CN105861674A - Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis - Google Patents
Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting gene mutation related to AML (acute myeloid leukemia) prognosis, a kit containing the primer combination and a method for detecting gene mutation related to AML prognosis. The primer combination covers all hotspot mutant sites of FLT3, NPM1, DNMT3A and CEBPA genes, and has the advantages of high specificity and wide covering range. The annealing temperatures of all the primers are close, so that the amplification stage can be completed at one time by one procedure, and the hotspot mutation can be subjected to combined parallel detection; and the detection efficiency is high. Besides, the forward and reverse amplification primers are adopted to directly carry out sequencing, and software is utilized to directly carry out mutation analysis, so that the method has the advantages of visual detection and low cost, thereby providing important references for therapeutic scheme determination and prognosis judgment of AML.
Description
Technical field
The present invention relates to gene engineering technology field, be particularly used for detecting the gene mutation relevant to AML prognosis
Primer, test kit and method.
Background technology
Acute myelocytic leukemia (acute myeloid leukemia, AML) is one group and originates from hematopoietic stem cell
Malignant hematologic disease, has height heterogeneity.At present, in AML evaluates the many factors of prognosis risk factor, cytogenetics is recognized
For being important independent prognostic factor, some specific chromosomal chromosome abnormalities, are considered as often guiding treatment and judging prognosis
Mark.But still having quite a few patient to can't detect chromosome abnormalities, these patients are either in therapeutic response or prognosis
Bigger variation is all there is in evaluation.Due to developing rapidly of Protocols in Molecular Biology, identify multiple relevant to AML successively
Molecular marker, this most fully demonstrates the important foundation that gene alteration is AML morbidity.Wherein FLT3-ITD, NPM1 and CEBPA
Gene mutation, as important risk stratification evaluation index, is included in acute myeloid leukocyte NCCN guide in 2010.
The sudden change of FLT3 producer, by destroying the increment of normal plasma cell, differentiation and apoptosis, the most in some cases can
Enough individually or with other combination of cytokines, support for leukaemia's offer of rising in value, be the important driving of hematological malignancy
Type suddenlys change.Its mutation type mainly has two kinds of forms: a kind of is that the internal series-connection of membrane-proximal region repeats (ITD) sudden change, often betides
Exons 14 or 15；Another kind is the point mutation of activation ring D835, often betides extron 20.FLT3-ITD sending out in AML
Raw rate is about 14%-32%, comes across each hypotype of FAB, wherein the most common with M5.Research display both at home and abroad is with FLT3-ITD sun
The patient of property is often accompanied by high peripheral white blood cell, high bone marrow blast ratio, be also embodied by relatively low CR lead, DFS, EFS
And OS, there is independent poor prognosis meaning.And the incidence rate of D835 point mutation is only about 7%, with leukemic prognosis phase
There is dispute in closing property, needs to be goed deep into further checking.
There is insertion mutation in NPM1 gene on 12 exons, changes the codon of NPM1 PROTEIN C end, anxious adult
Expressing up to about 35% in Acute myeloid leukemia patient, Aisan, less than America and Europe, is that the accounting rate found in AML is the highest
Sudden change, research finds that the incidence rate of NPM1 gene mutation also has heterogeneity in each hypotype of FAB, sees each Asia in addition to M3
Type.Although the type of NPM1 sudden change differs, but belongs to frameshift mutation, cause nuclear localization signal abnormal, so that nuclear phosphoprotein
By transposition in core in kytoplasm, this exception location not only can affect the normal function of protein, simultaneously to some tumor suppression base
Because regular path and the function of ARF, P53 also can produce certain impact.NPMl gene mutation is that AML prognosis is relatively good to be sentenced
Severed finger mark.The normal karyotype AML patient CR with NPM1 sudden change leads higher, and EFS, DFS, OS time is also obviously prolonged, and this may
More sensitive relevant to chemotherapeutics with NPM1 sudden change patient.In the patient suddenlyd change without FLT3-ITD, NPM1 suddenlys change the positive
Patient has good prognosis.
