CN104178557A - Detection method for leukemia fusion genes, primers, probes and gene chip thereof - Google Patents
Detection method for leukemia fusion genes, primers, probes and gene chip thereof Download PDFInfo
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Abstract
The invention discloses a method for detection of leukemia fusion genes through combination of multiplex RT-PCR and a gene chip. The method includes the steps of: 1) extracting total RNA of a sample cell; 2) subjecting RNA to specific reverse transcription to obtain cDNA; 3) conducting multiple RT-PCR amplification on the cDNA; and 4) detecting the PCR amplification product by the gene chip containing specific probes for detecting leukemia fusion genes. The invention also discloses the probes, the primers and the gene chip adopted by the method. A tube nested multiplex PCR is employed to amplify specific fusion gene fragments possibly contained in the sample, and then the fusion gene detection result can be obtained through gene chip hybridization and chip scanning image analysis. In this way, simultaneous detection of 27 or more different fusion forms of specific fragments of 15 leukemia fusion genes can be realized, thus greatly improving the detection efficiency of leukemia fusion genes, and being in favor of large-scale clinical screening.
Description
Technical field
The present invention relates to technique of gene detection field, particularly relate to a kind of method of multiple RT-PCR Polymorphism chip detection leukemia fusion gene, and the method specific probe, primer and gene chip used.
Background technology
Leukemia is a kind of malignant tumour of hemopoietic system, and annual morbidity is about 2.76 people/100,000 people, accounts for cancer 5% of the number of always falling ill, children and the 35 years old following adult's mortality of malignant tumors position of ranking the first.Leukemic generation development is a complicated process; often relate to the change of a plurality of genes; the sudden change or disappearance, the abnormal amplification of oncogene and the synergy of expression and a plurality of genes that comprise normal gene; in addition the pleiotropy of gene own and immune factor, whether final decision tumor phenotypes is expressed.
According to the course of disease, clinical manifestation leukemia, can be divided into two large class acute leukemia and chronic leukemias.According to parent cell source, acute leukemia can be divided into again to kemia (Acute Lymphoblastic Leukemia, ALL), acute myeloid leukaemia (Acute Myelogenous Leukemia, AML); Chronic leukemia is divided into chronic lymphatic leukemia (Chronic Lymphocytic Leukemia, CLL), chronic myelogenous leukemia (Chronic Myelogenous Leukemia, CML).Wherein, with acute leukemia common (70%), each age all can occur, and the male sex is more than women.Modal in grownup is AML and CML, ALL more commonly in children.
The leukemic generation of part may be relevant with the formed fusion gene of chromosome translocation, as chromosome translocation t (9; 22) (q34; Q11) BCR-ABL p190, t (4; 11) (q21; Q23) MLL-AF4, t (12; 21) (p13; Q22) TEL-AML1, t (1; 19) (q23; P13) E2A-PBX1 may be relevant to ALL; T (8; 21) (q22; Q22) AML1-ETO, inv (16) (p13; Q22)/t (16; 16) (p13; Q22) CBFB-MYH11, t (15; 17) (q22; Q12) PML-RARA, t (9; 11) (p22; Q23) MLL-AF9 and some other 11q23MLL gene rearrangement may be relevant to AML.The detection of the fusion gene that chromosome translocation forms, in conjunction with clinical morphocytology inspection, bone marrow smear inspection etc., can carry out the analysis of leukemic diagnosis, prognosis and treatment aspect.
The method that detects leukemia chromosome abnormalty mainly contains the karyotyping of conventional G banding chromosome, fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH) and round pcr.The karyotyping of conventional G banding chromosome remains the important means that chromosome disorder checks, it can reliable recognition chromosome number and the variation of structure, although have limitation in sensitivity and operability, such as leukemia cell's di is low, chromosome morphology is short and small, be often aobviously with unclear, conceal type transposition, make some complex structures or trickle change be difficult to accurate identification.FISH utilizes fluorescence labeling probe in conjunction with specific chromosome sequence, thereby is analyzed under the microscope.By fluorescence probe, can identify micro-even submicroscopic conceal type textural anomaly and chromosome number changes.FISH more can directly observe chromosome abnormalty with respect to karyotyping, and sensitivity is higher, is applicable to interkinesis medium cell, but it only can be analyzed the gene locus of known probe.It is quite general, not only useful to leukemic diagnosis that PCR detects acute leukemia, and be more suitable for the detection to leukemia MRD.These methods all have relative merits separately, but are all not suitable for the leukemia fusion gene examination of multiple sample.
