CN101955991A - Joint detection method of fusion genes related to leukemia and diagnosis kit - Google Patents
Joint detection method of fusion genes related to leukemia and diagnosis kit Download PDFInfo
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Abstract
The invention discloses a joint detection method of fusion genes related to leukemia and a diagnosis kit. The method comprises the following steps of: designing a primer and a probe through a fusion gene mRNA (the fusion genes related to the leukemia are SIL-TAL1(type II, type III), MLL-AF4(e9/e5,e9/e4) and CBFB-MYH11(type D)) related to the leukemia; hybridizing a probe and microsphere mixture formed by the covalent bonding of the probe and microspheres with an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification product; and adding phycoerythrin as streptavidin biotin to detect fluorescent signals of different microspheres so as to confirm whether a sample to be detected contains a leukemia fusion gene as well as the expression conditions of the fusion gene. The method and the kit of the invention have the advantages of high sensitivity and flux, rapid and accurate detection, and the like and can be used for simultaneously, qualitatively and quantitatively detecting various leukemia fusion genes.
Description
Technical field
The present invention relates to in-vitro diagnosis detection technique field, be specifically related to the liquid-phase chip associated detecting method and the diagnostic kit thereof of the relevant multiple fusion gene of leukemia.
Background technology
Leukemia is the hemopoietic system malignant tumour that causes owing to hemopoietic stem cell prosoplasia and neoplasm, is one of modal malignant tumour of China.Along with the continuous progress of leukemia research, find that many leukaemics have specific chromosome translocation, cause new fusion gene to produce, these aberrant genes have become dissimilar leukemic molecular biology specificity markers.WHO was about reducing some common aberrant genes the standard of leukemia basic diagnosis especially in the standard of leukemia classification in 2000.Therefore, in the molecular gene level the relevant fusion gene of leukemia is detected, not only can judge for leukemia diagnosis, somatotype, clinical treatment selection and prognosis provides important evidence, also detects the basis for the leukemia microresidual disease provides simultaneously.
Fusion gene common in the leukemia has: the BCR-ABL (b2a2) in the chronic myelocytic leukemia (CML), BCR-ABL (b3a2); BCR-ABL (e1a2) in the acute lymphoblastic leukemia (ALL), E2A-PBX1, TEL-AML1, MLL-AF4 (e10/e4, e9/e5, e9/e4); CBFB-MYH11 in the acute myeloblastic leukemia (AML) (type A, type D), AML1-ETO; PML-RARA (Lform), PML-RARA (S form) in the acute promyelocytic leukemia (APL); SIL-TAL1 in the T cell acute lymphoblastic leukemia (T-ALL) (typeII, type III).
At present, leukemia mainly is to rely on detection methods such as morphocytology, immunology, cytogenetics, molecular biology to diagnose and detect somatotype.Morphocytology is influenced by subjective factor, and the concordance rate of clinical diagnosis only is about 70%.Cytogenetic methods such as karyotyping and banding technique is at full genomic level examination chromosome translocation, the many chromosomal minor anomalies of omission easily.Immunological method has higher false positive rate and false negative rate, and can't accomplish early diagnosis.In the method for current domestic and international widely used molecular Biological Detection leukemia fusion gene, fluorescence in situ hybridization technique (FISH) can only be carried out qualitative detection, complicated operation; Quantitative fluorescent PCR exists the limitation that detects flux, so all can't really satisfy the needs that clinical diagnosis detects.Traditional solid phase biological chip (Biochip) technology exist repeatable poor, insufficient sensitivity good and the outstanding weakness of complex operation.Therefore need a kind of detection method clinically, can be rapidly, stablize, exactly leukemic multiple fusion gene carried out the associating parallel detection, a kind of so just novel detection technique of liquid-phase chip technology (xMAP).
