CN102643905A - Kit and method for detecting tamoxifen personalized medicine genetic polymorphism by use of pyrosequencing technique - Google Patents
Kit and method for detecting tamoxifen personalized medicine genetic polymorphism by use of pyrosequencing technique Download PDFInfo
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Abstract
The invention discloses a kit and method for detecting the tamoxifen personalized medicine genetic polymorphism by use of the pyrosequencing technique. The genetic polymorphism specifically refers to the single nucleotide polymorphism of CYP2D6*10(rs1065852) and (SULT1A1*2) (rs9282861). The kit comprises the primer shown by SEQ ID NO.3-8. The kit disclosed by the invention can realize accurate, quick and high-flux detection on CYP2D6*10(rs1065852) and (SULT1A1*2) (rs9282861) so as to realize safe, reasonable and effective personalized administration of the tamoxifen medicine.
Description
Technical field
The invention belongs to biology field, be specifically related to test kit and method that the tetra-sodium PCR sequencing PCR detects tamoxifen personalized medicine gene pleiomorphism.
Background technology
Tamoxifen is a kind of non-steroid estrogen antagonist medicine, is widely used in the patient with breast cancer's of estrogen receptor positive prevention and treatment at present.The metabolism of tamoxifen has multiple drug metabolism enzyme to participate in vivo, and about 10% tamoxifen is metabolized to active metabolite 4-hydroxy-n-demethyl tamoxifen (endoxifen) by CYP2D6.In Chinese population, the modal miopragia allelic mutation of CYP2D6 type is CYP2D6*10 (accounting for 53%).Accept in the therapy of TAM as assistant agent 200 patient with breast cancers, CYP2D6 poor metabolizer disease free survival is lower, thereby this and active metabolite endoxifen generate to slow down and cause the decline of curative effect consistent.In another 293 patient with breast cancers that receive treatment, carry the allelic T/T type of CYP2D6*10 patient and compare with C/T type or C/C type patient, 4-hydroxyl-tamoxifen in the blood (another active metabolite) is lower, and clinical effectiveness is relatively poor.The concentration of accepting active metabolite in patient with breast cancer's CYP2D6 genotype and the blood of tamoxifen treatment is closely related, and so recurrence rate and lifetime of remarkably influenced patient.Therefore U.S. FDA at first detects the genotype of CYP2D6 before suggestion patient in 2006 is accepting the tamoxifen treatment.
The ability of tamoxifen CE acceptor after the CYP2D6 metabolism is active metabolite 4-OH-tamoxifen strengthens greatly; This 4-OH-meta-bolites is by SULT1A1 sulfation metabolism inactivation, so the perhaps active curative effect for tamoxifen of SULT1A1 gene pleiomorphism has material impact.The enzymic activity that causes His213 (SULT1A1*2) reduces by 2 times and protein stability reduction, and causing the 4-OH-meta-bolites to be difficult to metabolism is active sulfate-tamoxifen, and the pharmacologically active of this secondary metabolite is higher by inference.Therefore, if this product can not form, chemotherapy effect and survival rate will reduce greatly.
In sum; CYP2D6*10 (rs1065852) and SULT1A1*2 (rs9282861) gene pleiomorphism are the principal elements that influences tamoxifen dosage requirements otherness, and the test kit of developing quick, efficient, accurate, convenient, economic detection CYP2D6*10 and SULT1A1*2 gene pleiomorphism will play positive pushing effect for the clinical individualized treatment of tamoxifen.
Tetra-sodium order-checking (Pyro sequencing) technology is a dna sequence analysis technology of new generation, and this technology need not be carried out electrophoresis, and dna fragmentation also need not fluorescent mark, is a kind of universal technology platform.Easy and simple to handle, characteristics such as low, the required sample size of detection cost is little, quick, accurate, high-throughput that this technology has meet clinical large sample and detect requirement.
