CN109055530A - A kind of mankind's aspirin resistance genetic polymorphism detection kit and its preparation method and application - Google Patents
A kind of mankind's aspirin resistance genetic polymorphism detection kit and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of mankind's aspirin resistance genetic polymorphism detection kit and its preparation method and application.The kit premixes reaction solution, positive reference substance and negative controls by the PCR for being respectively used for amplifying 3 gene pleiomorphisms of GPIIIa, GP1BA, PEAR1 relevant to mankind's aspirin resistance and forms, the PCR premix reaction solution includes specific primer sequence group, probe groups and the PCR reaction solution for expanding each site, and the ARMs primer of specificity of the specific primer group by common outer primer and with fluorescence labels forms.Kit of the present invention has many advantages, such as that high sensitivity, specificity is high, easy to operate, result is reliable for detecting mankind's aspirin resistance gene pleiomorphism, and detection can be completed in 1 hour, and result interpretation is simply objective.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of mankind's aspirin resistance genetic polymorphism detection reagent
Box and its preparation method and application.
Background technique
Aspirin is the basic pharmaceutical of anti-bolt after treating acute coronary syndrome and percutaneous coronary intervention,
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 cannot effectively inhibit the activity of blood platelet, i.e. aspirin resistance, incidence about 50%~60% and there are obvious kind
Race's otherness.Studies have shown that GPIIIa, GP1BA and PEAR1 gene polynorphisms play important work in aspirin resistance
With；
Platelet membrane glycoprotein IIIa gene is encoded by ITGB3 gene, the mutation of the 1565th amino acids of exon 2
(rs5918), i.e. T1565C (Leu59Pro), determines two kinds of allele of P1A1 and P1A2 respectively, GP IIIa P1A2 be Ah
Department woods resists oligogene, and after carrying saltant type P1A2 allele patient's row stenting, subacute stent thrombosis event occurs
Rate is 5 times of P1A1 homozygous wildtype patient, and the aspirin for generally requiring higher doses can be only achieved anticoagulant effect.
GP1BA alias GP1B (platelet glycoprotein 1B gene), the gene is relevant to aspirin, and there are two SNP
Point, wherein rs6065 (145Thr-Met) is related to the virulence of aspirin, CC genotype, and aspirin resistance risk increases；
CT and TT type, aspirin resistance risk reduce.
Platelet-endothelial aggregation receptor 1 gene (PEAR1) is the important gene of the regulation platelet function of discovered in recent years.
PEAR1 gene have multiple and different SNP sites, can influence platelet function mainly have the site rs2768759 and
The site rs12041331, wherein rs12041331 loci polymorphism be most study with the close phase of aspirin for treatment reactivity
The polymorphic site of pass.Rs12041331 site mutation is G > A, and the mutation rate in asian population is significantly higher than other crowds,
Wherein compared with GG homozygote, aspirin or the dual anti-treatment of aspirin combination clopidogrel, A equipotential base is either applied alone
Because the cardiovascular event risk and the death rate of carrier are significantly raised.
To sum up, aspirin resistance gene pleiomorphism is detected before using aspirin, for Patient genotype
Outcome prediction is carried out to patient, and high-risk patient is intervened in advance, to reduce patient medication risk, there is great significance.
Clinically following four mode is mainly taken to detect gene polynorphisms at present:
1.PCR- PCR sequencing PCR
2. high-resolution solubility curve method
3. chip hybridization methods
4.Taqman-qPCR method
PCR- PCR sequencing PCR sensitivity is low, and time-consuming for experiment, is not suitable for clinical expansion；Chip hybridization methods, detection sensitivity are low
And poor specificity is easy to appear false positive results；High-resolution solubility curve method is special to equipment requirement, and unsuitable clinic pushes away
Extensively, and detection sensitivity is not high.Taqman-qPCR method, detection sensitivity is high, but often due to site sequence reason, causes to examine
It is not high to survey specificity, and it carries out parting by Genotyping method, is distributed with and has to sample size and polymorphism
It asks, is not suitable for the too low site of the detection frequency of mutation.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of mankind's aspirin resistance gene pleiomorphism is examined
Test agent box and its preparation method and application.This kit has high sensitivity, high specific, low cost high throughput, operation letter
Just, the features such as experimental period is short can carry out aspirin resistance gene pleiomorphism in human genome DNA with fast accurate
Detection.
