is high, is unfavorable for clinical spread.
2, rflp analysis method: change based on the restriction enzyme enzyme recognition site that gene mutation causes, loses such as site or produces new
Site, expands a certain specific fragment by PCR, and recycling restricted enzyme carries out endonuclease reaction, electrophoresis observation fragment big
Little, but it has restriction enzyme site limitation, and it is difficult to high flux parallel analysis.3, quantitative fluorescent PCR: real-time fluorescence
Quantitative PCR is simple to operate quickly, tests highly sensitive, and result such as fast can quantify at the advantage, but to there is sample the most dirty for this technology
, easily there is cross reaction in dye, false positive rate is high.And common quantitative fluorescent PCR is only with one to four fluorescence channel, in
It is at most to detect 4 sites, it is impossible to detect while meeting multiple SNP site.4, gene chip (gene chip):
Gene chip is by micro-processing technology, by the gene probe of the particular sequence of ten hundreds of even million meters, arranges regularly
Fixing with on the holder such as silicon chip, slide, constitute a two-dimentional DNA probe array, utilize the biology of this kind of chip and labelling
Sample hybridizes, and the gene expression profile bio information of sample can be carried out qualitative and quantitative analysis.It can carry out high throughput testing,
But the method is costly, big to template demand, operation and data analysis are complicated, the longest.It is therefore desirable to set up a kind of same
Time quickly detect these three gene mutation method, for hospital and other medical institutions provide a kind of simple to operate, high specificity,
Highly sensitive, flux is high, the highly reliable and detection scheme of low cost.
Summary of the invention
It is an object of the invention to provide a kind of CYP2C19, CYP2C9 and VKORC1 gene type composite amplification system, this body
Cording has simple to operate, high specificity, highly sensitive, flux is high, the highly reliable and detection feature of low cost.
The application also provides for CYP2C19, CYP2C9 and VKORC1 gene parting detecting reagent, can quickly detect these three
The situation of gene mutation, provides medication guide for hospital and other medical institutions.
Meanwhile, the application also provides for the using method of this detection kit.
CYP2C19, CYP2C9 and VKORC1 gene type composite amplification system, described amplification system can expand 6 SNP simultaneously
Site, this system includes that the primer base sequences of 6 SNP site is as follows:
The primer base sequences in CYP2C19*2 gene 681G > A site is as follows:
Forward wild primers: 5 '-TTTCCCACTATCATTGATTATTTCCCG-3 '
Forward mutation type primer: 5 '-TTTCCCACTATCATTGATTATTTCCCA-3 '
Reversely general primer: 5 '-AACTAGTCAATGAATCACAGATACGC-3 '
The primer base sequences in CYP2C19*3 gene 636G > A site is as follows:
Forward wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTGG-3 '
Forward mutation type primer: 5 '-ATCAGGATTGTAAGCACCCCCTGA-3 '
Reversely general primer: 5 '-GATATTCACCCCATGGCTGTCTA-3 '
The primer base sequences in CYP2C19*17 gene-806C > T site is as follows:
Forward wild primers: 5 '-GCATTATCTCTTACATCAGAGATG-3 '
Forward mutation type primer: 5 '-GCATTATCTCTTACATCAGAGATA-3 '
Reversely general primer: 5 '-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '
The primer base sequences in CYP2C9*2 gene 430C > T site is as follows:
Forward wild primers: 5 '-GGGCTTCCTCTTGAACACG-3 '
Forward mutation type primer: 5 '-GGGCTTCCTCTTGAACACA-3 '
Reversely general primer: 5 '-GGGAGGATGGAAAACAGAGACTTAC-3 '
The primer base sequences in CYP2C9*3 gene 1075A > C site is as follows:
Forward wild primers: 5 '-GCTGGTGGGGAGAAGGTCAAT-3 '
Forward mutation type primer: 5 '-GCTGGTGGGGAGAAGGTCAAG-3 '
Reversely general primer: 5 '-AACCATCCTCTCTTTAAGTTTGC-3 '
The primer base sequences in VKORC1 gene-1 639G > A site is as follows:
Forward wild primers: 5 '-GCGTGAGCCACCGCACCC-3 '
Forward mutation type primer: 5 '-GCGTGAGCCACCGCACCT-3 '
Reversely general primer: 5 '-AGCAGGAGAGGGAAATATC-3 '.
