CN106381332A - Detection kit for detecting AML related gene group - Google Patents
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Abstract
A detection kit for detecting an AML related gene group includes primers for detecting the AML related gene group and reagents for next generation sequencing; the reagents include a dNTP solution, DNA polymerase, and Backman magnetic beads for separation or purification of nucleic acid. A sample of the detection kit is a tissue of a detected object, so long as the genomic DNA of the detected object can be extracted from the sample. The detection sample is preferably one or more of samples of bone marrow, blood and solid tumors.
Description
Technical field
The present invention relates to a kind of detection kit
Background technology
AML (acute myelocytic leukemia is acute myelocytic leukemia) is a leukemoid general name, faces
In bed, Acute Meyloid system leukemia can be divided into 8 kinds altogether of M0~M7.The Annual occurence rate of AML is approximately to have 2.3 people in every 100,000 people, man
Property slightly more than women, and the chance of age bigger generation is higher, and the chance that the people more than 65 years old obtains AML is about and is less than 65 years old
10 times of people.Past this over 20 years, the not too big change of its incidence rate.Also do not find real pathogenic because of but one
As think that heredity, radiation, chemical substance (as benzene), medicine and other professional exposures (as dense smoke, pigment, agrochemical) can
Can be relevant with the generation of AML.
The method of detection AML relates generally to cellular morphology, flow cyctometry, cytogeneticss, molecular biology at present.Its
The experimental technique of middle molecular biology mainly has nest-type PRC, generation sequencing etc..PCR method can not intuitively specifically be dashed forward
Become sequence, and the method for generation sequencing is affected by sequencing throughput it is impossible to disposably detect whole exons of multiple genes
Region.And secondary sequencing as a kind of high-throughout detection method, disposably can accurately measure the exon sequence of multiple genes
Row, make precisely to treat and are possibly realized.However, due to current document report AML related gene more than 100, to all of
Related gene is detected and targeted therapy was both uneconomical, also unrealistic, therefore how to filter out the base mostly concerned with AML
Because of group and provide and a kind of problem demanding prompt solution in prior art is become based on the detection kit of this gene group.
Content of the invention:
For solving aforementioned technical problem, the invention provides a kind of detection kit for detecting AML related gene group,
It is characterized in that described AML related gene group is by following 40 genomic constitutions:
Described detection kit is it is characterised in that described AML related gene group is by following 18 genomic constitutions:
Described detection kit, it is characterised in that including the primer for detecting described AML related gene group, described is drawn
The amplification scope of thing includes whole exon regions of the gene in described AML related gene group,
Described detection kit is it is characterised in that be used for detecting primer sequence such as table 2 institute of described AML related gene group
Show.
Table 2 primer sequence and related gene title
Described detection kit is it is characterised in that the amplification scope of described primer is preferred in whole exon regions
202 hot mutant site, described hot mutant site is as shown in table 1
Table 1 hot spot mutation list
Described detection kit is it is characterised in that include described primer and the reagent for secondary sequencing.
Described detection kit is it is characterised in that also include being available for extracting genomic DNA in detection object and make
Sequencing
Library reagent.
Described detection kit is it is characterised in that also include:DNTP solution, archaeal dna polymerase, for isolated or purified core
Reagent, positive control or the negative control reagent of acid.The described reagent for isolated or purified nucleic acid preferably employs Backman magnetic
Pearl.
Present invention also offers described AML related gene group and its encoding proteins answering in preparing AML diagnostic reagent
With.
Present invention also offers described AML related gene group and its encoding proteins are in the medicine of preparation treatment AML
Application.
202 hot spot mutations as table 1 that the present invention provides, are the conventional mutational site in this area, are preferably comprised
Indel mutational site (insert or lack the sequence causing and change insertion or deletion, indel), nonsense mutation position
Point, splice region mutational site or missense mutation site.
As described in Table 2 one group that the present invention provides detects the multiple of AML related gene exon region of the present invention
PCR primer, the amplification scope of described primer includes 18 exon regions with AML related gene, expands single obtaining
Segment length is 250bp.The multiple PCR primer being provided using the present invention, in conjunction with secondary sequencing technologies, can detect the present invention
Whole exon regions in described mutant gene group.
