CN106434943A - Kit and detection method thereof for SNP detection of aspirin individualized medication related genes - Google Patents
Kit and detection method thereof for SNP detection of aspirin individualized medication related genes 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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- 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 kit and a detection method thereof for SNP detection of aspirin individualized medication related genes. The kit consists of two forward primers and a reverse primer for detecting rs10306114 site of a PTGS1 gene, two forward primers and a reverse primer for detecting rs1057910 site of a CYP2C9 gene, two forward primers and a reverse primer for detecting rs2836051 site of a CYP2D6 gene, and two forward primers and a reverse primer for detecting rs12248560 site of a CYP2C19 gene. With the application of the kit provided by the invention, high-throughput detection can be conducted on the four sites and a genotyping results can be intuitively distinguished through software, so that the purposes of conducting quantitative control on aspirin measurement and reducing side effects to the greatest extent are achieved.
Description
Technical field
The present invention relates to the test kit of multiple gene detection and its detection method, especially a kind of for aspirin individuality
Change test kit and its detection method of medication related gene SNP detection.
Background technology
Aspirin is the basic pharmaceutical of thromboembolism preventing after treatment acute coronary syndrome and PCI,
It is widely used in the prevention of cardiovascular and cerebrovascular disease firsts and seconds.Clinical discovery some patientss are although long-term low dose takes Ah Si
Woods still can not effectively suppress hematoblastic activity, i.e. aspirin resistance, and its incidence rate about 50%～60% and existing substantially is planted
Race's gene pleiomorphism.
PTGS1 coding COX-1, PTGS1 gene pleiomorphism can cause base replacement and the change of promoter connecting portion, show
The function of impact intron or exon is write, changes the conformation of COX-1 albumen, make sensitivity inequality of the aspirin to COX-1
One, so as to affect the antiplatelet aggregative activity of aspirin.Rs10306114 site carries the individuality ratio of AG/GG genotype and takes
Individual with AA genotype occurs aspirin resistance risk to increase, aspirin can not play expected biological action or
Embolism class diseases can not be prevented.
CYP2C9*3 (rs1057910) is enzyme miopragia type allele, when alone clopidogrel, mutated genes
Carrier CYP2C9*3 occurs adverse cardiac events incidence rate substantially to increase；When clopidogrel is combined aspirin, then dash forward
Modification gene carrier CYP2C9*3 occurs main adverse cardiac events incidence rate substantially to reduce.
CYP2C19*17 (rs12248560) is that enzyme function strengthens allele, and its mutation frequency in population of China is
1.2%～3%, CYP2C19*17 carrier (i.e. CT and TT genotype) is when Aspirin and clopidogrel, and reaction increases
Strong and hemorrhage risk increases.
Contain numerous mononucleotide polymorphism sites on CYP2D6 gene, make heredity assume polymorphism, its metabolic response is in
Existing variation, the polymorphism of the enzyme is related with drug metabolism and drug effect.The individual of Rs28360521 pleomorphism site carries CC phase
The risk of gastrointestinal hemorrhage after medication be increased to carrying individuality CT, TT.
The method of detection gene pleiomorphism conventional at present has DNA direct sequencing, restriction fragment length polymorphism to divide
Analysis method (PCR-PFLP), high-resolution solubility curve (HRM), gene chip, Luminex, fluorescence quantitative PCR method etc..Sequencing
Technology is the goldstandard of generally acknowledged detection gene mutation, but its equipment cost height, detection cycle length, detection flux are low, detect spirit
The reasons such as the low technical operation to experimenter of sensitivity has high demands, result judgement complex steps, it is more difficult to form business-like diagnosis
Product；Restriction fragment length polymorphism analysis method detection sensitivity equally not high, complex operation step, the result of detection is still needed to
The checking again of generation sequencing is carried out, and the cross-contamination of PCR primer is easily caused especially when sample size is many and is easily gone out
Excessively there are false negative or false positive results in the existing insufficient or enzyme action of enzyme action, therefore cannot also be applied to clinically.Chip is examined
Survey because the accuracy of its testing result and repeatability poor, the shortcomings of experimental period is long, be also unsuitable for developing clinical detection reagent
Box.The detection method of quantitative fluorescent PCR has that low cost, sensitivity is high, high specificity, the advantages of as a result reproducible, is
Extraordinary a kind of detection meanss of detection SNP, but the Taqman sonde method probe Order Cost of routine is too high, multiple sites
While it is difficult to ensure that all primed probe all reach extraordinary expanding effect under same amplification condition when detecting.Therefore, anxious
Need to find a kind of simple and easy to do methods of genotyping.
