CN203530309U

CN203530309U - Kit for screening 31 kinds of blood disease-associated fusion genes

Info

Publication number: CN203530309U
Application number: CN201320688645.XU
Authority: CN
Inventors: 朱立文; 米国稳; 赵程; 侯然; 梁超; 王绪华; 方国伟; 黄士昂; 陈忠
Original assignee: BEIJING HAISITE CLINICAL INSPECTION Co Ltd
Current assignee: Beijing Haisite Medical Examine Laboratory Co ltd
Priority date: 2013-11-04
Filing date: 2013-11-04
Publication date: 2014-04-09
Anticipated expiration: 2023-11-04

Abstract

The utility model discloses a kit for screening 31 kinds of blood disease-associated fusion genes. The kit comprises a kit body, a kit cover and a liner arranged in the kit body, wherein the kit body is divided into an upper part and a lower part by the liner; small holes are formed in the liner; a reagent tube containing 32 reverse transcription specific primers, a reagent tube containing dNTP (deoxyribonucleoside triphosphate), a reagent tube containing reverse transcriptase, a reagent tube containing an RNA (ribonucleic acid) enzyme inhibitor, a reagent tube containing a reverse transcription buffer solution and a reagent tube containing a PCR (polymerase chain reaction) MIX reagent are arranged in the small holes; reagent tubes containing 110 PCR amplification primers respectively are placed in the kit body below the liner. The kit is simple in structure and reasonable in design, and can be conveniently popularized and used for clinical detection, and detection results can be rapidly and conveniently obtained when the kit is used for screening the 31 kinds of blood disease-associated fusion genes.

Description

Test kit for 31 kinds of hemopathy fusion genes of examination

Technical field

The utility model belongs to technical field of molecular biology, relates to a kind of test kit for 31 kinds of hemopathy fusion genes of examination.

Background technology

Development along with Protocols in Molecular Biology, scientific workers find that increasing hemopathic generation, development are relevant with gene unconventionality, found at present the gene relevant with leukemia reach hundreds of more than, by detecting these genes, can help hemopathic type, judging prognosis, guiding treatment, detection minimal residual leukemia (MRD).

The normal gene relevant with hemopathy detecting is as follows at present:

(1) CBFB/MYH11:Inv (16) is (p13q22) acute myeloid leukemia (AML) characteristic chromosome abnormalty, conventionally sees AML-M4Eo hypotype, accounts for total AML patient's 10%, wherein 50% occurs in AML-M4Eo.With Inv (16) patient (p13q22), conventionally there is good prognosis.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(2) AML1/ETO:t (8,21) (q22; Q22) reported first in 1973.AML1/ETO fusion gene appears in the AML that all t (8,21) are positive, also appears in the AML case of t (8, the 21) feminine gender with complex displaced simultaneously.T (8,21) appears in about 7% AML case, more common in young patient.It mainly appears in this hypotype of AML-M2, and nearly 20-40% case has t (8,21).At very rare AML-M1, in AML-M4 and t-AML, also there is report.T (8,21) dystopy is a sign preferably, and its existence makes this class case have good reaction to some medicines.Therefore the detected result based on cytogenetics and molecular genetics, can be the low good treatment plan of patient selection risk.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(3) PML/RARa:t (15,17) transposition is a typical characteristics of APL, and its transposition has caused PML gene on No. 15 karyomit(e)s and the fusion of the RARa gene on No. 17 karyomit(e)s, has produced a fusion gene PML/RARa.Due to the difference of position of fusion on PML gene, PML/RARa has three kinds of pattern of fusion, that is: bcr1 (55%), bcr2 (5%), bcr3 (40%).Whether the existence of detection PML/RARa fusion gene to be, the diagnosis to APL, and curative effect and the prognosis recurrence of judgement ATRA are all extremely important.The PML/RARa positive is indicating the possibility of recurrence strongly, and its persistence feminine gender indicates that patient has longer lifetime.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(4) E2A/PBX1:t (1:19) has caused E2A gene on No. 19 karyomit(e)s and the fusion of the PBX1 gene on No. 1 karyomit(e), produced fusion gene E2A/RBX1, at the children ALL of 5-6% and 3% adult ALL, detect, almost all appear in pre B cell ALL case.Carry the patient of this transposition, its clinical symptom is all more dangerous.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(5) MLL/AF4: at the infant ALL of 50-70% with have MLL/AF4 fusion gene in 5% children's and adult ALL case nearly.T (4,11) (q21; Q23) relevant with front B-ALL (CD79A+, CD19+, CD10-, CD24-), be differentiation antigen CD16 and CD65 and NG2 antigen coexpression with grain simultaneously.MLL/AF4 fusion gene is at all t (4; 11) in the case of displacement, exist, be also present in quite a few cytogenetics and t (4 do not detected; 11) in the case of displacement.In infant ALL, MLL/AF4 fusion gene is considered to the sign of prognosis mala.In adult ALL, be also a bad sign, but for adult ALL, the existence of this fusion gene seems to strengthen the curative effect of the Ara-C of high dosage.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(6) in case BCR/ABL: in surpassing 95% patient's CML leukemia cell, in the children ALL of adult ALL 30%(20-50%) and 2-10%, and the AML(lymphoma and the myelomatosis that are less than 2%), there is BCR/ABL fusion gene.In CML, BCR/ABL fusion gene is all p210 type, has 55% b3-a2 type in CML case, 40% b2-a2 type.B3-a2 and b2-a2 type difference simultaneously existing due to montage in 5% case.In addition, in patient CML of considerably less ph+, there is a kind of large BCR/ABL fusion gene e19-a2, i.e. u-bcr, p230 type BCR/ABL fusion gene.At the ALL of the adult ph+ of 30-50%, in the ALL case of 20-30% children ph+, BCR/ABL fusion gene is p210 type.The ALL patient's of 60% ph+ BCR/ABL fusion gene is p190 type in addition.In ALL, Ph positive and that occur thereupon, and BCR/ABL fusion gene is a very bad sign, and it affects the complete remission rate of Blood of Patients phase and possible production rate.The patient of this gene masculine, available imatinib mesylate is treated, and can carry out curative effect monitoring and small residual detection.