Therefore, some novel detection technique, especially biochip technologies have been there are.The great advantage that biochip technology detects be contain much information, operation automation, intellectuality, can detect rapidly and sensitively required gene, not only easy and simple to handle, expend also not high, therefore may be a kind of good detection method, being applied to routine clinical assessment and disease control aspect has very large prospect.There is the report that adopts the method for gene chip to detect leukemia fusion gene abroad, and domesticly not yet have a relevant report.But external main a few money chip limitation are also quite obvious, only for wherein a kind of fusion form of a few type of leukemia, and to relate to PCR part be all to adopt multitube nest-type PRC, and this is unfavorable for slightly having the screening of scale.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of method of multiple RT-PCR Polymorphism chip detection leukemia fusion gene, and it can improve the detection efficiency of leukemia fusion gene.
For solving the problems of the technologies described above, the method for multiple RT-PCR Polymorphism chip detection leukemia fusion gene of the present invention, step comprises:
1) extracting sample cell total rna;
2) RNA specificity reverse transcription step 1) being obtained is cDNA;
3) take cDNA as template, carry out multiple RT-PCR amplified reaction;
4) with whether containing corresponding leukemia fusion gene fragment in the genechip detection pcr amplification product that contains one or more specific probe that detects leukemia fusion gene.
Two of the technical problem to be solved in the present invention is to provide the specific probe for detection of leukemia fusion gene, and the sequence of this probe comprises the sequence shown in SEQ ID NO:31～80 or its complementary strand, and probe 5 ' is terminal modified amino.
Three of the technical problem to be solved in the present invention is to provide the gene chip for detection of leukemia fusion gene, the specific probe that this gene chip contains one or more above-mentioned detection leukemia fusion gene.
Four of the technical problem to be solved in the present invention is to provide the combination of primers that the RNA reverse transcription for above-mentioned multiple RT-PCR Polymorphism chip detection leukemia fusion gene method is cDNA, and described combination of primers has sequence or its complementary strand as shown in SEQ ID No:1～16.
Five of the technical problem to be solved in the present invention is to provide the combination of primers of multiple RT-PCR amplification cDNA, and described combination of primers has sequence or its complementary strand as shown in SEQ ID No:17～30.
The present invention adopts a pipe Nest multiplex PCR, and the specificity fusion gene fragment that may contain in amplified sample detects when having realized the specific fragment of 27 kinds of 15 leukemia fusion genes and above different fusion forms, comprising: t (8; 21) (q22; Q22) AML1-ETO, inv (16) (p13q22)/t (16; 16) (p13; Q22) CBFB-MYH11, t (15; 17) (q22; Q21) PML-RARA, t (11; 17) (q23; Q12) PLZF-RARA, t (5; 17) (q35; Q12) NPM1-RARA, t (9; 11) (p22; Q23) MLL-AF9, t (6; 11) (q27; Q23) MLL-AF6, t (11; 19) (q23; P13.3) MLL-ENL, t (11; 19) (q23; P13.1) MLL-ELL, t (10; 11) (p12; Q23) MLL-AF10, t (12; 21) (p13; Q22) TEL-AML1, t (1; 19) (q23; P13) E2A-PBX1, t (4; 11) (q21; Q23) MLL-AF4, del (1) (p32; P32) SIL-TAL1, t (9; 22) (q34, q11) BCR-ABL p190, p210 and p230, follow-up by gene chip hybridization and chip scanning image analysis, just can obtain fusion gene detected result, thereby greatly improve the detection efficiency of leukemia fusion gene, can be used for large-scale screening.
Accompanying drawing explanation
Fig. 1 is gene chip schematic diagram, and wherein A is the site plan of each probe of chip, and B is whole chip schematic diagram.
Fig. 2 is the chip hybridization result of K-562 clone.Wherein A is K-562 chip hybridization figure; B is the signal value distribution plan of each probe on chip, and dotted line is threshold value (background signal and negative signal averaging+3SD).
Fig. 3 is multiple RT-PCR specificity gel electrophoresis result.Wherein, M swimming lane is DL2000DNA Marker; Swimming lane 1-6 is clone, is respectively KASUMI-1, NB-4, ME-1, THP-1, K-562, REH; Swimming lane 7-14 is sample, is respectively PML-RARA S-form, MLL-AF4, MLL-ENL, MLL-ELL, MLL-AF6, MLL-AF10, E2A-PBX1, BCR-ABL p190; The negative contrast of swimming lane 15; Swimming lane 16 is H
2o.
