CN108949967A - With the specific primer and kit of liquid-phase chip technology detection cardiovascular disease medicine gene pleiomorphism - Google Patents
With the specific primer and kit of liquid-phase chip technology detection cardiovascular disease medicine gene pleiomorphism Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a kind of primer sets for detecting 15 kinds of cardiovascular drugs correlation target gene polymorphisms simultaneously with liquid-phase chip technology, including 15 groups of primers, wherein every group of primer includes a pair of of specificity amplification primer for detecting a kind of target gene polymorphism and respectively for two ASPE primers of the wild type of the target gene and saltant type.The invention also discloses kits and its application method comprising these primer sets.Primer sets and its kit of the invention can expand target sequence in One_step PCR reaction, and combines MagPlex microballoon while detecting 15 kinds of common cardiovascular drugs correlation target gene polymorphisms, it is high to detect flux, cost performance is high, one-time detection can provide the medication guide of cardiovascular patient, provide laboratory foundation for clinical rational drug use;Kit of the invention has extraordinary sensitivity, stability and accuracy rate in practical applications, coincide with gene sequencing result good, and greatly reduce testing cost.
Description
Technical field
The present invention relates to biotechnologys and medical domain, and in particular to detects common cardiovascular disease using liquid-phase chip technology
The specific primer of medicine gene pleiomorphism, the invention further relates to the kits comprising these specific primers and kit to make
Use method.
Background technique
Caused by cardiovascular disease is heart and vascular disorder, including coronary heart disease (heart attack), cranial vascular disease (in
Wind), hypertension (blood pressure raising), peripheral vascular disease, rheumatic heart disease, congenital heart disease, heart failure and cardiac muscle
Disease.Cardiovascular disease is a kind of common disease for seriously threatening the mankind, particularly 50 years old or more the middle-aged and the old health.Currently, painstaking effort
Pipe is died of illness the first place of the total cause of death of Wang Zhan urban and rural residents, rural area 45.01%, city 42.61%.According to statistics, about three points
One of not die of disease itself, but die of Irrational Use of Drugs.The accurate personalized medicine of cardiovascular disease is safe medication
It ensures, alloing doctor is patient personalized selection medicament categories and dosage.
Clinical common treating cardiovascular disease drug includes clopidogrel, warfarin, nitroglycerin, aspirin etc.
Deng.Due to the presence of individual difference, different patients take identical drug therapeutic effect obtained and are not quite similar.More and more
Research finds that the inherent cause of patient is an important factor for influencing drug metabolism, absorption and excretion, currently, having been detected by tens of
Kind of the enzyme activity in different individuals is not very identical, with CYP2C9, VKORC1, CYP4F2, CYP2C19, PON1, CYP2D6,
The important gene relevant to drug absorption, distribution, metabolism and excretion such as ADRB1, ALDH2, GP1BA, PEAR1, AGTR1 it is single
Sweet acid polymorphism (SNP) is found successively, determines medicine by the detection to various cardiovascular drugs Target genes polymorphisms
The selection of object or dosage are the clinical precisely medication means of vital assistance.
CYP2C9 gene, VKORC1 gene and CYP4F2 gene pleiomorphism and warfarin personalized medicine correlation
Warfarin (warfarin) is Coumarins oral anticoagulation, be at present clinically it is most widely used take orally it is anticoagulant
One of drug, but clinical efficacy and adverse reaction individual difference are very big, and dosage is difficult to grasp.Studies have shown that vitamin K epoxidation
The gene pleiomorphism and cytochrome P450 gene (CYP2C9) of object reduction combined enzyme agent subunit I gene (VKORC1) are to influence
Most important two inherent causes of warfarin dosage individual difference.In addition, CYP4F2 gene polynorphisms (rs2108622) also with
Warfarin individual dose difference is related.
CYP2C19 gene pleiomorphism and clopidogrel personalized medicine correlation
Clopidogrel (Clopidogrel) is oral anti-diabetic agent object, it is dynamic to have been widely used for acute coronary at present
Arteries and veins syndrome (ACS) and PCI aftertreatment can effectively prevent the generation of the Ischemia Time of ACS patient.The drug is through internal
Irreversible combination can occur with platelet surface adp receptor P2Y12 after P450 enzyme bioconversion, so that ADP be inhibited to induce
The aggregation of blood platelet reaches anticoagulant effect.Therefore, cytochrome P450 gene (CYP2C19) is then to influence chlorine pyrroles thunder
The most important inherent cause of dosage individual difference.
The personalized medicine correlation-of ALDH2 gene pleiomorphism and nitroglycerin
ALDH2 is the main path of human body catalysis nitroglycerin bioconversion, which has dehydrogenase and esterase simultaneously
Catalytic activity, esterase active can be by generating NO for nitroglycerin denitration, and then causes blood vessel dilatation.ALDH2 gene position
In the position 12q24.2 of No. 12 chromosome, overall length 43,438bp shares 13 exons, encodes by 517 amino acid residues
The polypeptide of composition, due to heredity, there are 3 kinds of situations in the enzyme gene type in crowd, has normal catalytic activity homozygous:
ALDH2*1/*1;The heterozygosis subtype of catalytic activity decline: ALDH2*2/*1;The homozygous that catalytic activity loses: ALDH2*2/*
2.Researches show that ALDH2 can improve prognosis, the Ischemic myocardium reperfusion injury of heart failure in recent years, after gene mutation
It causes enzymatic activity to decline, increases the risk of coronary heart disease (CAD) and myocardial infarction (MI).
CYP2D6, ADRB1, PON1, GP1BA, PEAR1, AGTR1 gene polynorphisms are related to cardiovascular disease medicine
Property
CYP2D6 and ADRB1 gene polynorphisms are related with the curative effect of beta receptor blocking agent, PON1 gene polynorphisms
(rs662) related with the curative effect of clopidogrel or adverse reaction, GP1BA gene polynorphisms (rs2243093) and aspirin
Antiplatelet drug effect is related, and PEAR1 is related with aspirin resistance and prognosis, AGTR1 gene polynorphisms (rs5186) and
Hypertension has correlation.
