CN109486943A - For detecting and the primer sets of aspirin resistance related gene polymorphic site and kit and its application - Google Patents
For detecting and the primer sets of aspirin resistance related gene polymorphic site and kit and its application Download PDFInfo
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Abstract
The present invention relates to beyond body nucleic acid detection technique fields, specifically, providing a kind of for detecting and the primer sets of aspirin resistance related gene polymorphic site and kit and its application.Provided by the present invention for detecting primer sets and kit with aspirin resistance related gene polymorphism site, COX1, COX2, GPIIIa, PEAR1, P2Y1, GPIa and PAI-1 gene pleiomorphism can detecte.High sensitivity can be detected accurately down to 0.1ng/ μ L genomic DNA；Specificity is good, and up to 300ng/ μ L genomic DNA will not generate non-specific amplification；Entire fluorescent PCR detection process only needs can be completed for 90 minutes, testing result intuitively easy interpretation.
Description
Technical field
The present invention relates to beyond body nucleic acid detection technique fields, support for detecting with aspirin in particular to one kind
The primer sets and kit in decorrelation gene polymorphic site and its application.
Background technique
Aspirin is widely used in the secondary prevention of cardiovascular and cerebrovascular disease as antiplatelet first-line drug.However,
There are individual difference, still there is cardiovascular and cerebrovascular ischemic thing after some patientss Aspirin in different crowd again in aspirin
That is, there is aspirin resistance in part.Aspirin resistance incidence 5%~45%, mechanism is related with gene pleiomorphism.
Gene relevant to aspirin resistance mainly has at present: in thromboxane activation pathway encode cyclooxygenase (COX) gene,
GPIIb/IIIa acceptor gene, Platelet endothelial cell are aggregated receptor 1 (PEAR1) gene, platelet surface adp receptor gene
P2Y1, platelet glycoprotein GPIa gene and endothelial cell type Plasminogen activator PAI-1 gene.Therefore,
Carrying out genotype detection to aspirin resistance related gene facilitates the personalized medicine of aspirin.
Mainly there are Sanger PCR sequencing PCR, chip method, quantitative fluorescent PCR etc. about the method for genetic test at present.
PCR sequencing PCR is the goldstandard of genetic test, however there are two distinct disadvantages for this method: first is that cumbersome, time-consuming
It is long；Second is that subsequent need to handle PCR product, it is easy pollution.
Chip method need to first carry out a wheel PCR amplification, then PCR product is hybridized with probe, according to the strong of hybridization signal
Degree judges genotype.The shortcomings that chip method, mainly has: needing to operate PCR product, easily causes pollution；Its
It is secondary, it is as a result not easy interpretation, is easy to appear false positive.
Fluorescent quantitative PCR technique is widely used in detection in Gene Mutation and Genotyping.This method can be divided into high-resolution
Solubility curve analytical technology (high resolution melting analysis, HRM), amplification refractory mutation system
(amplification refractory mutation system, ARMS) and Taqman sonde method.HRM specificity is not
Height, and the requirement to instrument and equipment is high, at present using few in clinical detection.ARMS utilizes the end the 3' last bit base of PCR primer
Must the principle that could effectively expand complementary with its template DNA, design primer appropriate for different bases to detect difference
Gene pleiomorphism.This method is easy to operate, and specificity and sensitivity are high, reproducible, in clinical detection gene mutation and gene
It is widely applied in polymorphism.But there are obvious shortcomings when detecting gene pleiomorphism for this method, first, detection flux is low, complete
Two pipe PCR reaction solutions need to be respectively set in one pattern detection, and a pipe is expanded and detected to wild-type template, and another pipe is to prominent
Modification template is expanded and is detected；Second, result interpretation is not intuitive, result need to be judged according to △ CT；Third, cannot
Completely eliminate non-specific amplification.
Therefore, developing one kind to multiple sites while can detect, time saving and energy saving, and specificity is high, testing result
More accurate method is particularly important.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of for detecting 7 and aspirin resistance related gene polymorphism
The technical issues of primer sets of SNP site, especially PAI-1 gene loci difficulty detect.
The second object of the present invention is to provide above-mentioned primer sets in preparation for detecting in aspirin resistance product
Using.