Wherein said secondary sequencing reagent is reagent commonly used in the art, as long as disclosure satisfy that, gained sequence is entered
The requirement of the secondary sequencing of row.Described preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention more preferably also include making the genomic DNA extracting in detection object be available for survey
The reagent in the library that sequence uses, this kind of reagent is reagent commonly used in the art, as long as disclosure satisfy that multiplex PCR to genome
The requirement of DNA mass.Described preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention preferably also includes:DNTP solution, archaeal dna polymerase, for isolated or purified core
Acid is Backman magnetic bead.
Sample of the present invention is the tissue of detection object, as long as the gene of detection object can be extracted from detection sample
Group DNA.Described detection sample is preferably one or more of bone marrow, blood, solid tumor sample it is therefore preferable to bone marrow
Sample.
A kind of using method of detection kit of the present invention, it comprises the following steps:
(1) utilize detection kit of the present invention to extract the bone marrow of detection object, extract genomic DNA;
(2) with multiple PCR primer, the exon region of 18 AML related genes described in invention in genomic DNA is entered
Row amplification;
(3) fragment of amplification gained in step (2) is made the library being available for the sequencing of Ion Torrent microarray dataset;
(4) gained DNA library in step (3) is sequenced, obtains lower machine data;
(5) bioinformatic analysis are carried out to the lower machine data in (4), then understood using medical science unscrambling data storehouse
Make and diagnosing.
The concrete operation method of each step above refers to kit specification.
Reagent used by the present invention and raw material are all commercially available.
The positive effect of the present invention is:On the basis of existing AML related gene, filter out and one group of AML phase
Correlation gene group, using the primer of this gene group design, in conjunction with secondary sequencing technologies (also known as high throughput sequencing technologies), it is possible to achieve
Outside morphology and flow cyctometry, auxiliary diagnosis are carried out to AML, and can be further with testing result to pre- for disease
Make assessment afterwards and the action effect for related targeting medicine makes anticipation.Provided by the present invention for examination AML dependency basis
Because of the detection kit of the gene group of mutation, have the advantages that detection efficiency height, recall rate are high.Particularly the present invention is excellent further
Choosing by 18 genomic constitution AML related gene groups, carried out further preferably in 18 genomic constitution AML related gene groups,
On the basis of reducing test kit cost, improving detection efficiency, maintain basic with the AML related gene group of 40 genomic constitutions
Identical high detection rate (close to 95%).Additionally, through filtering out 202 hot spot regions conduct in 18 genes further
The preferred scope of PCR primer amplification, can simplify design of primers further, while keeping high detection rate, improve further
Detection efficiency, reduction test kit cost.
Specific embodiment:
Further illustrate the present invention below by the mode of embodiment, but therefore do not limit the present invention to described reality
Apply among a scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product description selects.
Material and method:
In hematopathy hospital of the Chinese Academy of Medical Sciences and consonance magnificent medical diagnosiss center in December, 2015 in May, 2016
In the patient of diagnosis, according to following standard, we enter Line Continuity and enter group (240).
Inclusion criteria is:It is diagnosed as the patient of acute myelocytic leukemia (AML) through morphocytology and flow cyctometry.
The diagnostic result of 240 patients is as shown in table 2:
Table 3 patient diagnosis the results list
Collection 3mL Bone Marrow of Patients extraction genomic DNA is to be measured, and this detection is through the human relations of hematopathy hospital of the Chinese Academy of Medical Sciences
The approval of reason committee.
Sample process:In sample, genome utilizes Tiangeng DNA extraction kit (article No.:DP318-03) it is stripped, tool
Body operational approach is referring to the operation instructions of test kit.
A kind of detection kit for detecting AML related gene group of embodiment
Detect the sequence of primer (hereinafter referred to as multiple PCR primer) and the related gene such as table 2 of described AML related gene group
Shown, described detection kit includes described multi-primerses and the reagent for secondary sequencing.
With multiple PCR primer, genomic DNA is expanded, Examination on experimental operation is referring to the use of Life company test kit
Description.
The structure in library:Storehouse examination built by the Ion Torrent platform that the DNA fragmentation that amplification is obtained is produced using Life company
Agent box carries out the structure of sequencing library, and Examination on experimental operation is referring to the operation instructions of Life company test kit.
High-flux sequence:The sequencing library building is carried out height after Water-In-Oil process on Ion Torrent platform
Flux is sequenced, Water-In-Oil processing method and high-flux sequence method refer to high-flux sequence instrument Ion Torrent and it is joined
The operation instructions of complete equipment One Touch.