Fig. 4 is gel electrophoresis figure and the chip hybridization result of the different dilution of KASUMI-1 clone gradient.Wherein A is the gel electrophoresis result of the different dilution of KASUMI-1 gradient; B is that KASUMI-1 respectively dilutes chip hybridization figure corresponding to gradient; C is KASUMI-1 dilution gradient 10
-3with 10
-4the signal value distribution plan of corresponding each probe, dotted line is threshold value (background signal and negative signal averaging+3SD).
Fig. 5 is the result with multiple RT-PCR Polymorphism chip detection part leukemia clinical sample of the present invention.
Embodiment
Below by specific embodiment, and by reference to the accompanying drawings, the present invention is described in further detail.The following example is intended to illustrate rather than limit the present invention.
The leukemia fusion gene of embodiment 1 multiple RT-PCR Polymorphism chip detection K-562 clone
1. cell total rna extracting
Get K-562 clone, adopt the method for Trizol, according to Invitrogen company's T rizol specification sheets, extract cell total rna, the concrete steps of extraction are as follows:
(1) go bail for and have the sample 400 μ l in Trizol, add 400 μ l chloroforms, mix, room temperature is placed 5 minutes;
(2) 15000rpm is centrifugal 10 minutes, the colourless supernatant liquor of centrifugal rear absorption;
(3) in above-mentioned supernatant, add the Virahol of equal-volume precooling, put upside down and mix rear room temperature placement 10 minutes;
(4) 15000rpm is centrifugal 10 minutes, inhales and abandons supernatant;
(5) in above-mentioned centrifuge tube, add 70% ethanol of 300 μ l precoolings, centrifugal 5 minutes of 15000rpm, inhales and abandons supernatant; Room temperature is placed 10 minutes, makes ethanol volatilization;
(6) adding 30-60 μ l DEPC(diethylpyrocarbonate) water mixes dissolving, quantitatively, the OD of RNA
260/ OD
2801.8～2.0;
(7) get the total RNA of 2 μ g, for next step specificity reverse transcription reaction.
2.RNA specificity reverse transcription is cDNA
Reverse transcription reaction system is 25 μ l, comprise: the total RNA of 2 μ g, each species specificity reverse transcriptase primer of 2.5～3pmol (in Table 1, being synthesized by Shanghai Jierui Biology Engineering Co., Ltd), 200U M-MLV reversed transcriptive enzyme, 25U RNA enzyme inhibitors, 1mM dNTP, 10mM DTT(dithiothreitol (DTT)), 50mM Tris-HCl(pH8.3), 75mM KCl, 3mM MgCl
2.
Reaction conditions: RNA reacts 5 minutes prior to 70 ℃ with reverse transcriptase primer, is placed on ice, sneaks into other reacted constituents and hatches 1 hour in 42 ℃.
Table 1 reverse transcriptase primer sequence
3. multiplex PCR amplification
Take cDNA as template, carry out the amplification of two-wheeled Nest multiplex PCR.
First round PCR system 20 μ l, comprising: 1 μ l cDNA, the various PCR primers of 3pmol (in Table 2, being synthesized by Shanghai Jierui Biology Engineering Co., Ltd), 4pmol SP6,11mM Tris-HCl(pH8.3), 55mM KCl, 1.5mM MgCl
2, 15%DMSO(dimethyl sulfoxide (DMSO)), 0.4mM dNTP and 1.25U ExTaq HS enzyme.PCR program: 25 circle circulations, comprising: 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ are extended 1 minute; Loop ends, 72 ℃ are extended 7 minutes.
Second takes turns PCR system 20 μ l, comprising: 1 μ l first round PCR product, 10mM Tris-HCl(pH8.3), 50mM KCl, 1.5mM MgCl
2, 0.2mM dNTP, 2pmol T7 and 2pmol SP6(mark vitamin H) and 1U ExTaq HS enzyme.PCR program: 30 circle circulations, comprise 95 ℃ of sex change 30 seconds, anneal 40 seconds for 50 ℃, 72 ℃ are extended 1 minute; Loop ends, 72 ℃ are extended 7 minutes.PCR product is kept at 4 ℃.
Table 2 multiplex PCR upstream primer sequence
4. chip preparation
By 2 * sampling liquid dilution for synthetic probe (being synthesized by Shanghai Jierui Biology Engineering Co., Ltd), use full-automatic point sample instrument, according to the chip schematic diagram point sample of Fig. 1, each probe repeats 3 times.Point sample gene probe sequence is in Table 3(SEQ ID NO:31～80), all probes are all modified with amino at 5 ' end.This gene chip contains the sites such as positive quality control site, negative Quality Control site and blank simultaneously.