The third object of the present invention is to provide a kind of for detecting and aspirin resistance related gene polymorphic site
Kit lacks a kind of product for quickly, accurately detecting aspirin resistance gene pleiomorphism to alleviate in the prior art.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
It is a kind of for detect with the primer sets of aspirin resistance related gene polymorphic site, primer sets include following 7 groups
Primer pair or at least one set of primer pair or probe in 7 groups of probes；
Primer pair includes: for detecting the primer pair in the site COX1 gene-1 676G > A, for detecting COX2 gene -765G
The primer pair in the site > C, the primer pair for detecting the site GPIIIa gene 1565T > C, for detect PEAR1 gene -3996G >
The primer pair in the site A, the primer pair for detecting the site P2Y1 gene 893C > T, for detecting the site GPIa gene 807C > T
Primer pair and for detecting PAI-1 gene -6754G > site 5G primer pair；
Probe includes: wild-type probe for detecting the site COX1 gene-1 676G > A and saltant type probe, for examining
Survey COX2 gene -765G > site C wild-type probe and saltant type probe, for detecting the site GPIIIa gene 1565T > C
Wild-type probe and saltant type probe, for detect PEAR1 gene -3996G > site A wild-type probe and saltant type probe,
For detect the site P2Y1 gene 893C > T wild-type probe and saltant type probe, for detecting the site GPIa gene 807C > T
Wild-type probe and saltant type probe and for detecting PAI-1 gene -6754G > site 5G wild-type probe and mutation
Type probe；
Primer pair for detecting the site COX1 gene-1 676G > A includes shown in SEQ ID NO.1 and SEQ ID NO.2
Nucleotide sequence；It include SEQ ID NO.5 and SEQ ID NO.6 for detecting COX2 gene -765G > site C primer pair
Shown in nucleotide sequence；Primer pair for detecting the site GPIIIa gene 1565T > C includes SEQ ID NO.9 and SEQ ID
Nucleotide sequence shown in NO.10；For detect PEAR1 gene -3996G > site A primer pair include SEQ ID NO.13 and
Nucleotide sequence shown in SEQ ID NO.14；Primer pair for detecting the site P2Y1 gene 893C > T includes SEQ ID
Nucleotide sequence shown in NO.17 and SEQ ID NO.18；Primer pair for detecting the site GPIa gene 807C > T includes SEQ
Nucleotide sequence shown in ID NO.21 and SEQ ID NO.22；For detecting PAI-1 gene -6754G > site 5G primer pair
Including nucleotide sequence shown in SEQ ID NO.25 and SEQ ID NO.26；
Wild-type probe for detecting the site COX1 gene-1 676G > A includes nucleotides sequence shown in SEQ ID NO.3
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.4；For detecting COX2 gene -765G > site C open country
Raw type probe includes nucleotide sequence shown in SEQ ID NO.7, and saltant type probe includes nucleotide shown in SEQ ID NO.8
Sequence；Wild-type probe for detecting the site GPIIIa gene 1565T > C includes nucleotides sequence shown in SEQ ID NO.11
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.12；For detecting PEAR1 gene -3996G > site A
Wild-type probe includes nucleotide sequence shown in SEQ ID NO.15, and saltant type probe includes core shown in SEQ ID NO.16
Nucleotide sequence；Wild-type probe for detecting the site P2Y1 gene 893C > T includes nucleotides sequence shown in SEQ ID NO.19
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.20；For detecting the open country in the site GPIa gene 807C > T
Raw type probe includes nucleotide sequence shown in SEQ ID NO.23, and saltant type probe includes nucleosides shown in SEQ ID NO.24
Acid sequence；It include nucleotide shown in SEQ ID NO.27 for detecting PAI-1 gene -6754G > site 5G wild-type probe
Sequence, saltant type probe include nucleotide sequence shown in SEQ ID NO.28；
Wherein, 5 ' ends of wild-type probe and the saltant type probe are connected separately with fluorophor；
The fluorophor of the wild-type probe of any gene and the fluorophor of saltant type probe are different；
3 ' ends of wild-type probe and the saltant type probe are connected separately with quenching group.
The specifying information in the gene polymorphic site detected in the present invention is as shown in table 1 below:
1 gene polymorphic site information of table
| Gene | SNP sequence number | Gene pleiomorphism |
| COX1 | rs1330344 | -1676G>A |
| COX2 | rs20417 | -765G>C |
| GPIIIa | rs5918 | 1565T>C |
| PEAR1 | rs12041331 | -3996G>A |
| P2Y1 | rs1065776 | 893C>T |
| GPIa | rs1126643 | 807C>T |
| PAI-1 | rs1799768 | -675 4G>5G |
The particular sequence of 7 groups of primer pairs and 7 groups of probes in the present invention is as shown in table 2:
27 groups of primer pairs of table and 7 groups of probe sequences
Said gene site covers the relevant oligogene site of current aspirin resistance, by more to these sites
State property is detected, and is helped to carry out auxiliary judgment to patient's Aspirin curative effect, is realized personalized medicine.
Wherein, there are G repetitive sequences for PAI-1 gene polynorphisms location proximate, lead to wild type and saltant type probe region
Indexing is not high, and specificity is bad.The present invention replaces common dimethylene sulfoxide with tetramethylene sulfoxide, optimizes PCR buffering
Liquid component；Routine dNTPs is replaced with thermal starting dNTPs, optimizes dNTPs component.The present invention solves the high site GC probe not
The technical problem of easy parting, the primer pair provided and probe correctly can identify and detect above-mentioned 7 SNP sites.
The present invention is by carrying out Genotyping, due in a kind of detection of gene, wild type to SNP using Taqman probe
Probe is different with the fluorophor of saltant type probe, so the detection of single SNP can be completed in a single tube, signal is clear
Accurate stable.
In being preferably carried out mode, primer sets include above-mentioned 7 groups of primer pairs and 7 groups of probes.
When being detected to 7 SNP, it is only necessary to which detection can be completed in 7 single tubes, and result is easy interpretation, direct root
Result interpretation can be completed according to fluorescent amplification curve, avoid complicated calculating, it is time saving and energy saving.
In being preferably carried out mode, primer sets further include for detect the internal control primer of GAPDH gene to and internal reference visit
Needle, internal control primer is to including nucleotide sequence shown in SEQ ID NO.29 and SEQ ID NO.30；Internal reference probe includes SEQ
Nucleotide sequence shown in ID NO.31；Wherein, 5 ' ends of internal reference probe are connected with fluorophor, the fluorophor of internal reference probe
It is all different with the wild-type probe of any gene and the fluorogene of saltant type probe；3 ' ends of internal reference probe are connected with fluorescence
Quenching group.
3 internal control primer of table to and internal reference probe
| Number | Sequence (5'-3') |
| SEQ ID NO.29 | GGGCCACTAGGCGCTCA |
| SEQ ID NO.30 | AGCCACCCGCGAACTCA |
| SEQ ID NO.31 | CTCTCCCTCCGCGCAGCCG |
There may be partially or completely inhibiting in PCR reaction, amplification instrument there may be poor between the hole for being higher than allowed band, and
Artificial sample-adding mistake may also lead to the appearance of false negative result, so, it is eliminated in the present invention using internal reference system above-mentioned hidden
Suffer from, ensure that the accuracy of testing result.Internal control primer pair shown in SEQ ID NO.29-31 and internal reference probe are detected interior
The sequence selection of mark object is one section of GAPDH sequence of human genome DNA, and GAPDH sequence and target sequence are existed simultaneously in people
Genoid group, and with target gene to be checked without homology.Therefore it ensure that interior label primer only expands GAPDH sequence, without influencing
Target sequence amplification.
