CN103103251A - Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis - Google Patents
Kit for detecting FLT3-ITD (Fms-like tyrosine kinase 3-internal tandem duplication) gene mutation by using fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis Download PDFInfo
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Abstract
The invention relates to a kit for detecting ITD (internal tandem duplication) mutation in an FLT3 (Fms-like tyrosine kinase 3) gene juxtamembrane domain and in particular relates to a kit for detecting the FLT3-ITD in a clinical sample by using a fluorescence PCR (Polymerase Chain Reaction) capillary electrophoresis technology. The kit mainly comprises a PCR amplified reaction solution, a negative control product, a positive control product, a plurality of sealed reagent bottles or pipes which cover the kit as well as packaging boxes which are used for separating and concentratively packaging the reagent bottles or pipes, and the size and gene dosage of each amplified fragment are determined by using an electrophoresis method. The kit disclosed by the invention has high sensitivity and specificity in terms of detection and analysis of the FLT3-ITD in a whole blood or marrow sample and can be widely applied to the fields of judgment prognosis and guide treatment of leukemia and resurgence prevention of diseases.
Description
Technical field
The present invention relates to detect sequence internal series-connection repetition (the internal tandem duplications of FLT3 (Fms-like tyrosine kinase 3) gene membrane-proximal region, ITD) test kit of sudden change particularly relates to the test kit that detects FLT3-ITD in clinical sample with the fluorescent PCR capillary electrophoresis technique.Because this test kit has very high sensitivity and specificity to the determination and analysis of the FLT3-ITD in whole blood or marrow sample, can be widely used in the leukemia judging prognosis, a plurality of fields such as guiding treatment and predictive disease recurrence.
Background technology
FLT3 (Fms-like tyrosine kinase 3) is a member in III receptor family tyrosine kinase, and its sudden change is closely related with leukemic generation.FLT3 and its part (FL) play important regulating effect in the propagation of hematopoietic stem/progenitor and differentiation.FL is combined a series of Cellular Signaling Transduction Mediated approach of mediation with the extracellular domain of FLT3, regulate cell proliferation and activation.When the FLT3 producer suddenlys change, can cause the non-ligand dependent phosphorylation of FLT3, destroy propagation, differentiation and the apoptosis of normal plasma cell.The FLT3 sudden change has two kinds of forms: a kind of for occurring in sequence internal series-connection repetition (the internal tandem duplications of FLT3 gene membrane-proximal region, ITD), be called the FLT3-ITD sudden change, all there are polymorphism in the size of ITD and position, typical ITD is positioned at the 14th exon, also can involve the 14th intron and the 15th exon; A kind of for occurring in the point mutation of Tyrosylprotein kinase structural region, be called the FLT3-TKD sudden change.The FLT3-ITD sudden change that studies show that relevant for the FLT3-ITD sudden change at present the most often appears at acute myelocytic leukemia (acute myeloid leukemia, AML) in the patient, mutation rate is 15%～35%, secondly be myelodysplastic syndrome (myelodysplastic syndromes, MDS), mutation rate is about 2%～5%.FLT3-ITD is incidence extremely low (lower than 1%) in acute lymphoblastic leukemia (ALL) patient, and not yet finds the FLT3-ITD sudden change in chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), multiple myeloma, one-tenth human body cell leukemia.
AML is one group of malignant hematologic disease that originates from hemopoietic stem cell, has height heterogeneity.In the many factors that affects AML prognosis risk level, cytogenetics has been considered to most important independent prognostic factor, and some specific stain body chromosome abnormalities not only become the important symbol thing in the AML diagnosis, and often is considered to the sign of judging prognosis.It is abnormal that but about 40%～50% AML patient does not detect karyotype, and these patient's prognosis variations are larger, and survival rate 24%～42% in 5 years.Become so seek the normal AML patient's of caryogram prognosis molecule sign the focus that people pay close attention to.Application along with the Molecular genetic test method, scholars find that there is heterogeneity in the AML patient of normal karyotype, the abnormal expression that comprises transgenation or some specific gene, providing according to the FLT3 transgenation for AML patient's further prognosis classification and molecular targeted therapy is one of molecular marker that affects AML patient's prognosis of having found, can be used as the individual index of AML patient's prognosis mala.FLT3-ITD sudden change is modal a kind of mutation type in AML patient, and approximately there is the FLT3-ITD sudden change in 15%～35%AML case, and there is the FLT3-TKD sudden change in 7% the AML case of also having an appointment in addition.It is irrelevant with the AML prognosis that but most of research at present thinks that FLT3-TKD suddenlys change, and the FLT3-ITD sudden change is fallen ill with AML and it is closely related to develop, and is an important independent prognostic factor.The FLT3-ITD positive patient has than particular features or clinic, and the targeted therapy that is directed at present this sudden change has also entered different clinical experimental stages.And, also there is difference in different I TD patient's prognosis, this and ITD-AR (internal tandem allelic ratio, internal series-connection repetition allelotrope ratio) value is relevant, be ITD:WT (FLT3 wild type gene) ratio, the ITD-AR value increases may cause prognosis mala, so the ITD-AR value is also AML patient's very strong independent prognostic factor.
