CN103725787B - Fluorescent PCR (Polymerase Chain Reaction) detection kit for fusion genes of leukemia - Google Patents
Fluorescent PCR (Polymerase Chain Reaction) detection kit for fusion genes of leukemia Download PDFInfo
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Abstract
The invention provides a fluorescent PCR (Polymerase Chain Reaction) detection kit for fusion genes of leukemia. The kit comprises a PCR reaction liquid which comprises primers of a fusion gene PML/RARalpha of leukemia and a probe with sequences as follows: a PML/RARalpha upstream primer: TAGCATACCATGTAAAGGGACTGGCTG; a PML/RARalpha downstream primer: CGAAAGGAGACTTCGTGCGTTCCTT; and a PML/RARalpha probe: GCGACAACATAGGCTACCAGTGCGCTTCTA. Preferably, the kit further comprises another two PCR reaction liquids, one of which comprises a primer and a probe of the leukemia fusion gene E2a/PBX1 with sequences and the other of which comprises a primer and a probe of another leukemia fusion gene MLL/ENL with sequences. By applying the kit which is quick to operate, simple and convenient in method and good in detection effect, which is provided by the invention, positive and negative fusion genes of PML/RARalpha, E2A/PBX1 and MLL/ENL in the marrow tissues of a patient can be qualitatively detected, respectively, and thus help is provided for clinical diagnosis and subsequent medication of the leukemia.
Description
Technical field
The present invention relates to a kind of leukemia fusion gene fluorescence PCR detection reagent kit.
Background technology
Leukemia (leukemia) is the class general malignant tumour originating from hemopoietic system, has the heterogeneity of height, but its common feature is produce and gather a large amount of inmature or abnormal hemocyte in marrow.Compare with corresponding normal plasma cell, the cell of these exceptions has different biological characteristicses, often show as that multiplication capacity increases, dysdifferentiation or apoptosis reduce, and can express some cell surface molecules or secrete some soluble mediators thus play its malignant characteristics.Increase along with these are paracytic, suppression or the exhaustion of marrow normal hematopoiesis function and host immune function can be caused, and blood circulation can be entered and invade other any tissue and organs, the various clinical symptom finally causing anaemia, infection, hemorrhage and organ infiltration to cause and sign.Clinical appearance anaemia in various degree, hemorrhage, heating and liver spleen, lymphadenectasis, can threat to life.
Leukemia fusion gene (fusion gene) is leukemic molecular biology specificity marker.In recent years, due to the development of Protocols in Molecular Biology, the understanding that leukemia cellular elements genetics changes also is deepened continuously.Hitherto reported leukemia relates at least tens of kinds of fusion genes. and have realized that in most leukemia and there is chromosomal structural aberration, comprise disappearance, repetition, inversion, transposition etc., cause proto-oncogene and cancer suppressor gene structure variation, protooncogene activation or cancer suppressor gene inactivation, produce new fusion gene, encoding fusion protein.Some gene is regulating cell propagation, differentiation and the transcription factor of apoptosis, when gene morphs, directly affects downstream signaling pathway, causes that ability of cell proliferation strengthens, apoptosis obstacle, and dysdifferentiation etc., produce phenotype of leukemia.