(7) TEL/AML1:t (12; 21) (p13; Q22) transposition is in the news as far back as nineteen ninety-five.Report is subsequently found the transposition that this General Genetics cannot detect, and is transposition the most common in children ALL.Almost 25% children's ALL has this transposition.Its main positive patients appears at 1-12 between year, wherein maximum at 2-5 this age bracket in year.Its immunophenotyping of all cases is all pre B cell ALL.In addition, another feature of this class patient is that its white corpuscle is lower.T (12; 21) positive patient has good prognosis, and the time of its recurrence also can be more late.The patient of this gene masculine, can be used for curative effect monitoring and small residual detection.

(8) SIL/TAL1: the TAL1 gene being arranged on karyomit(e) 1p32 can cause at T cell unconventionality expression by multiple heritable variation.Wherein modal recombination form is the DNA disappearance of the about 90kb of TAL1 upstream region of gene, causes SIL and TAL1 to merge, micro-deleted all appearing in T-ALL case in this karyomit(e).The immunophenotype of SIL/TAL1 fusion gene and T-ALL is closely related.SIL/TAL1 fusion gene can detect in 26% children T-ALL case and adult's (being mainly Young Adults) T-ALL case of 16%.The relation of SIL/TAL1 fusion gene and T-ALL prognosis is also not very clear.This fusion gene can be for the auxiliary diagnosis of T-ALL, and this fusion gene can also be for curative effect monitoring and small residual detection.

(9) EVI1: be positioned karyomit(e) 3q26.In the acute myeloid leukemia model bringing out at Murine retroviral, EVI1 is the common insertion point of virus.Energy of EVI1 coding is in conjunction with the zinc finger protein of DNA, and its overexpression plays an important role in the generation of medullary system malignant tumour and development.In addition MDS and the CML relevant to its expression.The research of present stage shows the patient's of EVI1 high expression level prognosis mala, but its mechanism is not yet clear.

(10) HOX11: learn research according to cytogene, relate to the transposition of karyomit(e) 10q24 in the T-ALL of 4-7%.It is upper that HOX11 is positioned at 10q24 just, and by t (10; 14) (q24; Q11) transposition and t (7; 10) (q35; Q24) transposition activates.In B-ALL without the discovery of HOX11.In the bibliographical information of present stage, the prognosis of the children T-ALL of HOX11 high expression level has superiority compared with negative patient.In adult, also there is similar judgement.The HOX11 of low expression and negative patient no difference of science of statistics.

(11) MLL/AF10:MLL/AF10 is relevant to AML, shows as t (10 on karyomit(e); 11) (p12; Q23) transposition, is mainly seen in AML-M5 type patient, and children are common, and 80% patient is less than 3 years old.The patient's of the MLL/AF10 positive poor prognosis.

(12) MLL-AF9:t (9; 11) (p22; Q23) transposition mainly occurs in AML, is the modal transposition form of t in AML (11q23).The patient's of MLL-AF9 prognosis is poor.

(13) MLL/ENL:t (11; 19) (q23; P13.3) transposition is found in ALL, AML-M4, M5, M1, M2.Common to be less than the baby of 1 years old, median survival interval is 17.6 months.Transposition causes MLL-ENL fusion gene to form.Prognosis there is no definite explanation, and with the age, immunophenotype is relevant.

(14) MLL/ELL:t (11; 19) (q23; P13.1) account for 11q23 abnormal 3.8%, for AML characteristic is abnormal, the age take adult as main.White corpuscle 20 * 109/L, FAB somatotype M4 or M5, immunophenotype is CD13CD33, CD14CD15, CD11, HLA-DR expresses positive.Transposition causes MLL-ELL fusion gene to form.Prognosis mala, 2 years disease free survival rates 50%.

(15) MLL/AFX:t (X; 11) (q13; Q23) transposition only has some research researchs of external molecule to show that MLL-AFX can strengthen the self of hemopoietic stem cell and stop their maturations.Without prognosis related data.

(16) MLL/AF1q:t (1; 11) (q21; Q23) transposition, is mainly in AML.There is arguement in the prognosis saying about MLL/AF1q: in a research, the AF1q of high expression level is an independently poor prognosis factor; Another research shows the patient prognosis bona of the MLL-AF1q positive, whether because the formation of MLL-AF1q to have changed the expression of AF1q also unknown.

(17) MLL/AF1p:t (1; 11) (p32; Q23) transposition, ALL, AML, is all found in MDS.Median survival interval is 15 months.Prognosis is relevant to sex and somatotype, and women's median survival interval can reach 28 months, and the male sex is 11 months.Shortage is about the pertinent literature of prognosis.