Bioinformatic analysis:First, using Ion Report software (v4.6, Thermo Fisher, Carlsbad,
CA, USA) filter low-quality sequencing fragment, and qualified sequencing fragment is compared mankind's reference gene group hg19
(http://hgdownload.cse.ucsc.edu/downloads.html), using Torrent Variant Caller
(v4.6.0.7) subprogram detection mutational site, software parameter uses the setting of acquiescence.This software has been optimized for
Process the distinctive type of error of Ion Torrent microarray dataset.Finally the mutation found out is included with SNP and Indel use
ANNOVAR(http://annovar.openbioinformatics.org/en/latest/) software annotated, in annotation
Hold and include being mutated the position in genome, the gene of association, gene extron is numbered, nucleotide level makes a variation, corresponding egg
White level makes a variation, and this mutation is in dbSNP (http://www.ncbi.nlm.nih.gov/snp/), 1000Genomes data base
(http://www.1000genomes.org/) and cancer mutation database COSMIC (http://
Cancer.sanger.ac.uk/cosmic the annotation in), PolyPhen (http://genetics.bwh.harvard.edu/
) and SIFT (http pph2/://sift.jcvi.org/) function prediction result etc..Caused a disease using the screening of principle once further
Mutational site:(1) genomic locations according to mutation place and type, filters out and protein product sequence is not produced with the prominent of impact
Become;(2) utilize 1000Genomes data, annotate each mutation ratio in crowd, if ratio is less than or equal to 1%, no
It is considered pleomorphism site;(3) cancer databases COSMIC are retrieved, whether one mutation of inquiry is on the books in COSMIC, its
Occur in hematological system tumor;(4) predict whether this mutation affects egg using protein function forecasting software PolyPhen-2
Contour painting energy;(5) if after (2) (3) (4), a mutation meets " non-polymorphism ", " crossing described in hematological system tumor "
At least two in " impact protein function " this three, will be judged to may be related to disease.Finally, if a though mutation
So it is unsatisfactory for (5) but is recorded as detecting in tumor in COSMIC, then be read the mutation for interrogatory.Finally
If one mutation be expressly recited in the literature not with disease height correlation, be directly judged to hot spot mutation.
Statistical analysiss:Count the ratio that each gene carries pathogenic mutation, filter out mutation rate highest gene, use
Software is Excel.
Medical science is understood:With medical science unscrambling data storehouse, molecular pathology is made to the genic mutation type that bioinformatic analysis draw
Diagnosis.Medical science unscrambling data storehouse used is as follows:
(1) ASXL1 gene mutation all has been reported that in AML, CML, MDS, MPN patient, with AML overall patient's survival rate fall
Low phase closes (PMID:22417203).The not known medicine for ASXL1 gene at present.
(2) CEBPA is the important transcription factor safeguarding the differentiation of granulocyte hemopoietic system.CEBPA gene mutation is mainly seen in
AML patient, especially M1, M2 and M4, are currently known the AML patient prognosis bona of CEBPA diallele mutation.
(3) DNMT3A gene mutation is found in AML, MPN, MDS, ALL and Lymphoma.DNMT3A gene code DNA
Methylated transferase 3A, plays from the beginning methylated effect, the most prognosis malas of AML patient of DNMT3A gene mutation, but
The mutation of DNMT3A R882 position will not cause unfavorable result to catabasises AML patient.The daunorubicin of high dose may improve
Carry the survival rate of the AML patient of DNMNT3A mutation, but idarubicin is in the AML carrying DNMT3A gene exon23 mutation
Effect in patient is better than daunorubicin.DNA methylation inhibitor 5-Azacytidin may to DNMT3A mutation patient
Beneficial.(PMID:22417203;19776405;27476855)
(4) EZH2 gene mutation sees AML, ALL, CML, MDS, MPN, DLBCL and FL patient, and mutation patient's prognosis is not
Good.Comb the EZH2 albumen of protein family more EZH2 gene code, participate in apparent regulation process.EZH2 inhibitor EPZ-6438 opens
Begin to carry out II clinical trial phase (NCT01897571) in FL and DLBCL.(PMID:20081860;27447873)
(5) FLT3 gene code type III receptor tyrosine kinase protein, the propagation participating in regulation and control myeloid element is divided
Change, maturation and apoptosis etc..FLT3 gene mutation patient's prognosis malas, and can be independent of caryogram outside.A generation of FLT3 at present
Inhibitor has the secondary inhibitor of Sunitinib, Midostaurin, Lestaurtinib and Tandutinib to also have
Quizartinib, Sorafenib, Gilteritinib, Crenolanib and Ponatinib, wherein Sunitinib are targeting
The inhibitor of receptor tyrosine kinase FLT3, can be used for the cancer patient that treatment comes from FLT3 activated mutant (ITD, D835Y).