Described system can also expand an internal reference fragment Amel gene loci, also includes detecting this internal reference fragment Amel in this system
The primer base sequences of gene loci:
Forward primer: 5 '-CCCTGGGCTCTGTAAAGAATAG-3 '
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '.
Be added with on described primer modification or with modified base replace normal base, described in be modified to fluorescence group modify, phosphorylation
Modify, thiophosphorylation is modified, lock nucleic acid is modified or peptide nucleic acid(PNA) is modified.
Described primer 3 ' holds-2 to-15 to change 1 to 3 base or/and sequence after primer 3 ' holds-15 changes
Dynamic, described change includes that end increases other sequences, deletes portion distal end sequence, changing section base sequence.
Described 6 SNP site and internal reference fragment Amel gene loci are respectively by the fluorescent labeling of two kinds of colors, identical fluorescence mark
Note is considered as same group, and two groups are respectively as follows: first group of CYP2C19*2 gene 681G > A site, CYP2C19*3 gene 636G > A
Site, CYP2C19*17 gene-806C > T site；Second group of CYP2C9*2 gene 430C > T site, CYP2C9*3
Gene 1075A > C site, VKORC1 gene-1 639G > A site, Amel gene loci.
Described first group of fluorescent labeling is FAM, and second group of fluorescent labeling is HEX.
The fluorescent labeling of described 6 SNP site is positioned at 5 ' ends of reverse general primer, described internal reference fragment Amel gene loci
Fluorescent labeling be positioned at forward primer 5 ' end.
CYP2C19, CYP2C9 and VKORC1 gene parting detecting reagent, including PCR premixed solution (PCR Master
Mix), described PCR premixed solution includes above-mentioned amplification system.
Described premixed solution also includes thermal starting DNA Taq enzyme, UDG enzyme and 1.25 × Buffer.
Described detection kit also includes positive control dna, negative control and internal standard ROX500.
Described positive control dna is normal person DNA, and described negative control is aquesterilisa.
The using method of CYP2C19, CYP2C9 and VKORC1 parting detecting reagent.Mainly comprise the steps that
1) premixed solution subpackage
Vibration mixing premixed solution, the detection number subpackage carried out according to expectation, each PCR reaction tube subpackage 19 μ L；
2) template is added
1 μ L blood sample, or the Dried blood spots sample of addition diameter 1mm it is separately added in corresponding PCR reaction tube, or
1 μ L positive control, or 1 μ L negative control；
3) PCR amplification
Each reaction tube is put into PCR amplification instrument reactive tank, and arranging reaction system is 20 μ L；
PCR amplification is carried out by following response procedures:
4) genetic analyzer is used to detect amplified production by capillary electrophoresis.
Genetic analyzer detection (completing at detection zone)
1. prepared by detection sample
Preparation is mixed with the loading mixed liquor of molecular weight internal standard and Methanamide: (0.5 μ L molecular weight internal standard+8.5 μ L Methanamide) × detection
Sample number.The vortex oscillation mixing 10-15 second；
Methanamide and the internal standard mixture of each detection hole subpackage 9 μ L is given with pipettor；
Take 1 μ L amplified production to add in Methanamide and internal standard mixture, cover shrouding Jiao Gai.If desired with centrifuge
Bubble in sample is removed by of short duration being centrifuged；
Sample is placed 95 DEG C of degeneration 3 minutes, rapidly placement ice bath upper 3 minute.
2. detection
Detect according to genetic analyzer user's service manual step.It is 10 seconds, sample introduction voltage that detection suggestion arranges sample injection time
It it is 1800 seconds for 3kV, operation time.
3. data analysis
Associated documents are imported, including Panel, Bin, corresponding Analysis Method, ROX500 in GeneMapper software
Internal standard.Input sample source data (.fsa file), the file imported before selecting in related parameter choosing hurdle, analytical data.
Presence or absence according to length-specific amplified production i.e. can determine that whether sample specific site exists specific gene type.