Content of the invention
The technical problem to be solved is to provide a kind of quick, easy, economic, practical for aspirin
The test kit of personalized medicine related gene SNP detection and its detection method.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is：One kind is used for aspirin individuation
The test kit of medicine phases correlation gene SNP detection, including 2X qPCR mixed reaction solution, detects PTGS1 gene rs10306114 site
Primer mix, the primer mix in detection CYP2C9 gene rs1057910 site, detection the drawing of CYP2D6 gene rs28360521 site
The thing mix and primer mix in detection CYP2C19 gene rs12248560 site；Described detection PTGS1 gene rs10306114 position
The primer mix of point contains and detects two specific forward primer of the SNP site and a reverse primer, described detection
The primer mix in CYP2C9 gene rs1057910 site contain detect two specific forward primer of the SNP site and one anti-
To primer, the primer mix in described detection CYP2D6 gene rs28360521 site contain detect two of the SNP site special
Property forward primer and a reverse primer, the primer mix in described detection CYP2C19 gene rs12248560 site contains detection
Two specific forward primer of the SNP site and a reverse primer；The detection PTGS1 gene rs10306114 site
Two specific forward primer and reverse primer sequences such as SEQ ID NO.3, SEQ ID NO.4, a SEQ ID NO.5 institute
Show, two specific forward primer in the detection CYP2C9 gene rs1057910 site and reverse primer sequences are such as
Shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, the detection CYP2D6 gene rs28360521 site
Two specific forward primer and reverse primer sequences such as SEQ ID NO.9, SEQ ID NO.10, a SEQ ID NO.11 institute
Show, two specific forward primer in the detection CYP2C19 gene rs12248560 site and reverse primer sequences
As shown in SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14.
Buffer containing PCR, MgCl in described 2X qPCR mixed reaction solution2, dNTPs, Taq archaeal dna polymerase, two
Respectively with the universal fluorescent probe of FAM and HEX labelling, the universal fluorescent probe sequence such as SEQ ID NO.1 institute of the FAM labelling
Show, the universal fluorescent probe sequence of the HEX labelling is as shown in SEQ ID NO.2.
The detection method of the above-mentioned test kit for aspirin personalized medicine related gene SNP detection, including with
Lower step：The genomic DNA of sample to be detected is extracted, is then divided into 4 reaction tubes (a, b, c, d) and is detected, first every
2X qPCR mixed reaction solution is separately added in individual reaction tube, then adds detection PTGS1 gene in the first reaction tube (a)
The primer mix in rs10306114 site；The primer in detection CYP2C9 gene rs1057910 site is added in the second reaction tube (b)
mix；The primer mix in detection CYP2D6 gene rs28360521 site is added in the 3rd reaction tube (c)；In the 4th reaction tube (d)
The primer mix in detection CYP2C19 gene rs12248560 site is added, is finally added in 4 reaction tubes (a, b, c, d) to be checked
The genomic DNA of test sample sheet, carries out the fluorescent quantitative PCR of sample of nucleic acid, carries out the scanning of fluorescence signal at 37 DEG C, uses
Endpiont Genotyping module in LightCycler 480Software release software is believed according to two kinds of fluorescence
Number intensity and ratio automatic discrimination is carried out to genotype.