DNMT3A sudden change incidence rate in acute myelocytic leukemia patient is up to 20%～30%, and the DNMT3A positive is right
The treatment of AML and prognosis, especially have great importance to the patient of normal karyotype.Through the most large it is demonstrated experimentally that prominent
Become and be mostly focused on exon 23, the missense mutation of the 882nd arginic gene loci of coding, but its at exon 11～
The mutation type occurred in 22 is various.It is only second to the mutation type of FLT3 Yu NPM1 as incidence rate in AML patient, is different from
Other the two kinds high-risk impacts in AML that suddenly change, DNMT3A suddenlys change under patient is in long-term complete incidence graph state, it would still be possible to protect
Hold the positive.But the aberrant methylation that DNMT3A is caused, affects epigenetics regulation, and the generation with AML is closely related, is near
One of target spot of leukemia treating over Nian, selects medicine or stem cell transplantation to have directive function to clinicist.
CEBPA gene mutation sees the AML patient of about 5%～15%, in the majority with M1, M2 hypotype.Its sudden change mainly has two
The form of kind: one is the frameshift mutation of N-end, causes normal p42 protein translation to terminate in advance, from the beginning of another start codon
Translate into the p30 albumen of truncate；Two is that the base that C-end occurs is inserted, lacks, repeats and replaced, and usually sudden change in frame is produced
Raw hypoproteinosis DNA combines and homodimer activity.Patient both can occur single mutation (CEBPA single mutation,
CEBPAsm), it is possible to there is double sudden change (CEBPA double mutation, CEBPAdm).Therefore, if CEBPA gene occurs prominent
Becoming, the C/EBP α protein structure of its coding or abnormal expression will cause grain system dysdifferentiation, immature grain system precursor abnormal
Propagation, even causes the generation of acute myelocytic leukemia.At present, increasing research display is only with CEBPAdm's
AML patient just has good prognosis, CEBPAdmPatient CR leads and is better than CEBPAsmWith CEBPA wild type patient, multiplicity
Display is with CEBPAdmAML patient OS, EFS and RFS the highest, carry out accurate examination to contribute to clinicist being patient to it
Select optimal therapeutic scheme.
In the outer widely used molecular Biological Detection leukemia gene mutation method of Present Domestic, fluorescence in situ hybridization
Technology (FISH) can only carry out qualitative detection, operation complexity；Quantitative fluorescent PCR also exists the limitation of detection flux, for
CEBPA Yu NMP1 etc. do not have the situation of clear and definite mutantional hotspot, need to design multipair primer and probe, and cost is high and there is missing inspection
Probability, so all can't really meet the needs of clinical diagnosis detection.Therefore, need badly at present a kind of high specificity, quickly,
The method that the detection that low cost, loss are low suddenlys change with AML prognosis-related gene, the method can really meet clinical diagnosis inspection
The demand surveyed.
Summary of the invention
The present invention is directed to drawbacks described above present in prior art, the up-to-date and AML prognosis phase announced based on Genbank
The sequence of each mutant gene closed, has redesigned covering FLT3, NPM1, DNMT3A and CEBPA gene whole hot spot mutation position
The primer put and the method that FLT3, NPM1, DNMT3A and CEBPA gene mutation is carried out associating parallel detection.
To this end, one aspect of the present invention provides the primer combination of a kind of gene mutation relevant to AML prognosis for detection,
It is made up of the primer shown in SEQ ID NO.1-34.
In a preferred embodiment of the present invention, the primer combination of the present invention is made up of 1-4 group primer, wherein further
1st group is made up of the primer shown in SEQ ID NO.1-4, and the 2nd group is made up of the primer shown in SEQ ID NO.5-6, the 3rd group by
Primer composition shown in SEQ ID NO.7-26, the 4th group is made up of the primer shown in SEQ ID NO.27-34.
In further preferred embodiment of the present invention, the present invention is when carrying out PCR amplification, and the concentration of every primer is excellent
Elect 5 μMs as；When carrying out sequencing reaction, the concentration of every primer is preferably 3.2 μMs.
