CN108251527A - A kind of detection kit for detecting lymthoma related gene group - Google Patents
A kind of detection kit for detecting lymthoma related gene group Download PDFInfo
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Abstract
The sequencing kit for being used for screening lymthoma associated gene mutation the invention discloses one group and subsequent medicine unscrambling data library.Lymthoma related gene group 22 genes such as including MYD88, TP53.Sequencing kit includes the multiple PCR primer that related 22 gene whole extrons are expanded to lymthoma.The kit of screening lymthoma gene mutation provided by the invention has many advantages, such as that detection efficiency height, mutator broad covered area, recall rate are high；The medicine provided, which understands report, has the characteristics that accurate, authority.
Description
Technical field
The present invention relates to technical field of molecular biology, the detection further to screening lymthoma associated gene mutation tries
Agent box and its medicine unscrambling data library, and in particular to a kind of detection kit for detecting lymthoma related gene group.
Background technology
Lymthoma is one group of malignant tumour originating from lymph node or other lymphoid tissues, can be divided into Hodgkin's disease (referred to as
HD) increase with two major class of non-Hodgkin lymphoma (abbreviation NHL), the visible lymphocyte of histology and (or) histiocytic tumprigenicity
Raw, clinical the most typical with Silent Neuritis enlargement of lymph nodes, there are cachexia, fever and anaemia in the normal enlargement of liver and spleen, late period.Lymthoma
Cellular morphology is extremely complex, in the newly classification of WHO lymthomas in 2008, there is 80 hypotypes.Due to diseased region and range phase not to the utmost
Together, clinical manifestation is very inconsistent, and original site can be in lymph node, also can be in the lymphoid tissue outside knot, such as just tonsillotome, nasopharynx
Portion, gastrointestinal tract, spleen, bone or skin etc..The primary portion of knot lymphoid tissue, which becomes, is more common in NHL.
The method of detection lymthoma relates generally to cellular morphology, flow cyctometry, cytogenetics, molecular biosciences at present
It learns.Wherein the experimental method of molecular biology mainly has nest-type PRC, generation sequencing etc..PCR method cannot be obtained intuitively specifically
Mutant nucleotide sequence, and the method for generation sequencing is influenced by sequencing throughput, can not disposably detect all outer of multiple genes
Aobvious subregion.And the sequencing of two generations disposably can accurately measure the outer aobvious of multiple genes as a kind of detection method of high throughput
Subsequence makes it possible precisely to treat.
Invention content
The present invention is directed to be directed to the technological deficiency of the prior art, a kind of detection examination for detecting lymthoma related gene group is provided
Agent box is more limited to the diagnostic method for solving lymthoma in the prior art, is lacked from the angle of molecular biology and is diagnosed lymph
The foundation of knurl.
Another technical problem to be solved by the present invention is that the diagnostic method accuracy of lymthoma is relatively low in the prior art.
For realization more than technical purpose, the present invention uses following technical scheme：
A kind of detection kit for detecting lymthoma related gene group, the kit detect 22 genes as shown in Table 1
Catastrophe：
1 lymthoma gene group-list of table
。
Above-mentioned 22 genes, are made of 162 hot spot mutations, and mutational site is the mutational site of this field routine, compared with
Include goodly indel mutational sites (be inserted into or missing caused by sequence change insertion or deletion, indel),
Nonsense mutation site, splice region mutational site or missense mutation site, 22 gene extron sub-districts more preferably of the present invention
The hot spot mutation in domain is as shown in table 2.
2 hot spot mutation list of table
The hot spot mutation of 22 gene extron subregions of the present invention can be used for making lymthoma auxiliary diagnosis, this
The diagnostic method of field lymthoma routine has morphology, cytogenetics and flow cyctometry, and the method for the invention is preferably
Diagnosis is made to lymthoma from molecular biology angle.