Liquid-phase chip can carry out qualitative and quantitative analysis to a small amount of sample, has high-throughput, easy and simple to handle, good reproducibility, highly sensitive, outstanding advantage such as linearity range is wide.This system is that main matrix constitutes by many microballoons, in the middle of the manufacturing processed of microballoon, two kinds of different redness classification fluorescence have been mixed, ratio difference according to these two kinds of fluorescence, sphere matrix is divided into 100 kinds, 100 kinds of different probe molecules on can mark, can be simultaneously in the sample nearly 100 kinds of different target molecules detect.According to the difference that detects thing, microsphere surface can the various detection of nucleic acids probes of covalent attachment, add fluorescent mark when hybridization carries out.In same reaction system, can add different detection microballoons simultaneously, so just can utilize a spot of sample to carry out quick, high-throughout detection.After reaction finishes, by micro-fluidic technologies microballoon is lined up the single-row liquid-phase chip detector of flowing through fast, each microballoon can be arrived by two bundle laser detection simultaneously, and red laser excites the redness classification fluorescence on the microballoon, and the reaction that each is different makes a distinction and qualitative; Green laser then excites the fluorescent mark that is combined on the sample to be tested to carry out quantitatively.When the good sample to be tested of mark and the probe on the specific microballoon combined, the two bundle light that laser excited all can be detected.At last,, the average fluorescent strength on the specific microballoon be can automatic statistical analysis draw, thereby the kind and the quantity of thing determined to detect by the high speed digital signal processor of computer.
The present invention is based on the high-throughput of liquid-phase chip technology, easy and simple to handle, good reproducibility, highly sensitive, outstanding advantage such as linearity range is wide, leukemic multiple fusion gene is carried out the associating parallel detection, can on clinical detection, obtain better application.
Summary of the invention
The technical issues that need to address of the present invention provide the associated detecting method and the diagnostic kit of the relevant fusion gene of a kind of leukemia.This method and test kit comprise (the type II to SIL-TAL1, type III), MLL-AF4 (e9/e5, e9/e4), the multiple fusion gene joint-detection of CBFB-MYH11 (type D), can carry out clinical classification to T cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), simultaneously the patient be carried out observation of curative effect, prognosis and small residual disease and carry out dynamic monitoring.This detection method and diagnostic kit have advantages such as highly sensitive, high specific, split hair caccuracy, detection be rapid.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
In one aspect of the invention, provide the associated detecting method of the relevant fusion gene of a kind of leukemia, may further comprise the steps:
(1) comprise 3 kinds of carboxyl microballoon beads that difference is fluorescence-encoded, numbering is respectively 11,15,17; Distinguish the mRNA designed specific dna probe of covalent attachment on every kind of microballoon at 3 kinds of fusion genes of chromosome translocation formation in the leukemia;
(2) at the mRNA of the formed 3 kinds of fusion genes of chromosome translocation in the different type of leukemia, design the upstream and downstream primer respectively,, amplify corresponding product by reverse transcription PCR to different fusion gene mRNAs;
(3) contain the reverse transcription amplification product hybridization of the microballoon of specific dna probe and fusion gene mRNA after, add Streptavidin-phycoerythrin (Streptavidin-PE), detect fluorescent signal by liquid-phase chip method xMAP;
(4) fluorescent signal with detected fluorescent signal and internal control gene compares, thereby determines whether to contain the relevant fusion gene of leukemia in the test sample, and/or the expression situation of fusion gene in the sample.
Microballoon described in the above detection method is that mean diameter is 5.6 μ m, combines the polyphenyl alkene microballoon of different fluorescence dyes, i.e. color-code microballoon (color-coded beads); The fusion gene that chromosome translocation forms in the leukemia is at following multiple fusion gene that can independent assortment: and SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4), CBFB-MYH11 (type D).
