CN103525925A - Pair of specific primers and probe for detection of CYP2C19 gene chip - Google Patents
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Abstract
The invention relates to the molecular biology field, and discloses a specific oligonucleotide probe for detection of SNP loci of rs4244285681G/A and rs4986893636G/A of a CYP2C19 gene and a pair of specific primers for amplification of the CYP2C19 gene during detection of the above SNP loci. The probe can hybridize with different genotypes of the CYP2C19 gene specifically, and the genotypes comprise a CYP2C19*1 type, a CYP2C19*2 type and CYP2C19*3 type. The products can be used for detection of metabolic abilities for relevant medicines (such as plavix, omeprazole, voriconazole and the like) and of Chinese population, clinical medication schemes can be guided and adjusted, clinical personalized medication can be provided with a basis, the curative effects can be raised and the risks of toxic and side effects can be reduced.
Description
Technical field
The present invention relates to biology field and detection of nucleic acids field, specific design for the Auele Specific Primer of CYP2C19 gene of increasing to the specific probe with for CYP2C19 genechip detection.
Background technology
Cytochrome P 450 isozymes also claims drug metabolizing enzyme, and the superfamily being comprised of a series of structurally and functionally related enzymes is the Major Enzymes system of drug metabolism in human body.P450 enzyme system forms complicated, by gene diversity, is controlled.Current known 12 subtribes that have at least.Many P450 isozyme have genetic polymorphism, make corresponding enzymic activity show difference, also different to the metabolic capacity of medicine.According to the difference to medicament metabolism ability, crowd is divided into: the Four types such as ultrafast metabolism (UM), fast metabolism (EM), medium metabolism (IM), slow metabolism (PM).
CYP2C19 is that Cytochrome P450 (Cytochrome P450, CYP) enzyme is the important a member in the second subfamily.Medicine (as chlorine pyrroles thunder, proton pump inhibitor, anticonvulsive drug etc.) by CYP2C19 metabolism is different with patient's genotype, and its curative effect and side effect also have obvious difference.By detecting CYP2C19 genotype in patient DNA, judge patient's drug metabolic rate type, can help doctor's selecting properly medicine and rationally adjust drug dose, improve drug use validity, and reduce toxic side effect.In Chinese, CYP2C19 allelotrope is mainly * 1, * 2, * 3 types.* the kinase inactive of 2, * 3 allelotrope codings, the incidence of the slow metabolism causing thus in Chinese is about 35%.Measure at present the genotypic method of CYP2C19 and mainly adopt the methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), craft or automatic sequencing, sequence specific primers PCR, complex operation, sense cycle is long, detected result is difficult for accurately, being difficult to meet the requirement of Clinical Laboratory.
One of great Progress & New Products of the tool characteristics of the times that occurred in high-tech area in recent years is gene chip.With genechip detection CYP2C19 gene hypotype, performance genechip detection is simple to operate, fast, result is feature accurately, it is by the gene probe (oligonucleotide probe of lots of genes information in energy reflected sample, cDNA clone, PCR product etc.), be fixed in order solid support (as aldehyde radical, amino, sulfydryl, slide glass or the silicon chip of carboxyl isoreactivity base group modification, nylon membrane, nitrocellulose filter) the upper array that forms, by carrying out hybridization with actual sample (or amplified production), only need once test, the information that just can high-throughput obtains all genes to be checked.The feature of this Parallel testing, high information flux makes its application at aspects such as genetic expression, gene pleiomorphism detections have very strong advanced and obvious advantage.For instructing clinical personalized medication, have important practical significance.
Summary of the invention
The object of the present invention is to provide a kind of specificity amplification primer for detection of CYP2C19 gene SNP site to specific oligonucleotide hybridization probe.
The few core propylhomoserin of this specificity for detection of CYP2C19 gene SNP site probe can be used for detecting CYP2C19*2(681G/A, rs4244285) and CYP2C19*3(636G/A, rs4986893) site.