For achieving the above object, the technical solution adopted by the present invention are as follows:
A kind of mankind's aspirin resistance genetic polymorphism detection primer sets, the primer sets are by common outer primer and band
The ARMs primer composition of the specificity of fluorescence labels, particular sequence are as follows:
PEAR1-F1:5 '-TTGTGGACTTCTCTTCCTTC-3 ' SEQ ID NO.1
PEAR1-F2:5 '-VIC-ATCCCTTGTCATCGTCAGATATACG-3 ' SEQ ID NO.2
PEAR1-R1:5 '-GTCACCCTGCCAGAAAT-3 ' SEQ ID NO.3
PEAR1-R2:5 '-FAM-AAGATACATTGATGAAGAAGTTCAAT-3 ' SEQ ID NO.4
GP1BA-F1:5 '-TCCAAGAGCTCTACCTGAAAGGC-3 ' SEQ ID NO.5
GP1BA-F2:5 '-VIC-ATCCCTTGTCATCGTTAGTGCTCCTGAC-3 ' SEQ ID NO.6
GP1BA-R1:5 '-CTCAGTCAAGTTGTTGTTA-3 ' SEQ ID NO.7
GP1BA-R2:5’-FAM-AAGATACATTGATGATTGCGTGTGTGCA-3’SEQ ID NO.8
GPIIIa-F1:5 '-GACGCTGGGCATCATCATGG-3 ' SEQ ID NO.9
GPIIIa-F2:5 '-VIC-ATCCCTTGTCATCGTGGCCTTACAGT-3 ' SEQ ID NO.10
GPIIIa-R1:5 '-TGCAGCCGGCCCAGGGCTCCAG-3 ' SEQ ID NO.11
GPIIIa-R2:5 '-FAM-AAGATACATTGATGACAGAGTAGTCG-3 ' SEQ ID NO.12
Internal control F:5 '-CGGGACCTGACTGACTACCT-3 ' SEQ ID NO.13
Internal control R:5 '-GGCCATCTCTTGCTCGAAGT-3 ' SEQ ID NO.14.
A kind of mankind's aspirin resistance genetic polymorphism detection probe groups, the probe groups are by specific taqman-MGB
Probe and the lock nucleic acid probe composition for carrying quenching group, particular sequence are as follows:
Internal control-P:5 '-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3 ' SEQ ID NO.15
Wild type quenching group lock nucleic acid: 5 '-BHQ-ACGATGACAAGGGAT-3 ' SEQ ID NO.16
Saltant type quenching group lock nucleic acid: 5 '-BHQ-TCATCAATGTATCTT-3 ' SEQ ID NO.17.
A kind of mankind's aspirin resistance genetic polymorphism detection kit, by for expanding PEAR1, GP1BA, GPIIIa
PCR premix reaction solution, positive reference substance and the negative controls composition of 3 gene locis；
The concrete component of the PCR premix reaction solution is as follows:
(1) the PCR premix reaction solution for expanding PEAR1 gene loci includes: described for expanding PEAR1 gene
The primer in the site rs12041331, primer sequence is as shown in SEQ ID NO.1~SEQ ID NO.4；The drawing for internal control
Object sequence, sequence is as shown in SEQ ID NO.13~SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15~
Shown in SEQ ID NO.17；And PCR reaction solution composition；
(2) the PCR premix reaction solution for expanding GP1BA gene loci includes: described for expanding GP1BA gene
The primer in the site rs6065, primer sequence is as shown in SEQ ID NO.5~SEQ ID NO.8；The primer sequence for internal control
Column, sequence is as shown in SEQ ID NO.13~SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15~SEQ ID
Shown in NO.17；And PCR reaction solution composition；
(3) the PCR premix reaction solution for expanding GPIIIa gene loci includes: described for expanding GPIIIa gene
The primer in the site rs5918, primer sequence is as shown in SEQ ID NO.9~SEQ ID NO.12；The primer sequence for internal control
Column, sequence is as shown in SEQ ID NO.13~SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15~SEQ ID
Shown in NO.17；And PCR reaction solution composition；
The positive control includes: PEAR1 gene rs12041331 site mutation type plasmid, PEAR1 gene
The site rs12041331 wild plasmid, the site GP1BA gene rs6065 wild plasmid, the site gene rs6065 GP1BA are prominent
Modification plasmid, GPIIIa gene rs5918 site mutation type plasmid, GPIIIa gene rs5918 site wild plasmid and internal reference
Plasmid DNA；The negative control is the recombinant plasmid without reference gene and target gene site.
In above scheme, in PCR premix reaction solution, in probe groups the concentration of taqman-MGB probe be 50~
100nM, the concentration for carrying the lock nucleic acid probe of quenching group is 100~400nM；For expand each site without fluorescent base
Each primer concentration of group is 100~400nM, and each primer concentration with fluorophor is 50~100nM；For drawing for internal control
The concentration of object is 100~400nM.
In above scheme, the PCR reaction solution includes Premix qPCRmix and TE buffer.
In above scheme, the negative controls are made of PUC-19 plasmid empty carrier and TE buffer.
Above-mentioned mankind's aspirin resistance genetic polymorphism detection kit is supported in preparation for detecting mankind's aspirin
Application in anti-gene pleiomorphism product.
Mankind's aspirin resistance gene polymorphic is detected using mankind's aspirin resistance genetic polymorphism detection kit
The process of property, specifically comprises the following steps:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) PCR premix reaction solution and sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；The positive is set simultaneously
Control group and negative control group, then by reaction tube be placed in fluorescent PCR instrument carry out pcr amplification reaction, and acquire FAM, VIC and
ROX fluorescence signal；
(3) after pcr amplification reaction, carrying out result judgement according to fluorescence signal initial line situation collected (be see the table below
1)。
In above scheme, it is 0.1~100ng/ μ L that the sample DNA to be detected, which is diluted to concentration, in step (1), then into
Row pcr amplification reaction.
In above scheme, the condition of pcr amplification reaction described in step (2) are as follows: 95 DEG C of 1 minute initial denaturations；15 circulations:
95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, collect fluorescence.