Another aspect of the present invention provides the test kit of a kind of gene mutation relevant to AML prognosis for detection, its bag
Combine containing primer of the present invention.
In a preferred embodiment of the present invention, the present invention is when carrying out PCR amplification, and the concentration of every primer is preferably 5 μ
M；When carrying out sequencing reaction, the concentration of every primer is preferably 3.2 μMs.
Another aspect of the present invention provides the primer sets of the present invention and is combined in preparation for detecting the gene relevant to AML prognosis
Application in the test kit of sudden change.
Further aspect of the present invention provides primer of the present invention combination, or test kit of the present invention is in detection
The application in gene mutation relevant to AML prognosis.
Last aspect of the present invention provides the method for a kind of detection gene mutation relevant to AML prognosis, its comprise with
Lower step:
1, testing sample DNA is obtained；
2, use primer of the present invention to combine, or use test kit of the present invention, with what step 1 obtained
DNA is template, carries out PCR amplification；
3, pcr amplification product is after electroresis appraisal, carries out sequencing reaction；
4, sequencing reaction product is purified, after degeneration, upper sequencer；
5, order-checking peak figure is analyzed, the gene mutation type relevant to AML prognosis is identified.
In a preferred embodiment of the present invention, the present invention is when carrying out PCR amplification, and the concentration of every primer is preferably 5 μ
M；When carrying out sequencing reaction, the concentration of every primer is preferably 3.2 μMs.
In further preferred embodiment of the present invention, the present invention, before carrying out sequencing reaction, uses shrimp alkalescence phosphorus
The amplified production that step 2 is obtained by acid enzyme (Alkaline Phosphatas (Shrimp)) and excision enzyme (Exonuclease I)
Carry out digestion process, to remove the impurity outside double-stranded DNA.
Seen from the above description, compared with prior art, the present invention possesses following advantage.
1, the up-to-date each mutant gene sequence relevant to AML prognosis that the present invention announces based on Genbank, redesigns
Covering the primer of FLT3, NPM1, DNMT3A and CEBPA gene whole hot mutant site, primer specificity is strong, coverage rate
Extensively.
2, the annealing temperature of the whole primers designed by the present invention is all at similar level, and the amplification stage can use same program
Once complete, the hot spot mutation of FLT3, NPM1, DNMT3A and CEBPA gene can be carried out associating parallel detection, detection efficiency
High.
3, the present invention uses forward and reverse amplimer directly to check order, and is directly carried out the analysis suddenlyd change by software, inspection
Survey directly perceived, low cost.
In sum, the invention provides a kind of quickly, easy, detect the gene relevant to AML prognosis accurately, intuitively
The method of sudden change, can be the reference frame that therapeutic scheme determines and Index for diagnosis offer is important of acute myelocytic leukemia.
Accompanying drawing explanation
Fig. 1: saltant type DNMT3A and wild type order-checking peak comparison diagram.
Fig. 2: saltant type CEBPA and wild type order-checking peak comparison diagram.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, it is intended to is used for illustrating rather than restriction originally
Invention.It should be pointed out that, to those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to this
Bright carrying out some improvement and modification, these improve and modify and fall under the scope of the present invention too.
Embodiment 1: be designed for detecting the primer of the gene mutation relevant to AML prognosis
According to the sequence of each mutant gene relevant to AML prognosis that GenBank announces, utilize Primer Premier
5.0 primer-design softwares carry out design of primers, and designed primer is closed by Sangon Biotech (Shanghai) Co., Ltd.
Become.Meanwhile, in order to reduce order-checking Tu Di peak, peak, the purity of sequencing primer should be ensured to greatest extent.It is concrete that the present invention designs
Primer situation is as shown in table 1.
The primer information slip of the gene mutation that table 1, present invention detection is relevant to AML prognosis
* CEBPA gene only has a single exon, and digitized representation primer quantity not expands exon number herein.
Primer designed by the present invention is not only for the Hotspot region of FLT3, NPM1 gene, and substantially covers
Multiple saltation zone in 11～23 exons of DNMT3A gene, and substantially covers the total length of CEBPA gene.