Fusion gene detects has extremely important meaning to leukemic diagnosis.Find simple cell Morphologic classification by clinical practice, the subjective composition of tester is comparatively large, and mutual coincidence rate and accuracy have a definite limitation.Along with developing rapidly of Cytobiology and molecular biology technology and deepening continuously to leukemia study of incident mechanism, recognize gene in leukemia pathogenic process and character mutation significant to each leukemoid Clinics and Practices, therefore propose leukemia MICM somatotype.The research of nearly 2 years leukemia molecule features achieves obvious progress, especially forms fusion gene to chromosome translocation, has some as the dissimilar leukemic molecular biology specificity marker of diagnosis and the unique foundation determining diagnosis.Leukemia fusion gene can pass through reverse transcription PCR (RT-PCR) technology and be detected, and contributes to evaluating leukemic acute degree, clone's characteristic and somatotype, makes the more scientific and standardization of leukemic diagnosis typing." medical institutions' Clinical laboratory test catalogue " of Ministry of Health's promulgation in 2007, requirement is wherein had to utilize the leukemia fusion gene inspection of RT-PCR or real-time round pcr, relate generally to the inspection of 6 kinds of fusion genes, comprise BCR/ABL, PML/RAR а, AML1/MPSI/EV11, DEK/CAN, AML1/MTG8, E2A/PBX1.The comparable traditional cytology of RT-PCR and clinical symptom occur early 5 ~ 8 months, can detect 1 × 10
6a leukemia cell in individual karyocyte, has the unrivaled specificity of other method and susceptibility in leukemic early diagnosis.
Fusion gene is picked up to survey also has very important effect to leukemia treating and Index for diagnosis.The Prognostic significance of cytogenetics somatotype and disease is close, is of great significance for the selection and judging prognosis tool instructing clinical personalized therapy program.Acute leukemia has PML/RAR а, and the prognosis of CBFB/MYH11, MLL/ENL fusion gene is better, and chemotherapy is complete, and remission rate is high, can long-term remission or healing, does not advocate to do hematopoietic stem cell transplantation in early days; And for there being MLL abnormal (containing mll gene in fusion gene), or containing AML(acute myelocytic leukemia) in MYC/IgH fusion gene, or containing ALL(acute lymphoblastic leukemia) in BCR/ABL fusion gene, then patient is poor to chemotherapy side effect, recurrence rate is high, advises its actively row hematopoietic stem cell transplantation of having ready conditions then.There is the detection of fusion gene, can instruct when just controlling and select long-term treatment regimen scientifically and rationally, avoid unnecessary insufficient therapy or over-treatment.
Fusion gene detects conventional method and mainly comprises leukemia cell's chromosome karyotype analysis, chromosome fluorescence in-situ hybridization (Fluorescent In Situ Hybridazation, FISH) technology and polymerase chain reaction technology (Polymerase ChainReaction, PCR).
The positive rate of chromosomal G-banding karyotyping technology depends on the hyperplasia rate of leukemia cell to be checked, usually be subject to the impact of detection technique condition and manual operation factor, every part detect sample need at least to analyze 10 ~ 30 metaphase cell, and due to the cell in sample be not also all homogeneous tumour cell, therefore then can not carry out karyotyping when cell concentration is very few, there is certain difficulty in the identification of some complicacy chromosome translocation simultaneously technically.But karyotyping technology has advantage in the chromosome abnormalties such as detection disappearance and numerical aberration, is detect the requisite means of ALL patient's chromosome abnormalty.
Chromosome fluorescence in-situ hybridization is a kind of nonradioactivein situhybridization technology grown up on the basis of original radioactive in situ hybridization technology phase late 1980s.Be probe with fluorescently-labeled known single-chain nucleic acid, according to the principle of base complementrity, hybridize with single-chain nucleic acid unknown in material to be checked, form the heteroduplex nucleic acid that can be detected, to determine and the nucleotide sequence of probes complementary position on chromosome and distribution.Fluorescence in situ hybridization has fast, detection signal is strong, hybrid specificities is high and can the feature such as multiple staining.In situ hybridization detection can be carried out to the tumour cell of interval or division early metaphase with the DNA probe of vitamin H or digoxigenin labeled, can not only detect the karyomit(e) of metaphase, and the abnormal karyotype (comprising chromosome translocation) of inerphosei cells can be measured sensitively and carry out qualitative, quantitative and positioning analysis.But due to expensive, clinical application is subject to a definite limitation.