Described 3 specific dna probes that are covalently bonded on the microballoon comprise following sequence (wherein 5 ' end contains amido modified):
SIL-TAL1 (type II, type III): 5 '-AminolinkerC12 AGTTACGCTGCGGTGTGGTC-3 ', shown in SEQ ID NO.1;
MLL-AF4 (e9/e5, e9/e4): 5 '-AminolinkerC12 TATTGCTGTCAAAGGAGGCGG-3 ', shown in SEQ ID NO.2;
CBFB-MYH11 (type D): 5 '-AminolinkerC12 TGTCCTTCTCCGAGCCTCTTCA-3 ', shown in SEQ ID NO.3;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
Described at the designed upstream and downstream primer of the mRNA of fusion genes, comprise following sequence (wherein upstream primer 5 ' end contains biotin label):
SIL-TAL1 (type II, type III): upstream primer 5 '-biotin CCTGCAAACAGACCTCAGCTC-3 ', shown in SEQ ID NO.4; Downstream primer 5 '-CGAGGAAGAGGATGCACAC-3 ' is shown in SEQ IDNO.5;
MLL-AF4 (e9/e5, e9/e4): upstream primer 5 '-biotin TCCAGAGCAGAGCAAACAG-3 ', shown in SEQ ID NO.6; Downstream primer 5 '-CCTTGCTGAGAATTTGAGTG-3 ' is shown in SEQ ID NO.7;
CBFB-MYH11 (type D): upstream primer 5 '-biotin GAGGATGCATTAGCACAACAG-3 ', shown in SEQ ID NO.8; Downstream primer 5 '-GAAGCAACTCCTGGGTGTC-3 ' is shown in SEQ ID NO.9;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
The primer and the probe sequence of described internal control gene Abelson gene are as follows:
Upstream primer 5 '-biotin GCGCAAAATGTTGGAGATC-3 ' is shown in SEQ ID NO.10;
Downstream primer 5 '-GGAGCTTTTCACCTTTAGTTATGC-3 ' is shown in SEQ ID NO.11;
Probe 5 '-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3 ' is shown in SEQ ID NO.12;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
In another aspect of this invention, provide a kind of diagnostic kit that detects the relevant multiple fusion gene of leukemia, the comprised covalent attachment mixture of microspheres of leukemia fusion gene mRNA specific probe, the microballoon that combines the Abelson gene probe, the upstream and downstream primer of leukemia fusion gene mRNA, the upstream and downstream primer of Abelson gene, Streptavidin-phycoerythrin Streptavidin-PE, quality control product (negative control and positive control).
Quality control product described in the above test kit comprises positive control and negative control, wherein positive control is the mixed solution (comprising the plasmid that contains the Abelson gene) that contains various fusion gene plasmids, and the negative control product are not for containing the plasmid solution of fusion gene and Abelson gene; Described mixture of microspheres is according to the needs independent assortment of different test sample.
In another aspect of this invention, provide a kind of diagnostic kit that detects the relevant multiple fusion gene of leukemia, leukemia is carried out somatotype, early diagnosis, observation of curative effect, the application in the dynamic monitoring of prognosis and small residual disease detecting vitro samples.
Because the present invention has utilized liquid-phase chip technology, make detection method and test kit have outstanding advantages such as highly sensitive, high specific, high-throughput, good stability, detection be rapid, accurate, can carry out qualitative and detection by quantitative to leukemia fusion gene, can on clinical detection, obtain better application.
Embodiment
Describe the present invention in detail below in conjunction with specific embodiment, but can not be interpreted as limitation of the present invention.Described embodiment only provides and illustrates nucleic acid probe, test kit and making and methods for using them thereof and do not limited by it.Various during this time versions are expected in the scope of the present invention and described claim.
1. experiment material
Primer and probe are synthetic by invitrogen company; Trizol is available from invitrogen company; Reverse transcription cDNA synthetic agent box, PCR reagent are available from Fermentas company; Microballoon (surperficial carboxyl modified), the Streptavidin-phycoerythrin of different numberings are all purchased the company in QIAGEN; 1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) is purchased the company in Pierce; 2-(N-morpholine)-ethyl sulfonic acid (MES), N-lauroyl propylhomoserin sodium (Sarkosyl), tetramethyl ammonium chloride (TMAC) are all purchased the company in sigma.
