CN105483253A - AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit - Google Patents
AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit Download PDFInfo
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Abstract
The invention relates to an AML1 gene and ETO gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone for an AML1 gene is at least one of CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13, a selected BAC clone for an ETO gene is at least one of RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17, the DNA of a plasmid is obtained, and marking is conducted. The invention further discloses a reagent kit provided with the AML1 gene and ETO gene detection probe. By obtaining the optimal AML1 gene and ETO detection probe through screening, signal line counting is accurate and quick, and result repeatability is good.
Description
Technical field
The invention belongs to biotechnology, particularly relate to AML1 gene and ETO gene test probe and preparation method thereof and test kit.
Background technology
Acute myeloid leukemia t (8; 21) (q22; Q22) (AML1/ETO) is a kind of AML being usually expressed as neutrophil leucocyte and breaking up with maturation, is one of modal chromosomal structural abnormality of AML, accounts for 5% ~ 12% of AML; Account for 1/3 of the AML of chromosome abnormalities companion maturation.About have 8% adult and 12% variables in childhood acute myeloid leukemia patient this fusion gene visible, wherein with AMLM2 type (50%) and AMLM2b (90%) type common.T (8; 21) (q22; Q22) easy position influence AML1 gene (also known as RUNX1), this genes encoding CBFa and ETO gene.Genetic transcription thing accompanies t (8 at AML; 21) can detect in patient.The fracture of AML1 gene betides single intron inside.
AML1/ETO merges the cell and molecular genetics exception that are considered to a kind of prognosis bona, with t (8; 21) the usual chemotherapy side effect of AML is good, and remission rate is high, and consolidation uses HDAC (HDAra-C) the disease free survival phase long.If but abnormal with other moleculess, generate as a sex chromosome disappearance and del (9) (q22), KIT, FLT3, AML1-ETO9a splice variant etc. more easily cause leukemia, and affect prognosis.AML1-ETO can suppress GATA-1 acetylize, thus suppresses erythroid differentiation, and AML1/ETO has become the focus of research at present on the impact of its target gene and Rabdosia rubescens, imatinib mesylate targeted therapy.
CYTOGENETIC ANALYSIS OF ONE (karyotyping) is the detection t (8 the most often used; 21) method.But for cryptic translocation or complex translocation form, easily cause undetected.PCR method can only detect the gene order very among a small circle, so when gene order morphs, easily there is false negative result.FISH detection method can be used for AML1/ETO fusion gene in peripheral blood or marrow Interphase cells and detects, there is sensitivity, special, efficient and reproducible feature, it can disclose traditional CYTOGENETIC ANALYSIS OF ONE or all not detect submicroscopic exception by blood or bone marrow morphology inspection.Therefore, interval FISH can be used for acute myeloid leukemia t (8; 21) (q22; Q22) (AML1/ETO) diagnosis.At present, obtain great success abroad in AML1/ETO fusion gene context of detection, and domesticly to limit by each side condition, detection method is delayed, lacks corresponding molecular diagnosis agents, diagnostic reagent dependence on import, and reagent price is high, and testing cost is high, not easily promotes.
Fluorescence in situ hybridization (FluorescenceinsituhybridizationFISH) is a kind of nonradioactivein situhybridization technology grown up on the basis of original radioactive in situ hybridization technology phase late 1980s.This technology current has been widely used in the structural research of animal-plant gene group, the analysis of variance of karyomit(e) fine structure, viral infection assays, mankind's antenatal diagnosis, cancer genetics and genome evolution research and has treated many fields.The ultimate principle of FISH is probe with known labeling nucleic acid, according to the principle of base complementrity, carries out anisogamy with single-chain nucleic acid unknown in material to be checked, form the heteroduplex nucleic acid that can be detected.Because DNA molecular is linearly arrange along the karyomit(e) longitudinal axis on chromosome, thus can probe directly and karyomit(e) carry out hybridizing thus specific gene located on chromosome.Compared with traditional radio-labeling in situ hybridization, fluorescence in situ hybridization has fast, detection signal is strong, hybrid specificities is high and can the feature such as multiple staining, is therefore subject to common concern in molecular cytogenetics field.