Table 3 leukemia fusion gene probe sequence
Chip hybridization with develop a film
Before chip hybridization, use prehybridization solution (2 * SSC, 1mg/ml BSA, 0.2%SDS) to carry out prehybridization and process 1 hour, centrifuge dripping after washing.The first sex change 5 minutes at 95 ℃ of biotin labeled PCR product, ice bath is 5 minutes at once, after mixing, all transfers to the point sample region of chip with chip hybridization liquid, is placed in hybridizing box 46 ℃ of hybridization 12-15 hour.After having hybridized, with preheating washing lotion A (2 * SSPE, 1%SDS), washing lotion B (2 * SSPE, 1%SDS), washing lotion C (2 * SSPE, 1%SDS), respectively wash centrifuge dripping 5 minutes.Chip probe is arranged and is seen Fig. 1.
6. colour developing
Use PBS(phosphate buffer soln) by 1:100 dilution proportion Cy3-Streptavidin(CY3-Streptavidin), dripping on chip, room temperature lucifuge is hatched 30 minutes, cleans, and centrifuge dripping, notes lucifuge, and the chip after drying is standby in lucifuge box.
7. chip scanning image analysis and data processing
With
4000B confocal laser scanner (Axon Instruments, US) scanning chip, is used the monochromatic fluorescence of 532nm, 10 μ m resolving power.Image GenePixPro6.0 software analysis, extracts the fluorescence signal intensity that obtains each probe.Each probe repeats 3 times, calculates each fluorescence probe signal averaging.Using background signal and negative signal averaging+3SD(standard deviation) as the threshold value (Cutoff value) judging.
The chip detection result of K-562 clone and the signal value histogram of each probe are shown in Fig. 2.The chip hybridization signal and signal distributions corresponding to each probe that in Fig. 2, have shown K-562, visible effectively signal is probe p210-b3a2, BCR210 and ABL, can obtain thus K-562 correspondence and BCR-ABL p210b3a2 fusion form.
Embodiment 2 multiple RT-PCR detected results
Get KASUMI-1, NB4, ME-1, THP-1, REH and K-562 Leukemia Cell Lines, and t (15; 17) PML-RARA S type, t (4; 11) MLL-AF4, t (11; 19) MLL-ENL, t (11; 19) MLL-ELL, t (6; 11) MLL-AF6, t (10; 11) MLL-AF10, t (1; 19) E2A-PBX1, t (9; 22) BCR-ABL p190 positive sample, carries out cell total rna extracting, specificity reverse transcription reaction and multiplex PCR amplification test according to the 1st step to the 3 steps in embodiment 1.After off-test, get 3 μ lPCR products and detect with 2% agarose gel, electrophoresis result is shown in Fig. 3.
In Fig. 3, shown KASUMI-1, NB4, ME-1, THP-1, REH and K-562 Leukemia Cell Lines, and t (15; 17) PML-RARA S type, t (4; 11) MLL-AF4, t (11; 19) MLL-ENL, t (11; 19) MLL-ELL, t (6; 11) MLL-AF6, t (10; 11) MLL-AF10, t (1; 19) E2A-PBX1, t (9; 22) the multiple RT-PCR result of BCR-ABL p190 positive sample.What be positioned at 328bp is the band of reference gene gus gene, and an other band is the fusion gene fragment that each sample is corresponding.
Embodiment 3 sensitivity detect
Take KASUMI-1 cell as example, carry out sensitivity experiment.Initial R NA since 2 μ g with the negative leukemia cell of HL-60() RNA carry out 10 times of gradient dilutions, be minimumly diluted to 10
-4.Multiple RT-PCR Polymorphism chip detection, determines the sensitivity of KASUMI-1 cell, the results are shown in Figure 4.
The gel electrophoresis and the chip hybridization situation that in Fig. 4, have shown the different dilution of KASUMI-1 gradient.From gel electrophoresis and chip hybridization result, can find out, the detection sensitivity of KASUMI-1 is 10
-3, as long as exist 1 cell with AML1-ETO fusion gene just can to obtain by the inventive method in 1000 cells.
Embodiment 4 clinical samples detect
To 200 routine leukemia clinical samples, according to method described in embodiment 1, utilize multiple RT-PCR Polymorphism chip detection leukemia fusion gene, the results detailed in Table 4.