(PMID:27450971)
(6) IDH1 gene mutation is mainly seen in AML, MDS and MPN patient.IDH1 gene code Isocitrate dehydrogenase
1, participate in intracellular oxidative damage defense mechanism.For AML patient, merge possible prognosis during NPM1 mutation preferable.IDH1 inhibitor
AG-120 carries out Phase I clinical trial (NCT02632708) in AML patient.(PMID:22915641;27245312;
26660517;22417203)
(7) IDH2 gene mutation is mainly seen in AML, MDS and MPN patient.IDH2 gene code Isocitrate dehydrogenase
2, participate in intracellular oxidative damage defense mechanism.It is preferable that IDH2 gene mutation merges possible prognosis during NPM1 mutation.IDH2 inhibitor
AG-221 carries out Phase I clinical trial (NCT02632708) in AML patient.(PMID:22417203)
(8) KIT mutation occurs mainly in AML patient, and incidence rate is less than 5%.The TYR kinases of KIT gene code is subject to
Body plays an important role in the growth of hematopoietic cell and propagation.It is t (8 for caryogram;21) patient, KIT is mutated prognosis malas.
Imatinib is the targeted inhibition agent of KIT activated mutant.(PMID:26376137)
(9) KRAS is found in 43% MM patient.RAS albumen KRAS is primarily involved in RAS signal path.Clinical
The medicine of III phase has MEK162.(PMID:26282654)
(10) NPM1 mutation sees the AML patient of 25-35%, and the Phospoprotein NPM1 ribosomal generation of participation, center are little
The duplication of body, cell proliferation and TP53 signal path.For the AML patient of normal karyotype, NPM1 mutation is not accompanied by FLT3 simultaneously
Mutation, points out prognosis bona;NPM1 mutation can be used for the monitoring of minimal residual disease.NPM1 is mutated to the trouble of prognosis bona
Person, after first time chemotherapy is alleviated, row Allogeneic Hematopoietic Stem Cell Transplantation is unhelpful;For the elder patients of NPM mutation, tradition is strong
The chemotherapy of degree is beneficial.(PMID:26376137)
(11) NRAS mutation is found in about 15% AML patient and 43% MM patient.RAS albumen NRAS is primarily involved in
RAS signal path.In AML, if patient carries NRAS simultaneously, DNMT3A and NPM1 is mutated, then point out prognosis bona.RAS
Mutation patient is sensitive to cytosine arabinoside.(PMID:26376137)
(12) PTPN11 gene mutation is mainly seen in CML and AML, ALL and MPN patient.PTPN11 is protein-tyrosine phosphorus
Sour enzyme, all plays an important role in cell growth, differentiation, mitotic cycle and oncogenic transformation.Dodecane-
Trimethylamine can target PTPN11 (in grinding).
(13) RUNX1 mutation is mainly seen in medullary system tumor patient, is especially more common in AML patient.RUNX1 mutation MDS and
AML patient's prognosis and overall survival all can reduce.(PMID:19808697;22689681;21714648)
(14) SF3B1 gene mutation is mainly seen in CLL, in MDS and MPN patient, prognosis malas in CLL patient.?