The PCR amplification program is as follows：
The condition of denaturation is：Temperature is 94 DEG C, and the time is 15 seconds；
PCR amplification is made up of first stage and second stage；
First stage is made up of 10 amplification cycles, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Initial temperature is 61 DEG C, 0.5 DEG C of each wheel amplification cycles lapse of temperature, until amplification cycles knot
Bundle, the time is 60 seconds；
Second stage is made up of 29 wheel circulations, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Temperature is 55 DEG C, and the time is 60 seconds；
Signal scanning condition is：Temperature is 37 DEG C, and the time is 60 seconds.
The invention has the beneficial effects as follows：The test kit of the present invention can be quick, easy, economic, practical to gene type, and
And by comparing with generation sequencing technologies testing result, as a result goodness of fit up to more than 99%, is expected to divide in clinical rapid gene
It is used widely in type.Solve the problems, such as that other detection techniques existing are expensive, inefficiency and flux be not high.
Description of the drawings
Fig. 1 is two specificity forward directions using the detection PTGS1 gene rs10306114 site in test kit of the present invention
The testing result figure of primer and reverse primer to four samples.
Fig. 2 is generation sequencing (Sanger sequencing) figure of tetra- samples to be tested of 1-1,1-2,1-3,1-4 shown in Fig. 1.
Fig. 3 is two specificity forward directions using the detection CYP2C9 gene rs1057910 site in test kit of the present invention
The testing result figure of primer and reverse primer to four samples.
Fig. 4 is generation sequencing (Sanger sequencing) figure of tetra- samples to be tested of 2-1,2-2,2-3,2-4 shown in Fig. 3.
Fig. 5 is two specificity forward directions using the detection CYP2D6 gene rs28360521 site in test kit of the present invention
The testing result figure of primer and reverse primer to six samples.
Fig. 6 is the generation sequencing (Sanger of six samples to be tested of 3-1,3-2,3-3,3-4,3-5,3-6 shown in Fig. 5
Sequencing) figure.
Fig. 7 be using the detection CYP2C19 gene rs12248560 site in test kit of the present invention two specificitys just
To the testing result figure of primer and reverse primer to four samples.
Fig. 8 is generation sequencing (Sanger sequencing) figure of tetra- samples to be tested of 4-1,4-2,4-3,4-4 shown in Fig. 7.
Specific embodiment
With reference to the accompanying drawings and detailed description the present invention is described in further detail：
The present invention is used for the test kit of aspirin personalized medicine related gene SNP detection, anti-including 2X qPCR mixing
Answer liquid, two specific forward primer in detection rs10306114 site and a reverse primer, and detection rs1057910 position
Two specific forward primer of point and a reverse primer, and two specific forward primer in detection rs28360521 site
An and reverse primer, and two specific forward primer and a reverse primer in detection rs12248560 site.
Two specific forward primer in the detection rs10306114 site and the following institute of reverse primer sequences
Show：
A allele forward primer：GAAGGTGACCAAGTTCATGCTGAGCACCTACTACATGCTGGA SEQ ID NO.3
G allele forward primer：GAAGGTCGGAGTCAACGGATTGAGCACCTACTACATGCTGGG SEQ ID NO.4
Reverse primer：TGCACACAAATCTCCTGGTGCAGT SEQ ID NO.5
Two specific forward primer in the detection rs1057910 site and the following institute of reverse primer sequences
Show：
A allele forward primer：GAAGGTGACCAAGTTCATGCTGTGCACGAGGTCCAGAGATACA SEQ ID NO.6
C allele forward primer：GAAGGTCGGAGTCAACGGATTGCACGAGGTCCAGAGATACC SEQ ID NO.7
Reverse primer：AGGCTGGTGGGGAGAAGGTCAA SEQ ID NO.8
Two specific forward primer in the detection rs28360521 site and the following institute of reverse primer sequences
Show：
C allele forward primer：GAAGGTGACCAAGTTCATGCTGGATAGGTTGAGGCTGATCCTC SEQ ID NO.9
T allele forward primer：GAAGGTCGGAGTCAACGGATTATGGATAGGTTGAGGCTGATCCTT SEQ ID NO.10
Reverse primer：GGTTAGGGGAGGCAACCTGCT SEQ ID NO.11
Two specific forward primer in the detection rs12248560 site and the following institute of reverse primer sequences
Show：
C allele forward primer：GAAGGTGACCAAGTTCATGCTAATTTGTGTCTTCTGTTCTCAAAGC SEQ ID NO.12
T allele forward primer：GAAGGTCGGAGTCAACGGATTAATTTGTGTCTTCTGTTCTCAAAGT SEQ ID NO.13
Reverse primer：CGCATTATCTCTTACATCAGAGAT SEQ ID NO.14
Wherein, the forward primer is combined into by two parts sequence：5 ' ends of wild type forward primer are general sequence
Row GAAGGTGACCAAGTTCATGCT (SEQ ID NO.1), 3 ' ends are for recognizing the distinguished sequence of corresponding site；Saltant type forward direction
5 ' ends of primer are universal sequence GAAGGTCGGAGTCAACGGATT (SEQ ID NO.2), and 3 ' ends are for recognizing the spy of corresponding site
Different sequence.
