CN110423803A - Clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and kit - Google Patents
Clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and kit Download PDFInfo
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Abstract
The invention discloses a kind of clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and kits.The amplification system includes each two forward primers and a reversed fluorescent primer of 8 SNP detection sites, can expand 8 SNP sites simultaneously.The characteristics of system of the present invention is to realize that a tubular type expands 8 SNP detection sites, testing result, which can combine, carries out auxiliary diagnosis to clopidogrel, statin and aspirin personalized medicine by multi-fluorescence Arms-PCR technology.Specifically, the direct amplification of blood and blood card sample may be implemented in this amplification system, eliminates the step of extracting DNA；This amplification system can integrate UDG-dUTP anti-pollution measure, and product pollution can be effectively prevented.It is comprehensive, easy to operate, the specific height of detection architecture detection site of the present invention, high sensitivity, highly reliable, at low cost, have the ability of mass detection.
Description
Technical field
The present invention relates to one kind to be based on multiple fluorescence PCR technology and capillary electrophoresis technique.It is detected simultaneously using the technology
Clopidogrel, statin and aspirin drug metabolic rate 4 genes of relevant CYP2C19, ApoE, SLCO1B1 and PTGS1
Totally 8 loci polymorphisms provide reference in the personalized medicine of the diseases such as treatment angiocarpy to be clinical, belong to biomedical neck
Clinical molecular detection technique in domain.
Background technique
Cardiovascular disease incidence rate has the tendency that increasing year by year in the whole world, it has also become influence human health first kills
Hand.However current clinically common treating cardiovascular disease drug, due to individual difference, different patients take same dose
Drug therapeutic effect obtained is significantly different, there is certain puzzlement when this treats clinician using the drug.More
It is one of an important factor for causing drug effect individual difference come more research discovery hereditary variations.
Cardiovascular disease incidence rate has the tendency that increasing year by year in the whole world, it has also become influence human health first kills
Hand.However current clinically common treating cardiovascular disease drug, due to individual difference, different patients take same dose
Drug therapeutic effect obtained is significantly different, there is certain puzzlement when this treats clinician using the drug.More
It is one of an important factor for causing drug effect individual difference come more research discovery hereditary variations.The study found that clinically commonly using
Oral anticoagulation, lipid regulating agent and other preventive medicines such as aspirin etc., take curative effect or dosage and gene
Polymorphism is related.It with regard to clinically common a few major class drugs and its related gene of its medication may be influenced simply is retouched below
It states.
1, clopidogrel: clopidogrel is the most widely used antiplatelet of clinical treatment acute coronary syndrome
Drug, clopidogrel are prodrugs, need to can be just converted by the absorption of small intestine and the metabolism of liver cytochrome P 450
Active metabolite.Clopidogrel drug resistance is considered related with inherent cause, has sufficiently research shows that CYP2C19 enzyme
It plays an important role in the metabolism of clopidogrel.There are multiple polymorphic sites for CYP2C19 gene, lead to CYP2C19 between individual
There are apparent individual differences for enzyme activity.Wherein CYP2C19*2 and CYP2C19*3 site mutation can reduce the work of CYP2C19 enzyme
Property, both are mutated the phenotype that can explain > 99% Asians poor metabolizer and about 88% white people poor metabolizer.And
The mutation in the site CYP2C19*17 then obviously increases CYP2C19 enzymatic activity, and CYP2C19*17 carrier has significant chlorine pyrrole
Gray's strong reactivity and bleeding risk obviously increase.
2, statins: statins are Hydroxymethylglutaryl list acyl coenzyme A reductase inhibitors, to adjust blood lipid
Drug prevents for the firsts and seconds of cardiovascular disease and apoplexy.The drug correlation candidate gene of Statins predominantly influences
The Solute Carrier organic anion transporter 1B1 (SLCO1B1) of pharmacokinetics and the apo E for influencing its pharmacodynamics
(ApoE).Myopathy strong correlation caused by the gene pleiomorphism and Simvastatin, Pravastatin of SLCO1B1.The major physiological of ApoE
Function is by conjunction with ldl receptor and participating in LDL metabolic process, and the variation of ApoE gene can influence ldl receptor and combine activity
And lipid-metabolism is influenced, different polymorphism patients take statins curative effect and have differences.
3, aspirin: aspirin is many family's required medicines, there is good antipyretic effect, can not only be controlled
Treat cold, fever, headache toothache, it may also be used for treatment arthralgia, rheumatism etc..In addition, aspirin can inhibit platelet aggregation
Collection, so can also be used as anti-bolt Primary Care, be mainly used for preventing and treating ischemic heart disease, angina pectoris, cardiopulmonary infraction,
Cerebral thrombosis.