PCR- direct sequencing, PCR- pyrosequencing, fluorescence quantitative PCR method, PCR- are often used in the prior art
High-resolution melting curve method, ApoE gene method, PCR- restriction fragment length polymorphism method and solid chip
Method etc. detects the polymorphisms of one or more of cardiovascular drugs related genes, these methods mostly flexibility ratio is low, at high cost,
Flux is low, and the time is longer, and detecting instrument is expensive, is not suitable for clinical expansion, everyone reaches tens of arrive by the reagent cost once checked
Hundreds of dollars, and existing detection kit is not directed to Chinese human genetic disease spectrum of disease.Therefore, to specific contemporaneously or in parallel
Detecting the products of a variety of cardiovascular drugs related gene polymorphisms, there are great demands.
Summary of the invention
In view of the deficienciess of the prior art, the present inventor is by Luminex liquid-phase chip technology and allele
Specific primer extends (allele specific primer extension, ASPE) reaction and combines, and designs and constructs and contains
There are CYP2C9, VKORC1, CYP4F2, CYP2C19, PON1, CYP2D6, ADRB1, ALDH2, GP1BA, PEAR1, AGTR1 gene
The analog sample in mutational site establishes a kind of novel detection side that can detect cardiovascular drugs related gene polymorphism simultaneously
Method.
The first aspect of the present invention provides a kind of 15 kinds of cardiovascular drugs correlations of liquid-phase chip technology while detection
The primer sets of target gene polymorphism, including 15 groups of primers, wherein every group of primer includes for detecting a kind of target gene polymorphism
A pair of of specificity amplification primer and respectively for the target gene wild type and saltant type two ASPE primers;
Target gene polymorphism detected is respectively as follows: CYP2C9-C430T rs1799853, CYP2C9-A1075C
rs1057910、VKORC1-G1639Ars9923231、CYP4F2-C1347Trs2108622、CYP2C19-
G681Ars4244285、CYP2C19-G636Ars4986893、CYP2C19-C806T rs12248560、PON1-Q192R
rs662、CYP2D6-C2850T rs16947、CYP2D6-C100T rs1065852、ADRB1-G1165C rs1801253、
ALDH2-G504A rs671, GP1BA-T5C rs2243093, PEAR1 G > A rs2768759 and AGTR1-A1166C
rs5186;
Sequence for detecting the primer sets of the loci polymorphism of the target gene is as follows:
For detecting the specificity amplification primer such as sequence SEQ ID NO.1 and SEQ of CYP2C9-C430T rs1799853
Shown in ID NO.2, ASPE primer is as shown in sequence SEQ ID NO.3 and sequence SEQ ID NO.4;
For detecting the specificity amplification primer such as sequence SEQ ID NO.5 and SEQ of CYP2C9-A1075C rs1057910
Shown in ID NO.6, ASPE primer is as shown in sequence SEQ ID NO.7 and sequence SEQ ID NO.8;
For detecting the specificity amplification primer such as sequence SEQ ID NO.9 and SEQ of VKORC1-G1639A rs9923231
Shown in ID NO.10, ASPE primer is as shown in sequence SEQ ID NO.11 and sequence SEQ ID NO.12;
For detect CYP4F2-C1347T rs2108622 specificity amplification primer such as sequence SEQ ID NO.13 and
Shown in SEQ ID NO.14, ASPE primer is as shown in sequence SEQ ID NO.15 and sequence SEQ ID NO.16;
For detect CYP2C19-G681A rs4244285 specificity amplification primer such as sequence SEQ ID NO.17 and
Shown in SEQ ID NO.18, ASPE primer is as shown in sequence SEQ ID NO.19 and sequence SEQ ID NO.20;
For detect CYP2C19-G636A rs4986893 specificity amplification primer such as sequence SEQ ID NO.21 and
Shown in SEQ ID NO.22, ASPE primer is as shown in sequence SEQ ID NO.23 and sequence SEQ ID NO.24;
For detect CYP2C19-C806T rs12248560 specificity amplification primer such as sequence SEQ ID NO.25 and
Shown in SEQ ID NO.26, ASPE primer is as shown in sequence SEQ ID NO.27 and sequence SEQ ID NO.28;
For detecting the specificity amplification primer such as sequence SEQ ID NO.29 and SEQ of PON1-Q192R (A/G) rs662
Shown in ID NO.30, ASPE primer is as shown in sequence SEQ ID NO.31 and sequence SEQ ID NO.32;
For detecting the specificity amplification primer such as sequence SEQ ID NO.33 and SEQ of CYP2D6-C2850T rs16947
Shown in ID NO.34, ASPE primer is as shown in sequence SEQ ID NO.35 and sequence SEQ ID NO.36;
For detecting the specificity amplification primer such as sequence SEQ ID NO.37 and SEQ of CYP2D6-C100T rs1065852
Shown in ID NO.38, ASPE primer is as shown in sequence SEQ ID NO.39 and sequence SEQ ID NO.40;
For detecting the specificity amplification primer such as sequence SEQ ID NO.41 and SEQ of ADRB1-G1165C rs1801253
Shown in ID NO.42, ASPE primer is as shown in sequence SEQ ID NO.43 and sequence SEQ ID NO.44;
For detecting specificity amplification primer such as the sequence SEQ ID NO.45 and SEQ ID of ALDH2-G504A rs671
Shown in NO.46, ASPE primer is as shown in sequence SEQ ID NO.47 and sequence SEQ ID NO.48;
For detecting specificity amplification primer such as the sequence SEQ ID NO.49 and SEQ ID of GP1BA-T5C rs2243093
Shown in NO.50, ASPE primer is as shown in sequence SEQ ID NO.51 and sequence SEQ ID NO.52;
For detecting the specificity amplification primer such as sequence SEQ ID NO.53 and SEQ of PEAR1 G > A rs2768759
Shown in ID NO.54, ASPE primer is as shown in sequence SEQ ID NO.55 and sequence SEQ ID NO.56;
For detecting specificity amplification primer such as the sequence SEQ ID NO.57 and SEQ ID of AGTR1-A1166C rs5186
Shown in NO.58, ASPE primer is as shown in sequence SEQ ID NO.59 and sequence SEQ ID NO.60.
In a preferred embodiment of the invention, the ASPE primer includes positioned at the Tag sequence at 5 ' ends and positioned at 3 '
The specific primer sequence two parts for target gene polymorphism at end, and the base of 3 ' end first of the ASPE primer
With target gene complementary pairing to be detected.