Few core propylhomoserin probe sequence is as follows, during use, can from following three sequences, select one:
When described detection site is 681A, specific oligonucleotide probe sequence is for containing one of following nucleotide sequence:
（1）SEQ?ID?No.1：5'-ATTATTTCCC
AGGAACCCAT-3'；
（2）SEQ?ID?No.2：5'-TGATTATTTCCCAGGAACCCAT-3'；
（3）SEQ?ID?No.3：5'-TATTTCCCAGGAACCCAT-3'；
When described detection site is 681G, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（4）SEQ?ID?No.4：5'-ATTATTTCCCGGGAACCC-3'；
（5）SEQ?ID?No.5：5'-TTATTTCCCGGGAACCCAT-3'；
（6）SEQ?ID?No.6：5'-GATTATTTCCCGGGAACCCATA-3'；
When described detection site is 636G, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（7）SEQ?ID?No.7：5'-ACCCCCTGGATCCAGGTA-3'；
（8）SEQ?ID?No.8：5'-GCACCCCCTGGATCCAGGT-3'；
（9）SEQ?ID?No.9：5'-ACCCCCTGGATCCAGGTAAG-3'；
When described detection site is 636A, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（10）SEQ?ID?No.10：5'-ACCCCCTGAATCCAGGTAAG-3'；
（11）SEQ?ID?No.11：5'-GCACCCCCTGAATCCAGG-3'；
（12）SEQ?ID?No.12：5'-AGCACCCCCTGAATCCAG-3'。
Preferably, when described detection site is 681A, specific oligonucleotide probe is in SEQ ID No.1-3; When described detection site is 681G, specific oligonucleotide probe is in SEQ ID No.4-6; When described detection site is 636G, specific oligonucleotide probe is in SEQ ID No.7-9; When described detection site is 636A, specific oligonucleotide probe is in SEQ ID No.10-12.
Preferred, at the 5' of above-mentioned specific oligonucleotide probe end, also comprise one section of 5' amino (NH
2) the Poly dT(polydeoxy thymidine acid of modifying), length is 5-25.The 5' end that is specific oligonucleotide probe also comprises the poly-poly-deoxythymidylic acid of one section of 5-25.
While detecting above-mentioned CYP2C19 gene SNP site, the Auele Specific Primer using for these two sites of CYP2C19rs4244285681G/A and CYP2C19rs4986893636G/A of increasing is to as follows:
When described detection site is 681G/A, described primer pair sequence contains one of following nucleotide sequence:
(1) upstream SEQ ID No.13:5'-CTTGGCATATTGTATCTATACCTTT-3'
Downstream SEQ ID No.14:5'-CAAAACACAAATGATGCCTACAAAT-3'; Or,
(2) upstream SEQ ID No.15:5'-TACAACCAGAGCTTGGCATA-3'
Downstream SEQ ID No.16:5'-TACACTGACGAACGCATAAA-3'; Or,
(3) upstream SEQ ID No.17:5'-ACAATAAAAATTTCCCCATC-3'
Downstream SEQ ID No.18:5'-GTCCAGTTCCTCATTACGAA-3';
Described detection site is 636G/A, and described primer pair sequence contains one of following nucleotide sequence:
(4) upstream SEQ ID No.19:5'-CACCCTGTGATCCCACTTTC-3'
Downstream SEQ ID No.20:5 '-TCTGTCGGTACCCCACTTAT-3 '; Or,
(5) upstream SEQ ID No.21:5'-CCAATCATTTAGCTTCACCC-3'
Downstream SEQ ID No.22:5'-AAGGTGATTTGAGGTTTCGG-3', or,
(6) upstream SEQ ID No.23:5'-CACTTTCATCCTGGGCTG-3'
Downstream SEQ ID No.24:5'-ATGAAACCACTGTCGGGG-3'.