Hybridize probe used roughly can to classify three classes: 1) the special repetitive probe of karyomit(e), the such as probe of α satellite, satellite III class, its hybridization target position is often greater than 1Mb, not containing interspersed repeat sequence, be combined closely with target position, hybridization signal is strong, is easy to detect; 2) whole chromosome or chromosomal region specific probe, it is made up of extremely different nucleotide fragments on section a certain on item chromosome or karyomit(e), special large fragment can be obtained by the karyomit(e) be cloned in phage and plasmid; 3) specificity position probe, is made up of one or several cloned sequence.
The fluorescein-labelled of probe can adopt directly and the method for indirect labelling.Indirect labelling adopts biotin-labelled DNA probe, fluorescein avidin is associated with lotus root or Streptavidin detects after hybridization, Avidin-Biotin-luciferin complexes can also be utilized simultaneously, fluorescent signal be amplified, thus the fragment of about 500bp can be detected.And direct labelling method is by fluorescein directly and probe nucleotide or phosphopentose skeleton covalent attachment, or when nick-translation label probe, fluorescein ribonucleoside triphosphote is mixed.Direct labelling method step when detecting is simple, and Clinical practice is convenient.
And at present AML1/ETO gene FISH is detected, also lack the detection kit that specificity is high.
Summary of the invention
An object of the present invention is to provide a kind of AML1 gene and ETO gene test probe and preparation method thereof, prepared probe can be used for detecting AML1 gene and ETO gene appearance, namely AML1 gene and ETO gene test is detected, realize direct observation signal in cell and karyomit(e), there is good specificity.
The technical scheme realizing above-mentioned purpose is as follows.
A preparation method for AML1 gene and ETO gene test probe, comprises the following steps:
(1) the BAC clone chosen for AML1 gene is at least one in CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13, and the BAC clone chosen for ETO gene is at least one in RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17;
(2) respectively plasmid is extracted to clone, obtain plasmid DNA, quantitatively;
(3) use fluorescein-labelled plasmid DNA, the fluorescein that the plasmid DNA for same gene marks is identical, and the color of the fluorescein marked with the detection probes for ETO gene for AML1 gene is not identical, to obtain final product.
Wherein in an embodiment, the BAC clone of described AML1 probe is CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13.
Wherein in an embodiment, the BAC clone of described ETO probe is RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17.
Wherein in an embodiment, mark fluorescent element selects fluorescence dye known in the art, and preferably, fluorescein is Alexa
fITC, Alexa
rhodamine, TexasRed, pacific
dEAC.
Wherein in an embodiment, the mark of gene probe can adopt method of the prior art by corresponding fluorescein-labelled on double-strandednucleic acid, described method includes but not limited to: random priming, nick translation etc., mark gene probe can use commercially available nick translation labelling kit and random primer labelling kit, the NickTranslationKit of preferred abbott and Roche company.Step of the present invention (3) preferably adopts random priming, nick-translation method to carry out fluorescein-labelled to plasmid DNA.
Wherein in an embodiment, the temperature of described mark is 15 DEG C-18 DEG C, and the time of mark is 8-12 hour.
Another object of the present invention is to provide a kind of AML1 gene and ETO gene detecting kit.
Realize this object technical scheme as follows.
A kind of AML1 gene and ETO gene detecting kit, include above-mentioned AML1 gene and ETO gene test probe.
Wherein in an embodiment, also include the COTHumanDNA for closed tumor-necrosis factor glycoproteins, and DAPI counterstain.