In being preferably carried out mode, fluorophor is selected from any one of FAM, VIC, TET, HEX, JOE or ROX,
In, the fluorophor of the wild-type probe of any gene is preferably FAM, the fluorophor of the saltant type probe of any gene
Preferably VIC, the fluorophor of internal reference probe are preferably ROX.The fluorophor of three kinds of probes is different two-by-two can be direct
It is distinguished from result, accurate rapid results.
In being preferably carried out mode, quenching group is selected from any one of NFQ, TAMRA, BHQ1 or BHQ2, preferably
NFQ。
In being preferably carried out mode, wild-type probe, saltant type probe and internal reference probe have been independently connected MGB and have repaired
Group is adornd, MGB modification group is connect with quenching group.MGB modification group itself does not generate fluorescence, therefore can substantially reduce this
The intensity of bottom signal, while can be by the T of probemValue improves 10 DEG C or so, therefore same TmValue, with MGB modification group
Probe can than general T aqman probe design it is shorter so that probe identification have Single nuclear polymorphism site when,
Specificity is stronger.
Above-mentioned primer sets are preparing the application in the product for detecting aspirin resistance.Wherein, product be reagent or
Kit.
It is a kind of for detecting and the kit of aspirin resistance related gene polymorphic site, including above-mentioned primer sets.It should
Kit can detect 7 SNP site polymorphisms, cover oligogene relevant to aspirin resistance position at present
Point, one pipe PCR of this kit complete a SNP site detection, and whole stopped pipe operation avoids the risk of cross contamination, according to
Amplification curve directly carries out result interpretation, and the easy interpretation of visual result, this kit specificity is good, no non-specific amplification,
The genomic DNA of 300ng/ μ L will not generate non-specific amplification.The kit high sensitivity can detect down to 0.1ng/ μ L base
Because of a group DNA sample.
In being preferably carried out mode, the kit further include thermal starting archaeal dna polymerase, PCR reaction buffer 5 ×,
Thermal starting dNTPs and sterile water.Thermal starting dNTPs is similar to natural dNTPs, and thermal starting dNTPs is in the end 3' with thermo-labile guarantor
Protect base group modification.The nucleotide incorporation that the presence of the modification group prevents archaeal dna polymerase to be catalyzed, until in thermal activation step
Remove nucleosides acid protecting group.Kit spy is improved jointly using thermal starting archaeal dna polymerase and thermal starting dNTPs double action
It is anisotropic.
To whether there is whether pollution and quantitative fluorescent PCR reaction are normally carried out during monitoring operation, preferably
In embodiment, kit further includes positive control and blank control.Positive control is respectively COX1-1676G, COX1-
1676A、COX2-765G、COX2-765C、GPIIIa 1565T、GPIIIa 1565C、PEAR1-3996G、PEAR1-3996A、
P2Y1 893C, P2Y1 893T, GPIa 807C, GPIa 807T, PAI-1-6754G and PAI-1-6755G recombinant plasmid dna
Mixture.Blank control is Tris-HCl buffer (10mM).
In order to improve PAI-1 polymorphic site detection specificity, present invention optimizes PCR reaction buffer 5 ×, packet
It includes: 40-60mM Tris-HCl, 200-300mM KCl, 40-60mM (NH4)2SO4、15-25mM MgCl2, 5-8M glycine betaine, 5-
10% tetramethylene sulfoxide and 15-30% glycerol, pH7.5-8.5.The content of tetramethylene sulfoxide and glycerol is percent by volume.
In preferred embodiment, PCR reaction buffer 5 × include: 50mM Tris-HCl, 250mM KCl,
50mM (NH4)2SO4、20mM MgCl2, 6M glycine betaine, 5% tetramethylene sulfoxide and 20% glycerol, pH=8.0.
The present invention provides the method using mentioned reagent box detection aspirin resistance related gene polymorphism, including following
Step:
(1) human blood extracting genome DNA.
(2) fluorescent quantitative PCR: by step (1) extract DNA be added to people COX1, COX2, GPIIIa, PEAR1,
PCR amplification is carried out in P2Y1, GPIa and PAI-1 genetic polymorphism detection kit reaction solution, and acquires each channel fluorescence signal.
(3) interpretation of result
Kit and sample availability are determined first after the completion of amplification.All fluorescence signal channel C t of positive control
Value≤32, amplification curve has obvious Exponential growth stage；The channel blank control FAM, VIC, ROX is bent without amplification curve, or amplification
Line is straight line or slight oblique line, and without obvious Exponential growth stage, no Ct value or value >=38 Ct judge that kit is effective.Internal standard gene
ROX channel C t value < 38, amplification curve has obvious Exponential growth stage, and judgement sample is effective.
Testing result is determined under the premise of kit and effective sample.Value≤36 wild-type probe Ct, mutation
When type probe is without Ct value, it is judged as wild type；When wild-type probe is without Ct value, when saltant type probe Ct value≤36, it is judged as prominent
Modification；When value≤36 wild-type probe Ct, when saltant type probe Ct value≤36, it is judged as heterozygous.
The present invention provides detection people COX1, COX2, GPIIIa, PEAR1, P2Y1, GPIa and PAI-1 gene pleiomorphisms
Kit, with Sanger PCR sequencing PCR methods comparison result, the results showed that, the method for the present invention is with Sanger PCR sequencing PCR consistency
100%, the method for the present invention is accurate, reliable.
