CN102796813A - Assay kit and method for testing CYP1B1 gene polymorphism through pyrosequencing method - Google Patents

Abstract

The invention discloses an assay kit and a method for testing CYP1B1 gene polymorphism through a pyrosequencing method. The assay kit is used for testing the CYP1B1 gene polymorphism, specifically rs1056836(C) G mononucleotide polymorphism. The assay kit comprises a primer, such as SEQIDN.2-4. The assay kit can be used for testing the CYP1B1 gene polymorphism in an accurate, quick and high flux manner, and thus, the individualized drug administration of substrate anti-microtubule like chemotherapeutics and the like can be realized safely, reasonably, effectively.

Description

The tetra-sodium PCR sequencing PCR detects the test kit and the method for CYP1B1 gene pleiomorphism

Technical field

The invention belongs to biology field, be specifically related to test kit and method that the tetra-sodium PCR sequencing PCR detects the CYP1B1 gene pleiomorphism.

Background technology

The anticancer spectrum of anti-microtubule class medicine is very wide, one of the most widely used chemotherapeutic.Taxol is participated in the polymerization of tubulin, generates MAP, makes the tubulin subunit molecular increase to 60～76kDa.Its form of microtubule after the taxol effect changes, and is not connected with karyomit(e), makes cell surface projection occur, and suppresses mitotic division, from the performance antineoplastic action.Tumour cell G2 late period and M phase are comparatively responsive.Medicine commonly used has taxol, Docetaxel, vinealeucoblastine(VLB), vincristine(VCR) and vinorelbine etc.Paclitaxel treatment ovarian cancer, mammary cancer have good result, and treatment prostate cancer, upper digestive tract cancer, cellule type and lung cancer in non-cellule type are had good prospects.

CYP1B1 be a kind of in many tumor tissues relevant with hormone the CYP450 enzyme of specificity overexpression, can cause antitumor drug metabolism inactivations such as Docetaxel, produce resistance.CYP1B1 C4326G sudden change causes CYP1B1 mRNA and protein expression to increase, thereby the taxanes curative effect of medication is reduced.Accept in taxol/Dx treatment the patient with breast cancer; With 100 months served as to observe terminal point; The sudden change patient (Leu432Val, Val432Val) median survival interval is 30 months, and wild-type patient's (Leu432Leu) median survival interval does not reach (P=0.037) yet.

In sum; The CYP1B1 gene pleiomorphism can be used as the level of signification of the outcome prediction of taxanes chemotherapeutics etc., and the test kit of developing quick, efficient, accurate, convenient, economic detection CYP1B1 gene pleiomorphism will play positive pushing effect for the clinical individualized treatment of CYP1B1 substrate medicine (like taxanes chemotherapeutics etc.).

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

The present invention aims to provide test kit and the method that a kind of tetra-sodium PCR sequencing PCR detects CYP1B1 gene (SEQ ID NO.1) polymorphum; To realize quick, easy, accurate, efficient, practical, economic detection CYP1B1 gene pleiomorphism, be beneficial to clinical individuation and use the substrate medicine of CYP1B1 (like the taxanes chemotherapeutics etc.).

In order to achieve the above object, technical scheme provided by the invention is:

The test kit that detects the CYP1B1 gene pleiomorphism is sent out in said tetra-sodium order-checking, comprises following primer:

(1) amplimer:

Upstream primer: 5 '-GCG CTT CTC CAG CTT TGT GCC T-3 ' (SEQ ID NO.2);

Downstream primer: 5 '-AGG TCC TTG TTG ATG AGG CCA-3 ' (SEQ ID NO.3);

Wherein, 5 ' of downstream primer carries out biotin labeling;

(2) sequencing primer: 5 '-GGT CTG TGA ATC ATG ACC-3 ' (SEQ ID NO.4);

In practical application, can make appropriate change to above-mentioned primer, reach more than 95% as long as guarantee designed primer and above-mentioned primer homology; Other reagent and solution are the conventional reagent of PCR and the order-checking of DNA tetra-sodium in the test kit.

Said application mentioned reagent box detects the method for CYP1B1 gene pleiomorphism, comprises the steps:

(1) DNA extraction;

(2) polymerase chain reaction:

Prepare 50 μ l pcr amplification systems, comprise: 10 * PCR buffer, 5.0 μ l, dNTP 1.5 μ l, CYP1B1-upstream primer 0.5 μ l, CYP1B1 downstream primer 0.5 μ l, rTaq0.5 μ l, water 40 μ l, template 2 μ l; Cycling program is: 95 ℃ of preparatory sex change of 5min; Successively 95 ^oC 30S, 50 ^oC 30S, 72 ^oC 30S carries out 35 circulations; 72 ^oC keeps 5min, finally remains on 4 ℃, gets 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 to CYP1B1 rs1056836 (C>G) target sequence, and this target sequence comprises: wild-type CACTGAA (SEQ ID NO.5) and mutant CAGTGAA (SEQ ID NO.6); The sheet segment length who amplifies is 201bp.