Summary of the invention
CYP2D6*10 (rs1065852) and SULT1A1*2 (rs9282861) gene pleiomorphism are the main factors that influences tamoxifen dosage difference between individuals.The present invention provides a kind of medication gene C YP2D6 (SEQ ID NO.1) of the clinical tamoxifen personalized medicine treatment of influence and the test kit and method of SULT1A1 (SEQ ID NO.2) polymorphum of detecting, to realize quick, easy, accurate, efficient, practical, economic detection tamoxifen personalized medicine genes involved SNP.
In order to achieve the above object, technical scheme provided by the invention is:
A kind of tetra-sodium PCR sequencing PCR detects the test kit of tamoxifen personalized medicine gene pleiomorphism, comprises following primer:
(1) amplimer:
CYP2D6*10 (rs1065852) upstream primer: 5 '-GTA GTG AGG CAG GTA TGGG-3 ' (SEQ ID NO.3);
SULT1A1*2 (rs9282861) upstream primer: 5 '-GGA GAT TCA AAA GAT CCTGGA-3 ' (SEQ IDNO.4);
CYP2D6*10 (rs1065852) downstream primer: 5 '-CAG GAC CTC CTC CCT CAC CTGGTC-3 ' (SEQ IDNO.5);
SULT1A1*2 (rs9282861) downstream primer: 5 '-TTC ATC TCC TTG AAC GAC G-3 ' (SEQ IDNO.6);
Wherein, 5 of downstream primer ' carry out biotin labeling;
(2) sequencing primer:
CYP2D6*10 (rs1065852) sequencing primer: 5 '-GCA GGG GGC CTG GTG-3 ' is (SEQIDNO.7);
SULT1A1*2 (rs9282861) sequencing primer: 5 '-TGG AGT TTG TGG GGC-3 ' (SEQ ID NO.8);
Other reagent and solution are the conventional reagent of PCR and the order-checking of DNA tetra-sodium in the test kit.
A kind of application rights requires 1 described test kit to detect the method for tamoxifen medication gene pleiomorphism, comprises the steps:
(1) DNA extraction;
(2) polymerase chain reaction:
Prepare 50 μ lPCR amplification systems, comprise: 10 * PCR buffer, 10.0 μ l, dNTP 3.0 μ l, upstream primer 1.0 μ l, downstream primer 1.0 μ l, rTaq1.0 μ l, water 30.0 μ l, template 4.0 μ l; According to following loop parameter the amplification appearance is set: 95 ℃ of preparatory sex change of 5min; Then successively at 95 ℃ of 30S, 52 ℃ of 30S, 72 ℃ of 30S carry out 38 circulations; Keep 5min at 72 ℃ again, finally remain on 4 ℃, get amplified production;
(3) tetra-sodium order-checking strand sample purifying;
(4) tetra-sodium order-checking and interpretation of result.
Test kit of the present invention is analyzed and is detected CYP2D6*10 (rs1065852) target sequence and SULT1A1*2 (rs9282861) target sequence; Wherein, CYP2D6*10 (rs1065852) target sequence comprises: wild-type GGTAGCG (SEQ ID NO.9) and mutant AGTAGCG (SEQ ID NO.10), and the fragment length that amplifies is 212bp; SULT1A1*2 (rs9282861) target sequence comprises: wild-type GCTCCCTG (SEQ ID NO.11) and mutant ACTCCCTG (SEQ ID NO.12), the sheet segment length who amplifies is 93bp.
Owing to designed the high primer of specificity, and selected suitable method, test kit of the present invention to be applicable to tamoxifen personalized medicine gene is carried out rapid detection, can be widely used in the gene test of tamoxifen personalized medicine solution formulation clinically.Compared with prior art, it uses the tetra-sodium sequencing technologies can carry out the short dna sequential analysis quickly and accurately, is convenient to make up the normalizing operation flow process; Have characteristics such as high-throughput, low cost; The PCR product can directly be used for order-checking, need not carry out secondary treatments such as product purification, operates very easyly, and required sample size is little.