Buffer containing PCR, MgCl in described 2X qPCR mixed reaction solution2, dNTPs, Taq archaeal dna polymerase, two
Respectively with the universal fluorescent probe of FAM and HEX labelling, the universal fluorescent probe sequence such as SEQ ID NO.1 institute of the FAM labelling
Show, the universal fluorescent probe sequence of the HEX labelling is as shown in SEQ ID NO.2.
The detection method of test kit of the present invention, amplification program is as follows：
The condition of denaturation is：Temperature is 94 DEG C, and the time is 15 seconds；
PCR amplification is made up of first stage and second stage；
First stage is made up of 10 amplification cycles, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Initial temperature is 61 DEG C, 0.5 DEG C of each wheel amplification cycles lapse of temperature, until amplification cycles knot
Bundle, the time is 60 seconds；
Second stage is made up of 29 wheel circulations, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Temperature is 55 DEG C, and the time is 60 seconds；
Signal scanning condition is：Temperature is 37 DEG C, and the time is 60 seconds.
The primer (primer) of the present invention is the important component part in PCR (polymerase chain reaction) technology, and it is one
Segment single stranded DNA, as the starting point of DNA replication dna, in nucleic acid synthetic reaction, is extended as each polynucleotide chain
Starting point and the polynucleotide chain that works, on 3 '-OH of primer, nucleotide is synthesized with diester linkage form, is therefore drawn
3 '-OH of thing must be free.FAM fluorescence is used in 5 ' universal sequences of wild type forward primer and 2X PCR mixed reaction solution
The universal sequence of dye marker is consistent；5 ' universal sequences of saltant type forward primer are glimmering with HEX with 2X PCR mixed reaction solution
The universal sequence of photoinitiator dye labelling is consistent；The universal sequence for carrying fluorochrome label is used for completing for fluorescent quantitative PCR
Fluorescence signal is provided afterwards.
The operation principle of the present invention is：Two specific forward primer and one has been separately designed reversely for each site
Primer.The base pair complementarity that two specific forward primer 3 ' hold last nucleotide to be detected with mutational site respectively,
When PCR (polymerase chain reaction) is expanded, joint reverse primer carries out specific amplification to DNA profiling to be measured.10 before PCR
The amplification of circulation employs Touchdown PCR, i.e. touchdown PCR, is a kind of PCR method after optimizing and improveing, refers to each
(or n) circulation reduces by 0.5 DEG C of (or n DEG C) annealing temperature, and until reaching a relatively low annealing temperature, (Touchdown anneals
Temperature), ensure that with this primer of different Tm values all can be combined with corresponding DNA profiling to be measured and carry out specific amplification.
In the PCR amplification procedure of subsequently carry out 29 circulations, in 2X PCR mixed reaction solution respectively with the two of FAM and HEX labelling
The PCR primer for producing during 10 wheel PCR amplification before the meeting of bar general probe is template, and cooperation reverse primer is produced and carries difference
The PCR primer of fluorochrome label.The last scanning for carrying out fluorescence signal at 37 DEG C, by software according to the strong of two kinds of fluorescence signals
Degree and ratio carry out automatic discrimination to genotype.