(18) PLZF/RARa:t (11; 17) (q23; Q21) transposition is specifically found in acute promyelocytic leukemic or acute nonlymphocytic leukemia M3 type.Function is still not clear.Prognosis obviously poor with have t (15; 17) the ANLL M3 type of transposition, its chemotherapy to ATRA is reactionless.

(19) MLL/AF6:t (6; 11) (q27; Q23) be transposition common in 11q23, long hair and AML, particularly M4 in M5, are also found in T-ALL.The non-constant of prognosis, almost, without alleviating, lifetime is short.

(20) part of MLL/MLL:11q23 series connection repeats (MLL-PTD), in the AML of 10% normal karyotype, detects MLL-PTD, can be used as the monitoring mark of stable MRD.Poor prognosis, clinical remission rate is low.

(21) TLS/ERG:t (16; 21) (p11; Q22) young patient is more common in transposition, and each hypotype of FAB is all shown in.Prognosis mala, may not reach completely and alleviate, and the recurrence rate in a year is high, and median survival interval is 22 months.

(22) TEL/PDGFR:t (5; 12) (q33; P13) fusion gene that transposition forms, relevant with STAT5 signal transduction pathway to Ras/ERK, the differentiation of induced dry-cell.Prognosis is still not clear, median survival interval <20 month (n=11).

(23) TEL/ABL:t (9; 12) (q34; P13) transposition is rare, to 2010, only has 19 examples to be reported in CML, and AML, in ALL.Quantity is few, from the case of report, prognosis mala.

(24) SET/CAN:t (9; 9) (q34; Q34) transposition, rare, effect is not clear.There is bibliographical information to claim SET-CAN can suppress hyperplasia, short differentiation.

(25) DEK/CAN:t (6; 9) (p23; Q34) transposition, by the DEK gene fusion that is positioned at CAN gene and the 6q23 of 9q34, CAN gene is a part for product nuclear Pore Complex, can transport RNA and protein through nuclear membrane.In AML, accounting for 2%, be mainly M2 type, is secondly M4.Initial description is to take in marrow normal basophilia to have previously MDS medical history as feature 20% patient.Young patient (20～30 years old).Poor prognosis.

(26) MLL/AF17:t (11; 17) (q23; Q12-21) transposition, is found in AML.Without prognosis pertinent literature.

(27) E2A/HLF:t (17; 19) (q22; P13) transposition produces E2A/HLF fusion gene, occurs in the ALL case of its minority.Poor prognosis, reactionless to dose-dense, lifetime is short.

(28) NPM/MLF1:t (3; 5) (q25.1; Q34) transposition, at MPS, MDS, ANLL(M2, M4, M6) in be found.The non-constant of prognosis, median survival interval is less than 1 year.

(29) NPM/RARa:t (5; 17) (q35; Q22) transposition betides acute nonlymphocytic leukemia, sees M3, i.e. acute promyelocytic leukemia.Case is few, and on the books only have 2 routine children's cases, and the recurrence time of 2 examples is all short, and prognosis may be pessimistic.

(30) NPM1/ALK:t (2; 5) (p23; Q35) transposition, the transposition of 2p23 occurs in the primary cutaneous type (ALCL) over half, a kind of senior non-George Hodgson lymphoma.Although betide in high-grade invasive tumor, be longer than 80% patient's lifetime 5 years.

(31) AML1/MDS1:t (3; 21) (q26; Q22) transposition betides CML (accounting for 1%), in ANLL and MDS, conventionally relevant to treatment.Prognosis mala, survival rate is low.

At present clinical normal employing cytogenetics detects analyzes the fusion gene that karyotype is identified, examination chromosomal structural aberration forms, but the method sense cycle is long, is difficult to the small chromosome translocation of identification, and Chang Buneng meets clinical needs.

Utility model content

In order to solve the problems of the technologies described above, the purpose of this utility model is to provide a kind of test kit for 31 kinds of hemopathy fusion genes of examination, can apply multiple PCR technique and in a detection system, different fusion genes be detected, can obtain fast, easily detected result simultaneously.

For achieving the above object, the technical scheme that the utility model is taked is: a kind of test kit for 31 kinds of hemopathy fusion genes of examination, comprise box body (1), lid (2) and be arranged on the liner (3) in box body (1), described liner (3) is divided into upper and lower two portions by described box body (1), described liner (3) is provided with aperture, the Reagent Tube (4) of 32 reverse transcription Auele Specific Primers is housed, the Reagent Tube (5) of dNTP is housed, the Reagent Tube (6) of reversed transcriptive enzyme is housed, the Reagent Tube (7) of RNA enzyme inhibitors is housed and the Reagent Tube (8) of reverse transcription damping fluid is housed, the Reagent Tube (9) that PCR MIX reagent is housed is arranged in described aperture, the Reagent Tube that 110 pcr amplification primers are housed is respectively placed in the box body of described liner (3) below.

Wherein, described test kit also comprises: the Reagent Tube (10) that deionized water is housed.

Wherein, described liner (3) is provided with 7 apertures, and described aperture is divided into two rows and is arranged in parallel on described liner (3).