Preferably, when described detection site is 681G/A, one of described primer pair sequence is following nucleotide sequence:
(1) upstream SEQ ID No.13:5'-CTTGGCATATTGTATCTATACCTTT-3'
Downstream SEQ ID No.14:5'-CAAAACACAAATGATGCCTACAAAT-3'; Or,
(2) upstream SEQ ID No.15:5'-TACAACCAGAGCTTGGCATA-3'
Downstream SEQ ID No.16:5'-TACACTGACGAACGCATAAA-3'; Or,
(3) upstream SEQ ID No.17:5'-ACAATAAAAATTTCCCCATC-3'
Downstream SEQ ID No.18:5'-GTCCAGTTCCTCATTACGAA-3'
When described detection site is 636G/A, one of described primer pair sequence is following nucleotide sequence:
(4) upstream SEQ ID No.19:5'-CACCCTGTGATCCCACTTTC-3'
Downstream SEQ ID No.20:5 '-TCTGTCGGTACCCCACTTAT-3 ', or,
(5) upstream SEQ ID No.21:5'-CCAATCATTTAGCTTCACCC-3'
Downstream SEQ ID No.22:5'-AAGGTGATTTGAGGTTTCGG-3', or,
(6) upstream SEQ ID No.23:5'-CACTTTCATCCTGGGCTG-3'
Downstream SEQ ID No.24:5'-ATGAAACCACTGTCGGGG3'.
Preferred, the nucleotide sequence 5 ' end of above-mentioned primer pair SEQ ID No.13～24 is modified with vitamin H, digoxin, fluorescein, fluorescein derivative, fluorescence molecule, alkaline phosphatase or horseradish peroxidase.
Above-mentioned primer pair and specific oligonucleotide probe can be applicable to detect CYP2C19 gene rs4244285681G/A and rs4986893636G/A SNP site, and the step of detection method comprises:
(1) prepare the genomic dna of testing sample;
(2) adopt the gene fragment that comprises 681G/A and 636G/A in polymerase chain reaction method amplification CYP2C19 gene, with above-mentioned primer pair, increase;
(3) gained amplified production is mixed with hybridization buffer;
(4) by above-mentioned mixed solution and the hybridization of CYP2C19 gene SNP detection chip; Described CYP2C19 gene SNP detection chip comprises solid support and is fixed on the specific oligonucleotide probe on described solid support;
(5) hybridization signal of detection chip.
The method that detects hybridization signal in step (5) is selected from the blue color reaction of tetrazole of alkaline phosphatase enzyme catalysis, the bromo-4-of 5-chloro-3-indolol-phosphoric acid-4-Tolylamine color reaction or the fluoroscopic examination of horseradish peroxidase enzyme catalytic.
Above-mentioned specific oligonucleotide probe can be used for preparing CYP2C19 gene SNP detection chip or test kit.
Above-mentioned primer pair can be used for preparing CYP2C19 gene SNP detection chip or test kit.
Above-mentioned CYP2C19 gene SNP detection chip can be used for detecting the genotype in 681G/A and 636G/A site.
The preparation of said gene chip can be carried out according to the conventional manufacture method of biochip.For example, if what solid support adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with to solution, then with point sample instrument, its point is being modified to slide or silicon chip, be arranged in predetermined sequence or array, then by placement, spend the night and fix, just can obtain gene chip of the present invention.If oligonucleotide probe is containing amido modified, " the gene diagnosis technology-on-radiation operational manual " that its preparation method also can reference: Wang Shenwu chief editor; Derisi; JL equals 1997 at " science " 278(5338): " inquiring into metabolism and the Genetic Control of genetic expression in genome " (Dersi that 680-686 delivers; JL; Iyer VR, Brown PO.Exploring the metablic and genetic control of gene expression on a genomic scale.Science.1997; 278 (5338): 680-686) and the vertical people of horse wait the biochip of editing, Chemical Industry Press.
Method for the preparation of the chromosomal DNA of pcr amplification is a lot, can from whole blood, prepare, also can preparation from tissue.Concrete grammar can be consulted document (Ding Zhennuo chief editor " clinical PCR gene diagnosis technology 》， world book publishing company).
By the key of the specific fragment of PCR method amplifying genom DNA, be design of primers.
Getting appropriate amplified production adds in hybridization buffer and gene chip hybridization.During with gene chip hybridization, can first gene chip and prehybridization damping fluid be carried out to prehybridization.
