CN104232765A - Primer pair and kit for detecting BCR-ABL fusion genes by using pyrosequencing method - Google Patents
Primer pair and kit for detecting BCR-ABL fusion genes by using pyrosequencing method Download PDFInfo
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- 238000012175 pyrosequencing Methods 0.000 title abstract description 4
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 238000012163 sequencing technique Methods 0.000 claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 9
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- 235000011180 diphosphates Nutrition 0.000 claims description 11
- 230000000977 initiatory effect Effects 0.000 claims description 11
- 238000003908 quality control method Methods 0.000 claims description 7
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Abstract
The invention discloses a primer pair and a kit containing the primer pair for detecting BCR-ABL fusion genes by using a pyrosequencing method. By adopting the kit, multiple fusion genes of BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2) can be detected at one time. By designing primers for mRNA of the BCR-ABL fusion genes, and carrying out sequencing analysis on a product after reverse transcriptase PCR, the kit can be used for rapid, accurate and high-flux detection on the BCR-ABL fusion genes.
Description
Technical field
The invention belongs to biology field, be specifically related to the primer pair that a kind of Manganic pyrophosphate complex initiation method detects BCR-ABL fusion gene, comprise the test kit of described primer pair and the application of this test kit.
Background technology
BCR-ABL fusion gene comprises the broad varietys such as BCR-ABL (b2a2), BCR-ABL (b3a2), BCR-ABL (e1a2), wherein BCR-ABL (b2a2), BCR-ABL (b3a2) are common in chronic myelocytic leukemia (CML), and BCR-ABL (e1a2) is fusion gene common in acute lymphoblastic leukemia (ALL).
In the method for the outer widely used molecular Biological Detection BCR-ABL fusion gene of Present Domestic, fluorescence in situ hybridization technique (FISH) can only carry out qualitative detection, complicated operation; Quantitative fluorescent PCR also exists the limitation detecting flux, so all really can't meet the needs of clinical diagnosis detection.Traditional solid phase biological chip (Biochip) or biochip technology also exist repeatable poor, insufficient sensitivity good and the outstanding weakness of complex operation.
Pyrosequencing techniques (Pyrosequencing) is DNA sequence analysis technology of new generation, possess the ability of simultaneously a large amount of sample being carried out to sequencing analysis, for high-throughput, low cost, carry out nucleotide analysis and Clinical Laboratory provides ideal technological operation platform quickly and intuitively.
Summary of the invention
Goal of the invention: for the technical problem existed in prior art, the present invention proposes the test kit that a kind of Manganic pyrophosphate complex initiation method detects BCR-ABL fusion gene, BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2) 3 kinds of fusion genes can be detected simultaneously, there is the advantages such as highly sensitive, high specific, split hair caccuracy, detection be rapid.
Technical scheme: for realizing above-mentioned technical purpose, the present invention proposes the primer pair that a kind of Manganic pyrophosphate complex initiation method detects BCR-ABL fusion gene, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), it is characterized in that, described primer pair comprises:
(1) for BCR-ABL (b3a2) gene,
Amplimer is:
Upstream primer: 5 '-TATGGGTTTCTGAATGTCATCG-3 ',
Downstream primer: 5 '-AACGAAAAGGTTGGGGTCATT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer is:
5′-CACTGGATTTAAGCAGAGTT-3′；
(2) for BCR-ABL (b2a2) gene:
Amplimer:
Upstream primer: 5 '-CGCAACGGCAAGAGTTACAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CTGACCATCAATAAGGAA-3′
(3)BCR-ABL(e1a2)：
Amplimer:
Upstream primer: 5 '-AACTCGCAACAGTCCTTCGAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CCTTCCATGGAGACG-3′；
Present invention further proposes the test kit that a kind of Manganic pyrophosphate complex initiation method detects BCR-ABL fusion gene, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), it is characterized in that, described test kit comprises quality control product and following primer:
(1)BCR-ABL(b3a2)：
Amplimer:
Upstream primer: 5 '-TATGGGTTTCTGAATGTCATCG-3 ',
Downstream primer: 5 '-AACGAAAAGGTTGGGGTCATT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CACTGGATTTAAGCAGAGTT-3′；
(2)BCR-ABL(b2a2)：
Amplimer:
Upstream primer: 5 '-CGCAACGGCAAGAGTTACAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CTGACCATCAATAAGGAA-3′；
(3)BCR-ABL(e1a2)：
Amplimer:
Upstream primer: 5 '-AACTCGCAACAGTCCTTCGAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CCTTCCATGGAGACG-3′。
Wherein, described quality control product comprises positive reference substance and negative controls, wherein, described positive reference substance is the mixed solution of the plasmid containing BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), and described negative controls is not containing the plasmid solution of BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2)
The invention allows for the application of mentioned reagent box in the reagent of preparation detection BCR-ABL fusion gene, wherein, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2).