This test kit is by PCR Master Mix, positive control dna (normal person), negative control (aquesterilisa), internal standard
ROX500 forms, and wherein PCR Master Mix key component comprises thermal starting DNA Taq enzyme, UDG enzyme, 1.25 × Buffer
And each site primer etc..Only need to add template (blood/blood card/DNA) during amplification in PCR Master Mix to be available on the machine
Reaction.Two forward primers that described test kit includes detecting gene C YP2C19*2 (rs4244285,681G > A) site and
One reverse fluorescent primer；Two forward primers and one in detection gene C YP2C19*3 (rs4986893,636G > A) site
Individual reverse fluorescent primer；Two forward primers in described detection gene C YP2C19*17 (rs12248560 ,-806C > T) site with
And a reverse fluorescent primer；Two forward primers in described detection gene C YP2C9*2 (rs1799853,430C > T) site with
And a reverse fluorescent primer；Two forward primers in described detection gene C YP2C9*3 (rs1057910,1075A > C) site
And a reverse fluorescent primer；Two forward primers in described detection gene VKORC1 (rs9923231 ,-1639G > A) site
And a reverse fluorescent primer；The forward fluorescent primer in internal reference fragment Amel site and reverse primer.
The feature of the inventive method:
1) use genetic analyzer by capillary electrophoresis detection amplified production:
Genetic analyzer detection platform is one of widely used main flow detection platform, by capillary electrophoresis to fluorescent labeling
Amplified production detect, the major technique advantage of detection is:
1., detection sensitivity is high.
Capillary electrophoresis is used in combination fluorescent dye and detection sensitivity is greatly improved, higher than sepharose electrophoresis detection sensitivity
More than 100 times.Amplified production can be detected more delicately, and clearly distinguish amplification wild type and saltant type.On the other hand,
Owing to detection sensitivity is high, provide bigger adjustment space for composite PCR amplified reaction.The more important thing is, due to detection
Highly sensitive, reduce the requirement to pcr amplification product amount, PCR reaction template consumption can be reduced, can reduce
PCR amplification cycles number, saves sample.
2., detection resolution is high.
In the 100-500bp detection range generally utilized can clearly, efficiently differentiate 1bp difference.The highest resolution
Make will not due to product size close to and produce erroneous judgement, it also avoid the result erroneous judgement that non-specific amplification causes.The opposing party
Face, high-resolution also makes to detect more site simultaneously and is possibly realized.
3., can be simultaneously to 4 kinds of fluorescence signal detections.
Further expand detection range so that detect more site simultaneously and be possibly realized.
4., detection speed fast (40 minutes), need operation less, can automatization's mass detection.
5., testing result may utilize software and automatically analyzes and sentence type.
2) expand multiple site by a MULTIPLE COMPOSITE PCR amplification system simultaneously.
This patent utilizes a MULTIPLE COMPOSITE PCR amplification system, it is achieved about CYP2C19, CYP2C9 and VKORC1
6 detection site of these three gene and the amplification of a control site.It is advantageous that:
1., manipulation strength and testing cost are greatly reduced.Only a PCR amplification and a genetic analyzer detection reaction are i.e.
Can complete all to detect.
2., Single tube amplification detects, and avoids the mistakes such as pollution and sample mix to the full extent.
3) improvement that site selects.
1., detection site is comprehensive, adds the detection in CYP2C19*17 site occurred frequently in Uygur nationality crowd, makes
Detection and genotyping is more comprehensive.
2., CYP2C19, CYP2C9 and VKORC1 gene pleiomorphism directly or indirectly affects clopidogrel and warfarin
Metabolism in vivo and anticoagulant effect.This test kit can be clopidogrel and warfarin both medicines
Body medication provides reference, provides more treatment information for clinician.
4) DS system and anti-pollution measure
This amplification system can directly use the sample such as blood, blood card to expand, and eliminates the step of DNA extraction, and operation is more
Add simplicity, be suitable for batch operation.Another system adds UDG-dUTP anti-pollution measure, can effectively prevent
Product pollution, it is to avoid cause false positive results.