The present invention provides the multiple PCR primer of one group of detection lymthoma related gene exon region of the present invention, institute
The amplification range for stating primer includes 22 exon regions with lymthoma related gene, and the individual chip length expanded is
250bp or so.Using multiple PCR primer provided by the invention, with reference to two generation sequencing technologies, mutation of the present invention can be detected
Whole exon regions in gene group.
The two wherein described generation sequencing reagents be reagent commonly used in the art, as long as disclosure satisfy that gained sequence into
The requirement of two generation of row sequencing.The preparation method of reagent thereof is this field customary preparation methods, preferably commercially available.
Detection kit of the present invention is more preferably further included to be made to survey by the genomic DNA extracted in detection object
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 field customary preparation methods, preferably commercially available.
Detection kit of the present invention preferably further includes：DNTP solution, archaeal dna polymerase, for isolated or purified core
Acid is Backman magnetic beads.
Sample of the present invention is the tissue for detecting object, as long as the gene of detection object can be extracted from detection sample
Group DNA.The detection sample is preferably one or more of marrow, blood, solid tumor sample, it is therefore preferable to marrow
Sample.
A kind of application method of kit of the present invention, includes the following steps：
(1) using the marrow of detection kit of the present invention extracting detection object, genomic DNA is extracted；
(2) with multiple PCR primer to the exon region of 22 lymthoma related genes described in invention in genomic DNA
It is expanded；
(3) segment of amplification gained in step (2) is made to the library being sequenced for Ion Torrent microarray datasets；
(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), is then understood using medicine unscrambling data library
Make diagnosis.
The concrete operation method of above each step refers to kit specification.
Reagent and raw material used in the present invention is commercially available.
The positive effect of the present invention is：It can be used for screening lymthoma associated gene mutation using provided by the invention
Gene group, with reference to high throughput sequencing technologies, can realize and lymthoma is assisted except morphology and flow cyctometry
Diagnosis makes assessment especially for disease prognosis and makes anticipation with special excellent to the function and effect for related target medicine
Gesture.Provided by the present invention for the gene group of screening lymthoma associated gene mutation detection kit have detection efficiency it is high,
The advantages that recall rate is high.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has 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 September, 2015 in April, 2017
In the patient of diagnosis, according to following standard, we enter group (345) into Line Continuity.
Inclusion criteria is：The patient of lymthoma is diagnosed as through cytomorphology and flow cyctometry.
The diagnostic result of 345 patients is as shown in table 3：
3 patient's diagnostic result list of table
Collection 3mL Bone Marrow of Patients extraction genomic DNAs are to be measured, which passes through the human relations of blood disease hospital of the Chinese Academy of Medical Sciences
The reason committee ratifies.
Sample process：Genome utilizes Tiangeng DNA extraction kit (article No. in sample：DP318-03 it) is stripped, has
Body operating method referring to kit operation instructions.
Genomic DNA is expanded with multiple PCR primer, Examination on experimental operation referring to Life companies kit use
Specification.
The structure in library：Library examination is built to the Ion Torrent platforms that the DNA fragmentation that amplification obtains is produced using Life companies
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 carried out 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 please refers to high-flux sequence instrument Ion Torrent and it is matched
The operation instructions of complete equipment One Touch.