Wherein, described 32 reverse transcription specific primer sequences are as follows:

FG15’-GCTGACATTGAT-3’

FG25’-ACTCAAGCTGTG-3’

FG35’-CCACGTGTGCCTCTC-3’

FG45’-GTATTGACAGTAT-3’

FG55’-GACACCTTGAAG-3’

FG65’-ATGGCTGGCTCAG-3’

FG75’-AGGTCATTTTGAG-3’

FG85’-TTCCTCCTCTGAG-3’

FG95’-CCGATCAATCTTT-3’

FG105’-CTGCCACACTT-3’

FG115’-GCATAACAGTTGT-3’

FG125’-AAGTCTCCAACAG-3’

FG135’-TTTGCACTCTGAT-3’

FG145’-CGGTAATCGATTTC-3’

FG155’-CCCGTAACATAAC-3’

FG165’-TAAGGCTGCTCT-3’

FG175’-TTGCCTGGTACT-3’

FG185’-GCCCATGCTCAG-3’

FG195’-CCAGCCTTGATG-3’

FG205’-TCTCCACGAAGT-3’

FG215’-TGTCCAGCGTAGC-3’

FG225’-GTGTTAACTCTT-3’

FG235’-GGCTATCCATACTT-3’

FG245’-TGGGTACCTCTC-3’

FG255’-TGGCAATGGAAAG-3’

FG265’-CTGCTGGGTGAG-3’

FG275’-AGCTGCTTGATG-3’

FG285’-CAGCGAACAATG-3’

FG295’-CCCTCCAGAAG-3’

FG305’-CTGCAGGAAGGT-3’

FG315’-CGGTAAGCTCTC-3’

FG325’-CCGATTACCTA-3’

Wherein, described dNTP, reversed transcriptive enzyme, RNA enzyme inhibitors, reverse transcription damping fluid and PCR MIX reagent are the conventional reagent in this area.

Wherein, the sequence of described 110 pcr amplification primers is as following table:

First round primer sequence:

FG335’-TTTGACTCCCATGATTCTG-3’

FG345’-AGGCCTTAGGCCAGCTTCTTCT-3’

FG355’-GAGCTGTCCGAGAGAGTGGAGAT-3’

FG365’-CCGCCTCTGTTAGCCTACTAC-3’

FG375’-AGCACTCCAATGGCAATAGT-3’

FG385’-GGGTTCTCTGGCAGCACAGAT-3’

FG395’-CCGCTGCGACACTTCATAG-3’

FG405’-ACACCCCAGTGTGTCCTGTATG-3’

FG415’-CCGCAATCTGCCACCTACTAC-3’

FG425’-AGCACCTCCCAATGGCAATAGT-3’

FG435’-GGATACCTTCTCTTCATCTGTGTC-3’

FG445’-CCTCCAGTGGCTGCTCTGTGT-3’

FG455’-CTGTTCTGTCATGGCTGCTACTG-3’

FG465’-TTGCCCTCTGTGCACCTAGTCT-3’

FG475’-5-TGGACTTTATCGGCACCATTAC-3’

FG485’-TTTAGAGCACGAACACAGATGG-3’

FG495’-GCCACGCGAATTCCGCTAAC-3’

FG505’-CGACCCGCCAACCAGAG-3’

FG515’-CGGTCCTGGTCCGGGCATATTT-3’

FG525’-GCCCACATGCTCCCTCAAGT-3’

FG535’-CACTTTCTGGATTTCAAACAGTC-3’

FG545’-AGCCGCCGAGTAGTTTTCATTGC-3’

FG555’-GATGGTCACTGGTCACTGTG-3’

FG565’-CGATCTTTACTCTGGTCCATATTC-3’

FG575’-GGGCGTTCAACCTCACTG-3’

FG585’-GTCTGCAACCTCCACTTTGTC-3’

FG595’-TCTCCTATCCCGTCTGAAATGTC-3’

FG605’-GGTCGGGTGGTTATGGCAATC-3’

FG615’-GTTCATGGCTGTTTGCTCTTC-3’

FG625’-CCGCCCTTGGGACCTACTAC-3’

FG635’-AGCACTCAGTCTCCAATGGCAATAGT-3’

FG645’-GAATTTGATCCGTTTGAAGATGTATC-3’

FG655’-CCAGATGAGAGTTTGGTAACTCTGT-3’

FG665’-TTCCCTGCCTCCTCTATTTAC-3’

FG675’-GCTTGAGGGCTTTAGACAATGAG-3’

FG685’-CACCGGGAAATCCAGTGAAC-3’

FG695’-CGCTCTCGTCGTGCGCAGAACT-3’

FG705’-GAGTCCTACATGCTGCTTATGTC-3’

FG715’-TTGTTCTGCTGGGCTTCACAC-3’

FG725’-GCTGTTGCTGACCAAAGAGGACTT-3’

FG735’-CATAGCTAATTGCTTCTCACTG-3’

FG745’-TGCCAAAGGTGTTAAGAAGATAG-3’

FG755’-GGCAAGATTTGGTGTGAGAT-3’

FG765’-CACCAAGCATCAAATGGAAATCTG-3’

FG775’-CCACTACGAAGAAGGATGAGAAG

FG785’-TGCCTCGATAGGTTTGCAGAC-3’

FG795’-CCACAGTGAGGCTGGCTGTATT-3’

FG805’-CAAACAAGCCAGCCCAGAG-3’

FG815’-GTGCAGGCCAGGTGGTAGCTC-3’

FG825’-AAGCCCTTGCAGCCCTCAC-3’