The present invention has following beneficial effect:
(1) the present invention is by screening optimum AML1/ETO fusion gene detection probes and combination thereof, FISH (FluorescenceIn-SituHybridization) method is adopted to detect AML1/ETO fusion gene, signal-count row is accurate, quick, and result is reproducible; Supplement the deficiency of clinical middle AML1/ETO fusion detection dependence on import, be conducive to screening the patient benefiting from targeted drug more, improve acute nonlymphocytic leukemia survival and Overall survival.
(2) preferably to clone detection specificity good, highly sensitive in the present invention.By the visual inspection of resetting large fragment, not easily omit complicated variation type, also have good distinctive to unknown fused type.
(3) test kit is merged by acute nonlymphocytic leukemia AML1/ETO of the present invention, understand AML1/ETO Fusion Strain from gene level to change, multi-signal type list reveals the tumour cell genetic diversity of solid tissue, can be implemented in the application in the field such as oncobiology, cytogenetics, help each molecular marker of comprehensive evaluation, the medication of adjuvant clinical targeted therapy and treatment plan are selected.
Accompanying drawing explanation
Figure 1A is be the schematic diagram of AML1 gene test probe sequence in embodiment 1.
Figure 1B is be the schematic diagram of ETO gene test probe sequence in embodiment 1.
Fig. 2 is human peripheral culturing cell sheet AML1 gene and ETO gene test probe FISH detected result figure in embodiment 1.
Fig. 3 is clinical Bone Marrow sample FISH detected result figure in embodiment 4, and wherein, detection signal type is that 2R2G, AML1/ETO fusion gene detects feminine gender.
Fig. 4 is clinical Bone Marrow sample FISH detected result figure in embodiment 4, and wherein, detection signal type is that 1R1G2F, AML1/ETO fusion gene detects the positive.
Embodiment
For the ease of understanding the present invention, will be described more fully the present invention below.The present invention can realize in many different forms, is not limited to embodiment described herein.On the contrary, provide the object of these embodiments be make the understanding of disclosure of the present invention more comprehensively thorough.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.Term "and/or" used in the present invention comprises arbitrary and all combinations of one or more relevant Listed Items.
The preparation of embodiment 1AML1 gene and ETO gene test probe
This implements the preparation method of described AML1/ETO detection probes, comprises the following steps:
Select the clone comprising goal gene AML1 and ETO and two terminal sequences, as illustrated in fig. 1 and 2.
GSPAML1 comprises the first probe, the second probe, the 3rd probe and four point probe and the 5th probe, and table specific as follows, it is bought in InvitrogenRP11BAC and CTDBAC clone bank.
GSPETO comprises the first probe, the second probe, the 3rd probe, four point probe and the 5th probe, and table specific as follows, it is bought in InvitrogenRP11BAC and CTDBAC clone bank.
Two groups of detection probes are below divided to prepare respectively.
AML1
ETO
(2) preparation of gene probe: the PlasmidMaxiKit using Qiagen company, the working method required to specifications carries out ultralow copy extraction of plasmid DNA respectively to different B AC clone, quantitative to plasmid DNA by the absorbancy measuring 260nm and 280nm place; Adopt autoclaved ultrapure water to dilute for 200ng/ul, adopt the centrifuge tube packing of 1.5ml, finally will obtain 5 kinds of plasmid DNA mixing for AML1 gene and ETO gene respectively ,-20 DEG C of sealings are preserved.
(3) by nick translation method, fluorescent mark is carried out to plasmid DNA cocktail, fluorescein for often kind of probe mark of ETO gene is Spectrum-Orange, and the fluorescein for often kind of probe mark of AML1 gene is Spectrum-GreendUTP.Adopt the NickTranslationKit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.
Join rear concussion mixing, mark 12 hours at 16 DEG C, then 80 DEG C hatch 10 minutes inactivators.Getting 5ul uses 2% sepharose to do electrophoresis, requires the band that there is disperse at about 300bp.