To sum up, the art of this patent route applications detects in CYP2C19, CYP2C9 and VKORC1 gene type.Mainly
Advantage includes: each sample only needs an augmentation detection reaction, very big relative to method based on common amplification electrophoresis detection
Reduce manipulation strength；Available result in 3 hours.
The present invention compared with prior art has following advantages and an effect:
1, the present invention is based on multiplex PCR and capillary electrophoresis technique, a tubular type amplification, simultaneously to clopidogrel and warfarin this
6 SNP site that two kinds of drug metabolisms are relevant detect, for the drug dose relevant with these three gene pleiomorphism
Guidance, both saved production cost and testing cost, improve again detection efficiency；In addition introducing Amel sex site is
Internal reference is for monitoring whole response system and the quality of assessment DNA profiling, it is to avoid false negative result.
2, adding CYP2C19*17 typing (ultra-rapid metabolism type) detection, typing function is more comprehensive；
3, the present invention can realize the direct amplification of blood and blood card, it is not necessary to any process, eliminates the step extracting DNA,
Integrate the UDG anti-pollution system of enzyme-dUTP simultaneously, shorten the sample process time, decrease the generation of pollution；
4, the present invention utilizes genetic analyzer detection platform, is detected fluorescently-labeled amplified production by capillary electrophoresis,
Highly sensitive, simple to operate, genotyping result intuitively easily interpretation.Whole flow process completed all to detect in 3 hours, manual operation
Time is less than 30 minutes；
5, the present invention possesses high-volume augmentation detection ability.
Accompanying drawing explanation
Fig. 1 is the blood amplified production result collection of illustrative plates of the capillary electrophoresis separation sample A of genetic analyzer,
Fig. 2 is the DNA cloning product result collection of illustrative plates of the capillary electrophoresis separation sample A of genetic analyzer.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1: a kind of test kit detecting CYP2C19, CYP2C9 and VKORC1 gene type
This test kit is by PCR Master Mix, positive control dna (normal person), negative control (aquesterilisa), internal standard ROX500
Composition, wherein PCR Master Mix key component comprise thermal starting DNA Taq enzyme (5U/ μ L, KAPA), UDG enzyme (5U/ μ L,
NEB), 1.25 × Buffer and each site primer etc..Each amounts of components is: when reaction system is 20 μ L, 1.25 × Buffer
Being 16 μ L, thermal starting DNA Taq enzyme is 0.4 μ L, and UDG enzyme 0.04 μ L, primer Mix are 0.58 μ L (each primer final concentration
100～200nM), template DNA or blood are 1-2 μ L, and aquesterilisa complements to 20 μ L.
The former sequence of primer of 6 SNP site is as follows:
The primer base sequences in CYP2C19*2 gene 681G > A site is as follows:
Forward wild primers: 5 '-TTTCCCACTATCATTGATTATTTCCCG-3 '
Forward mutation type primer: 5 '-TTTCCCACTATCATTGATTATTTCCCA-3 '
Reversely general primer: 5 '-AACTAGTCAATGAATCACAGATACGC-3 '
The primer base sequences in CYP2C19*3 gene 636G > A site is as follows:
Forward wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTGG-3 '
Forward mutation type primer: 5 '-ATCAGGATTGTAAGCACCCCCTGA-3 '
Reversely general primer: 5 '-GATATTCACCCCATGGCTGTCTA-3 '
The primer base sequences in CYP2C19*17 gene-806C > T site is as follows:
Forward wild primers: 5 '-GCATTATCTCTTACATCAGAGATG-3 '
Forward mutation type primer: 5 '-GCATTATCTCTTACATCAGAGATA-3 '
Reversely general primer: 5 '-ATCTCTCGGGCTGTTTTCCTTAGATAA-3’
The primer base sequences in CYP2C9*2 gene 430C > T site is as follows:
Forward wild primers: 5 '-GGGCTTCCTCTTGAACACG-3 '
Forward mutation type primer: 5 '-GGGCTTCCTCTTGAACACA-3 '
Reversely general primer: 5 '-GGGAGGATGGAAAACAGAGACTTAC-3 '
The primer base sequences in CYP2C9*3 gene 1075A > C site is as follows:
Forward wild primers: 5 '-GCTGGTGGGGAGAAGGTCAAT-3 '
Forward mutation type primer: 5 '-GCTGGTGGGGAGAAGGTCAAG-3 '
Reversely general primer: 5 '-AACCATCCTCTCTTTAAGTTTGC-3 '
The primer base sequences in VKORC1 gene-1 639G > A site is as follows:
Forward wild primers: 5 '-GCGTGAGCCACCGCACCC-3 '
Forward mutation type primer: 5 '-GCGTGAGCCACCGCACCT-3 '
Reversely general primer: 5 '-AGCAGGAGAGGGAAATATC-3 '
For preventing the mutually amplification between wild type from mutant sequences, the different SNP typing products of convenient differentiation, therefore, this reagent
In box, the primer sequence of 6 SNP site has carried out a series of specific modification on the basis of above-mentioned former sequence, is included in primer
3 '-2 to-15, ends change 1 to 3 base, sequence end after primer 3 ' holds-15 increases, deletes and replacement part
Base sequence, carries out fluorescent decoration (5 ' end FAM or HEX fluorescent labeling), lock nucleic acid modification, phosphorylation modification, sulfur generation
Phosphorylation modification, peptide nucleic acid(PNA) modification etc., be prevented effectively from the mutual amplification of same site difference SNP, strengthen the specificity of primer.
In this patent, the primer base sequences of 6 SNP site is as follows:
The primer base sequences in CYP2C19*2 gene 681G > A site is as follows:
Forward wild primers: 5 '-TTTCCCACTATCATTGACTATTTCCAG-3’
Forward mutation type primer: 5 '-TTTCCACTATCATTGATTATTTGCCA-3’
Reversely general primer: 5 '-FAM-AACTAGTCAATGAATCACAGATACGC-3 '
The primer base sequences in CYP2C19*3 gene 636G > A site is as follows:
Forward wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTAG-3’
Forward mutation type primer: 5 '-ATCAGGATTGTAAgCACCCCATGA-3’
Reversely general primer: 5 '-FAM-GATATTCACCCCATGGCTGTCTA-3’
The primer base sequences in CYP2C19*17 gene-806C > T site is as follows:
Forward wild primers: 5 '-GCATTATCTCTTACATCAGTGATG-3’
Forward mutation type primer: 5 '-GCATTATCTCTTaCATCAGAGCTA-3’
Reversely general primer: 5 '-FAM-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '
The primer base sequences in CYP2C9*2 gene 430C > T site is as follows:
Forward wild primers: 5 '-GGGCTTCCTCTTGAACATG-3’
Forward mutation type primer: 5 '-GGGCTTCCTCTTGAAGACA-3’
Reversely general primer: 5 '-HEX-GGGAGGATGGAAAACAGAGACTTAC-3 '
The primer base sequences in CYP2C9*3 gene 1075A > C site is as follows:
Forward wild primers: 5 '-GCTGGTGGGGAGAAGGTCGAT-3’
Forward mutation type primer: 5 '-GCTGGTGGGGAGATGGTCAAG-3’
Reversely general primer: 5 '-HEX-AACCATCCTCTCTTTAAGTTTGC-3 '
The primer base sequences in VKORC1 gene-1 639G > A site is as follows:
Forward wild primers: 5 '-GCGTGAGCCACCGCTCCC-3’
Forward mutation type primer: 5 '-GCGTGAGCCACCGCACGT-3’
Reversely general primer: 5 '-HEX-AGCAGGAGAGGGAAATATC-3 '
Note: 1. "-" list underscore represents each primer and holds 1 to 3 base of-2 to-15 changes at primer 3 '.
2. "=" double underline represents each primer and the sequence after-15 can be held to be modified at primer 3 ', increase including end
Other sequences, deletion portion distal end sequence, changing section base sequence.
3. small letter base represents that lock nucleic acid (LNA) is modified.
D2EHDTPA is carried out between forward wild primers 3 ' end-5 and-6 bit bases of 4.CYP2C19*17 gene-806C > T site
Change and modify, VKORC1 gene-1 639G > A site forward wild primers 3 ' end phosphorylation modification, CYP2C19*3 base
Because of 636G > A site forward wild primers 3 ' end-5 bases be peptide nucleic acid(PNA) modification etc..