Solid-phase hybridization between amplified production of the present invention and gene chip carries out according to the classical way of this area, and the general personnel in this area easily easily determine relevant damping fluid, probe and the optimum condition of sample concentration, prehybridization temperature, hybridization temperature and time etc. according to experience.Or also can be with reference to Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J. the chief editor's such as Pehanorm Brooker " molecular cloning experiment guide 》， Science Press.
Then according to marking signal, the acquisition of information such as the position on gene chip, intensity is treated measurement information.The method of detection gene chip hybridization signal of the present invention is alkaline phosphatase or the tetrazole blue (NBT) of horseradish peroxidase enzyme catalytic and the color reaction of the chloro-3-indolol-phosphoric acid-4-of the bromo-4-of 5-toluene amine salt (BCIP) or tetramethyl benzidine (TMB) based on being combined with each other with anti-biotin antibodies or avidin or streptavidin or anti digoxin antibody or anti-fluorescein antibody.Concrete grammar can be with reference to Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual ".If amplified production fluorophor mark, also can be with reference to obtaining and treat measurement information with fluorescence detection device (as laser confocal scanning instrument Scanarray3000 etc.).
Utilize the prepared CYP2C19 gene SNP detection chip of probe of the present invention to there is good signal to noise ratio, designed probe has good specificity, can hybridize with CYP2C19 different genotype specifically, comprise CYP2C19*1 type, CYP2C19*2 type and CYP2C19*3 type, can accurately distinguish all types of mutational sites, and described detection method step is simple, 2 SNP sites can a step have been detected.Adopt Auele Specific Primer pair of the present invention, can utilize gene amplification method, in specific amplification CYP2C19 gene, contain the target area of detecting target site.More convenient and the many uncertain factors having avoided existing in manual operation process of full-automatic crossover process.Probe provided by the invention, Auele Specific Primer to and test kit, can be used for detecting CYP2C19 genotype, judge that patient's medicine is (as Plavix, omeprazole, voriconazole) metabolic rate type, for clinical personalized medicine provides foundation, can improve curative effect, reduce toxic side effect risk, for providing immunotherapy targeted autoantibody that a kind of simple and easy to do solution is provided.
Accompanying drawing explanation
Fig. 1 is in embodiment 1, a kind of point sample array of specific probe on gene chip.
Fig. 2 is in embodiment 5, the photo of acquired results when the chip of use embodiment 1 and probe in detecting CYP2C19 gene.
Embodiment
Following examples are to more detailed description of the present invention, rather than limiting the scope of the invention.
The source of gene order used is NCBI (U.S. state-run biotechnology information center):
CYP2C19?681G/A?（rs4244285）
CYP2C19?636G/A?（rs4986893）
The preparation of embodiment 1 gene chip
Aldehyde group modified slide glass (production code member: BSM03011, Baiao Science and Technology Co. Ltd., Shanghai).The following probe of synthetic (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), water is dissolved as the solution of 100pmol/ul concentration, then uses 2 * point sample buffer(production code member: BST02010, Baiao Science and Technology Co. Ltd., Shanghai) geometric ratio mixing.Then described in the GSM417 point sample instrument by specification of ，Yong Affymetrix company method points system as the array of Fig. 1.Room temperature is placed and is spent the night.
Each probe sequence following (underscore is SNP site).
The specific oligonucleotide probe that detects 681A site is: NH2-TTTTTTTTTTTTTTTTATTATTTCCC
agGAACCCAT;
The specific oligonucleotide probe that detects 681G site is: NH2-TTTTTTTTTTTTTTTTATTATTTCCC
ggGAACCC;
The specific oligonucleotide probe that detects 636G site is: NH2-TTTTTTTTTTTTTTTTACCCCCTG
gaTCCAGGTA;
The specific oligonucleotide probe that detects 636A site is: NH2-TTTTTTTTTTTTTTTTACCCCCTG
aaTCCAGGTAAG.
Be that above-mentioned each probe is respectively SEQ ID No.1,4,7,10 in sequence table, and 5' end also comprise one section of 5' amino (NH
2) the 16 poly-Polyd T(polydeoxy thymidines acid of modifying).