7 SNP detection sites of this kit are significant related to aspirin resistance, and Clinical significance of detecting is clear, have preferable
Clinical value, facilitate aspirin personalized medicine.
The present invention provides for detect with the primer sets and kit of aspirin resistance related gene polymorphic site, with
The prior art is compared, the invention has the benefit that
(1) 7 aspirin resistance related genes can be detected simultaneously, and it is related that site covers current aspirin resistance
Oligogene site, detection site is more comprehensive.
(2) probe recognition detection can be carried out to the high site GC, PAI-1 gene loci (4G > 5G) is rich in high GC structure, visits
Needle correctly can be identified and be detected.
(3) kit specificity is improved jointly using thermal starting archaeal dna polymerase and thermal starting dNTPs double action.
(4) kit specificity is good, and the genomic DNA of 300ng/ μ L will not generate non-specific amplification；High sensitivity, can
It detects down to 0.1ng/ μ L genomic DNA sample.
(5) Genotyping is carried out to SNP, needs to be arranged two pipe PCR amplifications different from ARMS-PCR, the present invention only needs single tube
Single SNP detection can be completed, detect flux ratio ARMS-PCR high.
(6) result interpretation is easy, and without calculating △ Ct after PCR, only need to just can be carried out knot according to fluorescent amplification curve
Fruit interpretation.
(7) the whole stopped pipe of all reactions operates, and without further analysis of uncapping after PCR, avoids cross contamination
Risk.
(8) at low cost, provided detection method can be completed in 90 minutes detection, required time far below PCR sequencing PCR,
Chip method etc. is particularly suitable for clinic outpatient service detection.
(9) genetic test site clinical meaning is clear, clinical value with higher.
Detailed description of the invention
Fig. 1 is PAI-1 gene different probe sequence wild type sample amplification in embodiment 1；
Fig. 2 is this amplification of PAI-1 gene different probe series jump pattern in embodiment 1；
Fig. 3 is PAI-1 gene different probe concentration wild type sample amplification in embodiment 2；
Fig. 4 is PAI-1 gene different probe concentration saltant type sample amplification in embodiment 2；
Fig. 5 is PAI-1 gene difference organic compounds containing sulfur additive amplification in embodiment 3；
Fig. 6 is PAI-1 gene difference dNTPs component amplification in embodiment 4；
Fig. 7 is PAI-1 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Fig. 8 is PAI-1 gene 0.1ng/ μ L heterozygous sample amplification in embodiment 6；
Fig. 9 is COX1 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 10 is COX2 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 11 is GPIIIa gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 12 is PEAR1 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 13 is P2Y1 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 14 is GPIa gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 15 is PAI-1 gene 300ng/ μ L homozygous wildtype sample amplification in embodiment 6；
Figure 16 is COX1 gene 300ng/ μ L homozygous mutant sample amplification in embodiment 6；
Figure 17 is COX2 gene 300ng/ μ L homozygous mutant reference material amplification in embodiment 6；
Figure 18 is GPIIIa gene 300ng/ μ L homozygous mutant reference material amplification in embodiment 6；
Figure 19 is PEAR1 gene 300ng/ μ L homozygous mutant sample amplification in embodiment 6；
Figure 20 is P2Y1 gene 300ng/ μ L homozygous mutant reference material amplification in embodiment 6；
Figure 21 is GPIa gene 300ng/ μ L homozygous mutant sample amplification in embodiment 6；
Figure 22 is PAI-1 gene 300ng/ μ L homozygous mutant sample amplification in embodiment 6；
Figure 23 is COX1 gene 0.1ng/ μ L heterozygous mutant sample amplification in embodiment 6；
Figure 24 is COX2 gene 0.1ng/ μ L heterozygous mutant reference material amplification in embodiment 6；
Figure 25 is GPIIIa gene 0.1ng/ μ L heterozygous mutant reference material amplification in embodiment 6；
Figure 26 is PEAR1 gene 0.1ng/ μ L heterozygous mutant sample amplification in embodiment 6；
Figure 27 is P2Y1 gene 0.1ng/ μ L heterozygous mutant reference material amplification in embodiment 6；
Figure 28 is GPIa gene 0.1ng/ μ L heterozygous mutant sample amplification in embodiment 6；
Figure 29 is PAI-1 gene 0.1ng/ μ L heterozygous mutant sample amplification in embodiment 6.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method
It can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with
Intercombination forms new technical solution.
In the present invention, unless otherwise indicated, numberical range " a~b " indicates the contracting of any real combinings between a to b
Sketch form shows that wherein a and b is real number.Such as numberical range " 6~22 " indicate herein all listed " 6~22 " it
Between whole real numbers, " 6~22 " be these combinations of values breviary indicate.
" range " disclosed in this invention can be respectively one or more lower limits and one in the form of lower and upper limit
A or multiple upper limits.
In the present invention, unless otherwise indicated, each reaction or operating procedure can be carried out sequentially, can not also be in sequence
It carries out.Preferably, reaction method herein is that sequence carries out.
Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art
Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only to use
It is used, is but should not be understood as present invention is limited in any form in being described in more detail.
Quantitative fluorescent PCR system includes: primer, probe, PCR buffer, dNTPs, Taq enzyme.To improve PCR system
Can, probe optimizes sequence, probe dosage, PCR buffer components and dNTPs ingredient.PAI-1 gene in the present invention
Site is insertion mutation there are 4 continuous G, mutational site there are high GC structure, the SNP site, and insertion base is G, therefore,
Site optimization difficulty is larger, is mainly manifested in wild type and saltant type probe discrimination is not high, specificity is bad.Below with
For the site PAI-1, the detailed process of quantitative fluorescent PCR reaction system optimization is described in detail.