MDS patient FLT3-ITD sudden change is than the patient who there is no sudden change, the probability that the FLT3-ITD positive patient transfers AML to has the trend of increasing, progress has shortening trend for the time of AML, and shorten overall lifetime, after MDS transfers AML to, the FLT3-ITD positive rate increases, so FLT3-ITD is the high risk factor that MDS is converted into AML, is the prognosis mala factor.
To sum up, FLT3-ITD can be used as the individual index of AML and MDS patient's prognosis mala, transgenation detects and can help judging prognosis to FLT3-ITD, is used for guiding treatment, judgement curative effect and palindromia, and long-term survival rate and the life quality that increases the patient had very important meaning.
FLT3-ITD detection in Gene Mutation test kit (fluorescent PCR capillary electrophoresis) provides a kind of method of rapid detection FLT3-ITD transgenation.Present method at 14 exons of FLT3-ITD generation and the two ends design upstream and downstream primer of 15 exons, for the DNA sample that the FLT3-ITD transgenation occurs, the amplified production greater than normal fragment will occur respectively according to PCR design of primers principle.Present method determines whether to exist the FLT3-ITD transgenation by the length that capillary electrophoresis method detects the PCR product, and can calculate the ITD-AR value by software analysis, provide theoretical foundation to clinical diagnosis, prognosis judgement and molecular targeted therapy for AML and MDS.Compare common agarose gel electrophoresis, the method is sensitiveer, and can accurately judge clip size and the gene dosage of sudden change band.
Summary of the invention
The purpose of this invention is to provide a kind of test kit that uses the fluorescent PCR capillary electrophoresis technique to detect FLT3-ITD transgenation in the leukemia clinical sample.This test kit uses the ITD zone of PCR method amplification FLT3 by extracting the DNA of blood samples of patients or marrow, then uses the fluorescent PCR capillary electrophoresis technique that ITD clip size and dosage are detected.
In order to realize the present invention, we have adopted following technical scheme:
(1) use suitable foranalysis of nucleic acids software known FLT3 gene nucleotide series is carried out homology relatively and find out on the basis of homology segment, further use suitable primer-design software (for example Primer 3) to select and the design oligonucleotides primer.14 exons that occur at FLT3-ITD due to designed primer and the two ends of 15 exons, and there is no homology with the nucleotide sequence of other genetic expression mRNA, do not comprise any common endonuclease yet, so false negative and the false positive of having avoided FLT3-ITD to detect have improved the reliability and the accuracy that detect;
(2) use the synthetic required Oligonucleolide primers of DNA synthesizer, process with carrying out the ammonia solution after molecular sieve and fast protein liquid chromatography method (FPLC) purifying.Same synthetic required primer sequence, after the ammonia solution is processed respectively at the 6-FAM amidite of its 5 ' end mark as fluorescence generation group (reporter group).With the fluorescently-labeled primer of FPLC purification by chromatography.Primer and the probe that then, can use polyacrylamide gel (20%) electrophoretic method under Denaturing and spectrophotometry physical characterization to be synthesized;
(3) extract DNA from the peripheral blood that derives from the experimenter or marrow, then use the PCR reaction system that it is increased;
(4) provide and comprise (a) sample to be checked, (b) hot resistant DNA polymerase and (c) amplification reaction system of 2-deoxynucleoside triphosphate (dNTPs), and comprise the forward primer that (d) can be combined with article one complementary strand of double-stranded target polynucleotide to be checked, the reverse primer that (e) can be combined with the second complementary strand of double-stranded target polynucleotide to be checked.By the said target polynucleotide of one or many PCR amplification; By the fragment length of fluorescent PCR capillary electrophoresis technique with the detection target polynucleotide.