Alcohol settling carried out to marked product and concentrate, in 1.5ml centrifuge tube, adding sodium-acetate and dehydrated alcohol by following scheme successively, lucifuge, on ice preparation:
Marked product 45ul
Sodium-acetate (3mol/L) 5ul
Dehydrated alcohol 125ul
Mixing to be placed in-70 DEG C of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 DEG C of 13000rpm, carefully remove supernatant, do not stir precipitation, add 70% ethanol of 1ml, 4 DEG C 13000 revs/min centrifugal 15 minutes, carefully removes supernatant, do not stir precipitation, and lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSPAML1 and GSPETO gene probe, lucifuge ,-20 DEG C of storages.
4.GSPAML1 and GSPETO gene probe is verified: the hybridization solution using AML1 probe+ETO probe preparation, uses mankind's proper splitting lymphocyte in mid-term to drip sheet and carries out probe checking (detection method reference example 3).Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.As shown in Figure 3: the FISH results of hybridization figure of Metaphase Chromosome.Karyomit(e) corresponding position display red fluorescence and green florescent signal can be seen in figure.Danger signal represents GSPETO, and green represents GSPAML1.
Embodiment 2:AML1 and ETO gene detecting kit preparation method
AML1 and ETO gene detecting kit includes AML1 and ETO hybridization solution and DAPI counterstain two components, and wherein AML1 and ETO hybridization solution comprises GSPAML1 and GSPETO gene probe described in embodiment 1, for hybridizing the buffer composition of environment (promoting hybridization), closing the COTHumanDNA etc. of tumor-necrosis factor glycoproteins.DAPI counterstain is mainly used in the cell after hybridizing and redyes, and DAPI wherein can be combined with DNA, makes nucleus demonstrate blue-fluorescence, and the counterstain containing Ursol D can keep the stable of fluorescence.
Concrete formula is as follows:
Hybridization solution is prepared
A.DAPI counterstain is prepared
The Ursol D of 10mg is dissolved in the PBS of 1ml, regulates pH to be 9.0, adds 9ml glycerine, repeatedly shake mixing ,-20 DEG C of storages.The DAPI solution (0.1mg/ml) getting 2.5 μ l is dissolved in that 1ml is anti-to fade in liquid, repeatedly shakes mixing ,-20 DEG C of airtight preservations of lucifuge under lucifuge condition.
B. finished product assembling
| Ingredient names | Specification/10test | Quantity |
| Hybridization solution | 100 μ l/ manage | 1 pipe |
| DAPI counterstain | 100 μ l/ manage | 1 pipe |
| Specification sheets | 1 part |
The detection method of embodiment 3:AML1/ETO gene detecting kit
1, sample preparation
1.1 get peripheral blood or the centrifugal 5min of marrow 2 ~ 3ml (heparin sodium anti-freezing) 2000rpm, carefully remove supernatant.
1.2 hypotonic mediums (0.075mol/LKCl) adding 10ml, piping and druming mixing, leaves standstill 3min.
1.337 ± 1 DEG C of hypotonic 30min of water bath.
1.4 add Fresh fixative 1ml, and piping and druming mixing, room temperature pre-fixes 10min.
1.5 piping and druming mixings, the centrifugal 5min of 2000rpm.
1.6 remove supernatant, and precipitation adds Fresh fixative 5 ~ 10ml, and piping and druming mixing, room temperature leaves standstill 10min.
1.72000rpm centrifugal 5min, removes supernatant.
1.8 can repeat above washing step, until cell precipitation whitens wash clean (this step does not need room temperature to leave standstill 10min).
2, film-making
2.1 get a clean slide glass;
Getting 3 μ l suspensions after 2.2 re-suspended cells is added drop-wise on slide glass;
2.3 dry under room temperature;
2.4, by 10 × object lens observation of cell density under phase microscope, require cell zero lap, and the wild cell quantity of haplopia is advisable at 100 ~ 200.