FG835’-ACGAAGGCAGTCCAATTAAAGTAAC-3’

FG845’-CACACTTCAGGCAGCGTCTTC-3’

FG855’-AGCTCTCCCTCTACCATCAGAGATACT-3’

FG1415’-TTCTCGTCCAGCCCTTCTACC-3’

FG1425’-TTTTCCTCTTCTCGCCGTTTCA-3’

Second takes turns primer sequence:

FG865’-TGGGCTGTCCCAAGTTTGATG-3’

FG875’-TGCCTGCTGGATGTTGAC-3’

FG885’-TTAGCGCCAGCTGGTCTTG-3’

FG895’-GGCCTCGGCCAAGAAAAGAAGT-3’

FG905’-AGACCCAGATGGCACAGGATCAG-3’

FG915’-GGTTTCTGGGGAGTCGCTTTAACT-3’

FG925’-GAGTTCTAGCATTCGCATATCAG-3’

FG935’-TTCGACCGACTGGAGACATAC-3’

FG945’-GGACCACCATAGAAAAGAAGT-3’

FG955’-AGCAGCGCATGGAGTCCACTCAG-3’

FG965’-TGTCGGCAGGAATCCCAAATCT-3’

FG975’-GTAGAGAAGCCAGAGAAAACAC-3’

FG985’-AACTGCTGCCATTTGCCTGGTTGAT-3’

FG995’-TTCAGAGGTGTGTGCAGAG-3’

FG1005’-GGCAACACTACTGGCATGTTAC-3’

FG1015’-TCTAGGCCCACGAGCAGGTTTT-3’

FG1025’-CATGTTGTCTCTCAGCATCAG-3’

FG1035’-CCCGCTCCCCGCTGCAAAC-3’

FG1045’-AGACCGGTACCCCCTGAATAG-3’

FG1055’-CGCCTCCAGTCTCACTTGTC-3’

FG1065’-CTCATCGTGCGGAAGAGCTTAC-3’

FG1075’-AGCACCCTGGAGAGGAAGTTG-3’

FG1085’-TGGCTGGCAAGAGCTGAAAAC-3’

FG1095’-CCCCACATATGATTTGACTCTC-3’

FG1105’-CTTCCCCTGGGGAGAGAGTAAC-3’

FG1115’-GCGCATTATTCATTGTTTGAG-3’

FG1125’-CGTTGTCGGCGAATGAACTG-3’

FG1135’-CAGCGGCTATGGTGTAGACAG-3’

FG1145’-GGTGCCTTCTGGCCAGGTGATG-3’

FG1155’-GGACCGCCAAGAAAAGAAGT-3’

FG1165’-AGCAGATGGAGTCCACAGGATCAG-3’

FG1175’-GTTTTTGGTTTGGGTTACAGAACT-3’

FG1185’-GAGCAAAGATCAAAATCAAATGTT-3’

FG1195’-CTCCATTTCAGAGTCATTGTCGTTAT-3’

FG1205’-TGCTGGCAGCCATGGGAGCTCTC-3’

FG1215’-GCGATCCGCCAGCTCTAAC-3’

FG1225’-ACTGCGTTTGATGTCGTGTC-3’

FG1235’-CACGTTCCCCATTCCTCTGAC-3’

FG1245’-ACACCATTCCCTGTGTTGATTAT-3’

FG1255’-CCTCATTCAGGTCGTGAGCTCTAT-3’

FG1265’-CATGGGACCCACGTAGATGTAC-3’

FG1275’-AGCAGCACTCCCAAGAAGAAT-3’

FG1285’-GTCTCTCGCCATGGCACAAG-3’

FG1295’-TGAGGAACAAGAGAGCTTCTTTAC-3’

FG1305’-CGTCGAATGACCACCTGCTTC-3’

FG1315’-GAACATAGAGGATGGTGACTGTAAG-3’

FG1325’-GTGGGCATACGAGTCAGAGAGC-3’

FG1335’-GCCAGTGCCTACGATCTCCAT-3’

FG1345’-CAGCGCGCCTGACTGGAGAT-3’

FG1355’-CCCATAGTGCCAGTAGAGGAC-3’

FG1365’-GGTTCAGGGGTAGTGCATATT-3’

FG1375’-CTTGGGTCAATGGGCATTC-3’

FG1385’-AAAGGGTTCAGAACTTCTTATCATC-3’

FG1395’-CTACGACGGGGGTCTCCAC-3’

FG1405’-AGGTTCCGCTCTCGCACTT-3’

By above technical scheme, the beneficial effects of the utility model are as follows:

(1) test kit of the present utility model is simple in structure, reasonable in design, is convenient for carrying, preserves, not vulnerable to pollution.

(2) test kit of the present utility model is when 31 kinds of hemopathy fusion genes of examination, use 110 pcr amplification primers, by multiple PCR technique, in a detection system, different fusion genes is detected simultaneously, can obtain detected result fast, easily, be convenient to apply in clinical detection.

Accompanying drawing explanation

Fig. 1 is structural representation of the present utility model;

In figure, 1-box body, 2-lid, 3-liner, 4-is equipped with the Reagent Tube of 32 reverse transcription Auele Specific Primers, 5-is equipped with the Reagent Tube of dNTPs, 6-is equipped with the Reagent Tube of reversed transcriptive enzyme, and 7-is equipped with the Reagent Tube of RNA enzyme inhibitors, and 8-is equipped with the Reagent Tube of reverse transcription damping fluid, 9-is equipped with the Reagent Tube of PCR MIX reagent, and 10-is equipped with the Reagent Tube of deionized water.