Wherein, primers F LT3-ITD-F (SEQ ID NO.1), primers F LT3-ITD-R (SEQ ID NO.2), primer
FLT3-TKD-F (SEQ ID NO.3), primers F LT3-TKD-R (SEQ ID NO.4) are for detecting the internal series-connection of FLT3 gene
Repeat sudden change and D835 point mutation.Primer NPM1-F (SEQ ID NO.5), NPM1-R (SEQ ID NO.6) are used for detecting NPM1
Gene mutation.10 pairs of primers of DNMT3A gene, the most corresponding SEQ ID NO.7～NO.26, for detecting DNMT3A gene
All types of sudden changes, including the R882 hot spot mutation on 23 exons.Primer CEBPA-1F (SEQ ID NO.27), CEBPA-1R
(SEQ ID NO.28)、CEBPA-2F(SEQ ID NO.29)、CEBPA-2R(SEQ ID NO.30)、CEBPA-3F(SEQ ID
NO.31), CEBPA-3R (SEQ ID NO.32), CEBPA-4F (SEQ ID NO.33), CEBPA-4R (SEQ ID NO.34) collection
Middle amplification CEBPA known mutations generating region.
Additionally, all of sequencing primer of the present invention keeps consistent with pcr amplification primer thing, in order to carry out two-way order-checking, subtract
The probability of few missing inspection.Meanwhile, the annealing temperature of the whole primer of the present invention is all similar level about 57 DEG C, thus ensure that and expanding
The increasing stage can use same program once to complete.
Embodiment 2: gene mutation relevant to AML prognosis in sample is detected
(1) reagent and material
1, detection system PCR reactant liquor: 10 × PCR Buffer, dNTPs (2.5mM), LA Taq DNA
Polymerase、ddH2O etc..
2, order-checking system:
(1) PCR primer digestion reaction liquid: shrimp alkaline phosphotase (Alkaline Phosphatas (Shrimp)) and circumscribed
Enzyme (Exonuclease I), 1:1 mixes；
(2) order-checking refined solution: EDTA (125mmol), 85% dehydrated alcohol, 75% dehydrated alcohol, HIDI (highly deionization
Methanamide)；
(3) sequencing reaction liquid:Terminator V3.1cycle sequencing
Kit。
(2) workflow
1, PCR amplification
The preparation of detection system PCR reactant liquor is as shown in table 2.
Table 2 detection system PCR reactant liquor preparation table
| Composition | Volume (ul) |
| 10×PCR buffer | 2.5 |
| dNTPs(2.5mM) | 3.0 |
| Primer F(5μM) | 1.0 |
| Primer R(5μM) | 1.0 |
| LA Taq DNA Polymerase(5U/μL) | 0.3 |
| Template DNA | 2.5 |
| ddH2O | 14.7 |
| Total volume | 25.0 |
Wherein, DNA is that testing sample obtains after QIAamp DNA Blood Mini kit test kit extracts, primer sequence
Row are as described in Table 1.
Positive reference substance: containing the solution of patient DNA.
Negative controls: containing the solution of normal person DNA.
Blank: without solution or the 2.5ul ddH of sample DNA2O。
Amplification reaction condition: 96 DEG C of denaturations 2min；Annealing 30s → 72 DEG C, 96 DEG C of degeneration 30s → 58 DEG C extend 1min, altogether
Carry out 35 circulations；72 DEG C extend 5min.
2, electroresis appraisal
1.5% agarose gel electrophoresis, 150V, 30min, gel imaging system is observed.
3, Sanger order-checking
Take 1ul order-checking digestion reaction liquid and join in amplified production, rely on Alkaline Phosphatase hydrolysed residue
DNTP, Exonuclease I hydrolysing single nucleic acid, thus the impurity outside removing double-stranded DNA.
Digestion reaction condition: 37 DEG C, 60min；80 DEG C, 15min；4 DEG C of preservations.
Take 1ul digestion product to mix by the system of table 3 with forward and reverse primer of each sequencing primer respectively.