In another preferred embodiment of the invention, the specific primer sequence such as sequence that includes in the ASPE primer
Shown in SEQ ID NO.61-90.
The second aspect of the present invention provides above-mentioned primer sets and is being prepared by 15 kinds of liquid-phase chip technology while detection
Purposes in the kit of cardiovascular drugs correlation target gene polymorphism.
The third aspect of the present invention provides a kind of using liquid-phase chip technology while detecting multiple cardiovascular drugs phases
The kit of target gene polymorphism is closed, the kit includes primer sets described in claim 1 and 30 kinds of microballoons, wherein
The primer sets include 15 groups of primers, and every group of primer includes special for detecting a kind of a pair of target gene polymorphism
Property amplimer and respectively for the target gene wild type and saltant type two ASPE primers, the ASPE primer packet
It includes the Tag sequence for being located at 5 ' ends and is located at the specific primer sequence two parts for target gene at 3 ' ends, and the ASPE
The base of 3 ' end first of primer and target gene complementary pairing to be detected;With
Every kind of microballoon is respectively coupled a kind of addressable probes sequence with the Tag sequence complementary pairing of the ASPE primer respectively
Column.
In a preferred embodiment of the invention, kit of the invention further includes that streptavidin-phycoerythrin is miscellaneous
Hand over buffer, PCR reaction solution, archaeal dna polymerase and negative control.
In another preferred embodiment of the invention, include in the ASPE primer in kit of the invention is located at 3 '
The specific primer sequence at end is as shown in sequence SEQ ID NO.61-90.
In another preferred embodiment of the invention, the negative control in kit of the invention is distilled water+microballoon.
The third aspect of the present invention provides a kind of method using kit of the invention, and the method includes following steps
It is rapid:
(1) multiplexed PCR amplification reacts: extracting the DNA in sample as template, is drawn using the specific amplification in kit
Object prepares multi-PRC reaction system, and PCR amplification is carried out under the PCR amplification program of setting;
(2) purifying of PCR product: with dNTP, primer and list extra in excision enzyme and alkaline phosphatase enzymic digestion PCR product
Chain product;
(3) ASPE reacts: PCR product after purification is under the ASPE response procedures of design, the DNA in ASPE reaction system
Under the action of polymerase, when the target sequence detection site mutual added time of the primer base of 3 ' end first and amplification, extend anti-
It answers, if not complementary, extension will be terminated;
(4) hybridization reaction: taking the 25 diluted microballoons of μ l to 96 holes to hybridize plate, and the microballoon and deionization of equivalent is added in control wells
The 2.5 μ L of ASPE reaction product of step (3) is added in water, and suction is beaten uniformly, and hybridization reaction condition is 96 DEG C of denaturation 90s, 37 DEG C of incubations
30min;
(5) result detects: the microballoon after hybridization being resuspended in 100 μ L and contains 6.5 μ g/mL Streptavidins-algae red
In 1 × hybridization buffer of albumen, after 37 DEG C of incubation 20min, detected on 200 instrument of Luminex.
In a preferred embodiment of the invention, in step (1), multi-PRC reaction system are as follows: 50 μ l of total volume,
The total 50ng of gDNA, 2 μ l, 10 × PCR reaction buffer 5 μ l, TaqTM0.25 μ each 2.5mM of l, dNTP of Hot Start enzyme, totally 4 μ l,
Primer concentration is 10 μM, each 0.5 μ l, 30.75 μ l of deionized water;
The program of pcr amplification reaction are as follows: initial denaturation: 94 DEG C of 30s, circulation: 94 DEG C of 30s, anneal 57 DEG C of 30s, extends 72 DEG C
30s, totally 5 times, 94 DEG C of 30s, anneal 55 DEG C of 30s, extends 72 DEG C of 30s, totally 30 times, extends eventually: 72 DEG C of 10min;
In step (3), ASPE reaction system are as follows: 5 μ l, 400 μ of 2 μ l, 10 × PCR reaction buffer of PCR product after purification
Each 100 μM of Biotin-dCTP0.35 the μ l, dATP, dGTP, dTTP of M, dCTP 0.3 the μ l, Taq of totally 1 μ l, 100mMTMHot
0.1 μ l, TAG-ASPE each 500nM of primer of Start enzyme, totally 1 μ l, 13.55 μ l of deionized water;
The program of ASPE extension are as follows: initial denaturation: 94 DEG C of 90s;Circulation: 94 DEG C of 30s, 57 DEG C of 30s, 74 DEG C of 1min, 40
It is secondary.
Primer and kit provided by the invention can detect a variety of common cardiovascular diseases using liquid-phase chip technology simultaneously
Disease medicament related gene has following advantageous effects:
1. liquid-phase chip detection kit of the invention includes 15 pairs of specific primers, and combines MagPlex microballoon one
Step PCR reaction in simultaneously expand and detect 15 kinds of common cardiovascular drugs correlation target gene CYP2C9, VKORC1, CYP4F2,
The polymorphism of CYP2C19, PON1, CYP2D6, ADRB1, ALDH2, GP1BA, PEAR1, AGTR1, detection flux is high, cost performance
Height, one-time detection can provide the medication guide of cardiovascular patient, provide laboratory foundation for clinical rational drug use, have pole
Big clinical value.
2. the kit has extraordinary specificity, cross reaction is not present between the primer of design, probe sequence.
3. the kit has extraordinary sensitivity and stability in actually detected.
4. kit operating procedure of the invention is simple, 15 kinds of mutational sites can combine ASPE reaction by PCR simultaneously
Detection avoids the sample that may cause in repeated multiple times PCR operating process from polluting, to greatly improve Detection accuracy.
5. testing result and sequencing result coincide well, it can accurately detect that cardiovascular drugs correlation target gene is more
State property site type, and result is reliable and stable, compared with conventional detection SNP method, greatly reduces testing cost.
Detailed description of the invention
Following drawings is for illustrating a specific embodiment of the invention, rather than limits and be defined by the claims
The scope of the present invention.
Figure 1A -1D in Fig. 1 shows that liquid-phase chip technology allelic specific primer extends the principle of (ASPE).
Specific embodiment
After extensive and in-depth study, a variety of painstaking effort can be detected to the present inventor simultaneously by developing one kind for the first time
Primer sets, kit and the kit application method of tubing pharmaceutical relevant gene polymorphism.