Description of drawings
Fig. 1 is a CYP2D6*10GG tetra-sodium sequencing result of the present invention;
Fig. 2 is a CYP2D6*10GA tetra-sodium sequencing result of the present invention;
Fig. 3 is a CYP2D6*10AA tetra-sodium sequencing result of the present invention;
Fig. 4 is a SULT1A1*2GG tetra-sodium sequencing result of the present invention.
Embodiment
Below in conjunction with embodiment mentioned reagent box and detection method are described in detail.
Embodiment 1:
CYP2D6*10 (rs1065852) upstream primer: 5 '-GTA GTG AGG CAG GTA TGG G-3 ' (SEQ ID NO.3);
SULT1A1*2 (rs9282861) upstream primer: 5 '-GGA GAT TCA AAA GAT CCTGGA-3 ' (SEQ IDNO.4);
CYP2D6*10 (rs1065852) downstream primer: 5 '-CAG GAC CTC CTC CCT CAC CTGGTC-3 ' (SEQ IDNO.5);
SULT1A1*2 (rs9282861) downstream primer: 5 '-TTC ATC TCC TTG AAC GAC G-3 ' (SEQ ID NO.6);
CYP2D6*10 (rs1065852) sequencing primer: 5 '-GCA GGG GGC CTG GTG-3 ' is (SEQIDNO.7);
SULT1A1*2 (rs9282861) sequencing primer: 5 '-TGG AGT TTG TGG GGC-3 ' (SEQID NO.8);
1.DNA extract
1.1 reagent material is prepared with inspection work following before the experiment:
(1) inspection test kit quality guaranteed period and guarantee to have added ethanol in Wash Buffer 1 and 2, and beat and collude √ at the respective identification place on bottle; (2) Virahol (as not having, available absolute ethyl alcohol substitutes) and 75% ethanol; (3) pipe of the 1.5mL Eppendorf in the autoclaving validity period and all kinds of liquid-transfering gun head.
1.2 from 4 ℃ of refrigerators, take out the EDTA anticoagulant tube that whole blood is housed, mixing for several times turns upside down;
1.3 manage corresponding sample uniqueness sign marked at 1.5mL Eppendorf;
1.4 pipette the 1.5mL Eppendorf pipe that 900uL Cell Lysis Solution adds to sterilization respectively;
1.5 carefully pipette the 1.5mL EP pipe that the 300uL whole blood is transferred to the above-mentioned Cell of being added with Lysis Solution;
1.6 cover Eppendorf pipe lid, incubated at room 10min;
1.713, centrifugal 20 seconds of 000rpm room temperature;
1.8 take out the Eppendorf pipe, observe white precipitate;
1.9 open Eppendorf pipe lid, hand-held pipe bottom, the inclination EP mouth of pipe discards the red supernatant of part, red supernatant is exhausted as far as possible;
1.10 cover the Eppendorf pipe,, make white precipitate resuspended with finger attack EP pipe bottom;
Go in the above-mentioned Eppendorf pipe 1.11 pipette 300uL Nuclei Lysis Solution, cover pipe, mixing for several times turns upside down;
1.12 open the Eppendorf pipe, pipette 100uL Protein Precipitation Solution and go in the above-mentioned Eppendorf pipe, cover the pipe pipe, thermal agitation is 20 seconds on the vibrator; 13, the centrifugal 3min of 000rpm room temperature;
Transfer to the new 1.5mL of sterilization Eppendorf pipe 1.13 pipette supernatant;
Go into the EP pipe 1.14 pipette the 300uL Virahol, the lid upper tube cap, the mixing for several times that turns upside down, visible white cotton-shaped gDNA separates out;
1.15 13, the centrifugal 1min of 000rpm room temperature;
1.16 open the Eppendorf pipe, hand is pinched pipe bottom, inclination mouth of pipe supernatant discarded;
Add the Eppendorf pipe 1.17 pipette 300uL 75% ethanol, lid upper tube cap, the washing precipitation of softly turning upside down;
1.18 13, the centrifugal 1min of 000rpm room temperature;
1.19 open the Eppendorf pipe, hand-held pipe bottom, inclination mouth of pipe supernatant discarded;
1.20 on experiment table, place new filter paper, back-off Eppendorf pipe blots liquid, with the Eppendorf pipe uncap be sidelong air-dry;
1.21 range estimation deposition size adds 50～100ul DNA Rehydration Solution to deposition;
Nucleic acid concentration mensuration is carried out with the Nano-Space ultraviolet spectrophotometer in the dissolving back 1.22 spend the night; It is qualified that nucleic acid concentration is regarded as greater than 50ng/ul; Not enough like concentration, add ethanol deposit D NA once more, add an amount of DNA Rehydration Solution dissolving DNA then again.