In above scheme, the principle of result judgement described in step (3) is (as shown in table 6 below): 1. working as positive controls
There are FAM, VIC, ROX fluorescence signal initial line, for negative control group without FAM, VIC, ROX fluorescence initial line, sample to be detected has ROX
When fluorescence signal initial line, determines Success in Experiment, subsequent parting judgement can be carried out；2. according to FAM, VIC fluorescence of detection sample
Signal judges the parting of PEAR1: VIC fluorescence signal initial line PEAR1 gene rs12041331 site parting is homozygous wildtype；
FAM fluorescence signal initial line, PEAR1 gene rs12041331 site parting are homozygous mutant；VIC, FAM fluorescence signal rise
Line, PEAR1 gene rs12041331 site parting are heterozygous mutant；3. being sentenced according to FAM, VIC fluorescence signal of detection sample
The parting of disconnected GP1BA: VIC fluorescence signal initial line GP1BA gene rs6065 site parting is homozygous wildtype；FAM fluorescence signal
Initial line, GP1BA gene rs6065 site parting are homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, GP1BA gene
The site rs6065 parting is heterozygous mutant；4. judging the parting of GPIIIa according to FAM, VIC fluorescence signal of detection sample:
VIC fluorescence signal initial line GPIIIa gene rs5918 site parting is homozygous wildtype；FAM fluorescence signal initial line, GPIIIa base
Because the site rs5918 parting is homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, the site GPIIIa gene rs5918 parting are
Heterozygous mutant.
Testing principle such as Fig. 1 and Fig. 2 institute of mankind's aspirin resistance genetic polymorphism detection kit of the present invention
Showing, the ARMs primer with sequence label for carrying FAM fluorescence specific recognition and can expand the template of mutated-genotype, and
With the consumption of primer, more FAM fluorescence are released, at the end of circulation, each channel fluorescence is acquired, it is bent to generate fluorescence
Line；The ARMs primer with sequence label for carrying VIC fluorescence specific recognition and can expand the template of wild-type genotype, and
With the consumption of primer, more VIC fluorescence are released, at the end of circulation, each channel fluorescence is acquired, it is bent to generate fluorescence
Line.Internal control probe uses MGB probe, and specific recognition internal control corresponding sequence, by amplification, more and more ROX fluorescence are released
It releases.So final display only has internal control (ROX) and wild (VIC) glimmering when the genomic templates of detection are homozygous wildtypes
Light curve can normal initial line and curve it is S-type, initial line or initial line be not very low and not S-type for mutation (FAM) fluorescence curve.When
The genomic templates of detection are homozygous mutants, and final display only has internal control (ROX) and mutation (FAM) fluorescence curve can be normal
Initial line and curve is S-type, initial line or initial line be not very low and not S-type for wild (VIC) fluorescence curve.When the genome mould of detection
Plate is heterozygote, and finally showing internal control (ROX), mutation (FAM) and wild (VIC) fluorescence curve can normal initial line and song
Line is S-type.
Beneficial effects of the present invention are as follows: mankind's aspirin resistance genetic polymorphism detection kit of the present invention can
With simple and quick detection mankind's aspirin resistance typing gene polymorphisms, using ARMs primer binding specificity fluorescence labels
Progress PCR reaction realizes each gene polynorphisms parting need to only be can be completed by a multi-PRC reaction, each PCR
Contain internal control in reaction, guarantee the accuracy of testing result, prevents the appearance of false negative and false positive results；Each PCR is anti-
The lock nucleic acid locking for answering the tape label ARMs primer for carrying FAM or VIC fluorescence in system that can accordingly be had quenching group makes
Fluorescence is in cancellation state, in PCR reaction process, carries the tape label ARMs primer and cls gene mould to be checked of FAM or VIC fluorescence
Release fluorescence generates fluorescence curve and carries out result interpretation after plate specific binding；The present invention utilizes carrying fluorophor and label sequence
The specific ARMs primer of column combines the interior of the lock nucleic acid sequence for carrying quenching group, specific internal control probe and specificity
Primer is controlled, the specificity and accuracy of Genotyping can be greatly improved；Meanwhile two outer primers F1 and R1 can also be expanded
Increase, is further enriched with genomic templates, parting efficiency can be greatly improved, and then can detecte the genome sample of lower concentration
This, kit of the present invention is minimum to carry out polymorphic detection to 0.1ng genomic DNA；In addition, kit of the present invention
It is packed by the way of premixing, when use only needs that genomic DNA is added, while simplifying the response procedures of PCR, can be
The detection of aspirin resistance gene pleiomorphism is completed in 1 hour, result interpretation, interpretation knot directly can be carried out according to fluorescence curve
Fruit simplicity is objective, convenient for analysis.
Detailed description of the invention
Fig. 1 is the detection schematic diagram of kit of the present invention.
Fig. 2 is the detection schematic diagram of kit of the present invention.
Fig. 3 is PEAR1 wild type testing result.
Fig. 4 is PEAR1 heterozygous testing result.
Fig. 5 is PEAR1 homozygous mutant testing result.
Fig. 6 is GP1BA homozygous wildtype testing result.