If a) cell density and number suitable, continue step 3;
If b) cell has overlap, then add proper amount of fresh stationary liquid diluting cells suspension, after mixing, separately get 3 μ l suspension film-makings,
If c) cell density is low, then the centrifugal 5min of 2000rpm, carefully sucks appropriate supernatant liquor, separately gets 3 μ l suspension film-makings, dry after mixing, observes;
2.5 observe under phase microscope, if cell debris is too many, then needs to do pre-treatment and select suitable hybridising region;
Attention: each case at least needs many systems sheet, and cell drips sheet can be placed in the encloses container being placed with dehydrated alcohol, can preserve 1 year at-20 ± 5 DEG C.Remaining cell suspension can be preserved one month, so that film-making again if desired at 2 ~ 8 DEG C.
3, slide pre-treatment
The slide dripped is placed in room temperature 2 × SSC (PH7.0) solution by 3.1 soaks 2min;
3.2 successively in room temperature 70%, soaks 2min dehydration in the ethanol of 90%, 100%; Then take out slide, room temperature is dried.
4, sample and the same time variation of probe
4.1 take out hybridization solution from-20 DEG C of refrigerators, concussion mixing, brief centrifugation;
4.2 add the hybridization solution of 10 μ l to hybridising region, cover rapidly 18 × 18mm cover glass, and light pressure makes hybridization solution be uniformly distributed, and avoid producing bubble;
4.3 use rubber glue along cover glass edge mounting, cover the position that cover glass contacts with slide glass completely;
Slide is put into hybridization instrument by 4.4, moistening in situ hybridization instrument humidity bar, insert wet bar, cover hybridization instrument upper cover, " Denat & Hyb " program is set, sex change 78 DEG C 2 minutes, hybridize 37 DEG C and (if amixia instrument, alternative instrument can be used, as Thermostatic platform carries out sex change in 10 ~ 18 hours, Electric heat oven/or water-bath are hybridized, and should be noted that temperature is accurate and keep hybridization humidity).
5, post-hybridization washing and redying
5.1 first 30 minutes of washings, the washing lotion I prepared is put into 72 ± 1 DEG C of water-baths, washing lotion II room temperature is placed;
5.2 close hybridization instrument power supply, are taken out by slide, tear rubber glue gently off, remove cover glass (if cover glass is difficult to remove, can puts it in washing lotion I and slightly rock, be beneficial to it and come off);
5.3 slides put into 72 ± 1 DEG C of washing lotion I (1 × SSC/0.3%NP-40) 2 minutes;
5.4 take out slides, then to put it in room temperature washing lotion II (0.1%NP-40/2 × SSC) 30 seconds;
5.5 take out slides, then put it into room temperature 70%, 90%, in 100% ethanol each 2 minutes;
5.6 take out slide, dark place seasoning slide;
5.7 drip 10 μ lDAPI counterstains on dry 22x22mm cover glass, and reversion sample chips, makes cover glass contact with the target area of slide glass, and light pressure after reversion, avoids producing bubble, in the dark deposit, to be seen.
6, interpretation of result
Fluorescence associated and DAPI need observe with suitable filter block.Wherein, GSPAML1 probe display green; GSPETO probe is danger signal.
6.1 use suitable filter, find, count under 100 × object lens under 10 × object lens;
The focal length that 6.2 adjustment are suitable, has clear and definite concept to signal and background; Signaling point is because being positioned at cell; When extracellular exists fluorescent signal point, note distinguishing with Intracellular signals point, preferably can avoid this region and count;
The several cell compartment of 6.3 pan, requires that nuclear boundary is complete, DAPI even dyeing, core zero lap, green and danger signal point is clear; Bypass signal is weak and do not have the nuclear counting of signal specific or high background; The core of subjective discrimination is needed not count;
6.4 analyze from the upper left corner of selected zone, from left to right sweep, observe multiple visual field;
6.5 forward 100 × object lens to, adjusting focal length, find all signaling points in the different levels of core;
6.6 at each core inside counting signaling point; All signaling points in each core are found in focusing, counts two kinds of signals in a region, and only counting often kind of color has 1 or more FISH signal, does not have signal or only has a kind of core of color signal not count; Record the total cellular score (signal number is normal and abnormal) observed;
The negative threshold value of 6.7 setting.