Table 3 checks order system PCR reactant liquor preparation table
Sequencing reaction is as follows: 96 DEG C, 2min；96 DEG C, 10s → 55 DEG C, 5s → 60 DEG C, 90s, carry out 25 circulations altogether；15
DEG C preserve.
4, purification after sequencing reaction
Before purification process, need pre-cooling centrifuge to 4 DEG C.This step is will to remove target single-chain nucleic acid sheet in sequencing reaction system
Impurity outside Duan is eliminated as much as, the impurity impact on peak plot quality during to reduce by 3730 capillary electrophoresis.
Every 5ul reaction system adds 0.125mol/L EDTA-Na2Solution, 85% dehydrated alcohol 30ul, cover silicagel pad,
Fully vibration 3～5 minutes, centrifugal 3000g, 4 DEG C, 30 minutes.EDTA, as metal ion chelation agent, can react with order-checking PCR
Ions binding in system thus remove deionization.
After centrifugal end, remove supernatant, stop immediately during to 185g.70% dehydrated alcohol is added again in every hole
50ul, DNA fragmentation dissolubility in 70% ethanol is low, can be by being centrifugation down.Cover silicagel pad, fully vibration 3 minutes,
Centrifugal 3000g, 15 minutes, after centrifugal end, removes supernatant again by 4 DEG C.
5, degenerative treatments
After above-mentioned purified product lucifuge is ventilated 20 minutes, operating in Biohazard Safety Equipment, every hole adds 8ul HI-DI formyl
Amine carries out degenerative treatments.Denaturation program: 95 DEG C, 3min；4 DEG C of coolings.After degeneration terminates, upper sequenator (ABI 3730) is surveyed
Sequence.
6, interpretation of result
After obtaining order-checking peak figure, it is loaded into software Variant Reporter software v1.1 through strictly renaming, from
Move and compare with Genbank wild-type sequence, and use negative control to be controlled simultaneously.According to actual catastrophe, point
The change of analysis coded amino acid, comparison diagram sees Figure of description 1 and accompanying drawing 2.
In Fig. 1, sample FG95 is the order-checking peak figure finding DNMT3A sudden change, contrast wild type (ZP), and this sports missense and dashes forward
Becoming (R827C), have no report in existing COSMIC data base, clinical meaning wouldn't be clear and definite.
In Fig. 2, sample FG87 is the order-checking peak figure finding CEBPA sudden change, and contrast wild type (A570), this sports synonym
Sudden change (I48I), does not cause amino acid change, no pathogenicity.
Additionally, by reporting peak figure clear background, the signal value height that can find the present invention and be presented, greatly reduce prominent
The analysis difficulty become.
Claims (10)
1. one kind is combined for detecting the primer of the gene mutation relevant to AML prognosis, and it is by shown in SEQ ID NO.1-34
Primer forms.
Primer the most according to claim 1 combines, and it is made up of 1-4 group primer further, and wherein the 1st group by SEQ ID
Primer composition shown in NO.1-4, the 2nd group is made up of the primer shown in SEQ ID NO.5-6, and the 3rd group by SEQ ID NO.7-26
Shown primer composition, the 4th group is made up of the primer shown in SEQ ID NO.27-34.
Primer the most according to claim 1 and 2 combines, and wherein when carrying out PCR amplification, the concentration of every primer is 5 μMs；
When carrying out sequencing reaction, the concentration of every primer is 3.2 μMs.
4., for detecting a test kit for the gene mutation relevant to AML prognosis, it comprises institute any one of claim 1-3
The primer combination stated.
Test kit the most according to claim 4, wherein when carrying out PCR amplification, the concentration of every primer is 5 μMs；Entering
During row sequencing reaction, the concentration of every primer is 3.2 μMs.
6. the primer sets according to any one of claim 1-3 is combined in and prepares for detecting the gene mutation relevant to AML prognosis
Test kit in application.
7. the primer combination according to any one of claim 1-3, or the test kit described in claim 4 or 5 is in detection
The application in gene mutation relevant to AML prognosis.