Term
Liquid-phase chip
The term as used herein " liquid-phase chip " is a kind of biochip of novel concept.The core of the technology is micro-
Small polystyrene sphere (5.6vm) is encoded with the method for fluorescent staining, then by the microballoon of each color (or to be glimmering
Pumped FIR laser microballoon) probe, antigen or the antibody of particular detection object are directed on covalent cross-linking.In application, first for different detections
The coding microball of object mixes, and adds micro sample to be examined, carries out in the molecule of suspension target and microsphere surface crosslinking special
It combines anisotropicly, up to 100 kinds of different biologicallies can be completed at the same time in a reacting hole.Finally use LuminexTM
Analysis software is analyzed, and instrument is identified coding microball respectively and detected by two beam laser reports that the fluorescence of molecule is strong on microballoon
Degree.Because molecule hybridization or immune response are carried out in aaerosol solution, detection speed is exceedingly fast, and can be in a micro liquid
Up to 100 indexs are detected in state reaction system simultaneously.Experimental principle is referring to Fig. 1.
Allele-specific primers extend (ASPE)
" allele-specific primers extend (Allele Specific Primer to the term as used herein
Extension, ASPE) " it is a kind of sequence-specific enzymatic reaction technology based on solution, it can be used for measuring in single pipe more
A SNP.ASPE method is related to two stages, is enzymatic reaction first, target genotype is determined, then on microspheres with solid surface
Capture is to be detected.Using solution phase kinetics phase, this technology allows that (i.e. of the invention seeks with the microballoon of sequence mark
The microballoon of location probe sequence coupling) the new template (that is, ASPE amplified production of the invention) of detection.This is special with allele
It is completed with the help of the appropriate capture sequence of specific oligonucleotide connection.
The template of ASPE reaction is the PCR fragment of amplification.When designing extension primer, SNP should be present in allele spy
3 ' ends of specific primer, accurately to be hybridized, capture sequence (TAG sequence) should be located at 5 ' ends of primer.In allele spy
Specific primer extends in step, and polymerase is by mixing the dNTP (Biotin-dCTP) of biotin labeling come extension primer.Only when
Just extend when 3 ' ends of allele-specific primers are in conjunction with homoallele sequence, therefore, extension products include
TAG sequence+purpose target gene site+the amplified fragments comprising biotin labeling.
Present invention will be further explained below with reference to specific examples.It should be appreciated that these embodiments are merely illustrative this
The purpose of invention and the limitation of non-present invention.Test method without specific conditions in following embodiment, according to routine experiment
Condition such as Sambrook et al., condition described in Molecular Cloning:A Laboratory guide (fourth edition) or is built according to goods producer
The condition of view carries out.
Embodiment
Reagent used in the present invention and instrument are as follows:
DNA extraction kit TIANamp Genomic DNA Kit is purchased from Beijing Tiangeng biochemical technology Co., Ltd;It is multiple
PCR kit TaqTM Hot Start Version is purchased from TaKaRa company;PlatinumTMGenoType Tsp DNA polymerization
Enzyme, nucleic acid exoenzyme I- shrimp alkaline phosphotase (ExoSAP-IT), Biotin-dCTP, dNTP, streptavidin-phycoerythrin (SA-
) etc. PE it is purchased from Thermo Fischer Scient Inc.;LifeECO gene-amplificative instrament is purchased from Tou Jing Life Science Co., Ltd of China;
BioDrop protein nucleic acid analyzer is purchased from Hao Wo Biotechnology Co., Ltd;MagPlex-Tag microballoon and Luminex 200 are purchased
From Luminex company of the U.S..
Primer sets of the embodiment 1. for liquid-phase chip technology detection cardiovascular drugs related gene
The design of primer and probe:
According to No. rs base sequence in Genbank near 15 catastrophe points of searching of SNP disclosed on NCBI, use
6.0 primer-design software of Primer draws designed for the specificity in the following cardiovascular Common drugs correlation target gene site of amplification
Object: A1075C polymorphism rs1057910, VKORC1 of C430T polymorphism rs1799853, the CYP2C9 gene of CYP2C9 gene
C1347T polymorphism rs2108622, the CYP2C19 gene of G1639A polymorphism rs9923231, the CYP4F2 gene of gene
The C806T of G636A polymorphism rs4986893, the CYP2C19 gene of G681A polymorphism rs4244285, CYP2C19 gene is polymorphic
The C2850T polymorphism rs16947 of Q192R polymorphism rs662, the CYP2D6 gene of property rs12248560, PON1 gene,
G1165C polymorphism rs1801253, the ALDH2 gene of C100T polymorphism rs1065852, the ADRB1 gene of CYP2D6 gene
The G > A polymorphism rs2768759 of T5C polymorphism rs2243093, the PEAR1 gene of G504A polymorphism rs671, GP1BA gene
With the primer of the A1166C polymorphism rs5186 of AGTR1 gene.
The loci polymorphism of 15 kinds of target genes is directed to according to the design of the nucleotide sequence of the loci polymorphism of the above gene
Specific primer is used to expand the DNA fragmentation in 15 kinds of target gene sites, and is directed to the wild type and mutation in 15 kinds of target gene sites
Type separately designs ASPE primer sequence, amounts to 30.ASPE primer is made of Tag sequence+specific primer sequence.It is set in primer
During meter, to avoid that cross reaction occurs between joint-detection target gene, first have to avoid 15 pairs of specific primers it
Between non-specific amplification occurs, make the Tm difference of PCR forward primer and reverse primer less than 5 DEG C, while by preceding 5 PCR cycles
Annealing temperature improves 2 DEG C, is set as 57 DEG C;Its is secondary to avoid occurring to intersect between ASPE primer extending, when carrying out specificity experiments
If cross reaction occurs between ASPE primer, ASPE primer PrimerPlex software design high degree of specificity is redesigned
ASPE primer, catastrophe point be located at design primer 3 ' end, by ASPE reaction allow pcr amplification product be coupled ASPE capture sequence
Primer simultaneously extends;In addition, Tag sequence is designed according to designed ASPE specific primer segment, to reduce to the maximum extent
The secondary structure being likely to form between Tag sequence and ASPE specific primer segment.