Bioinformatic analysis：First, using Ion Report softwares (v4.6, Thermo Fisher, Carlsbad,
CA, USA) the low-quality sequencing segment of filtering, and qualified sequencing segment is compared to mankind's reference gene group hg19
(http://hgdownload.cse.ucsc.edu/downloads.html), use Torrent Variant Caller
(v4.6.0.7) subprogram detection mutational site, software parameters use the setting of acquiescence.This software has been optimized for
Handle the distinctive type of error of Ion Torrent microarray datasets.Finally include SNP and Indel to the mutation found out to use
ANNOVAR(http://annovar.openbioinformatics.org/en/latest/) software annotated, in annotation
Hold and include being mutated the position in genome, associated gene, gene extron number, nucleotide level variation, corresponding egg
White level makes a variation, and the mutation is in dbSNP (http://www.ncbi.nlm.nih.gov/snp/), 1000Genomes databases
(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..Principle screening is further used to cause a disease
Mutational site：(1) it according to the genomic locations and type where mutation, filters out and dashes forward to what protein product sequence did not had an impact
Become；(2) using 1000Genomes data, ratio of each mutation in crowd is annotated, if ratio is less than or equal to 1% not
It is considered polymorphic site；(3) cancer databases COSMIC is retrieved, whether one mutation of inquiry is on the books in COSMIC,
It appears in hematological system tumor；(4) predict whether the mutation influences egg using protein function forecasting software PolyPhen-2
Contour painting energy；(5) if after (2) (3) (4), a mutation meets " non-polymorphism ", " being crossed described in hematological system tumor "
At least two in " influence protein function " this three, will be judged to may be related to disease.Finally, if a though mutation
It is so unsatisfactory for (5) but is recorded as detecting in tumour in COSMIC, be then read the mutation for interrogatory.Finally
If a mutation is expressly recited highly relevant not with disease in the literature, it is directly judged to hot spot mutation.
Statistical analysis：The ratio that each gene carries pathogenic mutation is counted, the highest gene of mutation rate is filtered out, uses
Software is Excel.
Medicine is understood：Molecular pathology is made to the genic mutation type that bioinformatic analysis obtains with medicine unscrambling data library
Diagnosis.Medicine unscrambling data library used is as follows：
(1) ATM gene mutations are mainly seen in CLL and MCL, also there is a small amount of report in other hematological system tumors.ATM bases
Because being located at 11q, mutation can cause gene function to be lost, and mutant form is mainly frameshit, nonsense, missense and 11q missings.11q
ATM caused by missing is mutated CLL patient diseases and makes fast progress, and life cycle is short.The ATM germlines of heterozygosis are mutated in CLL diseases
Rapid progress in also play a significant role.Document report at present, ATM mutation do not have apparent prognosis meaning still in MCL.
(PMID：24584352；26314984；21933854；12697903；16461462)
(2) BCL2 genes mostly occur fusion, reset, abnormal expression leads to the generation of disease, and mutation sees a variety of lymthomas.
(PMID：28765546；28600336；28584254)
(3) CCND1 gene mutations are mainly seen in MM and Lymphoma, particularly common in MCL.CCND1 mutant forms can have
Gene Fusion, fragment deletion and point mutation.CCND1 mutation may lead to MCL patient to ibrutinib drug resistances.It is known
Palociclib can be used for the patient for the treatment of generation CCND1 activated mutants.(PMID：27713153；17299095)
(4) CCND3 gene mutations are mainly seen in NK-AML and lymthoma, and CCND3 gene mutations reset the positive in MYC
Burkitt Lymphomas are common, and mostly occur in end-stage disease.Spleen diffusivity red pulp small B-cell lymphoma patient can go out
Existing CCND3 gene high expressions and gene mutation.CCND3 encodes albumen and interacts with Runx1 and its negativity is adjusted.
The influence prognosis of inpatient with haematological diseases is still not clear in CCND3 gene unconventionalities.(PMID：28069605；28209658)
(5) CREBBP gene mutations are mainly seen in the ALL and Lymphoma of recurrence.Document report, in ALL patients with recurrent
CREBBP and KRAS mutation often occur simultaneously, and CREBBP mutation and KRAS mutation may be related with the recurrence of children ALL.(PMID：
25917266)
(6) CXCR4 gene mutations see MM, AML and Lymphoma, also seen in Waldenstrom's macroglobulinemia patient.