The most all reverse general primers all carry out FAM or HEX fluorescent labeling at 5 ' ends.
Embodiment 2: the amplification method that a kind of test kit detecting CYP2C19, CYP2C9 and VKORC1 gene type uses
Step 1: the extraction of Whole Blood Genomic DNA
Taking 200 μ L EDTA anticoagulation (sample A), poba gene is extracted in the explanation extracting test kit according to Whole Blood Genomic DNA
Group DNA.By the concentration of spectrophotometer measurement DNA, and it is stand-by to be diluted to 5-10ng/ μ L.
Step 2:PCR amplified reaction
1, PCR premixed solution subpackage (completes in reagent area in preparation)
Vibration mixing PCR premixed solution (PCR Master Mix), it is contemplated that carrying out 4 detection numbers, each PCR reaction tube divides
Fill 19 μ L.
2, add template (preparing district in specimen to complete)
Detection template is blood and the DNA of above-mentioned patient A, each adds template, 1 μ L blood in corresponding PCR reaction tube
Sample, 1 μ L DNA sample, 1 μ L positive control and 1 μ L negative control.
3, PCR amplifications (completing in amplification region)
Each reaction tube is put into PCR amplification instrument reactive tank, and arranging reaction system is 20 μ L.
PCR amplification is carried out by following response procedures:
Step 3: amplified production carries out blood capillary electrophoresis detection
Preparation is mixed with the loading mixed liquor of molecular weight internal standard and Methanamide: (0.5 μ L molecular weight internal standard+8.5 μ L Methanamide) × detection sample
Number.The vortex oscillation mixing 10-15 second；Methanamide and the internal standard mixture of each detection hole subpackage 9 μ L is given with pipettor；Take 1 μ L
Amplified production adds in Methanamide and internal standard mixture, covers shrouding Jiao Gai.If desired with the of short duration centrifugal general of centrifuge
Bubble in sample is removed；Sample is placed 95 DEG C of degeneration 3 minutes, rapidly placement ice bath upper 3 minute.According to genetic analysis
Instrument user's service manual step detects.Detection suggestion arrange sample injection time be 10 seconds, sample introduction voltage be 3kV, the operation time
It it is 1800 seconds.
Step 4: data analysis
Associated documents are imported, including Panel, Bin, corresponding Analysis Method, ROX500 in GeneMapper software
Internal standard.Input sample source data (.fsa file), the file imported before selecting in related parameter choosing hurdle, analytical data.
Step 5: the judgement of testing result
As it can be seen, Fig. 1 is sample A blood direct augmentation detection result figure, Fig. 2 is augmentation detection knot after this sample extraction DNA
Fruit figure, wherein in figure, wild type SNP is designated " WT ", and saltant type is then designated corresponding detection site title.Amel site
Male's sample is shown as " XY ", and women sample is then shown as " X ".By testing result figure can be seen that same sample with blood and
DNA both forms expand, testing result zero difference.
Each site genotyping result is as follows:
The genotype that can draw sample A is: CYP2C19*2/*2, CYP2C9*1/*1, VKORC1-1639A/A；CYP2C19*3
Being wild type with CYP2C19*17 site, CYP2C19*2 site is no mutant homozygote, is judged to the slow inactivation of clopidogrel,
Dose should be increased or change therapeutic strategy；Two detection site of CYP2C9 gene are wild type, and VKORC1 (-1639A/A) is
No mutant homozygote, patient must reduce dosage when using warfarin.
In sum, the technical key point of the present invention and feature, its object is to allow the insider knowing this technology will appreciate that this
Invention content and can implement with this.Present disclosure is not limited in the above embodiments, all according to the technology of the present invention think of
Think that the equivalence that essence is made changes or modifies, all within protection scope of the present invention.