To sum up, pharmacogenetic polymorphism can behave as the polymorphism of drug metabolic enzyme, the polymorphism of drug transporter, with
And active receptors or target spot polymorphism etc..These polymorphisms may cause drug effect and adverse reaction in many drug therapies
Individual difference, by the detection to patient's certain drug related locus genotype, the therapeutic scheme of each patient is formulated in guidance,
So that patient is obtained optimum therapeuticing effect, avoids adverse drug reaction, achieve the purpose that drug usage individuation.
There are many method currently used for detecting gene pleiomorphism, such as direct sequencing, fluorescence quantitative PCR method, genetic chip
Method, high-flux sequence etc..These detection methods have the defects that it is various different degrees of, as operating process is cumbersome, poor repeatability,
As a result it is not easy that interpretation, detection site are few, detection flux is low, detection cycle is long, testing cost height etc..1, direct sequencing: design
Sequencing primer extracts DNA and carries out PCR amplification and product sequencing.The major defect of this method is: (1) needing pair before PCR reaction
Sample extracts DNA, detects quality and concentration, complicated for operation, there is the risk for obscuring sample.(2) when detecting multiple sites
One sample needs to carry out multiple reactions, and testing cost is high, and the demand to template quantity is high, and manipulation strength is big, and be easy to cause behaviour
Make mistake and pollution.(3) PCR reaction after need to purify product and be sequenced chemical reaction and etc..It is exactly on the whole
Complex steps, the test period is long, at high cost, is unfavorable for clinic and is widely applied.2, rflp analysis method: caused based on gene mutation
Restriction enzyme enzyme recognition site change, if site is lost or generates novel site, by a certain specific fragment of PCR amplification,
Restriction enzyme is recycled to carry out endonuclease reaction, electrophoresis observes the size of segment, but it is with restriction enzyme site limitation, and
It is difficult to carry out high-throughput parallel analysis.3, quantitative fluorescent PCR: real-time fluorescence quantitative PCR is easy to operate quickly, tests sensitivity
Height, the advantages that as a result can fast quantifying, but the technology there are samples easy to pollute, Yi Fasheng cross reaction, false positive rate are high.And
Common quantitative fluorescent PCR can only utilize one to four fluorescence channel, then can only at most detect 4 sites, not be able to satisfy more
It is detected while a SNP site.4, genetic chip (gene chip): genetic chip be by micro-processing technology, will be ten hundreds of
The even gene probe of the particular sequence of million meters constitutes one regularly on the supports such as arrangement fixation and silicon wafer, slide
Two-dimentional DNA probe array is hybridized using this kind of chip with the biological sample of label, can to sample gene expression profile biology
Information carries out qualitative and quantitative analysis.It can carry out high-throughput detection, but this method is costly, big to template demand, operation
And data analysis is complicated, time-consuming.
It is hospital and other therapeutic machines it is therefore desirable to establish a kind of while quickly detecting the methods of these three gene mutations
Structure provides a kind of easy to operate, high specificity, high sensitivity, the detection scheme that flux is high, highly reliable and at low cost.
Summary of the invention
The object of the present invention is to provide a kind of polymorphic with clopidogrel, statin and aspirin personalized medicine related gene
Property detection composite amplification system.The system have easy to operate, high specificity, high sensitivity, flux it is high, it is highly reliable and at
This low detection feature.
A kind of genetic polymorphism detection PCR for the guidance of clopidogrel, statin and aspirin personalized medicine is compound
Amplification system can simultaneously expand following 8 detection sites: CYP2C19*2 gene in the PCR composite amplification system
The site 681G > A, the site CYP2C19*3 gene 636G > A, the CYP2C19*17 gene -806C > site T, ApoE gene 388T > C
Point, the site ApoE gene 526C > T, the site SLCO1B1 gene 521T > C, the site SLCO1B1 gene 388A > G, PTGS1 gene-