Some researches show that CXCR4 high expression is the index (PMID of prognosis mala in the children's acute marrow series leukemia patient of low danger：
27135782).Also some researches show that the C1013G gene mutations of CXCR4 are drug resistant mark in Waldenstrom's macroglobulinemia patient
Will (PMID：24711662).There are some researches prove the antagonist plerixafor of CXCR4 can effectively treat WHIM syndromes at present
The Panleukopenia of patient.(PMID：21890643)
(7) DNMT3A gene mutations are mainly seen in the AML of medullary system tumour, especially normal karyotype, are also found in other blood
Liquid disease or normal the elderly's (being happened in normal person, general load is relatively low, and independent mutation).DNMT3A is as one
A relevant gene of epigenetic, typically occurs in the Preleukemia stage, mutational load is generally higher.DNMT3A genes are main
Mutant form is point mutation, mostly occurs in the amino acid of R882, and most common mutant form is R882H.DNMT3A mutation are suffered from
The most prognosis mala of person.DNMT3A mutation patients may be benefited using the daunorubicin of high dose, and DNA methylation transferase is pressed down
Preparation Decitabine has sound response.(PMID：25426837；26755712；22417203；19776405)
(8) EP300 gene mutations are mainly seen in Lymphoma, MM and B-ALL.EP300 gene code E1A binding proteins
P300 is the transcription co-activation factor closely coupled with CBP, with a variety of transcription factors and histone acetyltransferase phase interaction
With.(PMID：10648382)
(9) EZH2 gene mutations see AML, ALL, CML, MDS, MPN, DLBCL and FL, are mutated patient's prognosis mala,
EZH2 inhibitor EPZ-6438 has started to carry out II clinical trial phases (NCT01897571) in FL and DLBCL patients.EZH2 is
One tumor suppressor gene encodes the EZH2 albumen of more comb protein families, participates in epigenetic regulation.(PMID：20081860；
27447873)
(10) FBXW7 gene mutations are mainly seen in lymphocytic leukemia and Lymphoma.NOTCH1/FBXW7 mutation are big
It occurs approximately in 67% T-ALL patient, NOTCH1/FBXW7 occurs in the T-ALL patient of no carrying RAS/PTEN mutation
Mutation prompting prognosis is preferable.FBXW7 mutation are often accompanied by+12 in CLL.FBXW7 is a tumor suppressor gene, and coding includes F-
The E3 ubiquitin ligases of Box and WD repetitive structure domains.(PMID：27060156；24113472；27247421；22547598；
24166518；24113472)
(11) JAK1 gene mutations are mainly seen in T-PLL, ALL patient.With the ALL patient ages of JAK1 mutation higher than not
With the ALL patient that JAK1 is mutated, and DFS and OS are poor.The albumen of JAK1 gene codes is a kind of nonreceptor tyrosine kinase,
Can phosphorylation stat protein, play a significant role in other cytokine signalings such as interferon.JAK inhibitor
Tofacitinib and ruxolitinib has good efficacy to JAK1 mutation patients.(PMID：26819051；24048415；
18362173)
(12) JAK3 gene mutations are mainly seen in AMKL (acute megakaryocytic leukemia) and ETP-ALL.In T-PLL patient
Middle JAK3 is mutated prognosis mala.Tasocitinib is mutated targeted drug for JAK3.JAK3 albumen is that non-receptor type tyrosine swashs
Enzyme participates in the growth, development, differentiation of cell, plays an important role in the development of T cell.(PMID：26493028)
(13) KRAS gene mutation is found in the diseases in the blood system such as AML, MDS, MPN, ALL, CLL, MM.Age it is smaller (<
60 years old) AML patient in the incidence of KRAS mutation be about 5%, the recall rate higher in M4；RAS mutation are still not enough in AML
Into Prognostic Factors are independent, the AML patient of RAS mutation is carried, is consolidated after alleviation using the cytarabine of high dose
Treatment may benefit.In adult T-ALL, there is jejune immunophenotype in RAS gene mutations ratio about 10%, tendency；Children
The patient OS that KRAS mutation occurs in ALL is reduced, and related to palindromia；The patient of RAS accesses mutation occurs in children ALL
It may be benefited using selumetinib.(PMID：15951308；27655895；27226433；25253770；21680795；
18701506)
(14) MYC is oncogene, which is more common in MM and Lymphoma.Research shows that BCL2/MYC doubles are hit