As shown in Figure 1, on slide glass, put Quality Control point, 681A probe (SEQ ID No.1), 681G probe (SEQ ID No.4), 636G probe (SEQ ID No.7), 636A probe (SEQ ID No.10-12 any).Wherein, 0,1,2,3 and 4 be respectively Quality Control probe, 636G probe, 636A probe, 681G probe and 681A probe.
The preparation of embodiment 2 chromosomal DNAs
Using Baiao Science and Technology Co. Ltd., Shanghai's blood DNA to extract test kit by specification operates as follows:
Adsorption column activation:
Adsorption column is placed in to collection tube, adds 500 μ L buffer B H1, standing 2-3min, 12,000rpm(9,500 * g) centrifugal 30s; Discard the waste liquid in collection tube, adsorption column relay and reclaimed in collector, in adsorption column, add 500 μ L buffer B H2,12,000rpm(9,500 * g) centrifugal 30s, discard the waste liquid in collection tube, adsorption column is put back in collection tube stand-by.
Schedule of operation:
(1) Proteinase K of 20 μ L is joined to 1.5mL centrifuge tube bottom with pipettor.
(2) blood sample of 200 μ L is joined in centrifuge tube.
(3) add 200 μ L buffer B L in centrifuge tube, vibration mixes 15s.
(4) 56 ℃ of insulation 10min of centrifuge tube.
(5) of short duration centrifugal by the drop of centrifuge tube lid under.
(6) add 200 μ L dehydrated alcohols, vibration mixes 15s.Of short duration centrifugal by the drop of centrifuge tube lid under.
(7) get the adsorption column having activated and be enclosed within 2mL collection tube, above-mentioned mixed solution is carefully added in adsorption column, the mouth of pipe of not getting wet.Lid upper tube cap, 12,000rpm(9,500 * g) centrifugal 1min, abandon or adopt the collection tube that fills waste liquid, adsorption column is inserted in new collection tube.
(8) carefully open the pipe lid of adsorption column, add 500 μ L buffer B W1, the mouth of pipe of not getting wet.Lid upper tube cap, 12,000rpm(9,500 * g) centrifugal 1min, outwell waste liquid, adsorption column is inserted in collection tube.
(9) carefully open the pipe lid of adsorption column, add 500 μ L buffer B W2, the mouth of pipe of not getting wet.Lid upper tube cap, 12,000rpm(9,500 * g) centrifugal 1min, outwell waste liquid, adsorption column is inserted in collection tube.
(10) adsorption column is inserted in collection tube.12,000rpm(9,500 * g) centrifugal 1min.
(11) adsorption column is inserted to the aseptic centrifuge tube of clean 1.5mL, discard the collection tube of dress waste liquid.Carefully open the pipe lid of adsorption column, add elutriant BE or the deionized water of 60 μ L.Room temperature (15～25 ℃) is placed 5min, and then 12,000rpm(9,500) centrifugal 1min.
(12) it is standby that the DNA that extracting is good places-20 ℃ of Refrigerator stores.
Embodiment 3 use primer provided by the invention is by the PCR method CYP2C19 gene fragment that increases
Entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthetic primer, primer information is as follows.
During for detection of 681G/A site, amplification the primer to sequence is:
Upstream SEQ ID No.13:5'-CTTGGCATATTGTATCTATACCTTT-3'
Downstream SEQ ID No.14:5'-CAAAACACAAATGATGCCTACAAAT-3 '
During for detection of 636G/A site, amplification the primer to sequence is:
Upstream SEQ ID No.19:5'-CACCCTGTGATCCCACTTTC-3'
Downstream SEQ ID No.20:5'-TCTGTCGGTACCCCACTTAT-3'
And the 5 ' end at primer pair is modified with vitamin H.