Fig. 7 is GP1BA heterozygous testing result.
Fig. 8 is GP1BA homozygous mutant testing result.
Fig. 9 is GPIIIa homozygous wildtype testing result.
Figure 10 is GPIIIa heterozygous testing result.
Figure 11 is GPIIIa homozygous mutant testing result.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1 prepares aspirin resistance gene detecting kit of the invention
One, design of primers and synthesis:
It include 6 primers in 3 each systems in site of PEAR1, GP1BA and GPIIIa；Draw two of them positive-sense strand upstream
Object F1 and F2, two antisense strand downstream primers R1 and R2, F1 and R1 are general primer, and F2 and R2 are band fluorophor and label sequence
The specific ARMs primer of column is screened by primer and PCR reaction condition, guarantees that primer pair F1R1 is enriched with template, and
F2 and R1, F1 and R2 can normally amplify the specific genotype PCR product with fluorescence；Meanwhile it being added in each gene loci
Internal control primer.
Particular sequence such as the following table 1:
1 primer sequence of table
| Title | Sequence | Number | Annotation |
| PEAR1-F1 | 5’-TTGTGGACTTCTCTTCCTTC-3’ | SEQ ID NO.1 | |
| PEAR1-F2 | 5’-VIC-ATCCCTTGTCATCGTCAGATATACG-3’ | SEQ ID NO.2 | The wild template of specific recognition |
| PEAR1-R1 | 5’-GTCACCCTGCCAGAAAT-3’ | SEQ ID NO.3 | |
| PEAR1-R2 | 5’-FAM-AAGATACATTGATGAAGAAGTTCAAT-3’ | SEQ ID NO.4 | Specific recognition mutagenesis template |
| GP1BA-F1 | 5’-TCCAAGAGCTCTACCTGAAAGGC-3’ | SEQ ID NO.5 | |
| GP1BA-F2 | 5’-VIC-ATCCCTTGTCATCGTTAGTGCTCCTGAC-3’ | SEQ ID NO.6 | The wild template of specific recognition |
| GP1BA-R1 | 5’-CTCAGTCAAGTTGTTGTTA-3’ | SEQ ID NO.7 | |
| GP1BA-R2 | 5’-FAM-AAGATACATTGATGATTGCGTGTGTGCA-3’ | SEQ ID NO.8 | Specific recognition mutagenesis template |
| GPIIIa-F1 | 5’-GACGCTGGGCATCATCATGG-3’ | SEQ ID NO.9 | |
| GPIIIa-F2 | 5’-VIC-ATCCCTTGTCATCGTGGCCTTACAGT-3’ | SEQ ID NO.10 | The wild template of specific recognition |
| GPIIIa-R1 | 5’-TGCAGCCGGCCCAGGGCTCCAG-3’ | SEQ ID NO.11 | |
| GPIIIa-R2 | 5’-FAM-AAGATACATTGATGACAGAGTAGTCG-3’ | SEQ ID NO.12 | Specific recognition mutagenesis template |
| Internal control F | 5’-CGGGACCTGACTGACTACCT-3’ | SEQ ID NO.13 | |
| Internal control R | 5’-GGCCATCTCTTGCTCGAAGT-3’ | SEQ ID NO.14 |
Specific combination is as follows:
Wherein SEQ ID NO.1 and SEQ ID NO.4 is for expanding the wild matrix in the site PEAR1 gene rs12041331
Section, amplified production 403bp, SEQ ID NO.2 and SEQ ID NO.3 are prominent for expanding the site gene rs12041331 PEAR1
Modification segment, amplified production 366p, SEQ ID NO.1 and SEQ ID NO.3 expand two kinds of genotype；
Wherein SEQ ID NO.5 and SEQ ID NO.8 is produced for expanding the site GP1BA rs6065 wild-type fragment, amplification
Object is 89bp, and SEQ ID NO.6 and SEQ ID NO.7 is for expanding GP1BA rs6065 site mutation matrix section, amplified production
Two kinds of genotype are expanded for 120bp, SEQ ID NO.5 and SEQ ID NO.7；
Wherein SEQ ID NO.9 and SEQ ID NO.12 is expanded for expanding the site GPIIIa rs5918 wild-type fragment
Product is 335bp, and SEQ ID NO.10 and SEQ ID NO.11 expands for expanding GPIIIa rs5918 site mutation matrix section
Volume increase object is 286bp, and SEQ ID NO.9 and SEQ ID NO.11 expands two kinds of genotype；
SEQ ID NO.13 and SEQ ID NO.14 is for expanding internal control DNA fragmentation, amplified production 136bp.
Two, probe design and synthesis:
It include the lock nucleic acid of 2 specific band quenching groups in 3 site reaction systems of PEAR1, GP1BA and GPIIIa
Probe and 1 internal reference specificity MGB probe, the lock nucleic acid probe of 2 specific band quenching groups are wherein examined for one for locking
The specific ARMs primer with VIC fluorophor and sequence label of wild type is surveyed, another for lock-in detection saltant type
Specific ARMs primer with FAM fluorophor and sequence label；Internal reference specificity MGB probe internal reference site for identification.