(double-hit) overall survival of Diffuse Large B-Cell Lymphoma (DLBCL) patient and prognosis are very poor.(PMID：
26462634)
(15) MYD88 gene mutations are mainly seen in CLL, Lymphoma and MM.Wherein L265P site mutations are found in 86-
100% WM patient, the MGUS patient of 10-87%, the central nervous system lymphoma patient of 36-38%, 6-21% spleen side
Edge area Lymphoma, activation B cell origin DLBCL patient, 9% gastric mucosa associated lymphoid tissue Lymphoma and
The CLL patient of 2.9-4%.Intracellular ligandin MYD88 has key effect in the response of congenital and adaptive immunity.It carries
The Lymphoma prognosis mala of MYD88L265P.(PMID：27034430)
(16) NOTCH1 gene mutations are found in ALL, CLL and MCL.There is influence albumen work(in the PEST structural domains of C-terminal
The mutation of energy can lead to the generation of disease.Mutation rates of the NOTCH1/FBXW7 in T-ALL is about 67%, is not being carried
It is preferable that NOTCH1/FBXW7 mutation prompting prognosis occurs in the T-ALL patient of RAS/PTEN mutation；NOTCH1 mutation occur in CLL
Rate is about 12.3%, is often accompanied by+12, prompts prognosis mala.The drug of clinical test phase has MK0752.(PMID：15472075；
24166518；21835957；24113472)
(17) NRAS gene mutations are mainly seen in the diseases in the blood system such as AML, CML, ALL, MPN, MM.Age it is smaller (<60
Year) AML patient in NRAS be mutated incidence be about 11%, and usually without FLT3-ITD be mutated；RAS dashes forward in AML
Change is still not enough to become independent prognostic factor, and when NPM1 merging DNMT3A mutation, prognosis is poor, if but occurring on this basis
NRAS-G12/13 is mutated, then prognosis has improves to a certain degree；The AML patient that document report is mutated to carrying RAS, makes after alleviation
Carrying out after treatment with the cytarabine of high dose may benefit.RAS mutation rates are about 54.5% in MM, wherein 81% is
Recurrent MM；NRAS is mutated the sensibility that may be decreased MM patient's bortezomib single therapy.NRAS mutation prompting prognosis in MDS
It is bad.RAS mutation rates are about 10% in adult T-ALL, and are inclined to and jejune immunophenotype occur；In adult T-ALL
RAS/PTEN mutation prompting prognosis malas；The children ALL patient that selumetinib may be mutated RAS accesses benefits.(PMID：
15951308；27276561；26376137；24335104；27655895；25253770；24166518；11524732；
14737077；23708912)
(18) PTEN gene mutations are mainly seen in ALL and Lymphoma.PETN is a tumor suppressor gene,
It plays an important role in PI3K-AKT accesses.Gene inactivation can activate PI3K-AKT accesses, lead to leukaemic to changing
Drug resistance is treated, the T-ALL Patients on Recurrence risks with PETN genes Exon7 mutation increase.PTEN inhibitor is Temsirolimus.
It is abnormal that EGFR inhibitor Cetuximab and mTOR inhibitors Everolimus can intervene the extremely caused signal paths of PTEN.
(PMID：19829307)
(19) SF3B1 gene mutations are mainly seen in MDS, CLL, MPN and MDS/MPN.Mutation of the SF3B1 in MDS occurs
Rate is about 20%, and the mutation rate higher in MDS-RS contributes to the diagnosis of MDS-RS, and related to good prognosis；MDS/
Often with JAK2V617F/CALR/MPL with generation, this contributes to the diagnosis of MDS/MPN-RS-T for SF3B1 mutation in MPN-RS-T；
SF3B1 mutation are related to ringed sideroblasts in MDS.Mutation rates of the SF3B1 in CLL is about 9%, is often accompanied by del
(11q), it is related to CLL progress and shorter OS.(PMID：21995386；21998214；27069254；22158541；
24113472；23415222)
(20) TET2 gene mutations see AML, MDS, MPN, MDS/MPN and Lymphoma, multidigit in Exon4 or
Exon12, principal mode are missense, nonsense and frameshift mutation.TET2 mutation tendency betides older patient, TET2 in AML
Mutation incidence in CN-AML (the normal AML of caryogram) patient is about 23%, prompts prognosis poor, such as merges FLT3-ITD and dashes forward
Change then prompts prognosis worse, and TET2 mutation and IDH are mutated usual mutual exclusion.TET2 mutation can be happened at about 20% MDS and
In 14% MPN.Prognosis bona is prompted in TET2 mutation in MDS, and prompts prognosis mala in MDS/MPN and sAML.