The human peripheral blood cell that random selecting 5 example is above or medullary cell, require to process according to sample process, prepare negative threshold reference sheet.Often open reference plate random counter 200 cells.Observe the green (AML1) in each core and red (ETO) signaling point.To overlap with green point or both close on and are designated as 1 when distance is less than a signal diameter and merge signal (yellow, F) when red; Otherwise be designated as 1 redness (R) and 1 green (G) signal.
Embodiment 4:AML1 gene and ETO gene detecting kit Clinical practice are evaluated
Use AML1 gene and ETO gene test probe combinations described in embodiment 1,2 groups of detection probes test kits described in embodiment 2, to 18 parts of clinical samples (it is made a definite diagnosis through pathology detection, specifically sees the following form), detect respectively.With commercial goods reagents ratio comparatively, detected result is completely the same, the specificity of reagent and highly sensitive.Fig. 3 and Fig. 4 is the detected result of the test kit of two groups of probes, and shown in Fig. 3, detection signal type is 2R2G, shows as normal signal type, and therefore, it is negative that result is judged as that AML1/ETO fusion gene detects.In Fig. 4, detection signal type is 1R1G2F, relative to normal signal type 2R2G, except a group chromosome R and G chrominance signal act normally (normal chromosomal), there is fracture in another group red R signal, fracture also appears in green G-signal, and generation R and G-signal merge simultaneously, therefore, result is judged as that AML1/ETO fusion gene detects the positive.
In the present invention, for AML1 gene and ETO gene, use two groups of probes to achieve gene appearance and fusion detection respectively, but probe combinations uses relatively, the use of combination clone probe, detection signal can be better.Probe length is longer in theory, and the fluorescent signal brightness obtained during actual detection is brighter, but because may relate to more polygene sequence, the signal complicacy possibility obtained increases, and also strengthens detecting the difficulty realized.The BAC of the probe groups detection probes of the probe groups for AML1 gene of the present invention, ETO gene clones its length and is respectively: 878Kb and 791Kb.
Know from above-mentioned laboratory test results, after molecular marker detection is carried out to these samples, molecule parting can be carried out according to detected result to sample, according to the meaning of Testing index, for clinical treatment formulation, medication selection and Outcome measure.
Each technical characteristic of described embodiment can combine arbitrarily, for making description succinct, all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this specification sheets is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. a preparation method for AML1 gene and ETO gene test probe, is characterized in that, comprise the following steps:
(1) the BAC clone chosen for AML1 gene is at least one in CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13, and the BAC clone chosen for ETO gene is at least one in RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17;
(2) respectively plasmid is extracted to clone, obtain plasmid DNA, quantitatively;
(3) use fluorescein-labelled plasmid DNA, the fluorescein that the plasmid DNA for same gene marks is identical, and the color of the fluorescein marked with the detection probes for ETO gene for AML1 gene is not identical, to obtain final product.
2. the preparation method of AML1 gene according to claim 1 and ETO gene test probe, is characterized in that, the described BAC clone for ETO gene is RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17.
3. the preparation method of AML1 gene according to claim 1 and ETO gene test probe, is characterized in that, the BAC clone for AML1 gene is CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13.
4. the preparation method of AML1 gene according to claim 1 and ETO gene test probe, is characterized in that, described fluorescein is Alexa
fITC, Alexa
rhodamine, TexasRed, pacific
or DEAC.
5. the preparation method of AML1 gene and ETO gene test probe according to any one of claim 1-4, it is characterized in that, step (3) adopts random priming or nick-translation method to carry out fluorescein-labelled to plasmid DNA, the temperature of described mark is 15 DEG C-18 DEG C, and the time of mark is 8-12 hour.
6. the AML1 gene that the preparation method according to any one of claim 1-5 obtains and ETO gene test probe.