Since multiple primers and template are present in the same reaction tube in multiplex PCR system, it is easy to make in experimentation
At interfering with each other, and dimer easy to form between primer even results in test and loses to influence the extension of ASPE primer
It loses.Therefore design of primers is the key that of the invention, and carries out the basis of Luminex liquid-phase chip detection.
The present inventor overcomes the difficult point of above-mentioned multiple PCR primer design, needs to search by BLAST before design of primers
Rope to exclude homologous section, and between guaranteeing primer pcr amplification product in 100-700bp;It will be more than 1 in above-mentioned eight pairs of primer collections
Weight PCR system carries out PCR amplification;If moiety site amplification efficiency is inadequate, its PCR primer concentration or design are adjusted, or
Person adjusts the primer combination of multiplex PCR, until be optimized to can compare it is balanced amplify all sites, complete PCR optimization.By
A large amount of experiment is screened and is improved to the specificity amplification primer and ASPE extension primer of design, last preferably to go out for expanding
Increase 15 pairs of specificity amplification primers (being shown in Table 1) of 15 target gene templates and 30 ASPE for extending target gene site extend
Primer (is shown in Table 2).
Table 1. amplifies the specificity amplification primer of the target sequence comprising target gene site
The specificity amplification primer for amplifying the target sequence comprising target gene site is by the raw work bioengineering in Shanghai
Technology Service Co., Ltd's synthesis, every primer after synthesis are configured to 10 μM of storage liquid with deionized water respectively.
2. 15 pairs of target gene AsPE extension primer sequences of table
Note: thickening font is detection site.
The ASPE extension primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and every after synthesis draws
Object is configured to the storage liquid of 500nmol/L with deionized water respectively.
It is more that the utilization liquid-phase chip technology of the invention of embodiment 2. detects multiple cardiovascular drugs correlation target genes simultaneously
The kit of state property
Kit of the invention includes:
1. the primer in embodiment 1 in designed Tables 1 and 2.
2. 30 kinds are coupled the MagPlex-Tag microballoon for having addressable probes sequence, the addressable probes sequence can be with ASPE
Tag sequence complementary pairing in extension primer.30 kinds of microballoon numbers and the anti-Tag sequence being coupled on microballoon are as shown in table 3:
3. 30 kinds of table are coupled the MagPlex-Tag microballoon for having addressable probes sequence (anti-Tag sequence)
30 kinds of MagPlex-Tag microballoons of selection are purchased from U.S. Luminex company, and concentration is 2.5 × 106A/ml is used
When be diluted to every kind of microballoon containing 2500, wherein addressable probes sequence pairs are mutual with the Tag sequence in ASPE primer together on microballoon
It recruits pair.
3. streptavidin-phycoerythrin hybridization buffer, PCR reaction solution, archaeal dna polymerase, negative control;Wherein strepto-
Avidin-phycoerythrin hybridization buffer is purchased from Thermo Fischer Scient Inc., and concentration is 6.5 μ g/mL, multi-PRC reaction liquid
For 10xbuffer, Taq is come from archaeal dna polymeraseTMHotStartVersion kit is purchased from TaKaRa company, negative control
For microballoon+deionized water of equivalent.
The primer sets of the invention target gene polymorphism related to the kit common cardiovascular drugs of detection of embodiment 3.
The primer sets of the present embodiment Application Example 1 and the kit of embodiment 2 have detected common cardiovascular drugs 15
Kind target gene polymorphism.
Steps are as follows for specific experiment:
1. multiplexed PCR amplification reacts: 14 parts of whole blood samples (blood sample derives from Dongguan Hou Jie hospital) is extracted altogether, according to experiment
It is required that separately setting a negative control, DNA is extracted.Multi-PRC reaction is prepared using the specificity amplification primer in 1 table 1 of embodiment
System (50 μ l):
Ingredient | Dosage (μ l) |
gDNA | 50ng(2μl) |
10 × PCR reaction buffer | 5 |
TaqTMHotStar | 0.25 |
DNTP (each 2.5mM) | 4 |
Upstream and downstream primer (10 μM) | Each 0.5 |
Deionized water | 30.75 |
PCR amplification parameter designing is as follows:
Initial denaturation: 94 DEG C of 30s
Circulation: 94 DEG C of 30s, anneal 57 DEG C of 30s, extends 72 DEG C of 30s, and totally 5 times
94 DEG C of 30s, anneal 55 DEG C of 30s, extends 72 DEG C of 30s, and totally 30 times
Extend eventually: 72 DEG C of 10min
PCR, which is carried out, for the primer that verifying designs reacts whether resulting PCR product is the target comprising target gene site
Sequence leaves and takes part PCR product and Guangzhou Ai Ji biology Co., Ltd is sent to be sequenced, and sequencing result and target PCR product coincide
Degree is good.
The purifying of 2.PCR product: after reaction, product can remain extra dNTP, primer and single stranded product to PCR,
The extension for seriously affecting subsequent ASPE can be removed these impurity using ExoSAP-IT.2.5μl ExoSAP-IT+5μ
37 DEG C of incubation 30min of l PCR product, with excessive primer, single stranded DNA, the dNTP of degrading, subsequent 80 DEG C of incubations 20min loses enzyme
It is living, purified pcr product.
3.ASPE reaction: the Taq in ASPE reaction system of PCR product after purificationTMUnder the action of HotStar polymerase,
When the target sequence detection site mutual added time of the primer base of 3 ' end first and amplification, it can just continue extension, if
Not complementary, extension will terminate.ASPE reaction is carried out by following optimizing reaction system:
Table 4.ASPE optimizing reaction system
The design of ASPE response parameter is as follows:
Initial denaturation: 94 DEG C of 90s;
Circulation: 94 DEG C of 30s, 57 DEG C of 30s, 74 DEG C of 1min, 40 times;
Optimized detection architecture shows that the primer specificity that the present invention designs is strong, and no cross reaction occurs.This hair
DCTP (Biotin-dCTP) concentration of biotin labeling is tieed up with the dCTP concentration proportion that do not label in bright ASPE reaction system
Holding 3: 1 is successful one of the key factor of detection architecture, and ratio is excessively high, influences ASPE primer and effectively extends, ratio is too low then
Fluorescence signal is weak.