PCR detects leukemia correlation fusion gene comparatively effectively and the method for sensitivity, except conventional PCR method, Chao Shi RT-PCR(nested reverse transcription PCR), real-time fluorescence quantitative PCR (real time quantitative PCR, RQ-PCR) has been applied to the qualitative and detection by quantitative of leukemia correlation fusion gene.Real-Time Fluorescent Quantitative PCR Technique (realtime quantitative PCR, RQ-PCR) not only can determine whether leukemia cell expresses fusion gene, and the different steps for the treatment of, the change of track fusion level can also be detected, i.e. MRD level.This technology current is in scientific research and be widely used clinically, also provide new means for leukemic molecular biology research, existing much clinical and laboratory researchers apply this technology and have carried out large quantifier elimination report in leukemia fusion gene detection and clinical application thereof.Consult domestic and foreign literature, application RQ-PCR detects leukemia fusion gene, be all detect the expression of its RNA in fact, what mostly adopt is the RQ-PCR technology of the combination of Taqman hydrolysis probes, hybridization probe and DNA dyestuff, and wherein using maximum is hydrolysis probes technology.Although each detection technique is variant in principle, the detection method of its instrument (real-time fluorescence quantitative PCR instrument) used and foundation is similar.
The domestic existing multiple test kit based on Real-Time Fluorescent Quantitative PCR Technique detection leukemia fusion gene is applied in clinical detection, but be limited to its primer probe sequence, this area also needs to develop a kind ofly detects the good and PCR detection kit of high comprehensive performance of leukemia fusion gene specificity.In addition, these test kits also because not arranging positive internal reference (namely mark), cannot monitor false negative; With the measure generally not preventing PCR primer to pollute.
Summary of the invention
First the present invention provides a kind of leukemia fusion gene fluorescence PCR detection reagent kit, comprises PCR reaction solution in described test kit, and the primer and the probe sequence that comprise leukemia fusion gene PML/RAR α in described PCR reaction solution are as follows,
PML/RAR α upstream primer: TAGCATACCATGTAAAGGGACTGGCTG,
PML/RAR α downstream primer: CGAAAGGAGACTTCGTGCGTTCCTT,
PML/RAR α probe: GCGACAACATAGGCTACCAGTGCGCTTCTA.
Preferably, also containing detecting the interior mark of leukemia fusion gene in described test kit, and also comprise interior target primer in described PCR reaction solution and probe sequence as follows,
Interior label sequence: CCAGCAATTAAATCCTATTTGCAAGCACTCTGTCATCCAGTTAGCTGACTCACGTA TTCGTAGCCACCCTTCTGGAGGTGCAATCTCAATTATGTCATCA,
Interior mark upstream primer: TCCTATTTGCAAGCACTCTGTCA,
Interior mark downstream primer: TCCAGAAGGGTGGCTACGAA,
Interior mark probe: CCAGTTAGCTGACTCACGT.
In a kind of embodiment of the present invention, also comprise the primer probe sequence of fusion gene E2A/PBX1 and/or the primer probe sequence of fusion gene MLL/ENL in described PCR reaction solution, its sequence is as follows,
E2A/PBX1 upstream primer: GGTGTAACAGGTAAAAAGACTGGTTC,
E2A/PBX1 downstream primer: TTGAGGAGGAGTACATCCAAGTCCTT,
E2A/PBX1 probe: TCTACAACATTTTGGCTACCAGGGCTTCTA;
MLL/ENL upstream primer: CGTGTAGACATGATTATTGACTGGAAC,
MLL/ENL downstream primer: TTGAAGGGAGTTCTTGCGCTCCTT,
MLL/ENL probe: TCTTCAACATTGGTTAAAAGAACTTCTA.