1 primer of embodiment/probe sequence selection
According to SNP sequence signature, 3 sets of primers, 5 sets of probes are designed, prepare PCR system respectively.It is detected with the above system wild
Raw type and mutated genes group DNA sample, compare the difference of amplification capability between different primers probe combinations, as a result such as Fig. 1 and Fig. 2
Shown, Fig. 1 is the result figure of PAI-1 gene different probe sequence amplification wild type sample, and Fig. 2 is PAI-1 gene different probe
The result figure of sequence amplification saltant type sample.By a large amount of screening experiments, best primer combination of probe (probe combinations are obtained
2), i.e. SEQ ID NO.25/SEQ ID NO.26 primer pair and SEQ ID NO.27/SEQ ID NO.28 probe is combined into group
PCR system in all combinations best performance, show as it is specific preferably signal value is high, therefore, select the combination carry out into
One step system is optimized and revised.
The selection of 2 probe different amounts of embodiment
Probe is respectively adopted FAM fluorescence and VIC fluorescence and carries out real-time monitoring to target sequence amplification.Concentration and probe concentration directly affects
Amplification fluorescent signal value, concentration and probe concentration is low, and fluorescence signal value is low, and concentration and probe concentration is high, and fluorescence signal is high, while non-specific signals
Also high.
On the basis of the above-mentioned optimal primer combination of probe screened, further by adjusting concentration and probe concentration, optimize body
It is performance.3 concentration and probe concentrations (a:0.1 μM, b:0.2 μM, c:0.3 μM) is set
PCR reaction system is prepared respectively.Wild type and mutated genes group DNA sample are detected with the above system, than less
With the difference of amplification capability under concentration and probe concentration.Test result is as shown in Figure 3 and Figure 4, wherein Fig. 3 is 3 concentration and probe concentrations to wild
The result figure that pattern is originally expanded, Fig. 4 are the result figure that 3 concentration and probe concentrations expand saltant type sample.
Experimental result is it is found that c group concentration and probe concentration, i.e. 0.3 μM of SEQ ID NO.27,0.3 μM of SEQ ID NO.28 probe
The PCR system amplification capability of composition is best, and signal value is high, and specificity is good.
The optimization of 3 PCR buffer composition of embodiment
Under normal conditions, nearby repetitive sequence (especially > 3 G repetitive sequence) can reduce probe to target to SNP site
The recognition capability of SNP site.For target detection gene PAI-1 (4G > 5G) due to the presence of repetitive sequence, specificity is poor, intolerant to
By high concentration (such as 300ng/ μ L) genome sample.Fail to improve its specificity by the conventional dimethylene sulfoxide of addition,
Therefore, it uses instead and adds a kind of new organic compounds containing sulfur-tetramethylene sulfoxide into PCR buffer.Tetramethylene sulfoxide mentions
The performance of high PCR specificity is better than dimethylene sulfoxide, but it is particularly critical accurately to screen its optium concentration.Tetramethylene sulfoxide is used
It measures excessive easily generate PCR to inhibit, dosage is too small, and system specificity improvement is limited.Therefore, which has been carried out most preferably
The screening of concentration.It is final to determine system best performance under 5% tetramethylene sulfoxide.
Two kinds of PCR reaction solutions, a kind of addition tetramethylene sulfoxide are prepared respectively, another kind adds dimethylene sulfoxide,
Its component is completely the same.300ng/ μ L wild type gene group sample is detected with the above system.
As a result as shown in figure 5, when testing result shows to detect wild type sample, the system added with dimethylene sulfoxide
Detect mutation fluorescence signal, system specificity is poor, and mutation fluorescence is not detected in the system added with tetramethylene sulfoxide
Signal, specificity are good.Therefore, the PCR system specificity for adding tetramethylene sulfoxide is good, is resistant to 300ng/ μ L genomic DNA.
The site PAI-1 expands wild type sample results under the different organic compounds containing sulfurs of table 4
| Additive | FAM | VIC | ROX |
| Tetramethylene sulfoxide | 26.32 | No Ct | 26.26 |
| Dimethylene sulfoxide | 26.27 | 33.14 | 26.14 |
4 dNTPs constituent optimization of embodiment
It is the common methods for improving PCR amplification specificity using thermal starting enzyme.To further increase PCR specificity, reagent
Box replaces routine dNTPs using thermal starting dNTPs.Thermal starting dNTPs and routine dNTPs is respectively adopted, prepares PCR reaction solution.
Wild type gene group sample is detected with the above reaction solution.
As a result as shown in fig. 6, testing result shows that the system when detecting wild type sample, added with conventional dNTPs is examined
Mutation fluorescence signal is measured, system specificity is poor, and mutation fluorescence letter is not detected in the system added with thermal starting dNTPs
Number, specificity is good.
The site PAI-1 expands wild type sample results under 5 difference dNTPs of table
| dNTPs | FAM | VIC | ROX |
| Thermal starting dNTPs | 26.35 | No CT | 26.23 |
| Conventional dNTPs | 25.56 | 36.89 | 25.66 |
In conclusion being adjusted to suitable concentration and probe concentration, optimization PCR buffering by screening suitable primer combination of probe
Liquid component, dNTPs etc. are explored by a variety of trials, and many-sided improved method combination is finally just able to achieve excellent systemic property
Energy.
Embodiment 5 is used to detect the kit of aspirin resistance related gene polymorphism
Kit using PCR reaction premixed liquid design, mainly by COX1 reaction solution, COX2 reaction solution, GPIIIa reaction solution,
PEAR1 reaction solution, P2Y1 reaction solution, GPIa reaction solution, PAI-1 reaction solution, positive control and blank control composition.Kit
It include PCR buffer, dNTPs, primer and probe, Taq enzyme in PCR reaction solution, concrete composition is shown in Table 6.
