CN107868823A - A kind of detection kit of detection MDS/MPN related gene groups - Google Patents
A kind of detection kit of detection MDS/MPN related gene groups Download PDFInfo
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Abstract
A kind of detection kit of detection MDS/MPN related gene groups, the technical scheme determine the gene group for examination MDS/MPN associated gene mutations first, and have therefrom screened 17 genes with high directivity.High throughput sequencing technologies are combined on this basis, it is possible to achieve auxiliary diagnosis are carried out to MDS/MPN outside morphology and flow cyctometry, making assessment especially for disease prognosis and make prejudging to the action effect for related target medicine has special advantage.Have the advantages that detection efficiency is high, recall rate is high provided by the present invention for the detection kit of the gene group of examination MDS/MPN associated gene mutations.
Description
Technical field
The present invention relates to technical field of molecular biology, further to the detection of examination MDS/MPN associated gene mutations
Kit and its medical science unscrambling data storehouse, and in particular to a kind of detection kit of detection MDS/MPN related gene groups.
Background technology
Myelodysplastic syndrome/bone marrow proliferative tumour (MDS/MPN) is one group of clinical, laboratory and morphological feature
Upper existing myelodysplastic syndrome (MDS) has the overlapped or compound medullary system tumour of bone marrow proliferative tumour (MPN) again.
MDS/MPN clinic, hematology have MDS and MPN a dual characteristicses, i.e. a system or polyphyly myeloid cell has that MDS's is invalid
There is the reduction of respective series haemocyte in hematopoiesis and morbid hematopoiesis, peripheral blood, and there is another system or polyphyly myeloid cell MPN occur again
Effective multiplication, cause peripheral blood respective series hematocytosis.However, the myeloproliferative disorder that its pathology is essential and simple
Syndrome (MDS) or bone marrow proliferative tumour (MPN) differ, and are not also the simple superposition of both diseases, but have
A kind of disease of particular pathologies feature and pathogenic factor.Marrow increases in the World Health Organization's (WHO) medullary system staging (2016)
Raw abnormal syndrome/bone marrow proliferative tumour (MDS/MPN) includes leukemia chronic myelo-monocytic (CMML), is not true to type slowly
Property myelogenous leukemia (aCML), juvenile myelomonocytic leukaemia (JCMML), MDS-MPN are with annular iron myelocyte and blood
Platelet increases the disease type such as (RARS-T) and unclassified MDS/MPN (MDS/MPN-U).
The MDS/MPN incidence of disease is about 1/,100,000 populations, and more accumulation the elderly, the median age is 72 years old, the ratio between men and women
For 2:1, median survival time is 42.3 months.The MDS/MPN cause of disease is still not clear, it is considered that heredity, radiation (radiotherapy), chemistry
Material (such as benzene, polyethylene), some drugses (such as chemotherapeutic) and environmental pollution and other professional exposures (such as dense smoke, pigment,
Agrochemical) may be relevant with MDS/MPN generation.
Detection MDS/MPN method relates generally to cell phychology, flow cyctometry, cytogenetics, molecule life at present
Thing.Wherein the experimental method of molecular biology mainly has nest-type PRC, generation sequencing etc..PCR method can not intuitively be had
The mutant nucleotide sequence of body, and the method for generation sequencing is influenceed by sequencing throughput, can not disposably detect the whole of multiple genes
Exon region.And two generations sequencing be used as a kind of high-throughout detection method, can disposably the multiple genes of accurate measurement outside
Aobvious subsequence, make it possible precisely to treat.Therefore, how cost-effectively to filter out MDS/MPN related gene group and carry
Become a reality urgent problem to be solved in technology for a kind of detection kit based on the gene group.
The content of the invention
A kind of it is contemplated that technological deficiency for prior art, there is provided detection of detection MDS/MPN related gene groups
Kit, more limited to solving the diagnostic method of MDS/MPN in the prior art, lack from molecular biology angle and diagnose MDS/
The technical problem of MPN foundation.
Another technical problem to be solved by the present invention is that MDS/MPN diagnostic method accuracy is relatively low in the prior art.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of detection kit of detection MDS/MPN related gene groups, kit detection 38 genes as shown in table 1
Catastrophe：
The MDS/MPN gene group-lists of table 1
Preferably, the detection kit includes the primer of 38 genes above, the amplification scope of the primer includes
Whole exon regions of 38 genes of the above.