The test kit that fluorescent PCR capillary electrophoresis technique provided by the present invention detects FLT3-ITD in clinical sample comprises: (1) pcr amplification reaction liquid, negative quality control product, positive quality control product and a plurality of reagent bottles or the pipe that seal, (2) separate and concentrate the packing box of these reagent bottles of packing or pipe, (3) determine size and the gene dosage of amplified fragments with electrophoretic method.
A preferred embodiment of the present invention, wherein pcr amplification reaction liquid comprises that 5 ' end is combined with forward primer, reverse primer, dNTPs, hot resistant DNA polymerase, PCR damping fluid and the water of fluorescence generation group, be characterised in that the primer sequence that uses is respectively: 5 '-FAM-TCTCCTCTTCATTGTCGTTTTA-3 ' (SEQ ID NO:1), 5 '-ATGGTGAGTACGTGCATTTTA-3 ' (SEQ ID NO:2), SEQ ID NO:1 wherein, SEQ ID NO:2 coupling can detect FLT3-ITD together.
Another preferred embodiment of the present invention, the primer sequence that can also use in pcr amplification reaction liquid is respectively: 5 '-FAM-TTCATTATTCTTTCCTCTATCTGC-3 ' (SEQ ID NO:3), 5 '-TGTTGCGTTCATCACTTTTC-3 ' (SEQ ID NO:4), SEQ ID NO:3 wherein, SEQ ID NO:4 coupling can detect FLT3-ITD together.
Another preferred embodiment of the present invention, the primer sequence that can also use in pcr amplification reaction liquid is respectively: 5 '-FAM-AACTGCCTATTCCTAACTGACTC-3 ' (SEQ ID NO:5), 5 '-TCCATAAGCTGTTGCGTTC-3 ' (SEQ ID NO:6), SEQ ID NO::5 wherein, SEQ ID NO:6 coupling can detect FLT3-ITD together.
Another preferred embodiment of the present invention, the primer sequence that can also use in pcr amplification reaction liquid is respectively: 5 '-FAM-GCCAGCTACAGATGGTACAGG-3 ' (SEQ ID NO:7), 5 '-AGCATTTTGACGGCAACC-3 ' (SEQ ID NO:8), wherein SEQ ID NO:7 and SEQ ID NO:8 coupling can detect FLT3-ITD together.
A preferred embodiment of the present invention, pcr amplification reaction liquid wherein is characterised in that in the PCR damping fluid of every 25 microlitres to add 2 Taq of unit enzymes that damping fluid comprises 10mM Tris-HCl (pH8.0), 150mM KCl, 3mM MgCl
2
Another preferred embodiment of the present invention, wherein the pcr amplification reaction system is by PCR damping fluid, hot resistant DNA polymerase, dNTPs, primer forms, the combined sequence that it is characterized in that primer is SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8.
A preferred embodiment of the present invention, wherein negative quality control product is deionized water, positive quality control product is the recombinant plasmid that carries the FLT3-ITD sequence, plasmid is formed by commercially available carrier cloning structure by the PCR product of upstream and downstream primer amplification, the combined sequence of the upstream and downstream primer that uses can be SEQ ID NO:1 and SEQ ID NO::2, or SEQ ID NO:3 and SEQ ID NO::4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8.
A preferred embodiment of the present invention; come FLT3 normal gene and FLT3-ITD and dosage variation in analyzing samples according to composite amplification product clip size; any method that can the separating acid fragment size is for example filtered; high performance liquid chromatography; electrophoresis; the methods such as affine collection all can be used for the present invention, wherein separate by the method for gel electrophoresis and quantitatively, preferably use capillary electrophoresis to separate FLT3 normal gene and FLT3-ITD.
A preferred embodiment of the present invention can FLT3 normal gene quantitative according to the accumulative total fluorescent value of primer extension product and the relative quantity of FLT3-ITD, and analyzes the ITD-AR value by calculating FLT3 normal gene and FLT3-ITD relative dosage.
A preferred embodiment of the present invention, wherein the sample to be checked of test kit can be selected from but be not limited to leukaemic's clinical sample.
A preferred embodiment of the present invention, wherein the target polynucleotide of test kit is from the FLT3 gene.
A preferred embodiment of the present invention, wherein the nucleic acid amplification system of test kit adopts polymerase chain reaction, and the circulation of said amplified reaction is that the polymerase chain reaction of repetition 30-50 time circulates.