Then by water dissolution and be diluted to 10pmol/ μ l.The pcr amplification template that the Taq enzyme (TaKaRa) of purchasing, 10 * damping fluid (TaKaRa), dNTP (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), pure water and embodiment 2 are obtained is pressed following formulated pcr amplification system:
Table 1
With PCR instrument (TC-96/G/H(b) pcr amplification instrument, rich day of Hangzhou) by following program, increase: 50 ℃ of 5min, 94 ℃ of 5min, then by 94 ℃ of 25sec, 48 ℃ of 40sec, 72 ℃ of 30sec do 35 circulations, last 72 ℃ of 5min.
The PCR product hybridization of the gene chip made from embodiment 1 and embodiment 3.
The hybridization colouring reagents box (BST03021) that adopts Baiao Science and Technology Co. Ltd., Shanghai to produce,
e-Hyb Full-automatic hybridization appliance (Baiao Science and Technology Co. Ltd., Shanghai: BSE03011), hybridize by the following method: hybridization colouring reagents box is alkaline phosphatase color reaction.
1) hybridization reaction solution preparation: draw 150ul hybridization solution, add each 10 μ L of amplified production in embodiment 3, mix.
2) by table 2, response procedures is set, by each reagent of table requirement consumption packing.And each reagent is put into specified location, and working procedure, hybridization color reaction is carried out automatically.
Table 2 hybridization system and response procedures
Embodiment 5
The detection of gene chip hybridization signal: be placed in BaioBE3.0 biochip identification reading instrument (Baiao Science and Technology Co. Ltd., Shanghai) above by hybridizing and washing complete gene chip, obtain detected result as shown in Figure 2 after scanning.Result shows that person under inspection's CYP2C19 genotype belongs to * 2/*2 type (636GG and 681AA), belongs to medicine slow inactivation.Detected result meets completely through sequencing result checking.
Effect detection, the primer and the probe that adopt the present invention to carry detect 20 routine samples, and adopt sequence measurement to verify.Result demonstration, 20 routine sequencing results are all consistent with detected result.
With SEQ ID No.2 or 3, replace SEQ ID No.1, SEQ ID No.5 or 6 replaces SEQ ID No.4, SEQ ID No.8 or 9 replaces SEQ ID No.7, SEQ ID No.11 or 12 replaces SEQ ID No.10, as shown in Figure 1, on putting on slide glass, Quality Control point, 681A probe, 681G probe, 636G probe and 636A probe, make gene detecting chip.It is the Poly dT(polydeoxy thymidine acid that 16 5' amino (NH2) modifies that the 5' end of each probe can also comprise a segment length), also can be containing polydeoxy thymidine acid.
During for detection of 681G/A site, amplification the primer is to selecting:
Upstream SEQ ID No.15:5'-TACAACCAGAGCTTGGCATA-3',
Downstream SEQ ID No.16:5'-TACACTGACGAACGCATAAA-3', or,
Upstream SEQ ID No.17:5'ACAATAAAAATTTCCCCATC3',
Downstream SEQ ID No.18:5'-GTCCAGTTCCTCATTACGAA-3'
During for detection of 636G/A site, amplification the primer is to selecting:
Upstream SEQ ID No.21:5'-CCAATCATTTAGCTTCACCC-3'
Downstream SEQ ID No.22:5'-AAGGTGATTTGAGGTTTCGG-3', or,
Upstream SEQ ID No.23:5'-CACTTTCATCCTGGGCTG-3'
Downstream SEQ ID No.24:5'-ATGAAACCACTGTCGGGG-3'.
And at the 5' of primer end biotin modification, detect effect with embodiment 5.