Preferably, the catastrophe of 17 genes of kit detection as shown in table 2：
The MDS/MPN gene group preference lists of table 2
Preferably, the detection kit includes the primer of 17 genes above, the amplification scope of the primer includes
Whole exon regions of 17 genes of the above.
Preferably, the detection kit includes the primer of 17 genes above, the amplification scope of the primer includes
177 hot mutant sites of the exon region of 17 genes of the above, 177 hot spot mutations and its with 17 bases
The corresponding relation of cause is as shown in table 3：
The hot spot mutation list of table 3
The hot spot mutation of 17 gene extron subregions of the present invention can be used for making auxiliary diagnosis to MDS/MPN,
Diagnostic method conventional this area MDS/MPN has morphology, cytogenetics and a flow cyctometry, and the method for the invention is preferable
Diagnosis is made to MDS/MPN from molecular biology angle in ground.
The present invention provides the multiple PCR primer of one group of detection MDS/MPN related genes exon region of the present invention, institute
Stating the amplification scope of primer includes 17 exon regions with MDS/MPN related genes.Utilize multiplex PCR provided by the invention
Primer, with reference to two generation sequencing technologies, whole exon regions in mutator group of the present invention can be detected.
Two wherein described generation sequencing reagents are reagent commonly used in the art, and gained sequence is entered as long as disclosure satisfy that
The requirement of the generation of row two sequencing.The preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention more preferably also includes the genomic DNA extracted in detection object being made being available for surveying
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.The 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 beads.
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.The detection sample is preferably the one or more in marrow, blood, solid tumor sample, it is therefore preferable to marrow
Sample.
A kind of application method of kit of the present invention, it comprises the following steps：
(1) using the marrow of detection kit of the present invention extracting detection object, genomic DNA is extracted；
(2) exon 1 with multiple PCR primer to 17 MDS/MPN related genes described in invention in genomic DNA
Domain is expanded；
(3) fragment of amplification gained in step (2) is made to the library for being available for Ion Torrent microarray datasets to be sequenced；
(4) gained DNA library in step (3) is sequenced, obtains lower machine data；
(5) bioinformatic analysis is carried out to the lower machine data in (4), then understood using medical science unscrambling data storehouse
Make diagnosis.
The concrete operation method of each step refers to kit specification above.
Reagent and raw material used in the present invention is commercially available.
The positive effect of the present invention is：Dashed forward using provided by the invention available for examination MDS/MPN related genes
The gene group of change, with reference to high throughput sequencing technologies, it is possible to achieve carried out outside morphology and flow cyctometry to MDS/MPN auxiliary
Diagnosis is helped, makes assessing especially for disease prognosis and makes anticipation with special excellent to the action effect for related target medicine
Gesture.Provided by the present invention for the gene group of examination MDS/MPN associated gene mutations detection kit have detection efficiency it is high,
The advantages that recall rate is high.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.In addition to being defined, institute in following examples
Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Material and method：
In blood disease hospital of the Chinese Academy of Medical Sciences and consonance magnificent medical diagnosis center in August, 2015 in March, 2017
In the patient of diagnosis, according to following standard, we enter Line Continuity and enter group (44).
Inclusion criteria is：Myelodysplastic syndrome/bone marrow proliferation is diagnosed as through cytomorphology and flow cyctometry
The patient of property tumour (MDS/MPN).
The diagnostic result of 44 patients is as shown in table 4：
The patient's diagnostic result list of table 4
Collection 3mL Bone Marrow of Patients extraction genomic DNAs are to be measured, and the human relations of blood disease hospital of the Chinese Academy of Medical Sciences are passed through in the detection
The reason committee ratifies.
Sample process：Genome is stripped using Tiangeng DNA extraction kit in sample, and concrete operation method is referring to examination
The operation instructions of agent box.
Genomic DNA is expanded with multiple PCR primer, Examination on experimental operation referring to Life companies kit use
Specification.
The structure in library：The Ion Torrent platforms that the DNA fragmentation obtained to amplification is produced using Life companies build storehouse examination
Agent box carry out sequencing library structure, Examination on experimental operation referring to Life companies kit operation instructions.