Beneficial effect: compared with prior art, the test kit that Manganic pyrophosphate complex initiation method of the present invention detects BCR-ABL fusion gene can realize quick, easy, detect BCR-ABL (b3a2), BCR-ABL (b2a2), BCR-ABL (e1a2) fusion gene efficiently, accurately, for for the preparation of detecting BCR-ABL (b3a2), BCR-ABL (b2a2), the reagent of BCR-ABL (e1a2) fusion gene provides theoretical basis.
Embodiment
Experiment material:
Primer is synthesized by invitrogen company; Trizol is purchased from invitrogen company; Reverse transcription cDNA synthetic agent box is purchased in Fermentas company; Multiple PCR reagent kit is purchased in QIAGEN company; Manganic pyrophosphate complex initiation reagent is purchased in QIAGEN company.Manganic pyrophosphate complex initiation method detects a test kit for BCR-ABL fusion gene, comprises quality control product and following primer:
(1)BCR-ABL(b3a2)：
Amplimer:
Upstream primer: 5 '-TATGGGTTTCTGAATGTCATCG-3 ',
Downstream primer: 5 '-AACGAAAAGGTTGGGGTCATT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CACTGGATTTAAGCAGAGTT-3′，
(2)BCR-ABL(b2a2)：
Amplimer:
Upstream primer: 5 '-CGCAACGGCAAGAGTTACAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CTGACCATCAATAAGGAA-3′
(3)BCR-ABL(e1a2)：
Amplimer:
Upstream primer: 5 '-AACTCGCAACAGTCCTTCGAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5 '-CCTTCCATGGAGACG-3 ', wherein 5 ' end of downstream primer carries out biotin labeling.
Wherein, quality control product (quality control product DNA) comprises positive reference substance and negative controls, positive reference substance is the mixed solution of the plasmid containing BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), and described negative controls is not containing the plasmid solution of BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2)
Detection method comprises the steps:
(1) RNA extracts:
Get 15 clinical samples, wherein, 1-10 sample is the clinical sample of chronic myelocytic leukemia (CML) patient, and 11-15 sample is the clinical sample of acute lymphoblastic leukemia (ALL) patient.The clinical sample of 1-15 patient extracts RNA respectively according to the following steps:
(1) lymphocyte extract: get fresh whole blood 2ml and add 1ml 3wt% Trisodium Citrate, after mixing at 4 DEG C the centrifugal 10min of 3000r/min, get pale yellow chromatograph on blood cell layer and be lymphocyte, obtain lymphocyte liquid;
(2) get 1 centrifuge tube (through DEPC water treatment) and add lymphocyte liquid 150 μ l in (1), then add 1ml Trizol, room temperature places 5min, makes its abundant cracking;
The centrifugal 5min of (3) 12,000r/min, gets supernatant;
(4) in supernatant, add 200 μ l chloroforms, after acute vibration mixing, room temperature places 15min;
Centrifugal 15min under (5) 4 DEG C 12,000g;
(6) draw upper strata aqueous phase, aqueous phase is transferred in another centrifuge tube;
(7) in aqueous phase, add 500 μ l Virahol mixings, room temperature places 5 ~ 10min;
Centrifugal 10min under (8) 4 DEG C 12,000g, abandons supernatant, and RNA is sunken at the bottom of pipe;
(9) in the pipe of step (8), add 1ml75% (v/v) ethanol, gentle vibration centrifuge tube, suspend precipitation;
(10) 4 DEG C of 8,000g centrifugal 5min, abandons supernatant as far as possible;
(11) room temperature is dried or vacuum-drying 5 ~ 10min;
(12) with 50ul RNase-free dH
2o dissolves RNA sample, 55 ~ 60 DEG C, 5 ~ 10min;
(13) the quantitative RNA concentration of OD value is surveyed.