What deserves to be explained is especially, in order to ensure the accuracy of FLT3-ITD detection method of the present invention, we also use the recombinant plasmid that carries the FLT3-ITD sequence as positive quality control product.By these positive quality control product, use test kit of the present invention to carry out under the same conditions Parallel testing, with detection precision and the accuracy of further checking test kit of the present invention, and as the additional quality control standard of production test kit of the present invention.
Present technique compared with prior art, advantage is: height 1. is quick on the draw, 2. DNA concentration can detect when being 10ng/ μ l can carry out accurate quantification to gene in conjunction with capillary electrophoresis and 3. can carry out analytic statistics 4. under a large amount of wild type genes exist to mutated genes and wild type gene ratio, also can detect the low frequency mutated genes.
Description of drawings
Fig. 1 shows a normal male sample multiplex amplification product electrophorogram.In figure, to be about the blue band of 330bp be the amplified production of a normal male sample to clip size.
Fig. 2 shows a normal female sample multiplex amplification product electrophorogram.In figure, to be about the blue band of 330bp be the amplified production of a normal female sample to clip size.
Fig. 3 shows a male sex AML patient's FLT3-ITD sample amplified production electrophorogram.In figure, clip size is about two blue bands of 330bp and 360bp, shows a male sex AML patient's FLT3-ITD sample amplified production.
Fig. 4 shows a women AML patient's FLT3-ITD sample amplified production electrophorogram.In figure, clip size is about two blue bands of 330bp and 360bp, shows a women AML patient's FLT3-ITD sample amplified production.
Fig. 5 shows the electrophorogram of amplified production of the FLT3-ITD of a MDS clinical samples.In figure, clip size is about two blue bands of 330bp and 360bp, the electrophorogram of the amplified production of the FLT3-ITD of a MDS clinical samples of demonstration.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: detection kit and use thereof
1, preparation comprises the test kit of following moiety: ITD PCR reaction solution A (including SEQ ID NO:1 and SEQ ID NO::2) (500 μ l/ pipe), ITD PCR reaction solution B (75 μ l/ pipe), ITD positive quality control product (50 μ l/ pipe), negative quality control product (50 μ l/ pipe).
2, collection of specimens, transport and preserve:
(1) collection of specimens: sample is peripheral blood or marrow.Extract person under inspection's venous blood or marrow 3-5ml, inject the Glass tubing that contains EDTA (disodium ethylene diamine tetraacetate) or sodium citrate anticoagulant, put upside down gently immediately Glass tubing and mix 5～10 times, make the abundant mixing of antithrombotics and venous blood.
(2) preserve: can detect immediately, preserve a week for 4 ℃ ,-20 ℃ of preservation perives can reach 1 year.
(3) transportation: sample transports and should adopt 0 ℃ of curling stone.
3, detecting step and interpretation of result:
Qiagen DNA extraction test kit is used in the DNA extraction suggestion.
(1) get 200 μ l peripheral bloods or marrow to the 1.5ml centrifuge tube.
(2) add the proteolytic enzyme of 20 μ l.
(3) add the Buffer AL of 200 μ l, vibrated 15 seconds.
(4) 56 ℃ of incubations 10 minutes.
(5) add 200 μ l ethanol, vibrated 15 seconds.
(6) mixed solution is sucked QIAamp Mini spin column, centrifugal 1 minute of 6000g.
(7) inner prop is put into a new sleeve pipe, added the Buffer AW1 of 500 μ l, centrifugal 1 minute of 6000g.
(8) inner prop is put into a new sleeve pipe, added the Buffer AW2 of 500 μ l, centrifugal 1 minute of 6000g.
(9) inner prop is put into a new 1.5ml centrifuge tube, centrifugal 1 minute of maximum centrifugal speed.
(10) add the Buffer AE of 200 μ l, standing 1 minute of room temperature, centrifugal 1 minute of 6000g.
(11) obtain 200 μ l DNA solutions
The QF-PCR operation steps:
1, for each PCR reaction system, mixing following composition is 25ul to cumulative volume.The yin and yang attribute contrast is set.
2, PCR reaction conditions: 95 ℃ of denaturations 15 minutes, then 94 ℃ 30 seconds, 62 ℃ 30 seconds, 72 ℃ 30 seconds, totally 30 circulations; Last 72 ℃ 5 minutes.