6 kit forms of table
6 kit Performance Evaluation of embodiment
Evaluation on specificity: taking 300ng/ μ L wild type gene group DNA sample, detected with prepared PCR reaction solution,
It is required that the channel FAM normally expands, Ct value≤36, VIC channel is without amplification, Ct value > 36 or without Ct value.
As a result as shown in fig. 7, testing result shows that kit expands the wild type gene group at concentrations up to 300ng/ μ L
DNA sample, the channel FAM normally expand, and fluorescence signal is not detected in the channel VIC.Illustrate, the kit specificity is good, is resistant to
By 300ng/ μ L genomic DNA sample.
Sensitivity evaluation: taking 0.1ng/ μ L heterozygous genomic DNA sample, detected with prepared PCR reaction solution,
It is required that the channel FAM, the channel VIC and ROX internal standard channel normally expand, and value≤36 Ct.
As a result as shown in figure 8, testing result shows kit amplification down to the heterozygous genomic DNA sample of 0.1ng/ μ L
This, the channel FAM, the channel VIC and ROX internal standard channel normally expand, and value≤36 Ct.Illustrating, the kit sensitivity is good,
It can detect down to 0.1ng/ μ L genomic DNA sample.
It is prominent to 7 SNP site homozygous wildtypes, homozygosis respectively with the kit containing positive control and negative control simultaneously
Modification (COX2, GPIIIa, P2Y1 site mutation frequency are lower, the Plasmid DNA simulation sudden change sample manually constructed) and inspection
It surveys limit reference material (heterozygous mutant sample) to be detected, as a result as shown in Fig. 9-Figure 29.
The detection of 7 kit of embodiment and the comparison of Sanger sequencing assay result
1, sample collection and extraction
Collect 100 whole blood samples, it is desirable that 2~8 DEG C of sample saved no more than 30 days.Using Wuhan friend's sesame friend's medical science and technology
(article No. YZYMT-028, Hubei Province Chinese tool is for 20180027) progress for the poba gene group DNA extraction kit of limited liability company's exploitation
The extraction of poba gene group DNA.After the completion of DNA extracting, with its purity of micro UV spectrophotometer measuring, it is desirable that sample
OD260/280Between 1.6-2.0.
2, kit uses
Kit is taken out from refrigerator, is balanced to room temperature, and each component sufficiently melts, of short duration centrifugation 10 seconds.According to 23 holes μ L/
Every kind of reaction solution is dispensed into PCR reaction tube by dispensed loading amount respectively.By the genomic DNA, positive control, blank pair of sample to be tested
According to, be separately added into the reaction tube for having been loaded with PCR reaction solution, additional amount be 2 holes μ L/.Cover PCR reaction lid, of short duration centrifugation.
PCR pipe is put into quantitative fluorescent PCR instrument, fluorescent PCR augmentation detection is carried out.Pcr amplification reaction condition are as follows:
First stage: 95 DEG C of denaturation 5min；
Second stage: 95 DEG C of 15s, 60 DEG C of 60s (stage collects fluorescence signal), 40 circulations.
As a result interpretation
After reaction, according to amplification curve, suitable baseline and fluorescence threshold delimited, obtains different channel C t values.
Kit availability deciding:
Positive control: value≤32 FAM, VIC, ROX channel C t, amplification curve have obvious Exponential growth stage.
Blank control: the channel FAM, VIC, ROX is straight line or slight oblique line, no Ct without amplification curve or amplification curve
Value or value >=38 Ct.
The judgement of sample availability:
Internal standard gene: ROX channel C t value < 38 in all pattern detections, amplification curve has obvious Exponential growth stage.
The judgement of testing result:
Pattern detection result is determined according to the following table 7, determines sample gene pleiomorphism.
7 result judgement of table
3, gene tester compares
Above 100 samples are detected with this kit and Sanger PCR sequencing PCR respectively, Sanger sequencing commission is military
The healthy Biotechnology Co., Ltd of Chinese Chinese mugwort carries out.Sample genotype is counted after the completion of detection.By table 8-14 testing result
It is found that two methods testing result coincidence rate 100%.Show that this kit test result is accurate and reliable.
8 COX1 gene PCR method of table and Sanger sequencing hair testing result
9 COX2 gene PCR method of table and Sanger sequencing hair testing result
10 GPIIIa gene PCR method of table and Sanger sequencing hair testing result
11 PEAR1 gene PCR method of table and Sanger sequencing hair testing result
12 P2Y1 gene PCR method of table and Sanger sequencing hair testing result
13 GPIa gene PCR method of table and Sanger sequencing hair testing result
14 PAI-1 gene PCR method of table and Sanger sequencing hair testing result
8 COX1, COX2, GPIIIa, PEAR1, P2Y1, GPIa and PAI-1 gene pleiomorphism of embodiment and aspirin support
Decorrelation evaluation
Clear Aspirin is screened from ischemic cerebral apoplexy patient as the patient of secondary prevention as this research
Sample (totally 506 people), wherein 321 people of male, 185 people of women, the age 41~82 years old.Platelet aggregation is detected using light turbidimetry
Rate.1) 0.5mmol/L AA makees inducer, platelet aggregation rate >=20%；2) 10 μm of ol/L ADP make inducer, platelet aggregation
Collection rate >=70%.Meet at least one of above-mentioned two, belongs to aspirin resistance (AR)；Two equal those who do not meet are aspirin
Non resistance (non-aspirin resistance, NAR).Patient is divided into two groups, i.e. AS group and NAR according to platelet aggregation rate
Group.As a result 207 people of AS patient, 299 people of NAR patient, AR incidence 40.91%.