According to above-mentioned steps, obtain the mRNA of 1-15 sample respectively.
(2) multiple reverse transcription pcr amplification:
As follows multiple reverse transcription pcr amplification is carried out to the mRNA of above-mentioned 1-15 sample:
(1) synthesis of cDNA first chain
1. get the centrifuge tube of a RNase-free, be placed on ice, set up following reaction system;
Total RNA 5～10μg/3μl
Oligo(dT)18Primer(0.5μg/μl) 1μl
RNase-free dH
2o adds to 12 μ l
Mix reaction solution gently, with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge at the bottom of pipe;
2. centrifuge tube was in 70 DEG C of incubations 5 minutes, took out rapidly to be afterwards placed in ice and to cool, with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge at the bottom of pipe;
3. centrifuge tube is positioned on ice, is sequentially added into following reaction solution;
5×reaction buffer 4μl
RNase Inhibitor(20U/μl) 1μl
10mM dNTPs Mix 2μl
Mix reaction solution gently, with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge at the bottom of pipe;
4. centrifuge tube was in 37 DEG C of incubations 5 minutes;
5. add 1 μ l RevertAidTM Reverse Transcriptase (200U/ μ l), final volume is 20 μ l;
6. centrifuge tube was in 42 DEG C of reactions 60 minutes;
7. centrifuge tube is in 70 DEG C of heating, 10 minutes termination reactions, and rapid taking-up is afterwards placed in ice and cools.
So, synthesis cDNA first chain, i.e. template cDNA (Template cDNA).
(2) multiplex PCR
What adopt is the Multiplex PCR kit of QIAGEN company, and system is as follows
Final volume is 50 μ l
Wherein, warm start TaqDNA enzyme, MgCl is contained in 2 × QIAGEN Multiplex PCR Master Mix
2with dNTP Mix; 10 × Primer mix is the mixture comprising above-mentioned three kinds of fusion gene primer pairs.
Pcr amplification program: 95 DEG C of 15min; 94 DEG C of 30s, 55 DEG C of 90s, 72 DEG C of 90s, 35 circulations; 72 DEG C of 10min; 4 DEG C of insulations.
By above-mentioned steps, obtain PCR primer.
(3) Manganic pyrophosphate complex initiation
First, fix PCR primer with microballon, biotin labeled PCR primer is mainly fixed on the Agarose microbead (Streptavidin Sepharose High Performance) of Streptavidin bag quilt by it, comprises the steps:
(1) jog bag is by the Agarose microbead of Streptavidin, until obtain homogeneous solution;
(2) in a test tube, mix the Agarose microbead total amount (2 μ l/ sample) of Streptavidin bag quilt and binding buffer liquid (40 μ l/ sample), add ultrapure water to certain volume, this volume and the follow-up summation will adding the volume of the biotin labeled PCR primer optimized are 80 μ l/ holes;
(3) solution of the certain volume obtained in (2) is added in 24 hole PCR orifice plates;
(4) arrange according to orifice plate, the biotin labeled PCR primer that interpolation 5 ~ 20 μ l have optimized, to each hole slot of PCR orifice plate, makes wherein solution contained by every hole be 80 μ l;
(5) use orifice plate bar lid sealing PCR orifice plate, guarantee not leak between hole slot;
(6) shaker mixer (1400rpm) continuous vibration PCR orifice plate at least 5 ~ 10 minutes are used.
Through above-mentioned steps, biotin labeled PCR primer is fixed on the Agarose microbead of Streptavidin bag quilt.