7. detect a test kit for acute nonlymphocytic leukemia AML1 and ETO fusion gene, it is characterized in that, include AML1 gene according to claim 6 and ETO gene test probe.
8. the test kit of acute nonlymphocytic leukemia AML1 according to claim 7 and ETO fusion gene, is characterized in that, also includes the COTHumanDNA for closed tumor-necrosis factor glycoproteins, and DAPI counterstain.
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| CN201511029404.4A CN105483253A (en) | 2015-12-30 | 2015-12-30 | AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit |
| PCT/CN2016/105713 WO2017114006A1 (en) | 2015-12-30 | 2016-11-14 | Aml1 gene and eto gene detection probe, preparation method therefor, and test kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834493A (en) * | 2017-03-03 | 2017-06-13 | 武汉康录生物技术股份有限公司 | A kind of AML1 ETO fusion quick detection probes of low cost and its preparation method and application |
| WO2017114006A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Aml1 gene and eto gene detection probe, preparation method therefor, and test kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978279A (en) * | 2012-09-27 | 2013-03-20 | 中国医学科学院血液病医院（血液学研究所） | Gene probe composition and kit for acute lymphocytic leukemia detection |
| CN103627787A (en) * | 2013-08-06 | 2014-03-12 | 中国医学科学院血液病医院（血液学研究所） | Probe kit for detecting target gene copy-number alteration in t(8;21) AML |
| CN104328210A (en) * | 2014-11-24 | 2015-02-04 | 山东省医学科学院基础医学研究所 | Primer and kit of loop-mediated isothermal amplification detection method of AML1/ETO fusion gene |
| CN105313160A (en) * | 2014-08-04 | 2016-02-10 | 刘以年 | Hand-operated strip cutter |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952485B (en) * | 2014-04-24 | 2016-04-27 | 山西医科大学 | RUNX1 gene break and copy number increase detection kit and preparation method thereof |
| CN105483253A (en) * | 2015-12-30 | 2016-04-13 | 广州安必平医药科技股份有限公司 | AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit |
-
2015
- 2015-12-30 CN CN201511029404.4A patent/CN105483253A/en active Pending
-
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- 2016-11-14 WO PCT/CN2016/105713 patent/WO2017114006A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978279A (en) * | 2012-09-27 | 2013-03-20 | 中国医学科学院血液病医院（血液学研究所） | Gene probe composition and kit for acute lymphocytic leukemia detection |
| CN103627787A (en) * | 2013-08-06 | 2014-03-12 | 中国医学科学院血液病医院（血液学研究所） | Probe kit for detecting target gene copy-number alteration in t(8;21) AML |
| CN105313160A (en) * | 2014-08-04 | 2016-02-10 | 刘以年 | Hand-operated strip cutter |
| CN104328210A (en) * | 2014-11-24 | 2015-02-04 | 山东省医学科学院基础医学研究所 | Primer and kit of loop-mediated isothermal amplification detection method of AML1/ETO fusion gene |
Non-Patent Citations (3)
| Title |
|---|
| HIGUCHI M ET AL: "Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia", 《CANCER CELL》 * |
| MARLETTA ET AL: "Chromosome anomalies in bone marrow as primary cause of aplastic or hypoplastic conditions and peripheral cytopenia: disorders due to secondary impairment of RUNX1 and MPL genes", 《MOLECULAR CYTOGENETICS》 * |
| 刘晓晓等: "融合蛋白AML1-ETO的生物学功能及致癌机制探讨", 《中国病理生理杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017114006A1 (en) * | 2015-12-30 | 2017-07-06 | 广州安必平医药科技股份有限公司 | Aml1 gene and eto gene detection probe, preparation method therefor, and test kit |
| CN106834493A (en) * | 2017-03-03 | 2017-06-13 | 武汉康录生物技术股份有限公司 | A kind of AML1 ETO fusion quick detection probes of low cost and its preparation method and application |
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| WO2017114006A1 (en) | 2017-07-06 |
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