The configuration of damping fluid and hybridization solution:
Coupling buffer, PH4.5 250ml
MES 4.88g；
1.5 * TMAC hybridization solution 250ml
5M?TMAC 225ml
20％?Sarkosyl 1.88ml
1M?Tris-HCl，PH8.0 18.75ml
0.5M?EDTA，PH8.0 3.0ml
H
2O 1.37ml；
1.5 * TMAC hybridization solution 250ml
5M?TMAC 150ml
20％?Sarkosyl 1.25ml
1M?Tris-HCl，PH8.0 12.5ml
0.5M?EDTA，PH8.0 2.0ml
H
2O 84.25ml；
The TE damping fluid, PH8.0 500ml
1M?Tris-HCl，PH8.0 5ml
0.5M?EDTA，PH8.0 1ml
H
2O 444ml
The liquid-phase chip combined parallel detecting method of the multiple fusion gene that embodiment 1:3 kind leukemia is relevant
Concrete detection method comprises the steps:
One. detect SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4), the preparation of the microballoon mixed solution of CBFB-MYH11 (typeD) fusion gene
1. according to following sequence synthetic oligonucleotide probe:
SIL-TAL1 (type II, type III): 5 '-AminolinkerC12 AGTTACGCTGCGGTGTGGTC-3 ', shown in SEQ ID NO.1;
MLL-AF4 (e9/e5, e9/e4): 5 '-AminolinkerC12 TATTGCTGTCAAAGGAGGCGG-3 ', shown in SEQID NO.2;
CBFB-MYH11 (type D): 5 '-AminolinkerC12 TGTCCTTCTCCGAGCCTCTTCA-3 ', shown in SEQ IDNO.3;
The Abelson gene: 5 '-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3 ', shown in SEQ ID NO.12;
Will more than contain amido modified oligonucleotide probe respectively with the 4 kinds of carboxyl microballoons coupling that is numbered 11,15,17, No. 21
Equilibrate to room temperature 2.1 take out the fresh dry powder EDC of an aliquot-20 ℃ preservation;
2.2 use dH
2O dissolve respectively SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4), the oligonucleotide probe of CBFB-MYH11 (type D), Abelson, concentration is 1mM (1nmol/ μ l);
2.3 at full speed vortex is numbered 4 kinds of carboxyl microballoons of 11,15,17, No. 21 and stores suspensions 3min at least respectively, produces the microballoon suspension of homogeneous;
2.4 get 2.5 * 10 respectively
6Microballoon stores in suspension to 4 centrifuge tube;
2.510,000g, centrifugal, 1-2min;
2.6 remove supernatant liquor, with 50 μ l 0.1M MES, the coupling buffer of pH4.5, resuspended microballoon, vortex concussion 20 seconds;
2.7 with 3 kinds of concentration is that the 1mM oligonucleotide probe is used dH respectively
2O was with 1: 10 dilution proportion, and making its concentration is 0.1nmol/ μ l;
2.8 every kind of probe adds 2 μ l (concentration is 0.1nmol/ μ l) in the corresponding microballoon of mixing, vortex mixed;
2.9 use dH
2The fresh EDC solution of O configuration 10mg/ml (noting: keep the EDC powder for drying, be convenient to the use of next step EDC);
2.10 add 2.5 μ l 10mg/ml EDC fresh EDC solution branch be clipped to (25 μ g final concentrations are about 0.5 μ g/ μ l) vortex mixing in 4 kinds of microballoons;
2.11 the room temperature lucifuge is hatched 30min;
2.12 use dH
2The EDC fresh solution of second part of 10mg/ml of O configuration (noting:, advise that each step coupling process all uses fresh EDC powder) if EDC powder deliquescence then should abandon;
2.134 respectively add the EDC fresh solution of 2.5 μ l 10mg/ml in the kind microballoon, the vortex mixing;
2.14 the room temperature lucifuge is hatched 30min;
2.154 plant in the coupling microballoon and respectively add 1ml 0.02%Tween-20;
2.16 10,000g, centrifugal, 1-2min;
2.17 remove supernatant, use the resuspended 4 kinds of coupling microballoons of 1ml 0.1%SDS respectively, the vibration mixing;
2.18 10,000g, centrifugal, 1-2min;
2.19 remove supernatant, add 100 μ l TE respectively, PH8.0, vortex mixed 20s, resuspended microballoon;
2.20 with 4 kinds of couplings of cell counter counting the microballoon of oligonucleotide probe;
A. use dH
2O diluted the coupling microballoon with 1: 100;
B. vortex shakes abundant mixing;
C. get 10 μ l to cell counter;
D. count the microballoon total amount of 4 big lattice in angle on the cell counter;
E. microballoon/μ l=(4 big lattice microballoon total amount) * 2.5 * 100 (extension rates).
3. the configuration of microballoon mixed solution
With above-mentioned coupling the microballoon of oligonucleotide probe, as following: SIL-TAL1 (type II, type III) probe microballoon 11, MLL-AF4 (e9/e5, e9/e4) probe microballoon 15, CBFB-MYH11 (type D) probe microballoon 17, Abelson probe microballoon 21, equal proportion is mixed, and the final concentration of various microballoons is 1500/μ l, and 2-8 ℃ keeps in Dark Place.