The site 842A > G；Contain the Primer composition for expanding the detection site in the described PCR composite amplification system:
The site ApoE gene 388T > C:
Positive wild primers: 5 '-GCGGTACTGCACCAGGCGGCCGCA-3 ',
Forward mutation assay type primer: 5 '-GCGGTACTGCACCAGGCGGCCGCG-3 ',
Reversed general primer: 5 '-TCGGAACTGGAGGAACAACTGAC-3 '；
The site ApoE gene 526C > T:
Positive wild primers: 5 '-CCCGGCCTGGTACACTGCCAGGCG-3 ',
Forward mutation assay type primer: 5 '-CCCGGCCTGGTACACTGCCAGGCA-3 ',
Reversed general primer: 5 '-TCGGAACTGGAGGAACAACTGAC-3 '；
The site CYP2C19*2 gene 681G > A:
Positive wild primers: 5 '-TTTCCCACTATCATTGATTATTTCCCG-3 ',
Forward mutation assay type primer: 5 '-TTTCCCACTATCATTGATTATTTCCCA-3 ',
Reversed general primer: 5 '-AACTAGTCAATGAATCACAGATACGC-3 '；
The site CYP2C19*3 gene 636G > A:
Positive wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTGG-3 ',
Forward mutation assay type primer: 5 '-ATCAGGATTGTAAGCACCCCCTGA-3 ',
Reversed general primer: 5 '-GATATTCACCCCATGGCTGTCTA-3 '；
CYP2C19*17 gene -806C > the site T:
Positive wild primers: 5 '-GCATTATCTCTTACATCAGAGATG-3 ',
Forward mutation assay type primer: 5 '-GCATTATCTCTTACATCAGAGATA-3 ',
Reversed general primer: 5 '-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '；
PTGS1 gene -842A > the site G:
Positive wild primers: 5 '-GATAACTGAGCACCTACTACATGCTGGA-3 ',
Forward mutation assay type primer: 5 '-GATAACTGAGCACCTACTACATGCTGGG-3 ',
Reversed general primer: 5 '-CAAGGTACTTATCTTTTCTAGCCCTC-3 '；
The site SLCO1B1 gene 388A > G:
Positive wild primers: 5 '-AGGTCGATGTTGAATTTTCTGATGAATT-3 ',
Forward mutation assay type primer: 5 '-AGGTCGATGTTGAATTTTCTGATGAATC-3 ',
Reversed general primer: 5 '-CACATGCTGGGAAATTGACAGAAAGTA-3 '；
The site SLCO1B1 gene 521T > C:
Positive wild primers: 5 '-GAATCTGGGTCATACATGTGGATATATGT-3 ',
Forward mutation assay type primer: 5 '-GAATCTGGGTCATACATGTGGATATATGC-3 ',
Reversed general primer: 5 '-TAGACAAAGGGAAAGTGATCATACA-3 '.
Following control sites: gender Amel gene, human identity identification position can also be expanded in the PCR composite amplification system
Point D5S818 and Th01 contains the Primer composition for expanding the control site in the described PCR composite amplification system:
Compare the site Amel:
Forward primer: 5 '-CCCTGGGCTCTGTAAAGAATAG-3 ',
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '；
Compare the site D5S818:
Forward primer: 5 '-GTGGTGTCCCAGATAATCTGTAC-3 ',
Reverse primer: 5 '-GGTGAATAACTCCAAATACTCC-3 '；
Compare the site Th01:
Forward primer: 5 '-AGGCTCTAGCAGCAGCTCATG-3 ',
Reverse primer: 5 '-CTGGAAATGACACTGCTACAACTC-3 '.
Replace normal base added with modification or with modified base in the sequence of the primer, it is described to be modified to fluorophor
Modification, phosphorylation modification, thiophosphorylation modification, lock nucleic acid modification or peptide nucleic acid modification.
The primer 3 ' holds -2 to -15 1 to 3 bases of change or/and the sequence after primer 3 ' holds -15 to carry out
Change, the change include that end increases other sequences, deletes portion distal end sequence, changing section base sequence.
Respectively by the fluorescent marker of two kinds of colors, identical fluorescent marker is considered as 8 SNP sites and 3 control sites
Same group, two groups are respectively as follows: first group of PTGS1 gene -842A > site G, the site CYP2C19*3 gene 636G > A, CYP2C19*
17 gene -806C > the site T, the site CYP2C19*2 gene 681G > A, the site D5S818；Second group of site Amel, ApoE gene
The site 388T > C, the site SLCO1B1 gene 521T > C, the site SLCO1B1 gene 388A > G, the site ApoE gene 526C > T,
The site Th01.
First group of fluorescent marker is FAM, and second group of fluorescent marker is HEX.
The fluorescent marker of 8 SNP sites is located at 5 ' ends of reversed general primer, the fluorescent marker of the control site
Positioned at 5 ' ends of forward primer.
The clopidogrel, statin and aspirin personalized medicine related gene joint-detection system, can expand 4 simultaneously
8 polymorphic sites and 3 control sites of a gene.1 is shown in Table comprising 11 sites and the grouping in system in the system:
1 detection site list of table
First group of fluorescent marker is FAM, and second group of fluorescent marker is HEX, wherein two groups of sites are from top to bottom
Sequence be that segment in actually detected result figure from small to large puts in order.