4. hybridization reaction: 30 kinds of MagPlex-Tag microballoons of selection be purchased from U.S. Luminex company, concentration be 2.5 ×
106A/ml is diluted to 100 every microlitre, and the 25 diluted microballoons of μ l (containing every kind of microballoon 2500) to 96 holes is taken to hybridize plate, right
The microballoon and deionized water of equivalent are added according to hole, 2.5 μ L of ASPE reaction product is added, suction is beaten uniformly.Whole operation process pays attention to
It is protected from light.Hybridization conditions: 96 DEG C of denaturation 90s, 37 DEG C of incubation 30min.
5. result detects: the microballoon after hybridization being resuspended in 100 μ L and contains 6.5 μ g/mL Streptavidins-algae red egg
In 1 white × hybridization buffer, detected on 200 instrument of Luminex after 37 DEG C of incubation 20min.
Three, testing result and data analysis
It is detected by Luminex analysis instrument, testing result is as shown in table 5.
Requirement of the instrument to fluorescent value (MFI) are as follows: (mutation MFI+ is wild for detection site MFI ratio=detection site MFI/
Raw MFI), MFI ratio > 0.75 or < 0.25 is homozygote, is heterozygote between 0.25-0.75.With kit of the invention
The resolution ratio of gained testing result is higher, and homozygous MFI ratio is generally higher than 0.9 or less than 0.1, and heterozygote is in 0.35-
Between 0.65, illustrate that the resolution ratio of testing result is substantially better than the genotyping standard of Luminex company offer, the present invention establishes
Detection kit and primer 30 kinds of genotype can be clearly distinguished with higher resolution, provide laboratory for clinical rational drug use
Foundation has great clinical value.
It is compared with PCR sequencing PCR detection with liquid-phase chip result, calculates classifying method testing result provided by the present invention
The rate of coincideing.This method detects the testing result of the 30 kinds of genotype in cardiovascular drugs correlation target gene site of 14 parts of whole blood samples
Reach 100% with the sequencing result rate of coincideing.It can be seen that liquid-phase chip detection primer group provided by the present invention and kit can be quasi-
Really detect cardiovascular drugs correlation target gene polymorphic position vertex type, and result is reliable and stable.
The analysis of the detection specificity of the primer of the invention of embodiment 4. and kit and clinical samples confirmatory experiment
For the specific ASPE primer energy of the invention for ensuring to design and in corresponding detection site can only extend anti-
It answers, the present embodiment carries out specific detection for single-site mutant, and ASPE primed probe and other mutational sites are equal as the result is shown
No cross reaction, 15 target genes that Luminex 200 detects 13 parts of samples the results are shown in Table 6, each allele carry frequency with
Hatmap is almost the same.All detection sample send Guangzhou Ai Ji biology Co., Ltd to be sequenced, Luminex testing result and survey
Sequence result is completely the same, shows that the ASPE primer specificity of primer and kit of the invention is high.
The primer sets of the invention of embodiment 5. and the analysis experiment of the detection sensitivity of kit
Diluted sample is detected according to the optimal reaction system of embodiment 3, DNA concentration between 100-0.75ng it
Between.As DNA concentration reduces, the MFI value of detection is gradually reduced, and detects all allele MFI ratios between different DNA concentrations
For rate difference less than 0.03, testing result is shown in Table 7.Detecting three times, which can clearly differentiate the minimum concentration of target point gene type, is
1.5ng illustrates that the detection sensitivity of primer sets and kit of the invention is high.
So far, temporarily without finding that the technology that can detect the common 15 kinds of target gene sites of cardiovascular drugs simultaneously is flat
Platform, primer sets of the invention and kit detection flux are high, and cost performance is high, has great clinical value, can be simultaneously
The drug genes polymorphisms such as warfarin, clopidogrel, nitroglycerin, aspirin, beta receptor blocking agent are detected, are provided for clinic
In time, accurate medication information, and have many advantages, such as that low cost, high specific, high sensitivity, easy to operate, combination is flexible.
Sequence table
<110>Dongguan Houjie Hospital
<120>with the specific primer and reagent of liquid-phase chip technology detection cardiovascular disease medicine gene pleiomorphism
Box
<130> L019PAF20180307
<160> 90
<170> PatentIn version 3.5
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<400> 1
tcagcaatgg aaagaaatgg 20
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gaagatagta gtccagtaag gt 22
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ttcttcatta acttctaatc ttacaagagg agcattgagg acc 43
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tacaacatct cattaacata tacaaagagg agcattgagg act 43
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aagtccagga agagattgaa 20
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cggtgatggt agaggttta 19
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tacttcttta ctacaattta caacggtggg gagaaggtca at 42
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ctttctcata ctttcaacta atttggtggg gagaaggt 38
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tgtcaccaag acgctaga 18
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ccatctgcaa ccttaattcc 20
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ttaaacaatc tactattcaa tcactgaaaa acaaccattg gccg 44
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taacttacac ttaactatca tctttgaaaa acaaccattg gcca 44
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ggaggtgatg ttggatact 19
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agaagctgga gaattgtgt 19
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aatctctaca atttctctct aatactcagg gtccggccac ac 42
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caataaacat tctttacatt ctcactcagg gtccggccac at 42
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aagcaggtat aagtctagga 20
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ggttgttgat gtccatcg 18
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ctttcttaat acattacaac atacagtaat ttgttatggg ttccc 45
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tcaaactctc aattcttact taatagtaat ttgttatggg ttcct 45
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gtgatcccac tttcatcct 19
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tgggatattc atttcctgtg 20
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<211> 42
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acaaatatct aactactatc acaaattgta agcaccccct gg 42
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ctttatcaaa ttctaattct caacattgta agcaccccct ga 42
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tgaacaggat gaatgtggta 20
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cagcagccta aacatgaaat 20
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acactcattt aacactattt cattgtgtct tctgttctca aagc 44
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acacttatct ttcaattcaa ttacgtgtct tctgttctca aagt 44
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gaatagacag tgaggaatgc 20
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aaccagtatg ccttcacaa 19
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tacattcaac actcttaaat caaaattttc ttgaccccta cttaca 46
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atactttaca aacaaataac acacattttc ttgaccccta cttacg 46
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aatcacggca gtggtgta 18
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gtgcagaatt ggaggtcat 19
<210> 35
<211> 45
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tactacttct ataactcact taaagcttca atgatgagaa cctgg 45
<210> 36
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actacttatt ctcaaactct aatagcttca atgatgagaa cctga 45
<210> 37
<211> 19
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<213>artificial sequence
<400> 37
ttggtagtga ggcaggtat 19
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actcaggact aactcatctt c 21
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tacttaaaca tacaaactta ctcactgggc tgcacgctac c 41