Claims (8)
1. one group of specific oligonucleotide probe for detection of CYP2C19 gene SNP site, is characterized in that, described specific oligonucleotide probe is for detection of the SNP site of CYP2C19rs4244285681G/A and CYP2C19rs4986893636G/A;
When described detection site is 681A, specific oligonucleotide probe sequence is for containing one of following nucleotide sequence:
（1）SEQ?ID?No.1：5'-ATTATTTCCC
AGGAACCCAT-3'；
（2）SEQ?ID?No.2：5'-TGATTATTTCCCAGGAACCCAT-3'；
（3）SEQ?ID?No.3：5'-TATTTCCCAGGAACCCAT-3'；
When described detection site is 681G, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（4）SEQ?ID?No.4：5'-ATTATTTCCCGGGAACCC-3'；
（5）SEQ?ID?No.5：5'-TTATTTCCCGGGAACCCAT-3'；
（6）SEQ?ID?No.6：5'-GATTATTTCCCGGGAACCCATA-3'；
When described detection site is 636G, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（7）SEQ?ID?No.7：5'-ACCCCCTGGATCCAGGTA-3'；
（8）SEQ?ID?No.8：5'-GCACCCCCTGGATCCAGGT-3'；
（9）SEQ?ID?No.9：5'-ACCCCCTGGATCCAGGTAAG-3'；
When described detection site is 636A, specific oligonucleotide probe sequence contains one of following nucleotide sequence:
（10）SEQ?ID?No.10：5'-ACCCCCTGAATCCAGGTAAG-3'；
（11）SEQ?ID?No.11：5'-GCACCCCCTGAATCCAGG-3'；
（12）SEQ?ID?No.12：5'-AGCACCCCCTGAATCCAG-3'。
Described in claim 1 for detection of the specific oligonucleotide probe of CYP2C19 gene SNP site, it is characterized in that,
When described detection site is 681A, one of specific oligonucleotide probe is following nucleotide sequence:
（1）SEQ?ID?No.1：5'-ATTATTTCCC
AGGAACCCAT-3'；
（2）SEQ?ID?No.2：5'-TGATTATTTCCCAGGAACCCAT-3'；
（3）SEQ?ID?No.3：5'-TATTTCCCAGGAACCCAT-3'；
When described detection site is 681G, one of specific oligonucleotide probe is following nucleotide sequence:
（4）SEQ?ID?No.4：5'-ATTATTTCCCGGGAACCC-3'；
（5）SEQ?ID?No.5：5'-TTATTTCCCGGGAACCCAT-3'；
（6）SEQ?ID?No.6：5'-GATTATTTCCCGGGAACCCATA-3'；
When described detection site is 636G, one of specific oligonucleotide probe is following nucleotide sequence:
（7）SEQ?ID?No.7：5'-ACCCCCTGGATCCAGGTA-3'；
（8）SEQ?ID?No.8：5'-GCACCCCCTGGATCCAGGT-3'；
（9）SEQ?ID?No.9：5'-ACCCCCTGGATCCAGGTAAG-3'；
When described detection site is 636A, one of specific oligonucleotide probe is following nucleotide sequence:
（10）SEQ?ID?No.10：5'-ACCCCCTGAATCCAGGTAAG-3'；
（11）SEQ?ID?No.11：5'-GCACCCCCTGAATCCAGG-3'；
（12）SEQ?ID?No.12：5'-AGCACCCCCTGAATCCAG-3'。
Described in claim 1 or 2 for detection of the specific oligonucleotide probe of CYP2C19 gene SNP site, it is characterized in that, its 5' end also comprises one section of poly-poly-deoxythymidylic acid of 5-25 that 5' is amido modified.
4. one group of primer pair for detection of CYP2C19 gene SNP site, is characterized in that, described primer pair is the Auele Specific Primer pair for increase CYP2C19rs4244285681G/A and CYP2C19rs4986893636G/A;
When described detection site is 681G/A, described primer pair sequence contains one of following nucleotide sequence:
(1) upstream SEQ ID No.13:5'-CTTGGCATATTGTATCTATACCTTT-3'
Downstream SEQ ID No.14:5'-CAAAACACAAATGATGCCTACAAAT-3';
(2) upstream SEQ ID No.15:5'-TACAACCAGAGCTTGGCATA-3'
Downstream SEQ ID No.16:5'-TACACTGACGAACGCATAAA-3';
(3) upstream SEQ ID No.17:5'-ACAATAAAAATTTCCCCATC-3'
Downstream SEQ ID No.18:5'-GTCCAGTTCCTCATTACGAA-3';
Described detection site is 636G/A, and described primer pair sequence contains one of following nucleotide sequence:
(4) upstream SEQ ID No.19:5'-CACCCTGTGATCCCACTTTC-3'
Downstream SEQ ID No.20:5'-TCTGTCGGTACCCCACTTAT-3'
(5) upstream SEQ ID No.21:5'-CCAATCATTTAGCTTCACCC-3'
Downstream SEQ ID No.22:5'-AAGGTGATTTGAGGTTTCGG-3'
(6) upstream SEQ ID No.23:5'-CACTTTCATCCTGGGCTG-3'
Downstream SEQ ID No.24:5'-ATGAAACCACTGTCGGGG-3'.