Embodiment

Below in conjunction with embodiment, embodiment of the present utility model is further described, advantage and disadvantage of the present utility model will be more clear along with description.But these embodiment are only exemplary, scope of the present utility model are not formed to any restriction.

The experimental technique using in following embodiment if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

As shown in Figure 1, the utility model provides a kind of test kit for 31 kinds of hemopathy fusion genes of examination, comprise box body (1), lid (2) and be arranged on the liner (3) in box body (1), described liner (3) is divided into upper and lower two portions by described box body (1), described liner (3) is provided with 7 apertures, described aperture is divided into two rows and is arranged in parallel on described liner (3), the Reagent Tube (4) of 32 reverse transcription Auele Specific Primers is housed, the Reagent Tube (5) of dNTP is housed, the Reagent Tube (6) of reversed transcriptive enzyme is housed, the Reagent Tube (7) of RNA enzyme inhibitors is housed, the Reagent Tube (8) of reverse transcription damping fluid is housed, the Reagent Tube (10) that the Reagent Tube (9) of PCR MIX reagent is housed and deionized water is housed is arranged in described aperture, the Reagent Tube that 110 pcr amplification primers are housed is respectively placed in the box body of described liner (3) below.

FG15’-GCTGACATTGAT-3’

FG25’-ACTCAAGCTGTG-3’

FG35’-CCACGTGTGCCTCTC-3’

FG45’-GTATTGACAGTAT-3’

FG55’-GACACCTTGAAG-3’

FG65’-ATGGCTGGCTCAG-3’

FG75’-AGGTCATTTTGAG-3’

FG85’-TTCCTCCTCTGAG-3’

FG95’-CCGATCAATCTTT-3’

FG105’-CTGCCACACTT-3’

FG115’-GCATAACAGTTGT-3’

FG125’-AAGTCTCCAACAG-3’

FG135’-TTTGCACTCTGAT-3’

FG145’-CGGTAATCGATTTC-3’

FG155’-CCCGTAACATAAC-3’

FG165’-TAAGGCTGCTCT-3’

FG175’-TTGCCTGGTACT-3’

FG185’-GCCCATGCTCAG-3’

FG195’-CCAGCCTTGATG-3’

FG205’-TCTCCACGAAGT-3’

FG215’-TGTCCAGCGTAGC-3’

FG225’-GTGTTAACTCTT-3’

FG235’-GGCTATCCATACTT-3’

FG245’-TGGGTACCTCTC-3’

FG255’-TGGCAATGGAAAG-3’

FG265’-CTGCTGGGTGAG-3’

FG275’-AGCTGCTTGATG-3’

FG285’-CAGCGAACAATG-3’

FG295’-CCCTCCAGAAG-3’

FG305’-CTGCAGGAAGGT-3’

FG315’-CGGTAAGCTCTC-3’

FG325’-CCGATTACCTA-3’

First round primer sequence:

FG335’-TTTGACTCCCATGATTCTG-3’

FG345’-AGGCCTTAGGCCAGCTTCTTCT-3’

FG355’-GAGCTGTCCGAGAGAGTGGAGAT-3’

FG365’-CCGCCTCTGTTAGCCTACTAC-3’

FG375’-AGCACTCCAATGGCAATAGT-3’

FG385’-GGGTTCTCTGGCAGCACAGAT-3’

FG395’-CCGCTGCGACACTTCATAG-3’

FG405’-ACACCCCAGTGTGTCCTGTATG-3’

FG415’-CCGCAATCTGCCACCTACTAC-3’

FG425’-AGCACCTCCCAATGGCAATAGT-3’

FG435’-GGATACCTTCTCTTCATCTGTGTC-3’

FG445’-CCTCCAGTGGCTGCTCTGTGT-3’

FG455’-CTGTTCTGTCATGGCTGCTACTG-3’

FG465’-TTGCCCTCTGTGCACCTAGTCT-3’

FG475’-5-TGGACTTTATCGGCACCATTAC-3’

FG485’-TTTAGAGCACGAACACAGATGG-3’

FG495’-GCCACGCGAATTCCGCTAAC-3’

FG505’-CGACCCGCCAACCAGAG-3’

FG515’-CGGTCCTGGTCCGGGCATATTT-3’

FG525’-GCCCACATGCTCCCTCAAGT-3’

FG535’-CACTTTCTGGATTTCAAACAGTC-3’

FG545’-AGCCGCCGAGTAGTTTTCATTGC-3’

FG555’-GATGGTCACTGGTCACTGTG-3’

FG565’-CGATCTTTACTCTGGTCCATATTC-3’

FG575’-GGGCGTTCAACCTCACTG-3’

FG585’-GTCTGCAACCTCCACTTTGTC-3’

FG595’-TCTCCTATCCCGTCTGAAATGTC-3’

FG605’-GGTCGGGTGGTTATGGCAATC-3’

FG615’-GTTCATGGCTGTTTGCTCTTC-3’

FG625’-CCGCCCTTGGGACCTACTAC-3’

FG635’-AGCACTCAGTCTCCAATGGCAATAGT-3’

FG645’-GAATTTGATCCGTTTGAAGATGTATC-3’

FG655’-CCAGATGAGAGTTTGGTAACTCTGT-3’