Preferred embodiment:
The site ApoE gene 388T > C:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site ApoE gene 526C > T:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site CYP2C19*2 gene 681G > A:
Positive wild primers: 5 '-TTTCCCACTATCATTGACTATTTCCAG-3 '
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-AACTAGTCAATGAATCACAGATACGC-3 '
The site CYP2C19*3 gene 636G > A:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-GATATTCACCCCATGGCTGTCTA-3 '
CYP2C19*17 gene -806C > the site T:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '
PTGS1 gene -842A > the site G:
Positive wild primers: 5 '-AACTGAGCACCTACTACATGCTGCA-3 ',
Forward mutation assay type primer:It is reversed to share
Primer: 5 '-CAAGGTACTTATCTTTTCTAGCCCTC-3 '；
The site SLCO1B1 gene 388A > G:
Positive wild primers: 5 '-AGGTCGATGTTGAATTTTCTGATGAAAT-3 '
Forward mutation assay type primer: 5 '-GATTAGGTCGATGTTGAATTTTCTGATGATTC-3 '
Reversed general primer: 5 '-HEX-CACATGCTGGGAAATTGACAGAAAGTA-3 '
The site SLCO1B1 gene 521T > C:
Positive wild primers:
Forward mutation assay type primer: 5 '-GAATCTGGGTCATACATGTGGATATGTGC-3 '
Reversed general primer: 5 '-HEX-TAGACAAAGGGAAAGTGATCATACA-3 '
Compare the site Amel:
Forward primer: 5 '-HEX-CCCTGGGCTCTGTAAAGAATAG-3 '
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '
Compare the site D5S818:
Forward primer: 5 '-FAM-GTGGTGTCCCAGATAATCTGTAC-3 '
Reverse primer: 5 '-GGTGAATAACTCCAAATACTCC-3 '
Compare the site Th01:
Forward primer: 5 '-HEX-AGGCTCTAGCAGCAGCTCATG-3 '
Reverse primer: 5 '-CTGGAAATGACACTGCTACAACTC-3 '.
Detection architecture of the present invention, using allele characteristic PCR (allele-specific PCR, ASPCR)
Purpose site is expanded in conjunction with the method for quantitative fluorescence PCR (Quantitive Fluorescent PCR, QF-PCR), is led to
The detection that Capillary Electrophoresis carries out amplified production is crossed, the detection to purpose site parting is completed.Each detection site is arranged
Three primers two kinds of partings are respectively set the special primer an of different length and the downstream primer of a fluorescent marker.
Every special primer in conjunction with the DNA profiling of corresponding genotype and can only be expanded.Complete PCR amplification and Capillary Electrophoresis inspection
After survey, it can determine that sample specific site with the presence or absence of spy by the presence or absence of specific fluorescent label, the amplified production of specific length
Determine genotype.
Detection corresponding to above-mentioned clopidogrel, statin and aspirin personalized medicine related gene joint-detection system
Kit, including enzyme mixation, amplification buffer, primer mixture, or the premixed liquid (PCR Master Mix) of the above ingredient.
The kit also includes following component: thermal starting DNA Taq enzyme, UDG enzyme and 2 × Buffer, positive control
DNA, negative control and internal standard ROX500.
The application method of the clopidogrel, statin and aspirin personalized medicine related gene joint-detection system,
Mainly comprise the steps that PCR amplification, genetic analyzer detection amplified production, data analysis, the judgement of testing result.
The characteristics of the method for the present invention:
1) pass through capillary electrophoresis detection amplified production using genetic analyzer:
Genetic analyzer detection platform is one of widely used mainstream detection platform, by Capillary Electrophoresis to fluorescence mark
The amplified production of note is detected, and the major technique advantage of detection is:
1. detection sensitivity is high.
Fluorescent dye, which is used in combination, in Capillary Electrophoresis greatly improved detection sensitivity, than agarose electrophoresis detection sensitivity
High 100 times or more.It can more delicately detect amplified production, and clearly distinguish amplification wild type and saltant type.On the other hand,
Due to detection sensitivity height, bigger adjustment space is provided for composite PCR amplified reaction.Importantly, sensitive due to detecting
Degree is high, reduces the requirement to pcr amplification product amount, can reduce PCR reaction template dosage, can reduce PCR amplification circulation
Number saves sample.
2. detection resolution is high.
In the 100-500bp detection range usually utilized can it is clear, efficiently differentiate 1bp difference.So high resolution
Rate makes that erroneous judgement will not be generated since primer size is close, also avoids the erroneous judgement of result caused by non-specific amplification.Another party
Face, high-resolution also to detect more sites simultaneously.
3. can be detected simultaneously to 2 kinds of fluorescence signals.Detection range further is expanded, so that detection is more simultaneously
Site is possibly realized.
4. detection speed fast (40 minutes) needs to operate less, can automate mass detection.
5. testing result can be automatically analyzed using software sentences type.
2) multiple sites can be expanded simultaneously by a MULTIPLE COMPOSITE PCR amplification system:
This patent utilizes a MULTIPLE COMPOSITE PCR amplification system, realizes with cardiovascular personalized medicine in relation to 4 genes 8
The detection of a polymorphic site.It is advantageous that:
1. greatly reducing manipulation strength and testing cost.Only a PCR amplification and a genetic analyzer detection react and are
Achievable all detections.