Described in claim 4 for detection of the primer pair of CYP2C19 gene SNP site, it is characterized in that, while being 681G/A for the described detection site that increases, described primer pair sequence is selected from one of following nucleotide sequence:
(1) SEQ ID No.13: upstream 5'-CTTGGCATATTGTATCTATACCTTT-3'
SEQ ID No.14: downstream 5'-CAAAACACAAATGATGCCTACAAAT-3 ';
(2) SEQ ID No.15: upstream 5'-TACAACCAGAGCTTGGCATA-3'
SEQ ID No.16: downstream 5'-TACACTGACGAACGCATAAA-3';
(3) SEQ ID No.17: upstream 5'-ACAATAAAAATTTCCCCATC-3'
SEQ ID No.18: downstream 5'-GTCCAGTTCCTCATTACGAA-3';
Described detection site is 636G/A, and described primer pair is selected from one of following nucleotide sequence:
(4) SEQ ID No.19: upstream 5'-CACCCTGTGATCCCACTTTC-3'
SEQ ID No.20: downstream 5-TCTGTCGGTACCCCACTTAT-3 '
(5) SEQ ID No.21: upstream 5'-CCAATCATTTAGCTTCACCC-3'
SEQ ID No.22: downstream 5'-AAGGTGATTTGAGGTTTCGG-3'
(6) SEQ ID No.23: upstream 5'-CACTTTCATCCTGGGCTG-3'
SEQ ID No.24: downstream 5'-ATGAAACCACTGTCGGGG3'.
Described in claim 4 or 5 for detection of the primer pair of CYP2C19 gene SNP site, it is characterized in that, the nucleotide sequence 5' end of described SEQ ID No.13～24 is modified with vitamin H, digoxin, fluorescein, fluorescein derivative, fluorescence molecule, alkaline phosphatase or horseradish peroxidase.
7. the specific oligonucleotide probe described in claim 1～3 any one is for the preparation of CYP2C19 gene SNP detection chip or test kit.
8. the primer pair described in claim 4～6 any one is for the preparation of CYP2C19 gene SNP detection chip or test kit.
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| CN103952481A (en) * | 2014-04-04 | 2014-07-30 | 宋竞岩 | Primer combination for detecting allele CYP2C19*3 and detection kit thereof |
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| CN105274097A (en) * | 2015-10-14 | 2016-01-27 | 北京晋祺生物科技有限公司 | CYP2C19*2 and CYP2C19*3 detection primer and detection system thereof |
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| CN103952481A (en) * | 2014-04-04 | 2014-07-30 | 宋竞岩 | Primer combination for detecting allele CYP2C19*3 and detection kit thereof |
| CN103952481B (en) * | 2014-04-04 | 2018-01-16 | 宋竞岩 | Primer for detecting allele C YP2C19*3 combines and detection kit |
| CN106047992A (en) * | 2015-04-01 | 2016-10-26 | 东洋钢钣株式会社 | Probe for CYP2C19*2 detection, and probe for CYP2C19*3 detection |
| JP2016192939A (en) * | 2015-04-01 | 2016-11-17 | 東洋鋼鈑株式会社 | Probe for detecting cyp2c19*2 and probe for detecting cyp2c19*3 |
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| CN108913766A (en) * | 2018-07-17 | 2018-11-30 | 浙江大学 | A kind of specific primer and probe and kit detecting depressed individuals chemical drug object therapeutic gene multisite mutation |
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| CN114262734A (en) * | 2021-11-23 | 2022-04-01 | 苏州华谦科技有限公司 | Detection method of Ticagrelor medication related cytochrome enzyme P450 family gene mutation site |
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