The present invention is had the following advantages that and effect compared with prior art：
1st, the present invention is using the primer of particular design, and fluorescence signal carries different fluorescence dyes by two kinds in PCR mixed liquor
The universal sequence of material labelling is provided, and without the need for individually designed specific probe, greatly reduces R＆D cycle and testing cost.
2nd, the present invention adopts special PCR program, greatly reduces as Tm value is too low and primer that is causing is existed with template
The combination in mistake site, so as to improve PCR efficiency and sensitivity, is that the adjustment of clinical aspirin dosage is provided reliably
Foundation.
3rd, the SNP site genotyping software that the present invention is adopted, can intuitively each site to be measured of automatic discrimination base
Because of type, without the need for by the troublesome calculation of Ct value, greatly reducing human error during result interpretation, and shortening result interpretation
Time, improve detection efficiency.
4th, the test kit of the present invention, can according to sample to be tested quantity number, flexibly from 8 connecting legs, 96 orifice plates or
384 orifice plate of person is detected, detection flux once is respectively 1 person-portion, 28 person-portions and 124 person-portions.
5th, the test kit of the present invention, it is possible to achieve high accuracy, efficient to rs10306114, rs1057910,
Rs28360521 and tetra- SNP site detections of rs12248560, so as to reach the quantified controlling to aspirin dosage, very
Also function to necessarily to the prevention to thrombotic disease, the selection of anticoagulant, the research and development of acenocoumarol medicine, thrombotic disease prognosis
Effect.
Embodiment 1
A kind of test kit for instructing aspirin personalized medicine related gene SNP detection includes：Including 2X qPCR
Mixed reaction solution, the primer mix in detection PTGS1 gene rs10306114 site, detection CYP2C9 gene rs1057910 site
Primer mix, the primer mix in detection CYP2D6 gene rs28360521 site and detection CYP2C19 gene rs12248560 site
Primer mix.
Two specific forward primer in the detection PTGS1 gene rs10306114 site and reverse primer sequences
As follows：
A allele forward primer：GAAGGTGACCAAGTTCATGCTGAGCACCTACTACATGCTGGA SEQ ID NO.3
G allele forward primer：GAAGGTCGGAGTCAACGGATTGAGCACCTACTACATGCTGGG SEQ ID NO.4
Reverse primer：TGCACACAAATCTCCTGGTGCAGT SEQ ID NO.5
Two specific forward primer in the detection CYP2C9 gene rs1057910 site and reverse primer sequences
As follows：
A allele forward primer：GAAGGTGACCAAGTTCATGCTGTGCACGAGGTCCAGAGATACA SEQ ID NO.6
C allele forward primer：GAAGGTCGGAGTCAACGGATTGCACGAGGTCCAGAGATACC SEQ ID NO.7
Reverse primer：AGGCTGGTGGGGAGAAGGTCAA SEQ ID NO.8
Two specific forward primer in the detection CYP2D6 gene rs28360521 site and a reverse primer sequence
Row are as follows：
C allele forward primer：GAAGGTGACCAAGTTCATGCTGGATAGGTTGAGGCTGATCCTC SEQ ID NO.9
T allele forward primer：GAAGGTCGGAGTCAACGGATTATGGATAGGTTGAGGCTGATCCTT SEQ ID NO.10
Reverse primer：GGTTAGGGGAGGCAACCTGCT SEQ ID NO.11
Two specific forward primer in the detection CYP2C19 gene rs12248560 site and a reverse primer sequence
Row are as follows：
C allele forward primer：GAAGGTGACCAAGTTCATGCTAATTTGTGTCTTCTGTTCTCAAAGC SEQ ID NO.12
T allele forward primer：GAAGGTCGGAGTCAACGGATTAATTTGTGTCTTCTGTTCTCAAAGT SEQ ID NO.13
Reverse primer：CGCATTATCTCTTACATCAGAGAT SEQ ID NO.14
Wherein, the forward primer is combined into by two parts sequence：5 ' ends of wild type forward primer are general sequence
Row GAAGGTGACCAAGTTCATGCT (SEQ ID NO.1), 3 ' ends are for recognizing the distinguished sequence of corresponding site；Saltant type forward direction
5 ' ends of primer are universal sequence GAAGGTCGGAGTCAACGGATT (SEQ ID NO.2), and 3 ' ends are for recognizing the spy of corresponding site
Different sequence.