2. Single tube amplification detects, the mistakes such as pollution and sample mix are avoided to the full extent.
3) site is comprehensive:
This system covers and clopidogrel, statin and 8, aspirin personalized medicine related gene polymorphism site,
Reference can be provided for the personalized medicine of a variety of drugs, provide more patient informations for clinician.
4) direct expansion system and anti-pollution measure:
This amplification system can directly use the samples such as blood, blood card to expand, and eliminate the step of DNA is extracted, operation is more
Add simplicity, is suitable for batch operation.It joined UDG-dUTP anti-pollution measure in another system, product pollution can be effectively prevented,
It avoids causing false positive results.
To sum up, the art of this patent route applications are more in clopidogrel, statin and aspirin personalized medicine related gene
State property joint-detection.Major advantage includes: that each sample only needs an augmentation detection reaction, available 8 positions of a tube reaction
The testing result of point, greatly reduces manipulation strength relative to other detection methods on the market；Result can be obtained in 4 hours.
The present invention has following advantages and effect compared with prior art:
1, the present invention is based on multiplex PCR and capillary electrophoresis technique, tubular type amplification, while to clopidogrel, statin
4 genes relevant with aspirin these three types drug metabolism, 8 SNP sites are detected, and are used for and this 4 gene polymorphics
The guidance of the related drug dose of property, had not only saved production cost and testing cost, but also improve detection efficiency；Furthermore it introduces
Amel gender site is that internal reference is used to monitor entire reaction system and assesses the quality of template, avoids false negative result, draws
Entering D5S818, Th01 two individual recognition sites is internal reference, the cross contamination between sample being likely to occur in monitoring operation.
2, CYP2C19*17 parting (ultra-rapid metabolism type) detection is increased, parting function is more comprehensive；
3, the direct amplification of blood and blood card may be implemented in the present invention, is not necessarily to any processing, eliminates the step for extracting DNA
Suddenly, while the anti-pollution system of UDG enzyme-dUTP is integrated, shortens the sample process time, reduces the generation of pollution；
4, the present invention utilizes sequenator detection platform, is examined by amplified production of the Capillary Electrophoresis to fluorescent marker
It surveys, high sensitivity is easy to operate, genotyping result intuitively easy interpretation.All detections, manual operation are completed in whole flow process 3 hours
Time was less than 30 minutes；
5, present invention detection flux is high, has high-volume augmentation detection ability.
Detailed description of the invention
Fig. 1 is the result map that genetic analyzer detects the blood amplified production of sample A,
Fig. 2 is the result map that genetic analyzer detects the DNA cloning product of sample A.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments.
Embodiment: a kind of clopidogrel, statin and aspirin personalized medicine related gene combined detection kit and
Its method that augmentation detection is carried out to blood and DNA sample
One, detection architecture
Kit includes PCR Master Mix, positive control, negative control, internal standard.Wherein PCR Master Mix master
Wanting component includes thermal starting DNA Taq enzyme, UDG enzyme, amplification buffer and each site primer etc..
According to ApoE gene principle, every special primer can only be in conjunction with the DNA profiling of corresponding genotype simultaneously
It is expanded.For this purpose, having carried out a series of specific changes or modification to each primer.In order to coordinate amplification efficiency, improve
Product peak type is convenient for capillary electrophoresis detection, has also carried out a series of specific changes or modification to primer.The present embodiment is adopted
It is as follows by the primer sequence improved and optimizated:
The site ApoE gene 388T > C:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site ApoE gene 526C > T:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site CYP2C19*2 gene 681G > A:
Positive wild primers: 5 '-TTTCCCACTATCATTGACTATTTCCAG-3 '
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-AACTAGTCAATGAATCACAGATACGC-3 '
The site CYP2C19*3 gene 636G > A:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-GATATTCACCCCATGGCTGTCTA-3 '
CYP2C19*17 gene -806C > the site T:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '
PTGS1 gene -842A > the site G:
Positive wild primers: 5 '-AACTGAGCACCTACTACATGCTGCA-3 ',
Forward mutation assay type primer:
Reversed general primer: 5 '-CAAGGTACTTATCTTTTCTAGCCCTC-3 '；
The site SLCO1B1 gene 388A > G:
Positive wild primers: 5 '-AGGTCGATGTTGAATTTTCTGATGAAAT-3 '
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-CACATGCTGGGAAATTGACAGAAAGTA-3 '
The site SLCO1B1 gene 521T > C:
Positive wild primers:
Forward mutation assay type primer: 5 '-GAATCTGGGTCATACATGTGGATATGTGC-3 '
Reversed general primer: 5 '-HEX-TAGACAAAGGGAAAGTGATCATACA-3 '
Compare the site Amel:
Forward primer: 5 '-HEX-CCCTGGGCTCTGTAAAGAATAG-3 '
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '
Compare the site D5S818:
Forward primer: 5 '-FAM-GTGGTGTCCCAGATAATCTGTAC-3 '
Reverse primer: 5 '-GGTGAATAACTCCAAATACTCC-3 '
Compare the site Th01:
Forward primer: 5 '-HEX-AGGCTCTAGCAGCAGCTCATG-3 '
Reverse primer: 5 '-CTGGAAATGACACTGCTACAACTC-3 '.