Table 4 screens respectively the statistics contrast of 200 routine leukemia clinical samples by method of the present invention and traditional method
From table 4, multiple RT-PCR Polymorphism chip detecting method of the present invention and traditional detection method (for example karyotyping, FISH and Standard PC R) compare, can reach 96.5% coincidence rate, the fusion gene type of incongruent 7 routine samples is because the scope that the present invention relates to does not comprise these fusion genes.The multiple RT-PCR Polymorphism chip detection result of part clinical sample refers to Fig. 5.
Claims (10)
1. the method for multiple RT-PCR Polymorphism chip detection leukemia fusion gene, is characterized in that, step comprises:
1) extracting sample cell total rna;
2) RNA specificity reverse transcription step 1) being obtained is cDNA;
3) take cDNA as template, carry out multiple RT-PCR amplified reaction;
4) with whether containing corresponding leukemia fusion gene fragment in the genechip detection pcr amplification product that contains one or more specific probe that detects leukemia fusion gene.
2. method according to claim 1, is characterized in that step 2), the combination of primers that RNA reverse transcription is cDNA has sequence or its complementary strand as shown in SEQ ID No:1～16.
3. method according to claim 1, is characterized in that, step 3) is carried out two-wheeled nido multiple RT-PCR amplified reaction.
4. method according to claim 3, is characterized in that, step 3), and first round PCR system 20 μ l, comprising: 1 μ l cDNA, the various PCR primers of 3pmol, 4pmol SP6,11mM Tris-HCl, 55mM KCl, 1.5mM MgCl
2, 15%DMSO, 0.4mM dNTP and 1.25U ExTaq HS enzyme; Second takes turns PCR system 20 μ l, comprising: 1 μ l first round PCR product, 10mM Tris-HCl, 50mM KCl, 1.5mM MgCl
2, 0.2mM dNTP, 2pmol T7 and 2pmol mark vitamin H SP6 and 1U ExTaq HS enzyme; First round PCR program comprises 25 circles circulations, and second takes turns PCR program comprises 30 circles circulations, and every circle circulation comprises: 95 ℃ of sex change 30 seconds, and 55 ℃ of annealing 40 seconds, 72 ℃ are extended 1 minute; After loop ends, all at 72 ℃, extend 7 minutes.
5. according to the method described in claim 1 or 3 or 4, it is characterized in that, step 3), PCR combination of primers has sequence or its complementary strand as shown in SEQ ID No:17～30.
6. method according to claim 1, is characterized in that, step 4), and the sequence of described specific probe comprises the sequence shown in SEQ ID NO:31～80 or its complementary strand, probe 5 ' is terminal modified amino.
7. for detection of the specific probe of leukemia fusion gene, it is characterized in that, the sequence of probe comprises the sequence shown in SEQ ID NO:31～80 or its complementary strand, and probe 5 ' is terminal modified amino.
8. for detection of the gene chip of leukemia fusion gene, it is characterized in that, contain one or more probe claimed in claim 7.
9.RNA reverse transcription is the combination of primers of cDNA, it is characterized in that, has sequence or its complementary strand as shown in SEQ ID No:1～16.
10. the combination of primers of multiple RT-PCR amplification cDNA, is characterized in that having sequence or its complementary strand as shown in SEQ ID No:17～30.
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| CN105838793A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primers, kit and method for qualitatively detecting leukaemia fusion genes |
| CN109439704A (en) * | 2018-11-13 | 2019-03-08 | 中山大学达安基因股份有限公司 | Detect leucocythemia related method and kit because of variation |
| CN112063698A (en) * | 2020-08-31 | 2020-12-11 | 福州艾迪康医学检验所有限公司 | Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene |
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| CN104561331A (en) * | 2015-01-21 | 2015-04-29 | 苏州云泰生物医药科技有限公司 | Primer and probe for detecting leukemia-related fusion gene and kit of primer |
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| CN105838793B (en) * | 2016-04-22 | 2019-07-12 | 上海荻硕贝肯生物科技有限公司 | Primer, kit and method for qualitative detection leukemia fusion gene |
| CN109439704A (en) * | 2018-11-13 | 2019-03-08 | 中山大学达安基因股份有限公司 | Detect leucocythemia related method and kit because of variation |
| CN112063698A (en) * | 2020-08-31 | 2020-12-11 | 福州艾迪康医学检验所有限公司 | Oligonucleotide and method for detecting expression level of NPM1-RARA fusion gene |
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