COX1 gene pleiomorphism genotype frequency is distributed in study population, and in AS group, wild type GG gene frequency is
16.72%.In NAS group, wild type GG gene frequency is 6.28%.The GG type genotype frequency of AS group is greater than NAS group, through counting
Credit is analysed, and COX1 gene loci genotype frequency distributional difference is statistically significant between two groups of patients.Therefore COX1-1676G etc.
Position gene is significant related to AR.
COX2 gene pleiomorphism genotype frequency is distributed in study population, and in AS group, heterozygote GC frequency is 19.00%,
No mutant homozygote CC frequency is 0%；In NAS group, heterozygote GC frequency is 10.11%, and no mutant homozygote CC frequency is 0%.AS
The GC type genotype frequency of group is all larger than NAS group, and through statistical analysis, COX2 gene loci genotype frequency is divided between two groups of patients
Cloth difference is statistically significant.Therefore COX2-765C allele is significant related to AR.
GPIIIa gene pleiomorphism genotype frequency is distributed in study population, and in AS group, heterozygote TC frequency is 5.92%,
No mutant homozygote CC frequency is 0%；In NAS group, heterozygote TC frequency is 1.72%, and no mutant homozygote CC frequency is 0%.AS group
TC type genotype frequency be all larger than NAS group, through statistical analysis, GPIIIa gene loci genotype frequency point between two groups of patients
Cloth difference is statistically significant.Therefore GPIIIa 1565C allele is significant related to AR.
PEAR gene pleiomorphism genotype frequency is distributed in study population, and in AS group, heterozygote GA frequency is 56.52%,
No mutant homozygote AA frequency is 19.32%；In NAS group, heterozygote GA frequency is 46.15%, and mutant homozygous AA frequency is
14.05%.GA the and AA type genotype frequency of AS group is all larger than NAS group, through statistical analysis, PEAR1 gene between two groups of patients
Loci gene type frequency distribution difference is statistically significant.Therefore PEAR1-3996A allele is significant related to AR.
P2Y1 gene pleiomorphism genotype frequency is distributed in study population, and in AS group, heterozygote CT frequency is 13.04%,
No mutant homozygote TT frequency is 0.48%；In NAS group, heterozygote CT frequency is 5.35%, and no mutant homozygote TT frequency is 0%.
CT the and TT type genotype frequency of AS group is all larger than NAS group, through statistical analysis, P2Y1 gene loci genotype between two groups of patients
Frequency distribution difference is statistically significant.Therefore P2Y1 893T allele is significant related to AR.
GPIa gene pleiomorphism genotype frequency is distributed in study population, and in AS group, heterozygote CT frequency is 47.83%,
No mutant homozygote TT frequency is 9.18%；In NAS group, heterozygote CT frequency is 37.79%, and no mutant homozygote TT frequency is
7.02%.CT the and TT type genotype frequency of AS group is all larger than NAS group, through statistical analysis, GPIa gene position between two groups of patients
Point gene type frequency distribution difference is statistically significant.Therefore GPIa 807T allele is significant related to AR.
PAI-1 gene pleiomorphism genotype frequency is distributed in study population, and in AS group, wild type -/- gene frequency is
43.96%；In NAS group, wild type -/- gene frequency is 28.43%.AS group -/- gene frequency be higher than NAS group, through counting
Credit is analysed, and PAI-1 gene loci genotype frequency distributional difference is statistically significant between two groups of patients.Therefore PAI-1-675 4G
Allele is significant related to AR.
Shown in concrete outcome table 15-21.
From the above experimental results, we know that COX1, COX2, GPIIIa, PEAR1, P2Y1, GPIa and PAI-1 gene and Ah Si
It is significant that woods resists correlation, carries out genotype detection to aspirin resistance related gene and facilitate aspirin individuation to use
Medicine.
The genotype and gene frequency of 15 COX1 polymorphic site of table compare
The genotype and gene frequency of 16 COX2 polymorphic site of table compare
The genotype and gene frequency of 17 GPIIIa polymorphic site of table compare
The genotype and gene frequency of 18 PEAR1 polymorphic site of table compare
The genotype and gene frequency of 19 P2Y1 polymorphic site of table compare
The genotype and gene frequency of 20 GPIa polymorphic site of table compare
The genotype and gene frequency of 21 PAI-1 polymorphic site of table compare
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Wuhan You Zhiyou medical science and technology limited liability company
<120>it is used for and the primer sets of detection aspirin resistance related gene polymorphic site and kit and its application
<160> 31
<170> PatentIn version 3.5
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ctgcactcaa aacaagaaac acttg 25
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gacttgaact cacttctgat tctgagg 27
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Claims (10)
1. a kind of for detecting and the primer sets of aspirin resistance related gene polymorphic site, which is characterized in that the primer
Group includes following 7 groups of primer pairs or at least one set of primer pair or probe in 7 groups of probes；
The primer pair includes: for detecting the primer pair in the site COX1 gene-1 676G > A, for detecting COX2 gene -765G
The primer pair in the site > C, the primer pair for detecting the site GPIIIa gene 1565T > C, for detect PEAR1 gene -3996G >
The primer pair in the site A, the primer pair for detecting the site P2Y1 gene 893C > T, for detecting the site GPIa gene 807C > T
Primer pair and for detecting PAI-1 gene -6754G > site 5G primer pair；
The probe includes: wild-type probe for detecting the site COX1 gene-1 676G > A and saltant type probe, for examining
Survey COX2 gene -765G > site C wild-type probe and saltant type probe, for detecting the site GPIIIa gene 1565T > C