High-flux sequence：The sequencing library built is subjected to height after Water-In-Oil is handled on Ion Torrent platforms
Flux is sequenced, Water-In-Oil processing method and high-flux sequence method refer to high-flux sequence instrument Ion Torrent and it is matched somebody with somebody
Complete equipment One Touch operation instructions.
Use kit testing result of the present invention：
The positive rate of 17 genes is as shown in table 5 below detected by 44 patients：
5 17 gene masculine rate lists of table
| Mutator | It is mutated case load | Mutation rate | Mutator | It is mutated case load | Mutation rate |
| NRAS | 11 | 25.00 | CBL | 2 | 4.55 |
| U2AF1 | 8 | 18.18 | CSF3R | 2 | 4.55 |
| SETBP1 | 7 | 15.91 | JAK2 | 2 | 4.55 |
| PTPN11 | 6 | 13.64 | KRAS | 2 | 4.55 |
| ASXL1 | 5 | 11.36 | KIT | 1 | 2.27 |
| RUNX1 | 5 | 11.36 | NPM1 | 1 | 2.27 |
| SRSF2 | 5 | 11.36 | SF3B1 | 1 | 2.27 |
| TET2 | 5 | 11.36 | TP53 | 1 | 2.27 |
| DNMT3A | 4 | 9.09 |
Show after statistics：(being summarized according to above table) as can be seen here, the method being sequenced using two generations is to marrow
Hyperplasia exception syndrome/bone marrow proliferative tumour (MDS/MPN) carries out auxiliary diagnosis, has that diagnosis is accurate, is contained much information
The characteristics of.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (9)
1. a kind of detection kit of detection MDS/MPN related gene groups, it is characterised in that kit detection is as shown in the table
38 genes catastrophe：
2. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 1, it is characterised in that described
Detection kit includes the primer of 38 genes above, and the amplification scope of the primer includes 38 all outer of gene above and shown
Subregion.
A kind of 3. detection kit of detection MDS/MPN related gene groups according to claim 1, it is characterised in that the examination
Agent box detects the catastrophe of 17 genes as shown in the table：
4. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 3, it is characterised in that described
Detection kit includes the primer of 17 genes above, and the amplification scope of the primer includes 17 all outer of gene above and shown
Subregion.
5. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 3, it is characterised in that described
Detection kit includes the primer of 17 genes above, and the amplification scope of the primer includes the exon 1 of 17 genes above
177 hot mutant sites in domain, 177 hot spot mutations and its corresponding relation such as following table institute with 17 genes
Show：
6. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 5, it is characterised in that described
Kit includes being used for the multiple PCR primer for expanding the mutator group exon region, and the examination for the sequencing of two generations
Agent.
7. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 5, it is characterised in that described
Kit include by extracted in detection object genomic DNA be made be available for sequencing library reagent.
8. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 5, it is characterised in that described
Kit includes dNTP solution, archaeal dna polymerase, for the reagent of isolated or purified nucleic acid, positive control or negative control.
9. the detection kit of a kind of detection MDS/MPN related gene groups according to claim 5, it is characterised in that described
Kit includes the database of gene group examining report, and the database contains the medical explanation to the hot spot mutation.
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Cited By (4)
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| CN109554461A (en) * | 2018-12-19 | 2019-04-02 | 天津协和华美医学诊断技术有限公司 | The biomarker and its detection kit of one group of detection primary HLH |
| 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 |
| CN111304331A (en) * | 2020-03-09 | 2020-06-19 | 南京实践医学检验有限公司 | MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof |
| CN113481289A (en) * | 2021-06-22 | 2021-10-08 | 天津见康华美医学诊断技术有限公司 | Primer composition for detecting sideroblastic red blood cell anemia and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| CN111304331A (en) * | 2020-03-09 | 2020-06-19 | 南京实践医学检验有限公司 | MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof |
| CN113481289A (en) * | 2021-06-22 | 2021-10-08 | 天津见康华美医学诊断技术有限公司 | Primer composition for detecting sideroblastic red blood cell anemia and application thereof |
| CN113481289B (en) * | 2021-06-22 | 2022-03-29 | 天津见康华美医学诊断技术有限公司 | Primer composition for detecting sideroblastic red blood cell anemia and application thereof |
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