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tcttactaat ttcaatactc ttacctgggc tgcacgctac t 41
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catcatctac tgccgcag 18
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<400> 42
cctacacctt ggattccg 18
<210> 43
<211> 43
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tctctttaaa cacattcaac aatattccgc aaggccttcc agg 43
<210> 44
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cttaacattt aacttctata acacttccgc aaggccttcc agc 43
<210> 45
<211> 18
<212> DNA
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gcaacgagcc aagatcat 18
<210> 46
<211> 19
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ataacgaagc ccagcaaat 19
<210> 47
<211> 42
<212> DNA
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catcttcata tcaattctct tattggctgc aggcatacac tg 42
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caaatacata atcttacatt cactggctgc aggcatacac ta 42
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gtcactggaa tccctatcag 20
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tggagacctc acagatgg 18
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cttaaactct acttacttct aattaggagg agaggcatga gga 43
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ctttatcaaa ttctaattct caacaggagg agaggcat 38
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ccttaactga gtggtctgag 20
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acagccatgt gattagcc 18
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cataatcaat ttcaactttc tactgcttgc attattgcag gaacc 45
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aataacaact cactatatca taacgcttgc attattgcag gaacc 45
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aagaaggagc aagagaacat 20
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ggttcagtcc acataatgc 19
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acttatttct tcactactat atcacacttc actaccaaat gagca 45
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attcaatact atctaacact tactcacttc actaccaaat gagca 45
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aagaggagca ttgaggacc 19
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aagaggagca ttgaggact 19
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ggtggggaga aggtcaat 18
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ggtggggaga aggtcaag 18
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tgaaaaacaa ccattggccg 20
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tgaaaaacaa ccattggcca 20
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ctcagggtcc ggccacac 18
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ctcagggtcc ggccacat 18
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agtaatttgt tatgggttcc c 21
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agtaatttgt tatgggttcc t 21
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attgtaagca ccccctgg 18
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attgtaagca ccccctga 18
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gtgtcttctg ttctcaaagc 20
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gtgtcttctg ttctcaaagt 20
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attttcttga cccctactta ca 22
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attttcttga cccctactta cg 22
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gcttcaatga tgagaacctg g 21
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gcttcaatga tgagaacctg a 21
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ctgggctgca cgctacc 17
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ctgggctgca cgctact 17
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<211> 19
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ttccgcaagg ccttccagg 19
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<211> 19
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ttccgcaagg ccttccagc 19
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ggctgcaggc atacactg 18
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<400> 84
ggctgcaggc atacacta 18
<210> 85
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aggaggagag gcatgagga 19
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<400> 86
aggaggagag gcat 14
<210> 87
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<400> 87
gcttgcatta ttgcaggaac c 21
<210> 88
<211> 21
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gcttgcatta ttgcaggaac c 21
<210> 89
<211> 21
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cacttcacta ccaaatgagc a 21
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cacttcacta ccaaatgagc a 21
Claims (10)
1. a kind of primer sets for detecting 15 kinds of cardiovascular drugs correlation target gene polymorphisms simultaneously with liquid-phase chip technology, including
15 groups of primers, wherein every group of primer includes a pair of of the specificity amplification primer and difference for detecting a kind of target gene polymorphism
Two ASPE primers of wild type and saltant type for the target gene;
Target gene polymorphism detected is respectively as follows: CYP2C9-C430T rs1799853, CYP2C9-A1075C
rs1057910、VKORC1-G1639A rs9923231、CYP4F2-C1347T rs2108622、CYP2C19-G681A
rs4244285、CYP2C19-G636A rs4986893、CYP2C19-C806T rs12248560、PON1-Q192R rs662、
CYP2D6-C2850T rs16947、CYP2D6-C100T rs1065852、ADRB1-G1165C rs1801253、ALDH2-
G504A rs671, GP1BA-T5C rs2243093, PEAR1 G > A rs2768759 and AGTR1-A1166C rs5186;
Sequence for detecting the primer sets of the target gene polymorphism is as follows:
For detecting specificity amplification primer such as the sequence SEQ ID NO.1 and SEQ ID of CYP2C9-C430T rs1799853
Shown in NO.2, ASPE primer is as shown in sequence SEQ ID NO.3 and sequence SEQ ID NO.4;
For detecting specificity amplification primer such as the sequence SEQ ID NO.5 and SEQ ID of CYP2C9-A1075C rs1057910
Shown in NO.6, ASPE primer is as shown in sequence SEQ ID NO.7 and sequence SEQ ID NO.8;
For detecting specificity amplification primer such as the sequence SEQ ID NO.9 and SEQ ID of VKORC1-G1639A rs9923231
Shown in NO.10, ASPE primer is as shown in sequence SEQ ID NO.11 and sequence SEQ ID NO.12;
For detecting specificity amplification primer such as the sequence SEQ ID NO.13 and SEQ ID of CYP4F2-C1347T rs2108622
Shown in NO.14, ASPE primer is as shown in sequence SEQ ID NO.15 and sequence SEQ ID NO.16;
For detecting specificity amplification primer such as the sequence SEQ ID NO.17 and SEQ ID of CYP2C19-G681Ars4244285
Shown in NO.18, ASPE primer is as shown in sequence SEQ ID NO.19 and sequence SEQ ID NO.20;
For detecting specificity amplification primer such as the sequence SEQ ID NO.21 and SEQ ID of CYP2C19-G636A rs4986893
Shown in NO.22, ASPE primer is as shown in sequence SEQ ID NO.23 and sequence SEQ ID NO.24;
For detecting the specificity amplification primer such as sequence SEQ ID NO.25 and SEQ of CYP2C19-C806T rs12248560
Shown in ID NO.26, ASPE primer is as shown in sequence SEQ ID NO.27 and sequence SEQ ID NO.28;
For detecting specificity amplification primer such as the sequence SEQ ID NO.29 and SEQ ID of PON1-Q192R (A/G) rs662
Shown in NO.30, ASPE primer is as shown in sequence SEQ ID NO.31 and sequence SEQ ID NO.32;
For detecting specificity amplification primer such as the sequence SEQ ID NO.33 and SEQ ID of CYP2D6-C2850T rs16947
Shown in NO.34, ASPE primer is as shown in sequence SEQ ID NO.35 and sequence SEQ ID NO.36;
For detecting specificity amplification primer such as the sequence SEQ ID NO.37 and SEQ ID of CYP2D6-C100T rs1065852
Shown in NO.38, ASPE primer is as shown in sequence SEQ ID NO.39 and sequence SEQ ID NO.40;
For detecting specificity amplification primer such as the sequence SEQ ID NO.41 and SEQ ID of ADRB1-G1165C rs1801253
Shown in NO.42, ASPE primer is as shown in sequence SEQ ID NO.43 and sequence SEQ ID NO.44;
For detecting the specificity amplification primer such as sequence SEQ ID NO.45 and SEQ ID NO.46 of ALDH2-G504A rs671
Shown, ASPE primer is as shown in sequence SEQ ID NO.47 and sequence SEQ ID NO.48;
For detecting specificity amplification primer such as the sequence SEQ ID NO.49 and SEQ ID of GP1BA-T5C rs2243093
Shown in NO.50, ASPE primer is as shown in sequence SEQ ID NO.51 and sequence SEQ ID NO.52;
For detecting specificity amplification primer such as the sequence SEQ ID NO.53 and SEQ ID of PEAR1 G > A rs2768759
Shown in NO.54, ASPE primer is as shown in sequence SEQ ID NO.55 and sequence SEQ ID NO.56;
For detecting specificity amplification primer such as the sequence SEQ ID NO.57 and SEQ ID of AGTR1-A1166C rs5186
Shown in NO.58, ASPE primer is as shown in sequence SEQ ID NO.59 and sequence SEQ ID NO.60.