In this specific embodiment, a kind of situation comprises the primer probe sequence of PML/RAR α and the primer probe sequence of E2A/PBX1 in described PCR reaction solution, whether this test kit can detect in sample to be tested containing these two kinds of fusion genes, when not containing this two kinds of fusion genes in sample to be tested, detected result is negative, and when in sample to be tested containing any one in both or two kinds time, detected result be the positive.The second situation comprises the primer probe sequence of PML/RAR α and the primer probe sequence of MLL/ENL in described PCR reaction solution, same, when not containing this two kinds of fusion genes in sample to be tested, detected result is negative, and when in sample to be tested containing any one in both or two kinds time, detected result be the positive.And the third situation is the primer probe sequence comprising the primer probe sequence of PML/RAR α, the primer probe sequence of E2A/PBX1 and MLL/ENL in described PCR reaction solution, accordingly, when not containing this three kinds of fusion genes in sample to be tested, detected result is negative, and when in sample to be tested containing any one in this three or multiple time, detected result be the positive.
In another kind of embodiment of the present invention, another kind of PCR reaction solution is also comprised in described test kit, comprise the primer probe sequence of leukemia fusion gene E2A/PBX1 or the primer probe sequence of fusion gene MLL/ENL in described another kind of PCR reaction solution, its sequence is as implied above.Easy understand, use test kit of the present invention, whether can clearly learn from the detected result of sample to be tested in sample to be tested containing (PML/RAR α and E2A/PBX1 in these two kinds of fusion genes, or PML/RAR α and MLL/ENL) any one, that is this test kit can judge feminine gender and the positive of these two kinds of fusion genes respectively.
In another kind of embodiment of the present invention, another two kinds of PCR reaction solutions are also comprised in described test kit, a kind of in described another two kinds of PCR reaction solutions comprises the primer probe sequence of leukemia fusion gene E2A/PBX1 and the another kind of primer probe sequence comprising leukemia fusion gene MLL/ENL, and its sequence is as implied above.Easy understand, this test kit can judge feminine gender and the positive of these three kinds of fusion genes respectively.Apply the test kit that operation provided by the invention is quick, method is easy, Detection results is good, qualitative detection can be carried out to the RNA of PML/RAR α, E2A/PBX1, MLL/ENL fusion gene in Bone Marrow of Patients tissue, thus offer help for leukemic clinical diagnosis and subsequent administrations.
In this specific embodiment, preferably, in described test kit, also comprise interior mark, and all comprise interior target primer and probe sequence in each PCR reaction solution in test kit, interior label sequence and interior target primer probe sequence as implied above.
In another kind of embodiment, described test kit comprises interior mark, 1 ~ 3 kind of PCR reaction solution, enzyme mixation, positive control and negative control.
Embodiment
Illustrate the present invention with following embodiment in the present invention, but these embodiments are not used for limiting the present invention.
Embodiment 1
The present embodiment provides the PCR detection kit of a kind of leukemia fusion gene PML/RAR α, E2A/PBX1, MLL/ENL.
1. mark (positive internal reference) in: for the segment length inserting pUC18T carrier is the recombinant chou of the DNA artificial sequence synthetic of 100 base pairs, i.e. plasmid, concentration is 1.00E+04copies/ml ~ 5.00E+04copies/ml, as the positive internal reference in PCR amplification system, prevent the false negative that the PCR interfering substance owing to may exist in sample causes; The sequence of 100 base pairs is as follows: 5 '-CCAGCAATTAAATCCTATTTGCAAGCACTCTGTCATCCAGTTAGCTGACTCACGTA TTCGTAGCCACCCTTCTGGAGGTGCAATCTCAATTATGTCATCA-3 ';
2. PCR reaction solution: 10 × PCR reaction buffer 5 μ l, 0.2mmol/L deoxyribonucleoside triphosphate, the upstream and downstream primer for target polynucleotide amplification of 0.2 μm of ol/L ~ 0.4 μm ol/L, the probe detected for target polynucleotide of 0.2 μm of ol/L ~ 0.4 μm ol/L, the upstream and downstream primer for interior mark fragment amplification of 0.2 μm of ol/L ~ 0.4 μm ol/L, 0.1 μm of ol/L ~ 0.2 μm ol/L for detecting interior target probe.Described 10 × PCR reaction buffer comprises the 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution of pH7.5,30mmol/L magnesium chloride solution, 500mmol/L Klorvess Liquid, 0.2% (volume/volume) Triton solution and 10% (volume/volume) formamide soln; Described deoxyribonucleoside triphosphate is dATP, dCTP, dUTP, dGTP or dTTP; Described primer and probe sequence for detecting PML/RAR α, E2A/PBX1, MLL/ENL is respectively:
PML/RAR α upstream primer: 5 '-TAGCATACCATGTAAAGGGACTGGCTG-3 ';
PML/RAR α downstream primer: 5 '-CGAAAGGAGACTTCGTGCGTTCCTT-3 ';
PML/RAR α probe: 5 '-GCGACAACATAGGCTACCAGTGCGCTTCTA-3 '.