Particular sequence such as the following table 2:
2 probe sequence of table
| Title | Sequence | Number | Annotation |
| Internal control-P | 5’-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3’ | SEQ ID NO.15 | Identify internal control |
| Mt-PNA-1 | 5’-BHQ-ACGATGACAAGGGAT-3’ | SEQ ID NO.16 | Saltant type amplimer fluorescence is quenched |
| Wt-PNA-1 | 5’-BHQ-TCATCAATGTATCTT-3’ | SEQ ID NO.17 | Wild-type amplification primer fluorescence is quenched |
Specific combination is as follows:
Wherein SEQ ID NO.15 specific recognition internal reference site；SEQ IDNO.16 specific recognition and lock-in detection open country
The specific ARMs primer with VIC fluorophor and sequence label of raw type；The detection mutation of SEQ ID NO.17 specific recognition
The specific ARMs primer with FAM fluorophor and sequence label of type.
Three, PCR premixes reaction solution configuration
(1) PEAR1PCR premixes reaction solution: by the specific primer for expanding the site PEAR1, primer sequence such as SEQ
Shown in NO:1~4 ID；Internal control primer, primer sequence is as shown in NO:13~14 SEQ ID；Probe groups, nucleotide sequence such as SEQ
Shown in NO:15~17 ID；PCR reaction solution composition, specific ingredient such as the following table 3:
3 PEAR1 gene rs12041331 site PCR reaction solution reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.1 | 100~400nM |
| SEQ ID NO.2 | 50~100nM |
| SEQ ID NO.3 | 100~400nM |
| SEQ ID NO.4 | 50~100nM |
| SEQ ID NO.13 | 100~400nM |
| SEQ ID NO.14 | 100~400nM |
| SEQ ID NO.15 | 50~100nM |
| SEQ ID NO.16 | 100~400nM |
| SEQ ID NO.17 | 100~400nM |
| TE buffer | Complement to 30 μ L |
(2) GP1BA PCR premixes reaction solution: by the specific primer for expanding the site GP1BA, primer sequence such as SEQ
Shown in NO:5~8 ID；Internal control primer, primer sequence is as shown in NO:13~14 SEQ ID；Probe groups, nucleotide sequence such as SEQ
Shown in NO:15~17 ID；PCR reaction solution composition, specific ingredient such as the following table 4:
4 GP1BA gene rs6065 site PCR reaction solution reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.5 | 100~400nM |
| SEQ ID NO.6 | 50~100nM |
| SEQ ID NO.7 | 100~400nM |
| SEQ ID NO.8 | 50~100nM |
| SEQ ID NO.13 | 100~400nM |
| SEQ ID NO.14 | 100~400nM |
| SEQ ID NO.15 | 50~100nM |
| SEQ ID NO.16 | 100~400nM |
| SEQ ID NO.17 | 100~400nM |
| TE buffer | Complement to 30 μ L |
(3) GPIIIa PCR premixes reaction solution: by the specific primer for expanding the site GPIIIa, primer sequence is such as
Shown in NO:9~12 SEQ ID；Internal control primer, primer sequence is as shown in NO:13~14 SEQ ID；Probe groups, nucleotide sequence
As shown in NO:15~17 SEQ ID；PCR reaction solution composition, specific ingredient such as the following table 5:
5 GPIIIa gene rs5918 site PCR reaction solution reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.9 | 100~400nM |
| SEQ ID NO.10 | 50~100nM |
| SEQ ID NO.11 | 100~400nM |
| SEQ ID NO.12 | 50~100nM |
| SEQ ID NO.13 | 100~400nM |
| SEQ ID NO.14 | 100~400nM |
| SEQ ID NO.15 | 50~100nM |
| SEQ ID NO.16 | 100~400nM |
| SEQ ID NO.17 | 100~400nM |
| TE buffer | Complement to 30 μ L |
Four, reference substance configures
Positive control include: concentration be respectively 10^4copy/ μ l PEAR1 gene rs12041331 site mutation type plasmid,
The site PEAR1 gene rs12041331 wild plasmid, the site GP1BA gene rs6065 wild plasmid, GP1BA gene
Rs6065 site mutation type plasmid, GPIIIa gene rs5918 site mutation type plasmid, the site GPIIIa gene rs5918 are wild
Type plasmid and internal reference Plasmid DNA and TE buffer；The plasmid sequence is that will not enumerate herein known to those skilled in the art
Its sequence.
Negative controls: PUC-19 plasmid empty carrier and TE buffer.
Five, kit is assembled
Kit includes the PCR premix reaction that 3 pipes detect PEAR1, GP1BA and GPIIIa gene loci polymorphism respectively
Liquid, 1 pipe positive reference substance, 1 pipe negative controls.The usage amount for calculating 20 person-portion specifications, prepares the ingredient in each pipe and assembling.