Then, start vacuum work station preparation work, comprise the steps:
(1) following reagent is prepared: approximately 50ml70% (v/v) ethanol; About 40ml denaturing soln (QIAGEN company); About 50ml1 × lavation buffer solution (QIAGEN company)); About 50ml ultrapure water; About 70ml ultrapure water;
(2) open vacuum pump: open vacuum switch, carry out testing experiment, to determine whether filter probe normally works;
(3) permeability of filter probe is detected before each use vacuum pump with ultrapure water, centrifuge tube previously prepd being installed ultrapure water is inserted in PCR device, filter probe is fallen in ultrapure water, ultrapure water, as being evacuated in 20 seconds, shows that filter probe is normal, can use, otherwise then need to change filter probe;
(4) take off the PCR orifice plate vibrated, sample is put into PCR device, carefully fall filter probe in PCR orifice plate, stop 15 seconds; Guarantee that solution is all sucked away, to catch the microballon containing fixed die plate;
(5) vacuum unit is moved to the first reagent trough containing 70% (v/v) ethanol, washing and filtering probe 5 seconds;
(6) vacuum unit is moved to the second reagent trough containing denaturing soln, washing and filtering probe 5 seconds;
(7) vacuum unit is moved to the 3rd reagent trough containing lavation buffer solution, washing and filtering probe 10 seconds;
(8) vacuum unit is raised more than 90 ° of vertical lines 5 seconds, discharge opeing from filter probe;
(9) grip vacuum unit on Q24 orifice plate, the vacuum switch on stopping device unsettled stop 5 seconds, allow negative pressure leave;
(10) by left and right jog vacuum unit, release pearl is in the orifice plate containing 3 kinds of sequencing primers;
(11) when vacuum unit cuts out, vacuum unit is transferred to the 4th reagent trough containing ultrapure water, vibrates 10 seconds;
(12) fall probe to another containing ultrapure water the 5th reagent trough in and apply vacuum, cleaning probe, with 70ml ultrapure water filter probe;
(13) vacuum unit is raised more than 90 ° of vertical lines 5 seconds, discharge opeing from filter probe;
(14) close the switch on vacuum unit, and be placed on static (P) position.
Change like this completes the preparation work of vacuum station, then starts DNA isolation strand and is discharged into by sample in PyroMark Q24 orifice plate, comprising the steps: particularly
(1) 80 DEG C of exact heat 2 minutes on orifice plate base PyroMark Q24 orifice plate being placed on preheating;
(2) take off orifice plate from orifice plate base, make sample at room temperature at least cool 5 minutes.
Through above-mentioned steps, sample is discharged in PyroMark Q24 orifice plate by cup.Then prepare PyroMark Q24 reagent, carry out in the steps below:
(1) open PyroMark Q24 test kit and take out the bottle containing enzyme and substrate lyophilized powder, and the test tube containing Nucleotide;
(2) dissolve and packing enzyme and substrate according to test kit specification sheets ultrapure water, all reagent re-uses after need recovering room temperature;
(3) according to the volume that computer calculation goes out, in agent bin, enzyme, substrate and Nucleotide A, T, G, C (agent bin uses at most 30 times, and agent bin must be dry before use) is added.
After getting out PyroMark Q24 reagent, PyroMark Q24 instrument is tested in the steps below:
(1) PyroMark Q24 software is opened, click new assay (newly testing) → new AQ assay (new AQ test) → input catastrophe point sequence in sequence to analyze (sequence to be analyzed), click Generate Dispensation Order (generation allocation order), click and preserve;
(2) click new run (newly running) → Instrument method (instrumental method) → 007, then click the grid that will check order, click and be saved to USB flash disk;
(3) by the USB port before the USB flash disk inserting instrument containing operating file;
(4) orifice plate heated is put on instrument;
(5) label surface of agent bin (enzyme, substrate and Nucleotide) is put into instrument towards oneself, orifice plate put into by open holes board support frame, closes orifice plate support saddle frame and instrument lid;
(6) select Run (operation), and press OK;
(7) after entering Run (operation), the file that will run is selected by select (selection);
(8) instrument end of run after confirming that operating file is saved to USB flash disk, by close, takes out USB flash disk;
(9) open instrument, take out agent bin, and it is cleaned repeatedly, discarded orifice plate;
(10) when instrument does not run, select shutdown (shutdown) from master menu and press OK;
(11) when It is now safe to turn off the instrument (now can safety shutdown) occurs, can close instrument, power switch is positioned at after instrument.