1.23 cover SD sample exclusive number once more at tube wall and pipe, and twine protection with scotch tape;
1.24 preserve nucleic acid sample to 4 ℃ refrigerator;
2. polymerase chain reaction
2.1 prepare 50 μ l pcr amplification systems (except the template interpolation) in the reagent area in preparation, each component and addition such as following table:
Annotate: upstream primer and downstream primer respectively have two kinds, and the 1.0 μ l here are meant that every kind of upstream primer adds 0.5 μ l, and every kind of downstream primer adds 0.5 μ l.
2.2 add 4.0 μ l in the sample preparations district to amplification system to filling the of short duration centrifugal back of gDNA template, in PCR tube wall marked sample uniqueness sign, pipe covers the marker detection item designation.PCR pipe concussion mixing, of short duration centrifugal on the desktop whizzer;
2.3 carry out pcr amplification reaction in amplification region, the amplification appearance be set according to following loop parameter:
2.4 in drop-down menu subsequently, select behind the setting program " tube ";
2.5 click the operation of " start " beginning instrument.
3. tetra-sodium order-checking strand sample purifying
3.1 reagent and instrument are prepared before the purifying:
Before carrying out the sample purifying, guarantee that all solution all reach room temperature; Open the precise temperature control process furnace, make temperature reach 80 ℃.
3.2 strand sample purification process:
3.2.1 in PSQ 96 plates, add 40 μ lAnnealing Buffer and 2～3 μ l sequencing primers (10uM) earlier;
3.2.2 abundant mixing Sepharose Beads on vibrator;
3.2.3 required Sepharose Beads amount (every sample 3 μ l calculate) is transferred to 1.5mL Eppendorf pipe;
3.2.4 in Sepharose Beads, add Binding Buffer, make average each sample that the volume the same with the PCR system arranged approximately, on vibrator with the abundant mixing of mixture;
3.2.5 Sepharose Beads mixture is added in about 40 μ l PCR products, and every sample adds 40 μ l;
3.2.6 under the normal temperature, on vibrator with PCR plate mixing 10 minutes;
3.2.7 in Vacuum prep workstation, add 180ml pure water, 120ml 70% ethanol, Denaturation Buffer and Washing Buffer in four liquid tanks successively;
3.2.8 outwell the waste liquid in the waste collection bucket that links to each other with vacuum pump;
3.2.9 open vacuum pump and the valve of Vacuum Prep Workstation, Vacuum Prep Tool cleaned in pure water 30 seconds;
3.2.10 Vacuum prep Tool is moved on in the PCR plate hole, grasps the Sepharose Beads that has combined biotin labeling nucleic acid;
3.2.11 pick up the PCR plate, whether inspection Beads all has been attracted on the Vacuum Prep Tool;
3.2.12 Vacuum Prep Tool was put into 70% ethanol 5 seconds;
3.2.13 Vacuum Prep Tool was moved on among the Denatureation Buffer 5 seconds;
Cleaned 10 seconds 3.2.14 again Vacuum Prep Tool is moved on among the Washing Buffer;
3.2.15 the outstanding Pyro Sptting plate that is placed on of Tool;
3.2.16Vacuum Prep Tool puts into the Sptting plate that contains sequencing primer, rotation is shaken, to discharge Sepharose Beads;
Be placed on the Thermo Plate 3.2.17 be placed with PSQ 96 plates of purifying sample, place 80 ℃ of process furnace to heat 2min, naturally cool to room temperature after the taking-up, carry out downstream Pyrosequencing reaction.