Embodiment 2
The PCR amplification side that a kind of test kit for instructing aspirin personalized medicine related gene SNP detection is adopted
Method is comprised the following steps：
The first step：Sample to be tested extracting genome DNA
(1) from patient, blood sample is extracted.
(2) DNA is obtained from blood, using the TGuide poba gene group of Tiangeng biochemistry (Beijing) company limited production
DNA extraction kit (OSR-M102) is extracted on its supporting TGuide M16 nucleic acid automatic extracting instrument.
Extraction process is as follows：
A. the mammalian whole blood sample of 200 μ l/400 μ l is added toward in sample cell, and adds 10 μ l/20 μ l E.C. 3.4.21.64s
Mix.
B. sample cell is placed in 4 position of hole of T-shaped frame.Operation 102 program of numbering (Whole Blood Genomic DNA extraction procedure),
Select corresponding sample size volume and final elution volume.
Second step：DNA is expanded
Using 10ng/10ul reaction system, concrete operations are：
1st, by a series of above-mentioned primers respectively with poba gene group DNA, 2X PCR mixed reaction solution, deionized water according to one
Certainty ratio mixing constitutes single reaction system, specifically see the table below：
| Reagent name | Volume (ul) |
| Genomic DNA (10ng) | 2ul |
| Primer mix | 1ul |
| 2X PCR mixed reaction solution | 5ul |
| Deionized water | 2ul |
2nd, PCR cycle is carried out to above-mentioned reaction system, and PCR cycle condition see the table below：
3rd step：Observed result
Table 1 be using the detection PTGS1 gene rs10306114 site in test kit two specific forward primer and
The genotype results of one reverse primer to four samples.
Table 1
As shown in figure 1, in the fluorescent quantitative PCR system in PTGS1 gene rs10306114 site, detecting A equipotential
5 ' the terminal sequence of specific forward primer of gene is consistent with the fluorescently-labeled primer sequence of FAM in 2*qPCR reaction mixture, inspection
Survey 5 ' terminal sequence of specific forward primer and the fluorescently-labeled primer sequence of HEX in 2*qPCR reaction mixture of G allele
Unanimously.In Fig. 1, X-axis represents the intensity of FAM fluorescence, and Y-axis represents the intensity of HEX fluorescence, tetra- samples of 1-1,1-2,1-3,1-4
Near X-axis, this testing result illustrates that only FAM fluorescence is detected, therefore genotype is AA homozygosis.
As shown in Fig. 2 tetra- samples of 1-1,1-2,1-3,1-4 only detect A allele in rs10306114 site,
Illustrate that genotype of four samples in rs10306114 site is AA homozygosis.Generation sequencing result and test kit testing result one
Cause.
Table 2 be using the detection CYP2C9 gene rs1057910 site in test kit two specific forward primer and
The genotype results of one reverse primer to four samples.
Table 2
As shown in figure 3, in the fluorescent quantitative PCR system in rs1057910 site, detecting the special of A allele
Property 5 ' terminal sequence of forward primer consistent with the fluorescently-labeled primer sequence of FAM in 2*qPCR reaction mixture, detect C allele
5 ' terminal sequence of specific forward primer is consistent with the fluorescently-labeled primer sequence of HEX in 2*qPCR reaction mixture.X-axis in Fig. 3
The intensity of FAM fluorescence is represented, Y-axis represents the intensity of HEX fluorescence, and 2-1 and two pattern detection results of 2-2 are near diagonal
Position, illustrate that FAM and two kinds of fluorescence of HEX are all detected, therefore genotype be AC heterozygosis；2-3 and two pattern detection knots of 2-4
Near X-axis, fruit illustrates that only FAM fluorescence is detected, therefore genotype is AA homozygosis.