Embodiment 2
Sample to be tested is carried out using the mankind's aspirin resistance genetic polymorphism detection kit prepared in embodiment 1
Detection.The present embodiment collects 100 normal human subject peripheral blood samples, extracts genomic DNA, uses mankind's aspirin resistance base
Because polymorphic detection kit detects aspirin resistance gene polymorphic implementations, specific operation process is as follows:
(1) blood sample extracting genome DNA: commodity in use extracts kit carries out extracting genome DNA, has extracted
TE buffer eluted dna is used at rear, and measures DNA concentration；Genomic DNA is diluted to 20ng/ μ l；
(2) 30 μ l PCR premix reaction solution and 20 μ l sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；PCR
Response procedures are as follows: 95 DEG C of 1 minute initial denaturations；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulations: 95 DEG C
5 seconds, 61 DEG C 32 seconds, collect fluorescence.
(3) PCR carries out result interpretation after reaction according to table 6.
6 result interpretation of table
| PEAR1 | FAM(-A) | VIC(-G) | ROX (internal reference) |
| GG (homozygous wildtype) | – | + | + |
| GA (heterozygous mutant) | + | + | + |
| AA (homozygous mutant) | + | – | + |
| GP1BA | FAM(-T) | VIC(-C) | ROX (internal reference) |
| CC (homozygous wildtype) | – | + | + |
| CT (heterozygous mutant) | + | + | + |
| TT (homozygous mutant) | + | – | + |
| GPIIIa | FAM(-C) | VIC(-T) | ROX (internal reference) |
| TT (homozygous wildtype) | – | + | + |
| TC (heterozygous mutant) | + | + | + |
| CC (homozygous mutant) | + | – | + |
Note: "+" represents deta Rn end point fluorescence value > 100,000, and amplification curve is S-type；It is whole that "-" represents deta Rn
Point fluorescent value < 100,000 or the non-S type of curve；
Interpretation result is as follows:
PEAR1 homozygous wild 33, sample, one of testing result is as shown in Figure 3；
39, PEAR1 heterozygosis sample, one of testing result is as shown in Figure 4；
28, PEAR1 homozygous mutation sample, one of testing result is as shown in Figure 5；
GP1BA homozygous wild 84, sample, one of testing result is as shown in Figure 6；
8, GP1BA heterozygosis sample, one of testing result is as shown in Figure 7；
8, GP1BA homozygous mutation sample, one of testing result is as shown in Figure 8；
GPIIIa homozygous wild 90, sample, one of testing result is as shown in Figure 9；
3, GPIIIa heterozygosis sample, one of testing result is as shown in Figure 10；
7, GPIIIa homozygous mutation sample, one of testing result is as shown in figure 11；
Above-mentioned 100 sample fluorescence quantitative detection results and sequencing result 100% are consistent.The above result shows that this kit
It is reliable for mankind's aspirin resistance genetic polymorphism detection result, but kit detection sensitivity of the present invention is much higher than sequencing
Method.Using kit of the present invention detection mankind's aspirin resistance gene pleiomorphism detection method have high sensitivity,
The advantages that specificity is high, easy to operate, result is reliable, in addition, this invention simplifies the response procedures of PCR, by routine PCR reaction
In annealing and extend step (annealing 30 seconds extend 1 minute) and be reduced to a step (only needing 61 degree to react 32 seconds), so can
Detection is completed in 1 hour, and the present invention directly can carry out result interpretation, visual result, interpretation simplicity according to fluorescence curve.
Embodiment 3
The lowest detection line of this kit is verified in the kit detection prepared using embodiment 1, specifically includes following operation
Step:
(1) using the sample of known PEAR1, GP1BA and GPIIIa gene corresponding site genotype in embodiment 2, each
It is a case each that wild type gene group, mutated genes group and heterozygous genome sample are chosen in site respectively, by above-mentioned Sample Dilution
To 10ng/ μ L, 1ng/ μ L, 0.5ng/ μ L, 0.25ng/ μ L, 0.1ng/ μ L, 0.05ng/ μ L, 0.025ng/ μ L, 0.001ng/ μ L；
(2) 30 μ l PCR premix reaction solution and 20 μ l sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；PCR
Response procedures are as follows: 95 DEG C of 1 minute initial denaturations；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulations: 95 DEG C
5 seconds, 61 DEG C 32 seconds, collect fluorescence；Each concentration gradient counterpoise reinspection of each sample is surveyed 20 times；
(3) PCR carries out result interpretation after reaction according to table 6；
(4) detection success rate (the parting as detection consistent with known type of the above-mentioned each concentration gradient of sample is counted
Success detects success rate=testing result consistent results quantity/20 × 100%), it is shown in Table 7~9.
7 PEAR1 gene rs12041331 site primer successful rate statistics of table
8 GP1BA gene rs6065 site primer successful rate statistics of table
9 GPIIIa gene rs5918 site primer successful rate statistics of table
The above results show that the present invention can carry out mankind's aspirin resistance to the DNA sample of concentration down to 0.1ng/ μ L
Genetic polymorphism detection.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Sequence table
<110>Wuhan Kang Lu Biotechnology Ltd.