E2A/PBX1 upstream primer: 5 '-GGTGTAACAGGTAAAAAGACTGGTTC-3 ';
E2A/PBX1 downstream primer: 5 '-TTGAGGAGGAGTACATCCAAGTCCTT-3 ';
E2A/PBX1 probe: 5 '-TCTACAACATTTTGGCTACCAGGGCTTCTA-3 '.
MLL/ENL upstream primer: 5 '-CGTGTAGACATGATTATTGACTGGAAC-3 ';
MLL/ENL downstream primer: 5 '-TTGAAGGGAGTTCTTGCGCTCCTT-3 ';
MLL/ENL probe: 5 '-TCTTCAACATTGGTTAAAAGAACTTCTA-3 '.
Described is respectively for detecting interior target primer probe sequence:
Interior mark upstream primer: 5 '-TCCTATTTGCAAGCACTCTGTCA-3 ';
Interior mark downstream primer: 5 '-TCCAGAAGGGTGGCTACGAA-3 ';
The sequence of interior mark probe is: 5 '-CCAGTTAGCTGACTCACGT – 3 ';
3. enzyme mixation: hot resistant DNA polymerase (Taq enzyme) 1U/ μ l ~ 5U/ μ l, uracil dna glycosylase (UNG enzyme) 0.5U/ μ l ~ 2U/ μ l; Wherein UNG enzyme has the function of the PCR primer of degraded containing dU, utilizes the dUTP in UNG enzyme and PCR reaction solution can play the effect of prevention PCR primer pollution;
4. positive control: be strong positive plasmid, its concentration is 1.00 ~ 5.00E+05copies/ml;
5. negative control: be sterile saline;
Embodiment 2
The present embodiment provides leukemia fusion gene PML/RAR α, E2A/PBX1, MLL/ENL detection kit in embodiment 1 for detecting the operation steps as unknown sample such as Bone Marrow of Patients tissues.
One, reagent prepares:
According to the quantity of sample to be tested, negative control, positive control, get the PCR reaction solution (38 μ l/ person-portion) of respective amount, enzyme mixation (2 μ l/ person-portion) and interior mark (0.5 μ l/ person-portion) in proportion, be fully mixed into PCR-mix, for subsequent use after brief centrifugation.
Two, sample process and application of sample
1) in different 5 PCR reaction tubess, add same sample to be tested, negative control, each 5 μ l of positive control;
2) leave standstill after 10 minutes, often pipe adds PCR-mix45 μ l, inhales and plays mixing 2-3 time, lid upper tube cap (after removing bubble), centrifugal 30 seconds of 2000rpm.
Three, in the enterprising performing PCR amplification of fluorescent quantitative PCR instrument
1) PCR reaction tubes is put into amplification instrument sample cell, sample to be tested title is set by correspondence order.
2) fluorescence detection channel is selected: select FAM passage (Reporter:FAM, Quencher:None) detection fusion gene; HEX or VIC passage (Reporter:VIC, Quencher:None) is selected to detect interior mark; Reference fluorescent (Passive Reference) is set to none.