As shown in figure 4,2-1 and two samples of 2-2 are detected simultaneously by A and two kinds of equipotential bases of C in rs1057910 site
Cause, so the genotype of 2-1 and two samples of 2-2 in rs1057910 site is AC heterozygosis；2-3 and two samples of 2-4 exist
Rs1057910 site only detects A allele, so the genotype of 2-3 and two samples of 2-4 in rs1057910 site
For AA homozygosis.Generation sequencing result is consistent with test kit testing result.
Table 3 be using the detection CYP2D6 gene rs28360521 site in test kit two specific forward primer and
The genotype results of one reverse primer to six samples.
Table 3
As shown in figure 5, in the fluorescent quantitative PCR system in CYP2D6 gene rs28360521 site, detecting C equipotential
5 ' the terminal sequence of specific forward primer of gene is consistent with the fluorescently-labeled primer sequence of FAM in 2*qPCR reaction mixture, inspection
Survey 5 ' terminal sequence of T allele-specific forward primer and the fluorescently-labeled primer sequence one of HEX in 2*qPCR reaction mixture
Cause.In Fig. 5, X-axis represents the intensity of FAM fluorescence, and Y-axis represents the intensity of HEX fluorescence, 3-1 and two pattern detection knots of 3-2
Near the position of X-axis, fruit illustrates that only FAM fluorescence is detected, therefore genotype is CC homozygosis；3-3 and two pattern detection of 3-4
As a result near cornerwise position, illustrate that FAM and two kinds of fluorescence of HEX are all detected, therefore genotype is CT heterozygosis；3-5 and 3-6
Near the position of Y-axis, two pattern detection results illustrate that only HEX fluorescence is detected, therefore genotype is TT homozygosis.
As shown in fig. 6,3-1 and two samples of 3-2 only detect C allele in rs28360521 site, so 3-1
It is CC homozygosis with genotype of two samples of 3-2 in rs28360521 site；3-3 and two samples of 3-4 are in rs28360521 position
C and two allele of T are detected simultaneously by point, so the genotype of 3-3 and two samples of 3-4 in rs28360521 site is
CT heterozygosis；3-5 and two samples of 3-6 only detect T allele in rs28360521 site, so 3-5 and two samples of 3-6
This genotype in rs28360521 site is TT homozygosis.Generation sequencing result is consistent with test kit testing result.
Table 4 is two specific forward primer using the detection CYP2C19 gene rs12248560 site in test kit
With genotype results of the reverse primer to four samples.
Table 4
As shown in fig. 7, in the fluorescent quantitative PCR system in CYP2C19 gene rs12248560 site, detection C etc.
5 ' the terminal sequence of specific forward primer of position gene is consistent with the fluorescently-labeled primer sequence of FAM in 2*qPCR reaction mixture,
5 ' terminal sequence of detection T allele-specific forward primer and the fluorescently-labeled primer sequence of HEX in 2*qPCR reaction mixture
Unanimously.In Fig. 7, X-axis represents the intensity of FAM fluorescence, and Y-axis represents the intensity of HEX fluorescence, tri- samples of 4-1,4-2 and 4-3
Near the position of X-axis, testing result illustrates that only FAM fluorescence is detected, therefore genotype is CC homozygosis；4-4 pattern detection is tied
Near cornerwise position, fruit illustrates that FAM and two kinds of fluorescence of HEX are all detected, therefore genotype is CT heterozygosis.
As shown in figure 8, tri- samples of 4-1,4-2 and 4-3 only detect C allele, institute in rs12248560 site
With tri- samples of 4-1,4-2 and 4-3 rs12248560 site genotype as CC homozygosis；Two samples of 4-4 exist
Rs12248560 site is detected simultaneously by C and two allele of T, so gene of the 4-4 sample in rs12248560 site
Type is CT heterozygosis.Generation sequencing result is consistent with test kit testing result.