FG665’-TTCCCTGCCTCCTCTATTTAC-3’

FG675’-GCTTGAGGGCTTTAGACAATGAG-3’

FG685’-CACCGGGAAATCCAGTGAAC-3’

FG695’-CGCTCTCGTCGTGCGCAGAACT-3’

FG705’-GAGTCCTACATGCTGCTTATGTC-3’

FG715’-TTGTTCTGCTGGGCTTCACAC-3’

FG725’-GCTGTTGCTGACCAAAGAGGACTT-3’

FG735’-CATAGCTAATTGCTTCTCACTG-3’

FG745’-TGCCAAAGGTGTTAAGAAGATAG-3’

FG755’-GGCAAGATTTGGTGTGAGAT-3’

FG765’-CACCAAGCATCAAATGGAAATCTG-3’

FG775’-CCACTACGAAGAAGGATGAGAAG

FG785’-TGCCTCGATAGGTTTGCAGAC-3’

FG795’-CCACAGTGAGGCTGGCTGTATT-3’

FG805’-CAAACAAGCCAGCCCAGAG-3’

FG815’-GTGCAGGCCAGGTGGTAGCTC-3’

FG825’-AAGCCCTTGCAGCCCTCAC-3’

FG835’-ACGAAGGCAGTCCAATTAAAGTAAC-3’

FG845’-CACACTTCAGGCAGCGTCTTC-3’

FG855’-AGCTCTCCCTCTACCATCAGAGATACT-3’

FG1415’-TTCTCGTCCAGCCCTTCTACC-3’

FG1425’-TTTTCCTCTTCTCGCCGTTTCA-3’

Second takes turns primer sequence:

FG865’-TGGGCTGTCCCAAGTTTGATG-3’

FG875’-TGCCTGCTGGATGTTGAC-3’

FG885’-TTAGCGCCAGCTGGTCTTG-3’

FG895’-GGCCTCGGCCAAGAAAAGAAGT-3’

FG905’-AGACCCAGATGGCACAGGATCAG-3’

FG915’-GGTTTCTGGGGAGTCGCTTTAACT-3’

FG925’-GAGTTCTAGCATTCGCATATCAG-3’

FG935’-TTCGACCGACTGGAGACATAC-3’

FG945’-GGACCACCATAGAAAAGAAGT-3’

FG955’-AGCAGCGCATGGAGTCCACTCAG-3’

FG965’-TGTCGGCAGGAATCCCAAATCT-3’

FG975’-GTAGAGAAGCCAGAGAAAACAC-3’

FG985’-AACTGCTGCCATTTGCCTGGTTGAT-3’

FG995’-TTCAGAGGTGTGTGCAGAG-3’

FG1005’-GGCAACACTACTGGCATGTTAC-3’

FG1015’-TCTAGGCCCACGAGCAGGTTTT-3’

FG1025’-CATGTTGTCTCTCAGCATCAG-3’

FG1035’-CCCGCTCCCCGCTGCAAAC-3’

FG1045’-AGACCGGTACCCCCTGAATAG-3’

FG1055’-CGCCTCCAGTCTCACTTGTC-3’

FG1065’-CTCATCGTGCGGAAGAGCTTAC-3’

FG1075’-AGCACCCTGGAGAGGAAGTTG-3’

FG1085’-TGGCTGGCAAGAGCTGAAAAC-3’

FG1095’-CCCCACATATGATTTGACTCTC-3’

FG1105’-CTTCCCCTGGGGAGAGAGTAAC-3’

FG1115’-GCGCATTATTCATTGTTTGAG-3’

FG1125’-CGTTGTCGGCGAATGAACTG-3’

FG1135’-CAGCGGCTATGGTGTAGACAG-3’

FG1145’-GGTGCCTTCTGGCCAGGTGATG-3’

FG1155’-GGACCGCCAAGAAAAGAAGT-3’

FG1165’-AGCAGATGGAGTCCACAGGATCAG-3’

FG1175’-GTTTTTGGTTTGGGTTACAGAACT-3’

FG1185’-GAGCAAAGATCAAAATCAAATGTT-3’

FG1195’-CTCCATTTCAGAGTCATTGTCGTTAT-3’

FG1205’-TGCTGGCAGCCATGGGAGCTCTC-3’

FG1215’-GCGATCCGCCAGCTCTAAC-3’

FG1225’-ACTGCGTTTGATGTCGTGTC-3’

FG1235’-CACGTTCCCCATTCCTCTGAC-3’

FG1245’-ACACCATTCCCTGTGTTGATTAT-3’

FG1255’-CCTCATTCAGGTCGTGAGCTCTAT-3’

FG1265’-CATGGGACCCACGTAGATGTAC-3’

FG1275’-AGCAGCACTCCCAAGAAGAAT-3’

FG1285’-GTCTCTCGCCATGGCACAAG-3’

FG1295’-TGAGGAACAAGAGAGCTTCTTTAC-3’

FG1305’-CGTCGAATGACCACCTGCTTC-3’

FG1315’-GAACATAGAGGATGGTGACTGTAAG-3’

FG1325’-GTGGGCATACGAGTCAGAGAGC-3’

FG1335’-GCCAGTGCCTACGATCTCCAT-3’

FG1345’-CAGCGCGCCTGACTGGAGAT-3’

FG1355’-CCCATAGTGCCAGTAGAGGAC-3’

FG1365’-GGTTCAGGGGTAGTGCATATT-3’

FG1375’-CTTGGGTCAATGGGCATTC-3’

FG1385’-AAAGGGTTCAGAACTTCTTATCATC-3’

FG1395’-CTACGACGGGGGTCTCCAC-3’

FG1405’-AGGTTCCGCTCTCGCACTT-3’

The concentration of above-mentioned every kind of primer is 5pmol/ul.