Note: 1. "-" list underscores represent each primer and hold 1 to 3 base of -2 to -15 changes in primer 3 '.
2. "=" double underline represents each primer and can be modified in the sequence after primer 3 ' holds -15, including end
End increases other sequences, deletes portion distal end sequence, changing section base sequence.
3. all reversed general primers of detection site carry out FAM or HEX fluorescent marker at 5 ' ends.
4. control site forward primer carries out FAM or HEX fluorescent marker at 5 ' ends.
Two, detection method
Step 1:PCR amplified reaction
1) PCR premixed solution packing (being completed in reagent area in preparation)
Oscillation mixes PCR premixed solution (PCR Master Mix), it is contemplated that carries out 4 testing numbers, each PCR reaction tube point
Fill 19 μ L.
2) template (completing in sample preparation area) is added
Detection template is blood sample and DNA sample, and template, 1 μ L blood sample, 1 μ L is added into corresponding PCR reaction tube
DNA sample, 1 μ L positive control and 1 μ L negative control.
3) PCR amplification (being completed in amplification region)
Each reaction tube is put into PCR amplification instrument reactive tank, setting reaction system is 20 μ L.
PCR amplification is carried out by 2 response procedures of table:
Table 2PCR amplified reaction program
Step 2: amplified production carries out capillary electrophoresis detection
Prepare the loading mixed liquor for being mixed with molecular weight internal standard and formamide: (+8.5 μ L formamide of 0.5 μ L molecular weight internal standard)
× test sample number, vortex oscillation mix 10-15 seconds；The formamide of 9 μ L is dispensed to each detection hole with pipettor and internal standard is mixed
Close object；It takes 1 μ L amplified production to be added in formamide and internal standard mixture, covers sealing plate Jiao Gai.Centrifugation can be used if necessary
The bubble in sample is removed in the of short duration centrifugation of machine；Sample is placed 95 DEG C to be denaturalized 3 minutes, is placed on ice bath 3 minutes rapidly.According to
Genetic analyzer user's service manual step detects.Detection suggest setting sample injection time be 10 seconds, sample introduction voltage be 3kV,
Runing time is 1800 seconds.
Step 3: data analysis
Import associated documents into GeneMapper software, including Panel, Bin, corresponding Analysis Method,
ROX500 internal standard.Input sample source data (.fsa file), the file imported before being selected in related parameter choosing column, analysis
Data.
Step 4: the judgement of testing result
As shown, Fig. 1 is the augmentation detection result figure of blood sample, Fig. 2 is the amplification figure of DNA sample, wherein
Wild type SNP is identified as " WT " in figure, and saltant type is then identified as " Mu ".The site Amel male's sample is shown as " XY ", women sample
This is then shown as " X ".
Each site genotyping result such as the following table 3:
3 pattern detection result of table
Thus the genotype of blood sample and DNA sample 4 genes, 8 polymorphic sites can be obtained respectively, as the result is shown
This system it is compatible on blood and DNA amplification and on result without influence.
Clinician can carry out personalized medicine to the drug that patient uses according to genotype detected.Specific
Property medication guide scheme such as the following table 4:
4 medication guide scheme of table
Sample Genotyping testing result based on the present embodiment, can the medication to the patient carry out the tune of following scheme
It is whole:
In conclusion technical key point and feature of the invention, its object is to the insiders of this technology known to allowing can
Understand the contents of the present invention and can be implemented with this.The contents of the present invention are not limited in the above embodiments, all according to this hair
Equivalent change or modification made by bright technical idea essence, all within protection scope of the present invention.
Sequence table
<110>Beijing Microread Gene Technology Co., Ltd.