3) quantitative fluorescent PCR reaction conditions is:
Table 1
4) interpretation of result
After reaction terminates, the automatic saving result of instrument, the software that instrument can be utilized to carry carries out automatic analysis (also can the starting value of manual regulation baseline, end value and threshold line value analyze), then records sample Ct value and definite value result.The intersection point of amplification curve and threshold line, is called Ct(and cycle threshold, the cycling numerical value experienced when the fluorescent signal referring in PCR reaction tubes reaches the threshold value of setting); If sample amplification curve is S-type, there is Ct value and Ct≤39, leukemia fusion gene (any one or more in PML/RAR α, E2A/PBX1, MLL/ENL three) can be judged to positive; If sample amplification curve is straight, without Ct value display (Undet) display, leukemia fusion gene (PML/RAR α, E2A/PBX1 and MLL/ENL three) can be judged to negative.
Embodiment 3
The present embodiment provides the PCR detection kit of another kind of leukemia fusion gene PML/RAR α, E2A/PBX1, MLL/ENL, uses each can specifically differentiation in above-mentioned three kinds of fusion genes of this test kit to be negative or positive.
The component of this test kit 1. ~ 5. in except component 2. except, all the other components are identical with the component in embodiment 1.
Component in the present embodiment 2. PCR reaction solution is three kinds of PCR reaction solutions, concrete, containing the primer of leukemia fusion gene PML/RAR α and probe sequence and interior target primer probe sequence in the first PCR reaction solution, containing the primer of leukemia fusion gene E2A/PBX1 and probe sequence and interior target primer probe sequence in the second PCR reaction solution, and primer containing leukemia fusion gene MLL/ENL in the third PCR reaction solution and probe sequence and interior target primer probe sequence.
Embodiment 4
The present embodiment provides test kit described in embodiment 3 for detecting the operation steps as unknown sample such as Bone Marrow of Patients tissues.
One, reagent prepares:
According to the quantity of sample to be tested, negative control, positive control, get the PCR reaction solution (38 μ l/ person-portion) of respective amount, enzyme mixation (2 μ l/ person-portion) and interior mark (0.5 μ l/ person-portion) in proportion, be fully mixed into PCR-mix, for subsequent use after brief centrifugation.Wherein, corresponding to PCR-mix that every part of sample to be tested is all different according to three kinds of different PCR reaction solution preparation three pipes (3 PCR reaction tubess), negative control and positive control also prepare the different PCR-mix of three pipes separately.
Two, sample process and application of sample
1) and in different PCR reaction tubess, add sample to be tested, negative control, each 10 μ l of positive control;
2) leave standstill after 10 minutes, often pipe adds each 40 ~ 45 μ l of its corresponding PCR-mix, inhales and plays mixing 2-3 time, lid upper tube cap (after removing bubble), centrifugal 30 seconds of 2000rpm.
Three, in the enterprising performing PCR amplification of fluorescent quantitative PCR instrument
1) PCR reaction tubes is put into amplification instrument sample cell, sample to be tested title is set by correspondence order.
2) fluorescence detection channel is selected: select FAM passage (Reporter:FAM, Quencher:None) detection fusion gene PML/RAR α, E2A/PBX1 and MLL/ENL; Select mark in HEX or VIC Air conduct measurement; Reference fluorescent (PassiveReference) is set to none.
3) quantitative fluorescent PCR reaction conditions is as described in Table 1.
4) interpretation of result
After reaction terminates, if sample amplification curve is S-type, there is Ct value and Ct≤39, corresponding leukemia fusion gene (PML/RAR α, E2A/PBX1 or MLL/ENL) can be judged to positive; If sample amplification curve is straight, without Ct value display (Undet) display, corresponding leukemia fusion gene (PML/RAR α, E2A/PBX1 or MLL/ENL three) can be judged to and be negative.