(PMID：21828143；19262601；19666869；22430270；21828143)
(21) TP53 gene mutations are mainly seen in MDS, AML and CLL.The MDS survivals of TP53 mutation reduce and big
More prognosis malas.Mutation rates of the TP53 in CLL about 14% is related to CLL progression of disease；17p- and/or TP53 mutation
CLL patient's poor prognosis, and for fludarabine, cyclophosphamide, rituximab therapeutic responses are poor.Make
There are cisplatin, fluorouracil, docetaxel, paclitaxel for TP53 and its relevant drug of access,
aspirin.MDS the or AML patients for carrying TP53 gene mutations may react preferable for dicitabine.(PMID：
21714648；27959731；23138133；23415222)
(22) WHSC1 gene mutations are mainly seen in Lymphoma and Myeloma, the Lymphoma In Children of WHSC1 mutation
Patient's prognosis mala.WHSC1 mutation indications children ALL has risk of recurrence.(PMID：23823660；25790293；26294725；
24076604)
Use kit testing result of the present invention：
The positive rate of 22 genes is as shown in table 4 below detected by 345 patients：
4 22 gene masculine rate lists of table
Show after statistics：(being summarized according to above table) is it can be seen that using the method that two generations were sequenced to acute
Myelocytic leukemia carries out auxiliary diagnosis, has the characteristics that diagnosis is accurate, is contained much information.
The embodiment of the present invention is 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., should all
It is included within protection scope of the present invention.
Claims (7)
1. a kind of detection kit for detecting lymthoma related gene group, it is characterised in that kit detection is as shown in the table
The catastrophe of 22 genes：
。
2. a kind of detection kit for detecting lymthoma related gene group according to claim 1, it is characterised in that described
Detection kit includes the primer of above 22 genes, and the amplification range of the primer includes all outer aobvious of above 22 genes
Subregion.
3. a kind of detection kit for detecting lymthoma related gene group according to claim 2, it is characterised in that described
Exon region includes 162 hot spot mutations, 162 hot spot mutations and its as follows with the correspondence of 22 genes
Shown in table：
。
4. a kind of detection kit for detecting lymthoma related gene group according to claim 3, it is characterised in that described
Kit includes the examination for expanding the multiple PCR primer of the mutator group exon region and being sequenced for two generations
Agent.
5. a kind of detection kit for detecting lymthoma related gene group according to claim 3, it is characterised in that described
Kit includes that the reagent for extracting the library that genomic DNA is made for sequencing in object will be detected.
6. a kind of detection kit for detecting lymthoma related gene group according to claim 3, it is characterised in that described
Kit include dNTP solution, archaeal dna polymerase, for the reagent of isolated or purified nucleic acid, positive control or negative control.
7. a kind of detection kit for detecting lymthoma related gene group according to claim 3, 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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| CN109735621A (en) * | 2019-02-13 | 2019-05-10 | 上海交通大学医学院附属瑞金医院 | A kind of lymthoma related gene inspecting reagent kit and the construction method for expanding sublibrary |
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| CN111575373A (en) * | 2020-04-28 | 2020-08-25 | 南京实践医学检验有限公司 | Kit and method for detecting acute lymphocytic leukemia gene mutation by targeting high-throughput sequencing based on multiple PCR |
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| CN112430658A (en) * | 2020-11-04 | 2021-03-02 | 上海交通大学医学院附属瑞金医院 | Detection kit for intranodal peripheral T cell lymphoma related gene and library building method |
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