<120>clopidogrel, statin and aspirin relevant drug metabolism Genotyping detection composite amplification system and reagent
Box
<141> 2019-08-07
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gcggtactgc accaggcggc cgca 24
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gcggtactgc accaggcggc cgcg 24
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tcggaactgg aggaacaact gac 23
<210> 4
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cccggcctgg tacactgcca ggcg 24
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cccggcctgg tacactgcca ggca 24
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tcggaactgg aggaacaact gac 23
<210> 7
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tttcccacta tcattgatta tttcccg 27
<210> 8
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tttcccacta tcattgatta tttccca 27
<210> 9
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aactagtcaa tgaatcacag atacgc 26
<210> 10
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atcaggattg taagcacccc ctgg 24
<210> 11
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atcaggattg taagcacccc ctga 24
<210> 12
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gatattcacc ccatggctgt cta 23
<210> 13
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gcattatctc ttacatcaga gatg 24
<210> 14
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gcattatctc ttacatcaga gata 24
<210> 15
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
atctctcggg ctgttttcct tagataa 27
<210> 16
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gataactgag cacctactac atgctgga 28
<210> 17
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gataactgag cacctactac atgctggg 28
<210> 18
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
caaggtactt atcttttcta gccctc 26
<210> 19
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
aggtcgatgt tgaattttct gatgaatt 28
<210> 20
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aggtcgatgt tgaattttct gatgaatc 28
<210> 21
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
cacatgctgg gaaattgaca gaaagta 27
<210> 22
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gaatctgggt catacatgtg gatatatgt 29
<210> 23
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
gaatctgggt catacatgtg gatatatgc 29
<210> 24
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tagacaaagg gaaagtgatc ataca 25
<210> 25
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ccctgggctc tgtaaagaat ag 22
<210> 26
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
atcagagctt aaactgggaa gctg 24
<210> 27
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
gtggtgtccc agataatctg tac 23
<210> 28
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ggtgaataac tccaaatact cc 22
<210> 29
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
aggctctagc agcagctcat g 21
<210> 30
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ctggaaatga cactgctaca actc 24
Claims (10)
1. a kind of genetic test PCR composite amplification system for the guidance of clopidogrel, statin and aspirin personalized medicine,
The PCR composite amplification system can expand 8 SNP detection sites simultaneously, and PCR composite amplification system includes 8 detection sites
Primer composition:
The site ApoE gene 388T > C:
Positive wild primers: 5 '-GCGGTACTGCACCAGGCGGCCGCA-3 ',
Forward mutation assay type primer: 5 '-GCGGTACTGCACCAGGCGGCCGCG-3 ',
Reversed general primer: 5 '-TCGGAACTGGAGGAACAACTGAC-3 '；
The site ApoE gene 526C > T:
Positive wild primers: 5 '-CCCGGCCTGGTACACTGCCAGGCG-3 ',
Forward mutation assay type primer: 5 '-CCCGGCCTGGTACACTGCCAGGCA-3 ',
Reversed general primer: 5 '-TCGGAACTGGAGGAACAACTGAC-3 '；
* 2 sites gene 681G > A:
Positive wild primers: 5 '-TTTCCCACTATCATTGATTATTTCCCG-3 ',
Forward mutation assay type primer: 5 '-TTTCCCACTATCATTGATTATTTCCCA-3 ',
Reversed general primer: 5 '-AACTAGTCAATGAATCACAGATACGC-3 '；
* 3 sites gene 636G > A:
Positive wild primers: 5 '-ATCAGGATTGTAAGCACCCCCTGG-3 ',
Forward mutation assay type primer: 5 '-ATCAGGATTGTAAGCACCCCCTGA-3 ',
Reversed general primer: 5 '-GATATTCACCCCATGGCTGTCTA-3 '；
The * 17 gene -806C > site T:
Positive wild primers: 5 '-GCATTATCTCTTACATCAGAGATG-3 ',
Forward mutation assay type primer: 5 '-GCATTATCTCTTACATCAGAGATA-3 ',
Reversed general primer: 5 '-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '；
PTGS1 gene -842A > the site G:
Positive wild primers: 5 '-GATAACTGAGCACCTACTACATGCTGGA-3 ',
Forward mutation assay type primer: 5 '-GATAACTGAGCACCTACTACATGCTGGG-3 ',
Reversed general primer: 5 '-CAAGGTACTTATCTTTTCTAGCCCTC-3 '；
The site SLCO1B1 gene 388A > G:
Positive wild primers: 5 '-AGGTCGATGTTGAATTTTCTGATGAATT-3 ',
Forward mutation assay type primer: 5 '-AGGTCGATGTTGAATTTTCTGATGAATC-3 ',
Reversed general primer: 5 '-CACATGCTGGGAAATTGACAGAAAGTA-3 '；
The site SLCO1B1 gene 521T > C:
Positive wild primers: 5 '-GAATCTGGGTCATACATGTGGATATATGT-3 ',
Forward mutation assay type primer: 5 '-GAATCTGGGTCATACATGTGGATATATGC-3 ',
Reversed general primer: 5 '-TAGACAAAGGGAAAGTGATCATACA-3 '.
2. PCR composite amplification system according to claim 1,3 control sites: property can also be expanded in the PCR system
Other Amel gene, human identity recognition site D5S818 and Th01, PCR composite amplification system includes the primer of 3 control sites
Composition:
Compare the site Amel:
Forward primer: 5 '-CCCTGGGCTCTGTAAAGAATAG-3 ',
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 '；
Compare the site D5S818:
Forward primer: 5 '-GTGGTGTCCCAGATAATCTGTAC-3 ',
Reverse primer: 5 '-GGTGAATAACTCCAAATACTCC-3 '；
Compare the site Th01:
Forward primer: 5 '-AGGCTCTAGCAGCAGCTCATG-3 ',
Reverse primer: 5 '-CTGGAAATGACACTGCTACAACTC-3 '.