<120>a kind of mankind's aspirin resistance genetic polymorphism detection kit and its preparation method and application
<160> 17
<210> 1
<211> 20bp
<212> DNA
<213>artificial sequence
400 > 1 of <
ttgtggactt ctcttccttc 20
<210> 2
<211> 25bp
<212> DNA
<213>artificial sequence
400 > 2 of <
atcccttgtc atcgtcagat ataca 25
<210> 3
<211> 17bp
<212> DNA
<213>artificial sequence
400 > 3 of <
gtcaccctgc cagaaat 17
<210> 4
<211>26bp
<212> DNA
<213>artificial sequence
400 > 4 of <
aagatacatt gatgaagaag ttcaag 26
<210> 5
<211>23bp
<212> DNA
<213>artificial sequence
400 > 5 of <
tccaagagct ctacctgaaa ggc 23
<210> 6
<211>28bp
<212> DNA
<213>artificial sequence
400 > 6 of <
atcccttgtc atcgttagtg ctcctgac 28
<210> 7
<211>19bp
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<213>artificial sequence
400 > 7 of <
ctcagtcaag ttgttgtta 19
<210> 8
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400 > 8 of <
aagatacatt gatgattgcg tgtgtgca 28
<210> 9
<211>20bp
<212> DNA
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400 > 9 of <
gacgctgggc atcatcatgg 20
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<211>26bp
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400 > 10 of <
atcccttgtc atcgtggcct tacagg 26
<210> 11
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400 > 11 of <
tgcagccggc ccagggctcc ag 22
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400 > 12 of <
aagatacatt gatgacagag tagtct 26
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400 > 13 of <
cgggacctga ctgactacct 20
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400 > 14 of <
ggccatctct tgctcgaagt 20
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<212> DNA
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400 > 15 of <
accaccacgg ccgagc 16
<210> 16
<211>15bp
<212> DNA
<213>artificial sequence
400 > 16 of <
acgatgacaa gggat 15
<210> 17
<211>15bp
<212> DNA
<213>artificial sequence
400 > 17 of <
tcatcaatgt atctt 15
Claims (10)
1. primer sets, which is characterized in that the ARMs of specificity of the primer sets by common outer primer and with fluorescence labels draws
Object composition, particular sequence are as follows: for expanding the specific primer sequence such as SEQ ID in the site PEAR1 gene rs12041331
Shown in NO.1 ~ SEQ ID NO.4, wherein 5 ' ends of primer shown in SEQ ID NO.2 carry VIC fluorophor, SEQ ID
5 ' ends of primer shown in NO.4 carry FAM fluorophor；For expanding the specific primer sequence in the site GP1BA gene rs6065
As shown in SEQ ID NO.5 ~ SEQ ID NO.8, wherein 5 ' ends of primer shown in SEQ ID NO.6 carry VIC fluorophor,
5 ' ends of primer shown in SEQ ID NO.8 carry FAM fluorophor；Specificity for expanding the site GPIIIa rs5918 is drawn
Object sequence is as shown in SEQ ID NO.9 ~ SEQ ID NO.12, and wherein it is glimmering to carry VIC for 5 ' ends of primer shown in SEQ ID NO.10
5 ' ends of light group, primer shown in SEQ ID NO.12 carry FAM fluorophor；Primer sequence such as SEQ ID for internal control
Shown in NO.13 ~ SEQ ID NO.14.
2. probe groups, which is characterized in that the probe groups are by specific taqman-MGB probe and the lock core for carrying quenching group
Acid probe composition, for nucleotide sequence as shown in SEQ ID NO.15 ~ SEQ ID NO.17, concrete form is as follows:
Internal control-P:5 '-ROX-ACCACCACGGCCGAGC-MGB-BHQ-3 '
Wild type quenching group lock nucleic acid: 5 '-BHQ-ACGATGACAAGGGAT-3 '
Saltant type quenching group lock nucleic acid: 5 '-BHQ-TCATCAATGTATCTT-3 '.
3. a kind of detection kit includes probe groups described in primer sets and claim 2 described in claim 1, which is characterized in that
The kit premixes reaction solution, positive reference substance and yin by the PCR for being respectively used for amplifying the site PEAR1, GP1BA, GPIIIa
Property reference substance composition；
The concrete component of the PCR premix reaction solution is as follows:
(1) the PCR premix reaction solution for expanding PEAR1 gene loci includes: described for expanding PEAR1 gene
The primer in the site rs12041331, primer sequence is as shown in SEQ ID NO.1 ~ SEQ ID NO.4；The drawing for internal control
Object sequence, sequence is as shown in SEQ ID NO.13 ~ SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15 ~
Shown in SEQ ID NO.17；And PCR reaction solution composition；
(2) the PCR premix reaction solution for expanding GP1BA gene loci includes: described for expanding GP1BA gene rs6065
The primer of point, primer sequence is as shown in SEQ ID NO.5 ~ SEQ ID NO.8；The primer sequence for internal control, sequence is such as
Shown in SEQ ID NO.13 ~ SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15 ~ SEQ ID NO.17 institute
Show；And PCR reaction solution composition；
(3) the PCR premix reaction solution for expanding GPIIIa gene loci includes: described for expanding GPIIIa gene rs5918
The primer in site, primer sequence is as shown in SEQ ID NO.9 ~ SEQ ID NO.12；The primer sequence for internal control, sequence
Column are as shown in SEQ ID NO.13 ~ SEQ ID NO.14；The probe groups, sequence such as SEQ ID NO.15 ~ SEQ ID
Shown in NO.17；And PCR reaction solution composition；
The positive control includes: comprising PEAR1 gene rs12041331 site mutation type plasmid, PEAR1 gene
The site rs12041331 wild plasmid, the site GP1BA gene rs6065 wild plasmid, the site gene rs6065 GP1BA are prominent
Modification plasmid, GPIIIa gene rs5918 site mutation type plasmid, GPIIIa gene rs5918 site wild plasmid and internal reference
Plasmid DNA；
The negative control includes the recombinant plasmid without reference gene and target gene site.