The back is cleaned 3.3 purifying finishes:
3.3.1 do not open vacuum pump and valve, use a small amount of pure water to clean Vacuum Prep Tool, the Beads that does not come off is on a small quantity eluted;
3.3.2 open vacuum pump and valve behind the replacing pure water again, clean Tool with about 300mL pure water;
3.3.3 turn off vacuum pump and valve, Vacuum Prep Tool to be sidelong, room temperature is dried;
3.3.4 clean the plastic channel of all splendid attire reagent solutions, dry naturally;
3.3.5 with wet cloth wiping purifier apparatus surface.
4. tetra-sodium order-checking
4.1 call in the run program file of aforementioned setting, click the drop-down key of " View ", select " Run ", calculate each reagent usage quantity of this experiment automatically according to software, add each reagent composition to the reagent storehouse;
4.2 put into the corresponding position of instrument to ready sample and reagent cabin, click " Run " beginning tetra-sodium order-checking of screen bottom righthand side;
4.3 after detecting completion, " close " key of at first clicking the software process status window is to preserve sequencing result.
5. the tetra-sodium sequencing result is analyzed
In " SNP Runs " folder, double-click mouse, open above-mentioned operating file, select " SNP mode ", click " Analyze All " key, all are detected sample carry out gene type assay; Select " AQ mode ", click " Analyze All " key, all are detected sample carry out the gene frequency analysis.To pattern detection CYP2D6*10 and SULT1A1*2 gene pleiomorphism, tetra-sodium detected result such as Fig. 1～4.Can find out, adopt test kit of the present invention and method, can simple and direct, intuitive and accurate CYP2D6*10 and SULT1A1*2 gene pleiomorphism be detected and interpretation.Fig. 1～3 prompting CYP2D6*10 are respectively wild-type homozygote, mutant heterozygote, mutant homozygote, and it is the wild-type homozygote that Fig. 4 points out SULT1A1*2.The clinician can be according to CYP2D6*10 and SULT1A1*2 gene pleiomorphism, curative effect and toxic side effects when judging the different genotype patient and using the tamoxifen treatment.
To sum up; The target sequence that the present invention is selected; And use test kit of the present invention and can realize quick, easy, accurate, efficient, practical, economic detection CYP2D6*10 and SULT1A1*2 gene pleiomorphism; Can satisfy the requirement of Clinical Laboratory real work, the individuation that is beneficial to tamoxifen is used.