3. PCR composite amplification system according to claim 1 or 2, added with modification or with modified base on the primer
Replace normal base, it is described to be modified to the modification of fluorescence group, phosphorylation modification, thiophosphorylation modification, lock nucleic acid modification or peptide
Nucleic acid modification.
4. PCR composite amplification system according to claim 1 or 2, the primer 3 ' holds 1 to 3 alkali of -2 to -15 changes
Base or/and primer 3 ' hold -15 after sequence be modified, it is described change include end increase other sequences, delete
Portion distal end sequence, changing section base sequence.
5. PCR composite amplification system according to claim 4,8 detection sites and 3 control sites are respectively by two
The fluorescent marker of kind of color, identical fluorescent marker are considered as same group, and two groups are respectively as follows: first group of PTGS1 gene -842A > G
Point, * 3 sites gene 636G > A, the * 17 gene -806C > site T, * 2 sites gene 681G > A, the site D5S818；Second group of Amel
Site, the site ApoE gene 388T > C, the site SLCO1B1 gene 521T > C, the site SLCO1B1 gene 388A > G, ApoE gene
The site 526C > T, the site Th01.
6. PCR composite amplification system according to claim 5, first group of fluorescent marker is FAM, second group of fluorescence mark
It is denoted as HEX；The fluorescent marker of the detection site is located at 5 ' ends of reversed general primer, the fluorescent marker position of the control site
In 5 ' ends of forward primer.
7. PCR composite amplification system according to claim 5, PCR composite amplification system include following Primer composition:
The site ApoE gene 388T > C:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site ApoE gene 526C > T:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-TCGGAACTGGAGGAACAACTGAC-3 '；
The site CYP2C19*2 gene 681G > A:
Positive wild primers: 5 '-TTTCCCACTATCATTGACTATTTCCAG-3 ',
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-AACTAGTCAATGAATCACAGATACGC-3 '；
The site CYP2C19*3 gene 636G > A:
Positive wild primers:
Forward mutation assay type primer:
Reversed general primer: 5 '-FAM-GATATTCACCCCATGGCTGTCTA-3 '；
CYP2C19*17 gene -806C > the site T:
Positive wild primers:
Forward mutation assay type primer:Reversed share is drawn
Object: 5 '-FAM-ATCTCTCGGGCTGTTTTCCTTAGATAA-3 '；
PTGS1 gene -842A > the site G:
Positive wild primers: 5 '-AACTGAGCACCTACTACATGCTGCA-3 ',
Forward mutation assay type primer:
Reversed general primer: 5 '-CAAGGTACTTATCTTTTCTAGCCCTC-3 '；
The site SLCO1B1 gene 388A > G:
Positive wild primers: 5 '-AGGTCGATGTTGAATTTTCTGATGAAAT-3 ',
Forward mutation assay type primer:
Reversed general primer: 5 '-HEX-CACATGCTGGGAAATTGACAGAAAGTA-3 '；
The site SLCO1B1 gene 521T > C:
Positive wild primers:
Forward mutation assay type primer: 5 '-GAATCTGGGTCATACATGTGGATATGTGC-3 ',
Reversed general primer: 5 '-HEX-TAGACAAAGGGAAAGTGATCATACA-3 '；
Compare the site Amel:
Forward primer: 5 '-HEX-CCCTGGGCTCTGTAAAGAATAG-3 ',
Reverse primer: 5 '-ATCAGAGCTTAAACTGGGAAGCTG-3 ',
Compare the site D5S818:
Forward primer: 5 '-FAM-GTGGTGTCCCAGATAATCTGTAC-3 ',
Reverse primer: 5 '-GGTGAATAACTCCAAATACTCC-3 ',
Compare the site Th01:
Forward primer: 5 '-HEX-AGGCTCTAGCAGCAGCTCATG-3 ',
Reverse primer: 5 '-CTGGAAATGACACTGCTACAACTC-3 '.
8. PCR composite amplification system according to claim 1 or 2, the amplification uses multiple ApoE gene
Amplification combines quantitative fluorescence PCR to expand, and the detection uses detection of the Capillary Electrophoresis to pcr amplification product,
9. a kind of clopidogrel, statin and aspirin personalized medicine related gene polymorphism detection kit, feature exist
In including any PCR composite amplification system of claim 1-8.
10. detection kit according to claim 9, the kit also includes following component: thermal starting DNA Taq enzyme,
UDG enzyme and 2 × Buffer, positive control dna, negative control and internal standard ROX500.
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| CN111996244B (en) * | 2020-10-28 | 2022-05-06 | 浙江绍兴鼎晶生物医药科技股份有限公司 | Composition for detecting single nucleotide polymorphism and application thereof |
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