Two. the preparation of sample
1-15 leukaemic's clinical sample extracts RNA respectively according to the following steps:
1. lymphocyte extracts: get fresh whole blood 2ml and add 1ml 3% Trisodium Citrate, 3000r/min is centrifugal behind the mixing, and 10min, gets that pale yellow chromatograph is lymphocyte on the blood cell layer by 4 ℃;
2. get 1 centrifuge tube (through the DEPC water treatment) adding lymphocyte liquid, 150 μ l. and add 1ml TRIZOL then, room temperature is placed 5min, makes its abundant cracking;
3.12 the centrifugal 5min of 000rpm gets supernatant;
4. add 200 μ l chloroforms among every 1ml Trizol, room temperature is placed 15min behind the acute vibration mixing;
5.4 ℃ 12, the centrifugal 15min of 000g;
6. draw the upper strata water, to another centrifuge tube;
7. add 500 μ l Virahol mixings among every 1ml Trizol, room temperature is placed 5-10min;
8.4 ℃ 12, the centrifugal 10min of 000g abandons supernatant, RNA is sunken to the pipe end;
9. by adding 1ml 75% ethanol among every 1ml Trizol, gentle vibration centrifuge tube, suspension precipitation;
10.4 ℃ 8, the centrifugal 5min of 000g abandons supernatant as far as possible;
11. room temperature is dried or vacuum-drying 5-10min;
12. useful 50ul H
2O, TE buffer or 0.5%SDS dissolving RNA sample, 55-60 ℃, 5-10min (H
2O, TE or 0.5%SDS must handle and high pressure with DEPC);
13. survey the quantitative RNA concentration of OD value.
Three. multiple RT-PCR
MRNA to above-mentioned 1-15 sample carries out the multiple reverse transcription pcr amplification as follows:
1.cDNA first chain is synthetic
1. get the Eppendorf tube of handling once DEPC, place on ice, set up following reaction system;
Total?RNA 5～10μg/3μl
Oligo(dT)18?Primer(0.5μg/μl) 1μl
EDPC-treated?water forword?to?12μl
The mixing reaction solution arrives the pipe end with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge gently;
2. centrifuge tube is in 70 ℃ of incubations 5 minutes, take out rapidly afterwards to place ice to cool off, with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge to the pipe end;
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
The mixing reaction solution arrives the pipe end with the reaction solution on the centrifugal a little collection tube wall of refrigerated centrifuge gently;
4. centrifuge tube was in 37 ℃ 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 ℃ of reactions 60 minutes;
7. centrifuge tube is in 10 minutes termination reactions of 70 ℃ of heating, and taking-up places ice to cool off rapidly afterwards;
2. multiplex PCR
2.1 according to following sequence synthesized primer thing:
SIL-TAL1 (type II, type III): upstream primer 5 '-biotin CCTGCAAACAGACCTCAGCTC-3 ', shown in SEQ ID NO.4; Downstream primer 5 '-CGAGGAAGAGGATGCACAC-3 ' is shown in SEQ IDNO.5;
MLL-AF4 (e9/e5, e9/e4): upstream primer 5 '-biotin TCCAGAGCAGAGCAAACAG-3 ', shown in SEQ ID NO.6; Downstream primer 5 '-CCTTGCTGAGAATTTGAGTG-3 ' is shown in SEQ ID NO.7;
CBFB-MYH11 (type D): upstream primer 5 '-biotin GAGGATGCATTAGCACAACAG-3 ', shown in SEQ ID NO.8; Downstream primer 5 '-GAAGCAACTCCTGGGTGTC-3 ' is shown in SEQ ID NO.9;
The Abelson gene: upstream primer 5 '-biotin GCGCAAAATGTTGGAGATC-3 ', shown in SEQ ID NO.10; Downstream primer 5 '-GGAGCTTTTCACCTTTAGTTATGC-3 ' is shown in SEQ ID NO.11.