8. a method for the gene mutation that detection is relevant to AML prognosis, it comprises the steps of
(1) testing sample DNA is obtained；
(2) use the primer combination according to any one of claim 1-3, or use the reagent described in claim 4 or 5
Box, as template, carries out PCR amplification with the DNA of acquisition in step (1)；
(3) pcr amplification product is after electroresis appraisal, carries out sequencing reaction；
(4) sequencing reaction product is purified, after degeneration, upper sequencer；
(5) order-checking peak figure is analyzed, the gene mutation type relevant to AML prognosis is identified.
Method the most according to claim 8, wherein when carrying out PCR amplification, the concentration of every primer is 5 μMs；Carry out
During sequencing reaction, the concentration of every primer is 3.2 μMs.
Method the most according to claim 9, wherein before carrying out sequencing reaction, uses shrimp alkaline phosphotase
The amplified production that step (2) is obtained by (Alkaline Phosphatas (Shrimp)) and excision enzyme (Exonuclease I) enters
Row digestion process, to remove the impurity outside double-stranded DNA.
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Cited By (12)
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| CN106148553A (en) * | 2016-08-31 | 2016-11-23 | 北京海思特临床检验所有限公司 | ALL prognosis-related gene mutation detection methods, primer and test kit |
| CN106381332A (en) * | 2016-08-31 | 2017-02-08 | 天津协和华美医学诊断技术有限公司 | Detection kit for detecting AML related gene group |
| CN107841538A (en) * | 2017-11-23 | 2018-03-27 | 合肥金域医学检验所有限公司 | For detecting the primer and detection method of CEBPA gene mutations |
| CN107964561A (en) * | 2017-11-23 | 2018-04-27 | 合肥金域医学检验所有限公司 | Detect the primer and detection method of CEBPA gene mutations |
| CN108220411A (en) * | 2017-11-01 | 2018-06-29 | 天津协和华美医学诊断技术有限公司 | A kind of PCR primer and detection method for detecting human gene CEBPA |
| CN109402252A (en) * | 2017-08-11 | 2019-03-01 | 上海交通大学医学院附属瑞金医院 | Acute myelogenous leukemia risk assessment gene marker and application thereof |
| CN109988832A (en) * | 2017-12-29 | 2019-07-09 | 上海新培晶医学检验所有限公司 | A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene |
| CN110511983A (en) * | 2019-08-07 | 2019-11-29 | 南京实践医学检验有限公司 | Method based on capillary electrophoresis quantitative detection FLT3-ITD gene mutation |
| CN110564828A (en) * | 2019-09-20 | 2019-12-13 | 上海艾迪康医学检验所有限公司 | primer, kit and method for detecting NPM1 gene mutation |
| CN111154881A (en) * | 2020-03-09 | 2020-05-15 | 南京实践医学检验有限公司 | Detection kit for gene mutation in acute myeloid leukemia and application |
| CN111560438A (en) * | 2020-06-11 | 2020-08-21 | 迈杰转化医学研究(苏州)有限公司 | Primer composition and kit for detecting AML prognosis related gene mutation and application thereof |
| CN112626215A (en) * | 2020-12-30 | 2021-04-09 | 武汉康圣达医学检验所有限公司 | AML prognosis related gene expression detection kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948829A (en) * | 2010-05-18 | 2011-01-19 | 北京大学人民医院 | Kit for detecting CEBPA gene mutation |
| CN101955994A (en) * | 2010-06-17 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation |
| CN103540659A (en) * | 2013-09-30 | 2014-01-29 | 武汉艾迪康医学检验所有限公司 | Method, primer and kit for detecting DNMT3A mutation site |