Owing to designed the high primer of specificity; And selected suitable method; Test kit of the present invention can carry out rapid detection to the CYP1B1 gene pleiomorphism, can be widely used in the gene test of personalized medicine solution formulation such as taxanes chemotherapeutics 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 CYP1B1 rs1056836 of the present invention (CC) tetra-sodium sequencing result;

Fig. 2 is CYP1B1 rs1056836 of the present invention (CG) tetra-sodium sequencing result;

Fig. 3 is CYP1B1 rs1056836 of the present invention (GG) tetra-sodium sequencing result.

Embodiment

Below in conjunction with embodiment mentioned reagent box and detection method are described in detail.

Embodiment 1:

CYP1B1-pyroF (upstream primer): 5 '-GCG CTT CTC CAG CTT TGT GCC T-3 ' (SEQ ID NO.2);

CYP1B1-pyroR (downstream primer): 5 '-AGG TCC TTG TTG ATG AGG CCA-3 ' (SEQ ID NO.3);

Sequencing primer: 5 '-GGT CTG TGA ATC ATG ACC-3 ' (SEQ ID NO.4);

1. DNA extraction

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:

2.2 add 2 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;

3.2.16 Vacuum 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 CYP1B1 gene pleiomorphism, tetra-sodium detected result such as Fig. 1 ~ 3.Can find out, adopt test kit of the present invention and method, can simple and direct, intuitive and accurate the CYP1B1 genotype be detected and interpretation.Fig. 1 ~ 3 prompting CYP1B1 are respectively wild-type homozygote, mutant heterozygote, mutant homozygote, and the clinician can be according to the CYP1B1 genotype, the curative effect when judging the different genotype patient and using CYP1B1 substrate medicine (like the taxanes chemotherapeutics etc.).

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 CYP1B1 genotype; Can satisfy the requirement of Clinical Laboratory real work, the individuation that is beneficial to CYP1B1 substrate (like the taxanes chemotherapeutics etc.) is used.

SEQUENCE?LISTING

 

< 110>Zhou Honghao

< 120>the tetra-sodium PCR sequencing PCR detects CYP1B1 gene pleiomorphism test kit and method

<160> 6

<170> PatentIn?version?3.3

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<211> 601

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taagaatttt?gctcacttgc?ttttctctct?ccacattaaa?caccaaacag?gtatcctgat 60

gtgcagactc?gagtgcaggc?agaattggat?caggtcgtgg?ggagggaccg?tctgccttgt 120

atgggtgacc?agcccaacct?gccctatgtc?ctggccttcc?tttatgaagc?catgcgcttc 180

tccagctttg?tgcctgtcac?tattcctcat?gccaccactg?ccaacacctc?tgtcttgggc 240

taccacattc?ccaaggacac?tgtggttttt?gtcaaccagt?ggtctgtgaa?tcatgaccca 300

stgaagtggc?ctaacccgga?gaactttgat?ccagctcgat?tcttggacaa?ggatggcctc 360

atcaacaagg?acctgaccag?cagagtgatg?attttttcag?tgggcaaaag?gcggtgcatt 420

ggcgaagaac?tttctaagat?gcagcttttt?ctcttcatct?ccatcctggc?tcaccagtgc 480

gatttcaggg?ccaacccaaa?tgagcctgcg?aaaatgaatt?tcagttatgg?tctaaccatt 540

aaacccaagt?catttaaagt?caatgtcact?ctcagagagt?ccatggagct?ccttgatagt 600

g 601

 

<210> 2

<211> 22

<212> DNA

< 213>homo sapiens

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gcgcttctcc?agctttgtgc?ct 22

 

<210> 3

<211> 21

<212> DNA

< 213>homo sapiens

<400> 3

aggtccttgt?tgatgaggcc?a 21

 

<210> 4

<211> 18

<212> DNA

< 213>homo sapiens

<400> 4

ggtctgtgaa?tcatgacc 18

 

<210> 5

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cactgaa 7

 

<210> 6

<211> 7

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cagtgaa 7

 

 

Claims (2)

1. the test kit that detects the CYP1B1 gene pleiomorphism is sent out in a tetra-sodium order-checking, it is characterized in that, comprises following primer:

(1) amplimer:

Upstream primer: 5 '-GCG CTT CTC CAG CTT TGT GCC T-3 ';

Downstream primer: 5 '-AGG TCC TTG TTG ATG AGG CCA-3 ';

Wherein, 5 ' of downstream primer carries out biotin labeling;

(2) sequencing primer: 5 '-GGT CTG TGA ATC ATG ACC-3 '.

2. an application rights requires 1 described test kit to detect the method for CYP1B1 gene pleiomorphism, it is characterized in that, comprises the steps:

(1) DNA extraction;

(2) polymerase chain reaction:

Prepare 50 μ l pcr amplification systems, comprise: 10 * PCR buffer, 5.0 μ l, dNTP 1.5 μ l, CYP1B1-upstream primer 0.5 μ l, CYP1B1 downstream primer 0.5 μ l, rTaq0.5 μ l, water 40 μ l, template 2 μ l; Cycling program is: 95 ℃ of preparatory sex change of 5min; Successively 95 ^oC 30S, 50 ^oC 30S, 72 ^oC 30S carries out 35 circulations; 72 ^oC keeps 5min, finally remains on 4 ℃, gets amplified production;

(3) tetra-sodium order-checking strand sample purifying;

(4) tetra-sodium order-checking and interpretation of result.

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