2.2PCR reaction system is as follows
cDNA 10μl
10×PCR?Buffer 10μl
MgCl2(25mmol/L) 4μl
dNTPs(10mM) 0.5μl
Taq?Polymerase(5u/μl) 0.5μl
Primer?mix 11μl
dd?H
2O 14μl
Final volume is 50 μ l
Pcr amplification program: 95 ℃ of 10min; 95 ℃ of 30s, 64-50 ℃ of 1min, 72 ℃ of 30min; Preceding 14 circulations, every circulation primary, annealing temperature reduces by 1 ℃, back 21 circulations, annealing temperature remains in 50 ℃ carries out; 72 ℃ of 7min; 4 ℃ of insulations.
Four. the hybridization of oligonucleotide probe and PCR product
1. choose the oligonucleotide probe mixture of microspheres that disposes in the step 1;
2. vortex shakes 20s, mixing microballoon;
3. preparation microballoon working fluid is 150 microballoons/μ l with 1.5 * TMAC hybridization solution dilution coupling microballoon to concentration.(annotate: the microballoon working fluid of each reaction needed 33 μ l);
4. vortex shakes 20s, mixing microballoon working fluid;
5. in sample aperture, positive control hole, negative control hole, add 33 μ l microballoon working fluids respectively;
6. add the pcr amplification product and the TE solution (PH is 8.0) of 1-15 patient's sample in each sample well respectively, cumulative volume is 17 μ l;
7. inhale up and down with the volley of rifle fire and beat gentle mixing reaction solution;
8. cover the reaction lid and avoid evaporation, under 95-100 ℃, hatch 1-3min, make the oligonucleotide in the sample remove secondary structure;
9. incubation reaction plate 15min under hybridization temperature;
10. prepare fresh detection mixed solution, with 1 * TMAC hybridization solution dilution Streptavidin phycoerythrin solution to 10 μ g/ml (the detection mixed solutions of each reaction needed 25 μ l);
11. add the detection mixed solution of 25 μ l to every hole, inhale up and down with the volley of rifle fire and beat gentle mixing;
12. under hybridization temperature, hatch 5min;
Above-mentioned steps can be programmed its merging by following scheme by the PCR instrument:
95℃，1-3min；
Hybridization temperature, forever;
13. on liquid-phase chip instrument (LiquiChip 200, QIAGEN company), keep hybridization temperature, each reacting hole is carried out check and analysis, measure fluorescence MFI value.
Five. detected result and analysis
Detected result (fluorescence MFI value) is as follows:
Interpretation of result:
1,5,9,10, No. 13 patient's types are leukemia fusion gene CBFB-MYH11 (type D) positive
2,7,8,11,14, No. 15 patient's types are leukemia fusion gene MLL-AF4 (e9/e5, e9/e4) positive
3,4,6, No. 12 patient's types are leukemia fusion gene SIL-TAL1 (type II, the type III) positive
Simultaneously, the fluorescence MFI value of the positive fusion gene of patient is compared with the fluorescence MFI value of confidential reference items Abelson gene, can be drawn the expression situation of fusion gene.
About after the those set forth of the present invention, those skilled in the art can do various modifications or variation to the present invention more than having read, and these equivalent form of values belong to the scope defined in the application's appended claims equally.
Sequence table
＜110〉Jiangsu Mai Dijiyin bio tech ltd
＜120〉associated detecting method and the diagnostic kit of the fusion gene that leukemia is relevant
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tattgctgtc?aaaggaggcg?g 21
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<223>(1)...(22)
<400>3
tgtccttctc?cgagcctctt?ca 22
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
cctgcaaaca?gacctcagct?c 21
<210>5
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
cgaggaagag?gatgcacac 19
<210>6
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
tccagagcag?agcaaacag 19
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
ccttgctgag?aatttgagtg 20
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
gaggatgcat?tagcacaaca?g 21
<210>9
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>9
gaagcaactc?ctgggtgtc 19
<210>10
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>10
gcgcaaaatg?ttggagatc 19
<210>11
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>11
ggagcttttc?acctttagtt?atgc 24
<210>12
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<223>(1)...(20)
<400>12
ctgaagggct?tcttccagat 20
Claims (9)
1. the associated detecting method of the fusion gene that a leukemia is relevant is characterized in that:
(1) comprises 3 kinds of carboxyl microballoon Beads that difference is fluorescence-encoded, distinguish the mRNA designed specific dna probe of covalent attachment on every kind of microballoon at the multiple fusion gene of chromosome translocation formation in the leukemia;
(2) at the mRNA of the formed multiple fusion gene of chromosome translocation in the different type of leukemia, design the upstream and downstream primer respectively,, amplify corresponding product by reverse transcription PCR to different fusion gene mRNAs; The fusion gene that chromosome translocation forms in the described leukemia is at following multiple fusion gene that can independent assortment: and SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4), CBFB-MYH11 (type D);
(3) contain the microballoon of specific dna probe and the reverse transcription amplification product bulk crossing of fusion gene mRNA, behind adding Streptavidin-phycoerythrin Streptavidin-PE, detect fluorescent signal by liquid-phase chip method xMAP;
(4) fluorescent signal with detected fluorescent signal and internal control gene compares, thereby determines whether there is leukemia fusion gene in the test sample, and the expression situation of fusion gene in the sample.