The amplified production electrophoresis
For each analysis, 1ul PCR product and 13.5ul Hi-DiTM formamide, 0.5ul GeneScan ROX-500Size standard mix.95 ℃ of heat denatured mixtures 5 minutes.Placed at least 1 minute on ice.Instantaneous centrifugal mixture.Loading ABI310/ABI3100 genetic analyzer, then application software is carried out interpretation of result.Collection of illustrative plates such as Fig. 1 as a result, Fig. 2, Fig. 3, Fig. 4, shown in Figure 5.Fig. 1 wherein, Fig. 2 is the interpretation of result collection of illustrative plates of normal male and normal female.
Embodiment 2: by QF-PCR increase FLT3 site detecting AML patient's FLT3-ITD
From AML patient's blood or sample of bone marrow, carry out the DNA extraction purifying according to the standard program of Qiagen DNA extraction test kit.The ITD PCR reaction solution A, the ITD PCR reaction solution B that provide in test kit are provided carry out amplified reaction and loading analysis according to test kit detection rules.Analysis collection of illustrative plates such as Fig. 3 of loading result, Fig. 4.As shown in the figure, wild-type and ITD site on the FLT3 gene.Produce the ITD sudden change at the FLT3 gene, the amplified band greater than wild-type fragment can occur.Thereby can rapid detection FLT3-ITD.
Embodiment 3: by QF-PCR increase FLT3 site detecting MDS patient's FLT3-ITD
From MDS patient's blood or sample of bone marrow, carry out the DNA extraction purifying according to the standard program of Qiagen DNA extraction test kit.The ITD PCR reaction solution A, the ITD PCR reaction solution B that provide in test kit are provided carry out amplified reaction and loading analysis according to test kit detection rules.Analysis collection of illustrative plates such as Fig. 5 of loading result.As shown in the figure, wild-type and ITD site on the FLT3 gene.Produce the ITD sudden change at the FLT3 gene, the amplified band greater than wild-type fragment can occur.Thereby can rapid detection FLT3-ITD.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (6)
1. the test kit of FLT3-ITD in a test sample, test kit comprises: (1) pcr amplification reaction liquid, negative quality control product, positive quality control product and a plurality of reagent bottles or the pipe that seal, (2) separate and concentrate the packing box of these reagent bottles of packing or pipe, wherein pcr amplification reaction liquid comprises that 5 ' end is combined with the forward primer of fluorescence generation group, reverse primer, dNTPs, hot resistant DNA polymerase, PCR damping fluid and water, (3) determine size and the gene dosage of amplified fragments with electrophoretic method, it is characterized in that forward and reverse primer of using sequence can for:
5’-FAM-TCTCCTCTTCATTGTCGTTTTA-3’、5′-ATGGTGAGTACGTGCATTTTA-3′；
5’-FAM-TTCATTATTCTTTCCTCTATCTGC-3’、5′-TGTTGCGTTCATCACTTTTC-3′；
5’-FAM-AACTGCCTATTCCTAACTGACTC-3’、5’-TCCATAAGCTGTTGCGTTC-3’；
5’-FAM-GCCAGCTACAGATGGTACAGG-3’、5’-AGCATTTTGACGGCAACC-3’。
2. according to claim 1 test kit, be further characterized in that the PCR damping fluid comprises 10mM Tris-HCl (pH8.0), 150mM KCl, 3mM MgCl
2, add 2 Taq of unit enzymes in the PCR damping fluid of every 25 microlitres.
3. according to claim 1 test kit, be further characterized in that the pcr amplification reaction system is by PCR damping fluid, hot resistant DNA polymerase, dNTPs, primer forms, the combined sequence that it is characterized in that primer is SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8.
4. according to claim 1 test kit, be further characterized in that positive quality control product is the recombinant plasmid that carries the FLT3-ITD sequence, plasmid is formed by commercially available carrier cloning structure by the PCR product of upstream and downstream primer amplification, the combined sequence of the upstream and downstream primer that uses can be SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8.
5. according to claim 1 test kit is further characterized in that the nucleic acid amplification system of test kit adopts polymerase chain reaction, and the circulation of said amplified reaction is that the polymerase chain reaction of repetition 30-50 time circulates.
6. according to claim 1 test kit, be further characterized in that institute's test sample is the leukemia clinical samples such as whole blood or marrow.
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Application publication date: 20130515 |