In sum, present disclosure is not limited in the above embodiments, and the knowledgeable people in same area can
Can propose other embodiments within the technological guidance's thought of the present invention easily, but this embodiment is included in this
Within the scope of bright.
Claims (4)
1. a kind of for aspirin personalized medicine related gene SNP detection test kit, it is characterised in that including 2X
QPCR mixed reaction solution, the primer mix in detection PTGS1 gene rs10306114 site, detection CYP2C9 gene rs1057910 position
The primer mix of point, the primer mix in detection CYP2D6 gene rs28360521 site and detection CYP2C19 gene rs12248560
The primer mix in site；4 primer mix all reversely draw containing two specific forward primer and for detecting its SNP site
Thing, two specific forward primer in detection PTGS1 gene rs10306114 site and a reverse primer sequences such as SEQ ID
Shown in NO.3, SEQ ID NO.4, SEQ ID NO.5, just two specificitys in CYP2C9 gene rs1057910 site are being detected
To primer and reverse primer sequences as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, CYP2D6 is detected
Two specific forward primer in gene rs28360521 site and reverse primer sequences such as SEQ ID NO.9, a SEQ
Shown in ID NO.10, SEQ ID NO.11, two specific forward primer in CYP2C19 gene rs12248560 site are detected
And reverse primer sequences are as shown in SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14.
2. the test kit for the detection of aspirin personalized medicine related SNP according to claim 1, its feature exists
In Buffer containing PCR, MgCl in described 2X qPCR mixed reaction solution2, dNTPs, Taq archaeal dna polymerase, two use respectively
The universal fluorescent probe of FAM and HEX labelling, the universal fluorescent probe sequence of FAM labelling as shown in SEQ ID NO.1, HEX labelling
Universal fluorescent probe sequence as shown in SEQ ID NO.2.
3. the detection side of the test kit of aspirin personalized medicine related gene SNP detection is used for as claimed in claim 1
Method, it is characterised in that comprise the following steps：Extract the genomic DNA of sample to be detected, be then divided into 4 reaction tubes (a, b, c,
D) detected, be separately added into 2X qPCR mixed reaction solution first in each reaction tube, then added in the first reaction tube (a)
Enter to detect the primer mix in PTGS1 gene rs10306114 site；Detection CYP2C9 gene is added in the second reaction tube (b)
The primer mix in rs1057910 site；The primer in detection CYP2D6 gene rs28360521 site is added in the 3rd reaction tube (c)
mix；The primer mix in detection CYP2C19 gene rs12248560 site is added in the 4th reaction tube (d), finally in 4 reaction tubes
The genomic DNA of sample to be detected is added in (a, b, c, d), is carried out the fluorescent quantitative PCR of sample of nucleic acid, is carried out at 37 DEG C
The scanning of fluorescence signal, using the Endpiont Genotyping in LightCycler 480Software release software
Module carries out automatic discrimination according to the intensity of two kinds of fluorescence signals and ratio to genotype.
4. the detection of the test kit for aspirin personalized medicine related gene SNP detection according to claim 3
Method, it is characterised in that the PCR amplification program is as follows：
The condition of denaturation is：Temperature is 94 DEG C, and the time is 15 seconds；
PCR amplification is made up of first stage and second stage；
First stage is made up of 10 amplification cycles, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Initial temperature is 61 DEG C, 0.5 DEG C of each wheel amplification cycles lapse of temperature, until amplification cycles terminate, when
Between be 60 seconds；
Second stage is made up of 29 wheel circulations, and its condition is：
Degeneration：Temperature is 94 DEG C, and the time is 20 seconds；
Annealing+extend：Temperature is 55 DEG C, and the time is 60 seconds；
Signal scanning condition is：Temperature is 37 DEG C, and the time is 60 seconds.
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