2. the associated detecting method of the fusion gene that leukemia as claimed in claim 1 is relevant is characterized in that described microballoon is that mean diameter is 5.6 μ m, combines the polyphenyl alkene microballoon of different fluorescence dyes, i.e. color-code microballoon Color-coded beads.
3. the associated detecting method of the fusion gene that leukemia as claimed in claim 1 is relevant is characterized in that described 3 specific dna probes that are covalently bonded on the microballoon comprise following sequence, and wherein 5 ' end contains amido modified:
SIL-TAL1 (type II, type III): 5 '-AminolinkerC12AGTTACGCTGCGGTGTGGTC-3 ' is shown in SEQ ID NO.1;
MLL-AF4 (e9/e5, e9/e4): 5 '-Aminol inkerC12TATTGCTGTCAAAGGAGGCGG-3 ', shown in SEQ ID NO.2;
CBFB-MYH11 (type D): 5 '-AminolinkerC12TGTCCTTCTCCGAGCCTCTTCA-3 ' is shown in SEQ ID NO.3;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
4. the associated detecting method of the fusion gene that leukemia as claimed in claim 1 is relevant is characterized in that, describedly comprises following sequence at the designed upstream and downstream primer of the mRNA of fusion genes, and wherein upstream primer 5 ' end contains biotin label:
SIL-TAL1 (type II, type III): upstream primer 5 '-biotin CCTGCAAACAGACCTCAGCTC-3 ', shown in SEQ ID NO.4; Downstream primer 5 '-CGAGGAAGAGGATGCACAC-3 ' is shown in SEQ IDNO.5;
MLL-AF4 (e9/e5, e9/e4): upstream primer 5 '-b i ot i n TCCAGAGCAGAGCAAACAG-3 ', shown in SEQ ID NO.6; Downstream primer 5 '-CCTTGCTGAGAATTTGAGTG-3 ' is shown in SEQ ID NO.7;
CBFB-MYH11 (type D): upstream primer 5 '-biotin GAGGATGCATTAGCACAACAG-3 ', shown in SEQ ID NO.8; Downstream primer 5 '-GAAGCAACTCCTGGGTGTC-3 ' is shown in SEQ ID NO.9;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
5. the associated detecting method of the fusion gene that leukemia as claimed in claim 1 is relevant is characterized in that described internal control gene is the Abelson gene, and its primer and probe sequence are as follows:
Upstream primer 5 '-biotin GCGCAAAATGTTGGAGATC-3 ' is shown in SEQ ID NO.10;
Downstream primer 5 '-GGAGCTTTTCACCTTTAGTTATGC-3 ' is shown in SEQ ID NO.11;
Probe 5 '-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3 ' is shown in SEQ ID NO.12;
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence (containing complementary sequence) to 5 ';
Perhaps with above-mentioned sequence (containing complementary sequence) homology greater than 85% sequence;
Perhaps with the complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
6. diagnostic kit that detects the relevant fusion gene of leukemia, it is characterized in that, comprised the covalent attachment described in the claim 1 the leukemia fusion gene specific probe mixture of microspheres and combine the upstream and downstream primer of the microballoon of Abelson gene probe, the leukemia fusion gene mRNA described in the claim 1 and upstream and downstream primer, Streptavidin-phycoerythrin, the quality control product of Abelson gene.
7. the diagnostic kit of the fusion gene that detection leukemia as claimed in claim 6 is relevant is characterized in that described mixture of microspheres is according to the needs independent assortment of different test sample.