| CN104508143A (en) * | 2012-03-12 | 2015-04-08 | 纪念斯隆-凯特琳癌症中心 | Methods and compositions for the diagnosis, prognosis and treatment of acute myeloid leukemia |
-
2016
- 2016-04-27 CN CN201610268837.3A patent/CN105861674A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948829A (en) * | 2010-05-18 | 2011-01-19 | 北京大学人民医院 | Kit for detecting CEBPA gene mutation |
| CN101955994A (en) * | 2010-06-17 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation |
| CN104508143A (en) * | 2012-03-12 | 2015-04-08 | 纪念斯隆-凯特琳癌症中心 | Methods and compositions for the diagnosis, prognosis and treatment of acute myeloid leukemia |
| CN103540659A (en) * | 2013-09-30 | 2014-01-29 | 武汉艾迪康医学检验所有限公司 | Method, primer and kit for detecting DNMT3A mutation site |
Non-Patent Citations (6)
| Title |
|---|
| R KIHARA ET AL: "Comprehensive analysis of genetic alterations and their prognostic", 《LEUKEMIA》 * |
| SANAM LOGHAVI: "Clinical features of De Novo acute myeloid leukemia with concurrent DNMT3A, FLT3 and NPM1 mutations", 《JOURNAL OF HEMATOLOGY & ONCOLOGY》 * |
| WENGANG CHEN ET AL: "A rapid, one step assay for simultaneous detection of FLT3/ITD and NPM1 mutations in AML with normal cytogenetics", 《BRITISH JOURNAL OF HAEMATOLOGY》 * |
| 国家药典委员会编: "《中华人民共和国药典》", 30 November 2014, 中国医药科技出版社 * |
| 温旺荣: "《临床分子诊断学》", 30 April 2015, 广东科技出版社 * |
| 闫有圣: "《实用遗传病诊断剂产前诊断技术》", 31 July 2014, 科学技术文献出版社 * |
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| CN106148553A (en) * | 2016-08-31 | 2016-11-23 | 北京海思特临床检验所有限公司 | ALL prognosis-related gene mutation detection methods, primer and test kit |
| CN106381332A (en) * | 2016-08-31 | 2017-02-08 | 天津协和华美医学诊断技术有限公司 | Detection kit for detecting AML related gene group |
| CN109402252A (en) * | 2017-08-11 | 2019-03-01 | 上海交通大学医学院附属瑞金医院 | Acute myelogenous leukemia risk assessment gene marker and application thereof |
| CN108220411A (en) * | 2017-11-01 | 2018-06-29 | 天津协和华美医学诊断技术有限公司 | A kind of PCR primer and detection method for detecting human gene CEBPA |
| CN107841538A (en) * | 2017-11-23 | 2018-03-27 | 合肥金域医学检验所有限公司 | For detecting the primer and detection method of CEBPA gene mutations |
| CN107964561A (en) * | 2017-11-23 | 2018-04-27 | 合肥金域医学检验所有限公司 | Detect the primer and detection method of CEBPA gene mutations |
| CN109988832A (en) * | 2017-12-29 | 2019-07-09 | 上海新培晶医学检验所有限公司 | A kind of kit and its detection method of the mutation of detection AML patient's prognostic gene |
| CN110511983A (en) * | 2019-08-07 | 2019-11-29 | 南京实践医学检验有限公司 | Method based on capillary electrophoresis quantitative detection FLT3-ITD gene mutation |
| CN110564828A (en) * | 2019-09-20 | 2019-12-13 | 上海艾迪康医学检验所有限公司 | primer, kit and method for detecting NPM1 gene mutation |
| CN111154881A (en) * | 2020-03-09 | 2020-05-15 | 南京实践医学检验有限公司 | Detection kit for gene mutation in acute myeloid leukemia and application |
| CN111560438A (en) * | 2020-06-11 | 2020-08-21 | 迈杰转化医学研究(苏州)有限公司 | Primer composition and kit for detecting AML prognosis related gene mutation and application thereof |
| CN111560438B (en) * | 2020-06-11 | 2024-01-19 | 迈杰转化医学研究(苏州)有限公司 | Primer composition and kit for detecting AML prognosis related gene mutation and application thereof |
| CN112626215A (en) * | 2020-12-30 | 2021-04-09 | 武汉康圣达医学检验所有限公司 | AML prognosis related gene expression detection kit |
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