Claims (10)
1.CYP2C19, CYP2C9 and VKORC1 gene type composite amplification system, described amplification system can expand 6 SNP simultaneously
Site, this system includes following primer sequence: in this patent, the primer base sequences of 6 SNP site is as follows:
The primer base sequences in CYP2C19*2 gene 681G > A site is as follows:
Forward wild primers: 5 '-TTTCCCACTATCATTGATTATTTCCCG-3 ',
Forward mutation type primer: 5 '-TTTCCCACTATCATTGATTATTTCCCA-3 ',
Reversely general primer: 5 '-AACTAGTCAATGAATCACAGATACGC-3 '；
The primer base sequences in CYP2C19*3 gene 636G > A site is as follows:
Forward wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTGG-3 ',
Forward mutation type primer: 5 '-ATCAGGATTGTAAGCACCCCCTGA-3 ',
Reversely general primer: 5 '-GATATTCACCCCATGGCTGTCTA-3 '；
The primer base sequences in CYP2C19*17 gene-806C > T site is as follows:
Forward wild primers: 5 '-GCATTATCTCTTACATCAGAGATG-3 ',
Forward mutation type primer: 5 '-GCATTATCTCTTACATCAGAGATA-3 ',
Reversely general primer: 5 '-ATCTCTCGGGCTGTTTTCCTTAGATAA-3’；
The primer base sequences in CYP2C9*2 gene 430C > T site is as follows:
Forward wild primers: 5 '-GGGCTTCCTCTTGAACACG-3 ',
Forward mutation type primer: 5 '-GGGCTTCCTCTTGAACACA-3 ',
Reversely general primer: 5 '-GGGAGGATGGAAAACAGAGACTTAC-3 '；
The primer base sequences in CYP2C9*3 gene 1075A > C site is as follows:
Forward wild primers: 5 '-GCTGGTGGGGAGAAGGTCAAT-3 ',
Forward mutation type primer: 5 '-GCTGGTGGGGAGAAGGTCAAG-3 ',
Reversely general primer: 5 '-AACCATCCTCTCTTTAAGTTTGC-3 '；
The primer base sequences in VKORC1 gene-1 639G > A site is as follows:
Forward wild primers: 5 '-GCGTGAGCCACCGCACCC-3 ',
Forward mutation type primer: 5 '-GCGTGAGCCACCGCACCT-3 ',
Reversely general primer: 5 '-AGCAGGAGAGGGAAATATC-3 '.
Amplification system the most according to claim 1, described system can also expand an internal reference fragment Amel gene loci, this system
In also include the primer base sequences that detects this internal reference fragment Amel gene loci:
Forward primer: 5 '-CCCTGGGCTCTGTAAAGAATAG-3 ',
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '.
3., according to the amplification system described in claim 1,2, described primer is added with modification or replaces normal base with modified base,
The described modification of fluorescence group, phosphorylation modification, thiophosphorylation modification, lock nucleic acid modification or the peptide nucleic acid(PNA) of being modified to is modified.
4. according to the amplification system described in claim 1,2, described primer 3 ' hold-2 to-15 change 1 to 3 base or
Sequence after primer 3 ' holds-15 is modified, and described change includes that end increases other sequences, deletes portion distal end sequence
Row, changing section base sequence.
Amplification system the most according to claim 2, described 6 SNP site and internal reference fragment Amel gene loci are respectively by two
Planting the fluorescent labeling of color, identical fluorescent labeling is considered as same group, and two groups are respectively as follows: first group of CYP2C19*2 gene 681G > A
Site, CYP2C19*3 gene 636G > A site, CYP2C19*17 gene-806C > T site；Second group of CYP2C9*2
Gene 430C > T site, CYP2C9*3 gene 1075A > C site, VKORC1 gene-1 639G > A site, Amel
Gene loci.
Amplification system the most according to claim 1, described first group of fluorescent labeling is FAM, and second group of fluorescent labeling is HEX.
7.CYP2C19, CYP2C9 and VKORC1 gene parting detecting reagent, including PCR premixed solution, described PCR is pre-
Miscible fluid includes the arbitrary described amplification system of claim 1-6.
The most according to claim 7, detection kit, described premixed solution also includes thermal starting DNA Taq enzyme, UDG enzyme and 1.25
×Buffer。
Detection kit the most according to claim 6, described detection kit also includes positive control dna, negative control and
Internal standard ROX500.
The most according to claim 8, detection kit, described positive control dna is normal person DNA, and described negative control is
Aquesterilisa.
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