The present invention is detected by quantitative fluorescent PCR fusion gene and diagnoses leukemia, for clinical leukemia rapid detection open up new, succinctly detect approach easily.In addition, combination is optimized to PCR reaction system, utilizes UNG enzyme can degrade containing the feature of the DNA chain of dU, in PCR system, with the addition of UNG enzyme and dUTP, the pollution of previous PCR product can be prevented, prevent pattern detection false positive; In sample extraction process, add interior mark, mark monitoring DNA extraction and PCR reaction process in utilizing, whether monitoring reaction system is effective, prevents pattern detection false negative.Detected result can be used for leukemic auxiliary diagnosis and successive treatment effect judges, for clinical diagnosis and treatment provide molecular diagnosis foundation.Use test kit of the present invention to detect enterprise work reference material, yin and yang attribute reference material coincidence rate is 100%.And precision test shows: batch in and batch between reproducible, the variation coefficient <10% of detected result Ct value, concentration variation coefficient <50%.
Claims (7)
1. a leukemia fusion gene fluorescence PCR detection reagent kit, is characterized in that, comprises PCR reaction solution in described test kit, and the primer and the probe sequence that comprise leukemia fusion gene PML/RAR α in described PCR reaction solution are as follows,
PML/RAR α upstream primer: TAGCATACCATGTAAAGGGACTGGCTG,
PML/RAR α downstream primer: CGAAAGGAGACTTCGTGCGTTCCTT,
PML/RAR α probe: GCGACAACATAGGCTACCAGTGCGCTTCTA;
Also containing detecting the interior mark of leukemia fusion gene in described test kit, and also comprise interior target primer in described PCR reaction solution and probe sequence as follows,
Interior label sequence: 5 '-CCAGCAATTAAATCCTATTTGCAAGCACTCTGTCATCCAGTTAGCTGACTCACGTA TTCGTAGCCACCCTTCTGGAGGTGCAATCTCAATTATGTCATCA,
Interior mark upstream primer: TCCTATTTGCAAGCACTCTGTCA,
Interior mark downstream primer: TCCAGAAGGGTGGCTACGAA,
Interior mark probe: CCAGTTAGCTGACTCACGT.
2. test kit according to claim 1, is characterized in that, also comprise the primer probe sequence of fusion gene E2A/PBX1 and/or the primer probe sequence of fusion gene MLL/ENL in described PCR reaction solution, its sequence is as follows,
E2A/PBX1 upstream primer: GGTGTAACAGGTAAAAAGACTGGTTC,
E2A/PBX1 downstream primer: TTGAGGAGGAGTACATCCAAGTCCTT,
E2A/PBX1 probe: TCTACAACATTTTGGCTACCAGGGCTTCTA;
MLL/ENL upstream primer: CGTGTAGACATGATTATTGACTGGAAC,
MLL/ENL downstream primer: TTGAAGGGAGTTCTTGCGCTCCTT,
MLL/ENL probe: TCTTCAACATTGGTTAAAAGAACTTCTA.
3. test kit according to claim 1, it is characterized in that, another kind of PCR reaction solution is also comprised in described test kit, comprise the primer probe sequence of leukemia fusion gene E2A/PBX1 or the primer probe sequence of fusion gene MLL/ENL in described another kind of PCR reaction solution, its sequence is as shown in claim 2.
4. test kit according to claim 1, it is characterized in that, another two kinds of PCR reaction solutions are also comprised in described test kit, a kind of in described another two kinds of PCR reaction solutions comprises the primer probe sequence of leukemia fusion gene E2A/PBX1 and the another kind of primer probe sequence comprising leukemia fusion gene MLL/ENL, and its sequence is as shown in claim 2.
5. the test kit according to claim 3 or 4, it is characterized in that, all comprise interior target primer and probe sequence in the PCR reaction solution of the PCR reaction solution of the primer probe sequence containing described E2A/PBX1 and the primer probe sequence containing described MLL/ENL, and interior label sequence and interior target primer probe sequence are the sequence shown in claim 1.
6. according to the test kit in Claims 1 to 4 described in any one, it is characterized in that, described test kit comprises enzyme mixation, positive control and negative control.
7. test kit according to claim 5, is characterized in that, described test kit comprises enzyme mixation, positive control and negative control.
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