(4) interpretation of result:
Open Pyromark Q24 software, analyze the type of BCR-ABL (b3a2), BCR-ABL (b2a2), BCR-ABL (e1a2) fusion gene, analytical results is as following table:
Result shows, 1-5 patient is that BCR-ABL (b2a2) fusion gene is positive; 6-10 patient is that BCR-ABL (b3a2) fusion gene is positive; 11-15 patient is that BCR-ABL (e1a2) fusion gene is positive.
To sum up, apply test kit of the present invention can realize quick, easy, detect BCR-ABL (b3a2), BCR-ABL (b2a2), BCR-ABL (e1a2) fusion gene efficiently, accurately.
About after those set forth of the present invention more than having read, those skilled in the art can do various amendment or change to the present invention, comprising the various changes of primer, these equivalent form of values belong to the scope defined in the application's appended claims equally.
Claims (4)
1. the primer pair of a Manganic pyrophosphate complex initiation method detection BCR-ABL fusion gene, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), it is characterized in that, described primer pair comprises:
(1) for BCR-ABL (b3a2) gene,
Amplimer is:
Upstream primer: 5 '-TATGGGTTTCTGAATGTCATCG-3 ',
Downstream primer: 5 '-AACGAAAAGGTTGGGGTCATT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer is:
5′-CACTGGATTTAAGCAGAGTT-3′；
(2) for BCR-ABL (b2a2) gene:
Amplimer is:
Upstream primer: 5 '-CGCAACGGCAAGAGTTACAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CTGACCATCAATAAGGAA-3′
(3) for BCR-ABL (e1a2) gene:
Amplimer is:
Upstream primer: 5 '-AACTCGCAACAGTCCTTCGAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CCTTCCATGGAGACG-3′。
2. the test kit of a Manganic pyrophosphate complex initiation method detection BCR-ABL fusion gene, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), it is characterized in that, described test kit comprises quality control product and following primer:
(1) for BCR-ABL (b3a2) gene:
Amplimer is:
Upstream primer: 5 '-TATGGGTTTCTGAATGTCATCG-3 ',
Downstream primer: 5 '-AACGAAAAGGTTGGGGTCATT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CACTGGATTTAAGCAGAGTT-3′；
(2) for BCR-ABL (b2a2) gene:
Amplimer is:
Upstream primer: 5 '-CGCAACGGCAAGAGTTACAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CTGACCATCAATAAGGAA-3′
(3) for BCR-ABL (e1a2) gene:
Amplimer is:
Upstream primer: 5 '-AACTCGCAACAGTCCTTCGAC-3 ',
Downstream primer: 5 '-AGCGGCTTCACTCAGACCCT-3 ',
Wherein 5 ' end of downstream primer carries out biotin labeling;
Sequencing primer:
5′-CCTTCCATGGAGACG-3′。
3. test kit according to claim 2, it is characterized in that, described quality control product comprises positive reference substance and negative controls, wherein, described positive reference substance is the mixed solution of the plasmid containing BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2), and described negative controls is not containing the plasmid solution of BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2).
4. test kit according to claim 2 detects the application in the reagent of BCR-ABL fusion gene in preparation, wherein, described BCR-ABL fusion gene is any one or a few in BCR-ABL (b3a2), BCR-ABL (b2a2) and BCR-ABL (e1a2).
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103045590A (en) * | 2012-12-26 | 2013-04-17 | 武汉康圣达医学检验所有限公司 | Primer and method for detecting drug-resistant mutation point in BCR/ABL fusion gene ABL kinase zone by using same |
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| CN103045590A (en) * | 2012-12-26 | 2013-04-17 | 武汉康圣达医学检验所有限公司 | Primer and method for detecting drug-resistant mutation point in BCR/ABL fusion gene ABL kinase zone by using same |
Non-Patent Citations (1)
| Title |
|---|
| C. CAMERON YIN，ET AL: "Rapid clonal shifts in response to kinase inhibitor therapy in chronic myelogenous leukemia are identified by quantitation mutation assays", 《CANCER SCI》, vol. 101, no. 9, 31 October 2010 (2010-10-31), pages 2005 - 2010 * |
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