Claims (2)
1. the test kit of a tetra-sodium PCR sequencing PCR detection tamoxifen personalized medicine gene pleiomorphism is characterized in that, comprises following primer:
(1) amplimer:
Upstream primer: 5 '-GTA GTG AGG CAG GTA TGG G-3 ';
5′-GGA?GAT?TCA?AAA?GAT?CCT?GGA-3′；
Downstream primer: 5 '-CAG GAC CTC CTC CCT CAC CTG GTC-3 ';
5′-TTC?ATC?TCC?TTGAAC?GAC?G-3′；
Wherein, 5 of downstream primer ' carry out biotin labeling;
(2) sequencing primer: 5 '-GCA GGG GGC CTG GTG-3 ';
5′-TGG?AGT?TTG?TGG?GGC-3′。
2. an application rights requires 1 described test kit to detect the method for tamoxifen personalized medicine gene pleiomorphism, comprises the steps:
(1) DNA extraction;
(2) polymerase chain reaction:
Prepare 50 μ lPCR amplification systems, comprise: 10 * PCR buffr10.0 μ l, dNTP 3.0 μ l, upstream primer 1.0 μ l, downstream primer 1.0 μ l, rTaq1.0 μ l, water 30.0 μ l, template 4.0 μ l; Cycling program is: 95 ℃ of preparatory sex change of 5min; Successively at 95 ℃ of 30S, 52 ℃ of 30S, 72 ℃ of 30S carry out 38 circulations; 72 ℃ keep 5min, finally remain on 4 ℃, get amplified production;
(3) tetra-sodium order-checking strand sample purifying;
(4) tetra-sodium order-checking and interpretation of result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210094422.0A CN102643905B (en) | 2012-04-01 | 2012-04-01 | Kit and method for detecting tamoxifen personalized medicine genetic polymorphism by use of pyrosequencing technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201210094422.0A CN102643905B (en) | 2012-04-01 | 2012-04-01 | Kit and method for detecting tamoxifen personalized medicine genetic polymorphism by use of pyrosequencing technique |
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| CN104862308A (en) * | 2014-02-26 | 2015-08-26 | 文洁 | Kit for tamoxifen medication guidance |
| CN106244708A (en) * | 2016-08-30 | 2016-12-21 | 长沙三济生物科技有限公司 | The Pyrosequencing primer of qualitative detection CYP2D6 gene type to and test kit |
| CN107841546A (en) * | 2016-09-20 | 2018-03-27 | 宁波美丽人生医药生物科技发展有限公司 | TAM curative effect related gene genetic polymorphism detection kit and detection method |
| CN108018340A (en) * | 2016-10-31 | 2018-05-11 | 广州康昕瑞基因健康科技有限公司 | CYP2D6 gene detecting kits and detection method |
| CN108070659A (en) * | 2017-12-27 | 2018-05-25 | 中国医学科学院肿瘤医院 | Application of the SNP markers in TAM Adjuvant Endocrine Therapy patient with breast cancer's curative effects are predicted |
| CN108389625A (en) * | 2018-02-09 | 2018-08-10 | 元码基因科技（北京）股份有限公司 | Computer system for assessing relapse and metastasis risk after drug therapy |
| CN109295178A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the fluorescence in situ hybridization of SULT1A1 and mgmt gene SNP |
| CN113151441A (en) * | 2021-04-12 | 2021-07-23 | 湖南菲思特精准医疗科技有限公司 | Gene detection kit for beta receptor antagonist medication and method and application thereof |
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| CN108018340A (en) * | 2016-10-31 | 2018-05-11 | 广州康昕瑞基因健康科技有限公司 | CYP2D6 gene detecting kits and detection method |
| CN108070659A (en) * | 2017-12-27 | 2018-05-25 | 中国医学科学院肿瘤医院 | Application of the SNP markers in TAM Adjuvant Endocrine Therapy patient with breast cancer's curative effects are predicted |
| CN108070659B (en) * | 2017-12-27 | 2020-05-05 | 中国医学科学院肿瘤医院 | Application of SNP marker in predicting curative effect of TAM (prostate cancer) assisted endocrine therapy on breast cancer patient |
| CN108389625A (en) * | 2018-02-09 | 2018-08-10 | 元码基因科技（北京）股份有限公司 | Computer system for assessing relapse and metastasis risk after drug therapy |
| CN109295178A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | Detection method is sequenced in the fluorescence in situ hybridization of SULT1A1 and mgmt gene SNP |
| CN113151441A (en) * | 2021-04-12 | 2021-07-23 | 湖南菲思特精准医疗科技有限公司 | Gene detection kit for beta receptor antagonist medication and method and application thereof |
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