8. the diagnostic kit of the fusion gene that detection leukemia as claimed in claim 6 is relevant, it is characterized in that, described quality control product comprises positive control and negative control, and wherein positive control is the mixed solution that contains various fusion gene plasmids, comprises the plasmid that contains the Abelson gene; Negative control is not for containing the plasmid solution of fusion gene and Abelson gene.
9. the diagnostic kit of the fusion gene that a detection leukemia as claimed in claim 6 is relevant is detecting vitro samples, leukemia is carried out the application in the dynamic monitoring of somatotype, early diagnosis, observation of curative effect, prognosis and small residual disease.
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| CN201010137451.1A CN101955991B (en) | 2010-03-31 | 2010-03-31 | Joint detection method of fusion genes related to leukemia and diagnosis kit |
| PCT/CN2011/072139 WO2011120398A1 (en) | 2010-03-31 | 2011-03-30 | Joint detection method for leukemia fusion genes and diagnostic kits thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011120398A1 (en) * | 2010-03-31 | 2011-10-06 | 江苏迈迪基因生物科技有限公司 | Joint detection method for leukemia fusion genes and diagnostic kits thereof |
| CN103667453A (en) * | 2013-11-18 | 2014-03-26 | 福州艾迪康医学检验所有限公司 | Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes |
| CN104278093A (en) * | 2014-09-28 | 2015-01-14 | 南京百捷生物科技有限公司 | Primer pair and kit for detecting SIL-TAL1 fusion genes by pyrosequencing method |
| CN107151699A (en) * | 2017-06-01 | 2017-09-12 | 武汉艾迪康医学检验所有限公司 | Detect the kit and method of NUP214 ABL1 gene relative expression quantities |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480456A (en) * | 2021-12-21 | 2022-05-13 | 安徽医科大学第二附属医院 | Standard plasmid for detecting multiple fusion genes and detection kit |
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|---|---|---|---|---|
| WO2009124901A1 (en) * | 2008-04-07 | 2009-10-15 | Erasmus University Medical Center Rotterdam | Methods and kits for detecting tumor-specific fusion proteins |
| CN101624623A (en) * | 2008-07-11 | 2010-01-13 | 秦亚溱 | Kit for quantitatively detecting ABL mRNA level |
| GB2463401A (en) * | 2008-11-12 | 2010-03-17 | Caris Mpi Inc | Diagnostic methods using exosomes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101955991B (en) * | 2010-03-31 | 2014-05-28 | 江苏迈迪基因生物科技有限公司 | Joint detection method of fusion genes related to leukemia and diagnosis kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009124901A1 (en) * | 2008-04-07 | 2009-10-15 | Erasmus University Medical Center Rotterdam | Methods and kits for detecting tumor-specific fusion proteins |
| CN101624623A (en) * | 2008-07-11 | 2010-01-13 | 秦亚溱 | Kit for quantitatively detecting ABL mRNA level |
| GB2463401A (en) * | 2008-11-12 | 2010-03-17 | Caris Mpi Inc | Diagnostic methods using exosomes |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011120398A1 (en) * | 2010-03-31 | 2011-10-06 | 江苏迈迪基因生物科技有限公司 | Joint detection method for leukemia fusion genes and diagnostic kits thereof |
| CN103667453A (en) * | 2013-11-18 | 2014-03-26 | 福州艾迪康医学检验所有限公司 | Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes |
| CN103667453B (en) * | 2013-11-18 | 2015-06-17 | 福州艾迪康医学检验所有限公司 | Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes |
| CN104278093A (en) * | 2014-09-28 | 2015-01-14 | 南京百捷生物科技有限公司 | Primer pair and kit for detecting SIL-TAL1 fusion genes by pyrosequencing method |
| CN104278093B (en) * | 2014-09-28 | 2016-11-23 | 南京百捷生物科技有限公司 | Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion primer to and kit |
| CN107151699A (en) * | 2017-06-01 | 2017-09-12 | 武汉艾迪康医学检验所有限公司 | Detect the kit and method of NUP214 ABL1 gene relative expression quantities |
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|---|---|
| WO2011120398A1 (en) | 2011-10-06 |
| CN101955991B (en) | 2014-05-28 |
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