4. detection kit according to claim 3, which is characterized in that in the PCR premix reaction solution, in probe groups
The concentration of taqman-MGB probe is 50 ~ 100nM, and the concentration for carrying the lock nucleic acid probe of quenching group is 100 ~ 400nM；With
It is 100 ~ 400nM in each primer concentration without fluorophor for expanding each site, each primer concentration with fluorophor is equal
For 50 ~ 100nM；The concentration of primer for internal control is 100 ~ 400nM.
5. detection kit according to claim 3, which is characterized in that the PCR reaction solution includes Premix qPCRmix
With TE buffer.
6. any one of claim 3 ~ 5 detection kit is in preparation for detecting mankind's aspirin resistance gene pleiomorphism
Application in product.
7. applying according to claim 6, which is characterized in that specific detection method includes the following steps:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) PCR premix reaction solution and sample to be tested DNA fluorogenic quantitative detection: are added into reaction tube；Positive control is set simultaneously
Then reaction tube is placed in fluorescent PCR instrument and carries out pcr amplification reaction by group and negative control group, and it is glimmering to acquire FAM, VIC and ROX
Optical signal；
(3) after pcr amplification reaction, result judgement is carried out according to fluorescence signal initial line situation collected.
8. application according to claim 7, which is characterized in that be diluted to the sample DNA to be detected in step (1) dense
Degree is 0.1 ~ 100ng/ μ L, then carries out pcr amplification reaction.
9. application according to claim 7, which is characterized in that the condition of step (2) described pcr amplification reaction are as follows: 95 DEG C
1 minute initial denaturation；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32
Second, collect fluorescence.
10. application according to claim 7, which is characterized in that the criterion of step (3) described result judgement are as follows: 1. work as sun
Property control group has FAM, VIC, ROX fluorescence signal initial line, and negative control group is to be detected without FAM, VIC, ROX fluorescence initial line
When sample has ROX fluorescence signal initial line, determines Success in Experiment, subsequent parting judgement can be carried out；2. according to detection sample
FAM, VIC fluorescence signal judge the parting of PEAR1: the site VIC fluorescence signal initial line PEAR1 gene rs12041331 parting is
Homozygous wildtype；FAM fluorescence signal initial line, PEAR1 gene rs12041331 site parting are homozygous mutant；VIC, FAM are glimmering
The equal initial line of optical signal, PEAR1 gene rs12041331 site parting are heterozygous mutant；3. according to detection sample FAM,
VIC fluorescence signal judges the parting of GP1BA: VIC fluorescence signal initial line GP1BA gene rs6065 site parting is wild for homozygosis
Type；FAM fluorescence signal initial line, GP1BA gene rs6065 site parting are homozygous mutant；VIC, FAM fluorescence signal rise
Line, GP1BA gene rs6065 site parting are heterozygous mutant；4. being judged according to FAM, VIC fluorescence signal of detection sample
The parting of GPIIIa: VIC fluorescence signal initial line GPIIIa gene rs5918 site parting is homozygous wildtype；FAM fluorescence signal
Initial line, GPIIIa gene rs5918 site parting are homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, GPIIIa gene
The site rs5918 parting is heterozygous mutant.
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| CN111154842A (en) * | 2020-02-20 | 2020-05-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of aspirin resistance gene of human |
| CN111593100A (en) * | 2019-02-20 | 2020-08-28 | 葛猛 | Artificial simulated nucleic acid molecular beacon and kit for detecting GP1BA gene rs6065 site polymorphism |
| CN111893173A (en) * | 2020-07-22 | 2020-11-06 | 福州艾迪康医学检验所有限公司 | Primer, method and kit for detecting PEAR1 SNP locus |
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| CN111154842A (en) * | 2020-02-20 | 2020-05-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of aspirin resistance gene of human |
| CN111154842B (en) * | 2020-02-20 | 2023-04-14 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of aspirin resistance gene of human |
| CN111893173A (en) * | 2020-07-22 | 2020-11-06 | 福州艾迪康医学检验所有限公司 | Primer, method and kit for detecting PEAR1 SNP locus |
| CN113046424A (en) * | 2021-03-05 | 2021-06-29 | 为朔医学数据科技（北京）有限公司 | Preparation method of nucleic acid composition for PCR detection, nucleic acid composition prepared by preparation method and PCR detection method |
| CN113046424B (en) * | 2021-03-05 | 2022-12-27 | 为朔医学数据科技（北京）有限公司 | Preparation method of nucleic acid composition for PCR detection, nucleic acid composition prepared by preparation method and PCR detection method |
| CN116083562A (en) * | 2023-03-14 | 2023-05-09 | 北京寻医问译科技发展有限公司 | SNP marker combination and primer set related to aspirin resistance auxiliary diagnosis and application thereof |
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