In MDS, it is that refractory anemia increases (RARS) with ringed sideroblasts that SF3B1 is mutated modal hypotype, and overall survival
Rate improves.(PMID:21995386)
(15) SRSF2 mutation is mainly seen in the patients such as MDS, t-AML and CML, the MDS overall patient of SRSF2 mutation
Survival rate reduces, and the risk to AML conversion increases.(PMID:22389253)
(16) TET2 gene mutation is mainly seen in MDS, AML, CML.TET2 gene mutation MDS and CML patient's accumulation existence
Rate reduces.The TET inhibitor of approved has Hydrochlorothiazide and Succinic acid.(PMID:20644105;
21714648)
(17) TP53 gene mutation is mainly seen in MDS, AML, CLL.MDS Patients Patients' survival rate fall of TP53 gene mutation
Low and most prognosis malas.Act on TP53 and its medicine of path correlation have Cisplatin, Fluorouracil,
Docetaxel, Paclitaxel, Aspirin.(PMID:21714648)
(18) U2AF1 gene mutation is mainly seen in MDS, AML.U2AF1 mutation can lead to its protein level to rise, and changes
Its cut mode.The MDS patient of U2AF1 mutation easily progresses to secondary acute marrow series leukemia, but its overall survival is not had
Have an impact.(PMID:22158538)
In 240 patients listed by table 3, have 213 patients and be diagnosed through morphocytology and flow cyctometry diagnosis
For AML, the sample of bone marrow of above-mentioned 213 patients is detected using using detection kit provided in an embodiment of the present invention,
The sample of 200 patients is wherein had to obtain positive findingses (at least 1 positive in 18 genes) it is possible to determine that being inspection
Go out, recall rate 93.9%:
Detected by 200 patients, the positive rate of 18 genes is as shown in table 3 below:
3 18 gene masculine rate lists of table
Mutant gene | Mutation case load | Mutation rate | Mutant gene | Mutation case load | Mutation rate |
ASXL1 | 1 | 0.42% | NPM1 | 30 | 12.50% |
CEBPA | 51 | 21.25% | NRAS | 51 | 21.25% |
DNMT3A | 17 | 7.08% | PTPN11 | 14 | 5.83% |
EZH2 | 5 | 2.08% | RUNX1 | 8 | 3.33% |
FLT3 | 60 | 25.00% | SF3B1 | 3 | 1.25% |
IDH1 | 11 | 4.58% | SRSF2 | 2 | 0.83% |
IDH2 | 2 | 0.83% | TET2 | 11 | 4.58% |
KIT | 18 | 7.50% | TP53 | 7 | 2.92% |
KRAS | 25 | 10.42% | U2AF1 | 11 | 4.58% |
Show after statistics:(being summarized according to above table) as can be seen here, using secondary sequencing method to acute
Myelocytic leukemia carries out auxiliary diagnosis, have the characteristics that diagnosis accurately, obtain and contain much information.
And will for 202 hot spot mutations when optimize multi-primerses sequence further after, the detection kit obtaining exists
When the detection sample of above-mentioned 213 patients is detected, the recall rate obtaining is held essentially constant with the positive rate of each gene,
The hot spot mutation that the present invention screens further is described, can keep Detection results while further optimizing detection test kit.
It should be understood that after having read the above of the present invention, those skilled in the art can make various changing to the present invention
Move or change, these equivalent form of values equally fall within the application appended claims limited range.
Claims (10)
1. a kind of detection kit for detecting AML related gene group is it is characterised in that described AML related gene group is by following
40 genomic constitutions:
2. detection kit as claimed in claim 1 is it is characterised in that described AML related gene group is by following 18 genomes
Become:
3. detection kit as claimed in claim 1 or 2 is it is characterised in that include for detecting described AML related gene group
Primer, the amplification scope of described primer includes whole exon regions of the gene in described AML related gene group.
4. detection kit as claimed in claim 3 is it is characterised in that be used for detecting the primer sequence of described AML related gene group
Row are as follows with related gene title:
5. detection kit as claimed in claim 3 is it is characterised in that the amplification scope of described primer is whole exon 1s
Preferred 202 hot mutant site in domain, described hot mutant site is as follows with related gene title:
6. described detection kit as arbitrary in claim 3~5 it is characterised in that include as described in primer and be used for secondary sequencing
Reagent.
7. detection kit as claimed in claim 6 is it is characterised in that also include to extract genomic DNA in detection object
Make the reagent in the library being available for being sequenced.
8., it is characterised in that also including dNTP solution, archaeal dna polymerase, for separating for detection kit as claimed in claim 6
Or the reagent of purification of nucleic acid, positive control or negative control reagent.
9. application in preparing AML diagnostic reagent for the AML related gene group and its encoding proteins described in claim 1 or 2.
10. the AML related gene group described in claim 1 or 2 and its encoding proteins answering in the medicine of preparation treatment AML
With.
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Cited By (7)
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CN107881233A (en) * | 2017-10-31 | 2018-04-06 | 天津协和华美医学诊断技术有限公司 | A kind of detection kit for detecting myeloma related gene group |
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 |
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 |
CN110819710A (en) * | 2018-08-10 | 2020-02-21 | 珠海铂华生物工程有限公司 | High-throughput sequencing detection of myeloid tumors |
CN110982884A (en) * | 2019-12-05 | 2020-04-10 | 阅尔基因技术(苏州)有限公司 | Primer group, kit and method for detecting AML related gene mutation |
CN111020710A (en) * | 2018-10-10 | 2020-04-17 | 珠海铂华生物工程有限公司 | ctDNA high-throughput detection of hematopoietic and lymphoid tissue tumors |
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 |
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