2. primer sets as described in claim 1, wherein the ASPE primer includes positioned at the Tag sequence at 5 ' ends and positioned at 3 '
The specific primer sequence two parts for target gene polymorphism at end, and the base of 3 ' end first of the ASPE primer
With target gene complementary pairing to be detected.
3. primer sets as claimed in claim 2, wherein the specific primer sequence such as sequence for including in the ASPE primer
Shown in SEQ ID NO.61-90.
4. the primer sets as described in claim 1-3 are being prepared by liquid-phase chip technology while detecting 15 kinds of cardiovascular drugs
Purposes in the kit of related target gene polymorphism.
5. a kind of kit for detecting multiple cardiovascular drugs correlation target gene polymorphisms simultaneously using liquid-phase chip technology, institute
Stating kit includes primer sets described in claim 1 and 30 kinds of microballoons, wherein
The primer sets include 15 groups of primers, and every group of primer includes expanding for detecting a kind of a pair of specificity of target gene polymorphism
Increase primer and respectively for two ASPE primers of the wild type of the target gene and saltant type, the ASPE primer includes position
The Tag sequence held in 5 ' and specific primer sequence two parts for target gene positioned at 3 ' ends, and the ASPE primer
The base of 3 ' end first and target gene complementary pairing to be detected;With
Every kind of microballoon is respectively coupled a kind of addressable probes sequence with the Tag sequence complementary pairing of the ASPE primer respectively.
6. kit as claimed in claim 5, further include streptavidin-phycoerythrin hybridization buffer, PCR reaction solution,
Archaeal dna polymerase and negative control.
7. kit as claimed in claim 5, the specific primer sequence for being located at 3 ' ends for including in the ASPE primer is such as
Shown in sequence SEQ ID NO.61-90.
8. kit as claimed in claim 6, the negative control is distilled water+microballoon.
9. a kind of method using kit described in any one of claim 5 to 8, the described method comprises the following steps:
(1) multiplexed PCR amplification reacts: extracting the DNA in sample as template, is matched using the specificity amplification primer in kit
Multi-PRC reaction system processed carries out pcr amplification reaction;
(2) purifying of PCR product: with dNTP, primer and single-stranded production extra in excision enzyme and alkaline phosphatase enzymic digestion PCR product
Object;
(3) ASPE reacts: PCR product after purification is in ASPE reaction system under the action of archaeal dna polymerase, when 3 ' end of primer
The target sequence detection site mutual added time of first base and amplification carries out extension, if not complementary, extension will be whole
Only;
(4) hybridization reaction: taking the 25 diluted microballoons of μ l to 96 holes to hybridize plate, and the microballoon and deionized water of equivalent is added in control wells,
The 2.5 μ L of ASPE reaction product of step (3) is added, suction is beaten uniformly, and hybridization reaction condition is 96 DEG C of denaturation 90s, 37 DEG C of incubations
30min;
(5) result detects: the microballoon after hybridization being resuspended in 100 μ L and contains 6.5 μ g/mL streptavidin-phycoerythrin
1 × hybridization buffer in, after 37 DEG C of incubation 20min, detected on 200 instrument of Luminex.
10. method as claimed in claim 9, wherein
In step (1), multi-PRC reaction system are as follows: the total 50ng of 50 μ l, gDNA of total volume, 2 μ l, 10 × PCR reaction buffers
5 μ l, TaqTM0.25 μ each 2.5mM of l, dNTP of Hot Start enzyme, totally 4 μ l, primer concentration are 10 μM, each 0.5 μ l, deionized water
30.75μl;
The program of pcr amplification reaction are as follows: initial denaturation: 94 DEG C of 30s, circulation: 94 DEG C of 30s, anneal 57 DEG C of 30s, extends 72 DEG C of 30s,
Totally 5 times, 94 DEG C of 30s, anneal 55 DEG C of 30s, extends 72 DEG C of 30s, totally 30 times, extends eventually: 72 DEG C of 10min;
In step (3), ASPE reaction system are as follows: 2 μ l, 10 × PCR reaction buffer of PCR product, 5 μ l after purification, 400 μM
Each 100 μM of Biotin-dCTP 0.35 μ l, dATP, dGTP, dTTP, dCTP 0.3 the μ l, Taq of totally 1 μ l, 100mMTMHot
0.1 μ l, TAG-ASPE each 500nM of primer of Start enzyme, totally 1 μ l, 13.55 μ l of deionized water;
The program of ASPE extension are as follows: initial denaturation: 94 DEG C of 90s;Circulation: 94 DEG C of 30s, 57 DEG C of 30s, 74 DEG C of 1min, 40 times.
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