Wild-type probe and saltant type probe, for detect PEAR1 gene -3996G > site A wild-type probe and saltant type probe,
For detect the site P2Y1 gene 893C > T wild-type probe and saltant type probe, for detecting the site GPIa gene 807C > T
Wild-type probe and saltant type probe and for detecting PAI-1 gene -6754G > site 5G wild-type probe and mutation
Type probe；
It is described for detect the primer pair in the site COX1 gene-1 676G > A to include shown in SEQ ID NO.1 and SEQ ID NO.2
Nucleotide sequence；
It is described for detect COX2 gene -765G > site C primer pair to include shown in SEQ ID NO.5 and SEQ ID NO.6
Nucleotide sequence；
It is described for detect the primer pair in the site GPIIIa gene 1565T > C to include SEQ ID NO.9 and SEQ ID NO.10 institute
The nucleotide sequence shown；
It is described for detect PEAR1 gene -3996G > site A primer pair to include SEQ ID NO.13 and SEQ ID NO.14 institute
The nucleotide sequence shown；
It is described for detect the primer pair in the site P2Y1 gene 893C > T to include shown in SEQ ID NO.17 and SEQ ID NO.18
Nucleotide sequence；
It is described for detect the primer pair in the site GPIa gene 807C > T to include shown in SEQ ID NO.21 and SEQ ID NO.22
Nucleotide sequence；
It is described for detect PAI-1 gene -6754G > site 5G primer pair to include SEQ ID NO.25 and SEQ ID NO.26
Shown in nucleotide sequence；
It is described for detect the wild-type probe in the site COX1 gene-1 676G > A to include nucleotides sequence shown in SEQ ID NO.3
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.4；
It is described for detect COX2 gene -765G > site C wild-type probe to include nucleotides sequence shown in SEQ ID NO.7
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.8；
It is described for detect the wild-type probe in the site GPIIIa gene 1565T > C to include nucleotide shown in SEQ ID NO.11
Sequence, saltant type probe include nucleotide sequence shown in SEQ ID NO.12；
It is described for detect PEAR1 gene -3996G > site A wild-type probe to include nucleotide shown in SEQ ID NO.15
Sequence, saltant type probe include nucleotide sequence shown in SEQ ID NO.16；
It is described for detect the wild-type probe in the site P2Y1 gene 893C > T to include nucleotides sequence shown in SEQ ID NO.19
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.20；
It is described for detect the wild-type probe in the site GPIa gene 807C > T to include nucleotides sequence shown in SEQ ID NO.23
Column, saltant type probe includes nucleotide sequence shown in SEQ ID NO.24；
It is described for detect PAI-1 gene -6754G > site 5G wild-type probe to include nucleosides shown in SEQ ID NO.27
Acid sequence, saltant type probe include nucleotide sequence shown in SEQ ID NO.28；
Wherein, 5 ' ends of the wild-type probe and the saltant type probe are connected separately with fluorophor；
The fluorophor of the wild-type probe of any gene and the fluorophor of saltant type probe are different；
3 ' ends of the wild-type probe and the saltant type probe are connected separately with quenching group.
2. primer sets according to claim 1, which is characterized in that the primer sets include 7 groups of primer pairs and 7 groups of probes.
3. primer sets according to claim 1, which is characterized in that the fluorophor be selected from FAM, VIC, TET, HEX,
Any one of JOE or ROX；
Preferably, the fluorogene of the wild-type probe of any gene is FAM, and the fluorogene of saltant type probe is VIC；
Preferably, the quenching group is selected from any one of NFQ, TAMRA, BHQ1 or BHQ2, preferably NFQ；
Preferably, MGB modification group has been independently connected in the wild-type probe and the saltant type probe, and the MGB is repaired
Decorations group is connect with the quenching group.
4. primer sets according to claim 1-3, which is characterized in that the primer sets further include for detecting
The internal control primer of GAPDH gene to and internal reference probe；
The internal control primer is to including nucleotide sequence shown in SEQ ID NO.29 and SEQ ID NO.30；
The internal reference probe includes nucleotide sequence shown in SEQ ID NO.31；
Wherein, 5 ' ends of the internal reference probe are connected with fluorophor, and the fluorophor of internal reference probe is wild with any gene
The fluorogene of type probe and saltant type probe is all different；
3 ' ends of the internal reference probe are connected with fluorescent quenching group；
Preferably, the fluorophor is selected from any one of FAM, VIC, TET, HEX, JOE or ROX, preferably ROX；
Preferably, the quenching group is selected from any one of NFQ, TAMRA, BHQ1 or BHQ2, preferably NFQ；
Preferably, the internal reference probe is connected with MGB modification group, and the MGB modification group is connect with the quenching group.
5. the described in any item primer sets of claim 1-4 are preparing the application in the product for detecting aspirin resistance.
6. application according to claim 5, which is characterized in that the product is reagent or kit.
7. a kind of for detecting and the kit of aspirin resistance related gene polymorphic site, which is characterized in that the reagent
Box includes the described in any item primer sets of claim 1-4.
8. kit according to claim 7, which is characterized in that the kit further includes that the kit further includes heat
Start archaeal dna polymerase, PCR reaction buffer 5 ×, thermal starting dNTPs and sterile water；
Preferably, the kit further includes positive control；
Preferably, the kit further includes blank control.
9. kit according to claim 8, which is characterized in that the PCR reaction buffer 5 × it include: 40-60mM
Tris-HCl、200-300mM KCl、40-60mM(NH4)2SO4、15-25mM MgCl2, 5-8M glycine betaine, tetra- methylene of 5-10%
Base sulfoxide and 15-30% glycerol, pH7.5-8.5.
10. kit according to claim 9, which is characterized in that the PCR reaction buffer 5 × it include: 50mM
Tris-HCl、250mM KCl、50mM(NH4)2SO4、20mM MgCl2, 6M glycine betaine, 5% tetramethylene sulfoxide and 20% sweet
Oil, pH=8.0.
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