Wherein, described dNTPs is the conventional dNTPs reagent in this area, comprises dATP, dGTP, dTTP and dCTP, and concentration is 2.5mM.

Wherein, described reversed transcriptive enzyme is the reversed transcriptive enzyme of this area routine, and preferably, described reversed transcriptive enzyme is the Reverse Transcriptase M-MLV of Takara company.

Wherein, the RNA enzyme inhibitors that described RNA enzyme inhibitors is this area routine, preferably, the Ribonuclease Inhibitor that described RNA enzyme inhibitors is Takara company.

Wherein, 5 * Reverse Transcriptase M-MLV Buffer that described reverse transcription damping fluid is Takara company.

Wherein, described PCR MIX reagent is is the PCR MIX reagent of this area routine, described PCRMIX reagent preferably comprises Taq archaeal dna polymerase, dNTPs and MgCl2, wherein, the concentration of described Taq archaeal dna polymerase is 50units/ml, described dNTPs is respectively dATP, dGTP, dCTP, dTTP, and their concentration is 400 μ M, and the concentration of described MgCl2 is 3mM.

Detecting step is as follows:

(1) use ordinary method to extract the RNA of sample to be tested, the RNA of recommendation Beijing Tian Mo Science and Technology Development Co., Ltd. extracts test kit, operation steps is to specifications extracted, the concentration that extraction obtains is that the RNA of 50～100ng/ μ l can be directly used in next step reverse transcription reaction, also can be stored in-80 ℃;

(2) carry out reverse transcription reaction, reactions steps is as follows:

A. 2 μ l32 bar reverse transcription Auele Specific Primers are mixed in test tube with 5 μ l RNA, place 8min in 70 ℃, after proceed to ice chest and place 2～3min;

B. in test tube, add 2 μ l reverse transcription damping fluids, 2 μ l dNTPs, 0.5 μ l reversed transcriptive enzyme, 0.5 μ lRNA enzyme inhibitors, mix 42 ℃ of 45min; Obtain the cDNA product of reverse transcription;

C. divide three-wheel to carry out multiplex PCR amplification, the cDNA product+10 μ l deionized water that the template of first round pcr amplification reaction is reverse transcription, second to take turns with the template of third round pcr amplification reaction be first round product; The primer of three-wheel reaction is all Auele Specific Primer.

First round PCR primer:

FG33R1AB?CBFB

FG34R1A?MYH11

FG35R1B?MYH11

FG36R1C?MLL

FG37R1D?MLL

FG38R1C?AFX1

FG39R1D?AF6

FG40R1E?ELL

FG41R2ABC?MLL

FG42R2DEF?MLL

FG43R2A?AFP1

FG44R2B?AF17

FG45R2C?AF10

FG46R2D?AF10

FG47R2E?AF10

FG48R2F?MLL

FG49R3A?PBX1

FG50R3B?HIL

FG51R3D?SIL

FG52R3D?TAL1

FG53R3C?TEL

FG54R3C?AML1

FG55R4AB?AML1

FG56R4B?AML

FG57R4D?HOX11

FG58R4D?HOX11

FG59R4A?ETO

FG60R4C?TLS

FG61R4C?ERG

FG62R5ABC?MLL

FG63R5CDE?MLL

FG64R5A?AF4

FG65R5D?AF9

FG66R5C?AF9

FG67R5E?AF1Q

FG68R5B?ENL

FG69R6A?BCR

FG70R6B?BCR

FG71R6ABC?ABL

FG72R6C?TEL

FG73R6D?PDGFR

FG74R7A?DEK

FG75R7AB?CAN

FG76R7B?SET

FG77R7C?EV1

FG78R7C?EV1

FG79R8A?PLZF

FG80R8B?PML3

FG81R8C?PML3

FG82R8ABC?RARA

FG83R8DEF?NPM

FG84R8D?NPM

FG85R8F?MLF1

FG141ABCDE?E2A

FG142ABCDE?E2A

Second takes turns PCR primer:

FG86R1AB?CBFB

FG87R1A?MYH11

FG88R1B?MYH11

FG89R1C?MLL

FG90R1D?MLL

FG91R1C?AFX1

FG92R1D?AF6

FG93R1E?ELL

FG94R2ABC?MLL

FG95R2DEF?MLL

FG96R2A?AFP1

FG97R2B?AF17

FG98R2C?AF10

FG99R2D?AF10

FG100R2E?AF10

FG101R2F?MLL

FG102R3A?PBX1

FG103R3B?HIL

FG104R3D?SIL

FG105R3D?TAL1

FG106R3C?TEL

FG107R3C?AML1

FG108R4AB?AML1

FG5109R4B?AML

FG110R4D?HOX11

FG111R4D?HOX11

FG112R4A?ETO

FG113R4C?TLS

FG114R4C?ERG

FG115R5ABC?MLL

FG116R5CDE?MLL

FG117R5A?AF4

FG118R5D?AF9

FG119R5C?AF9

FG120R5E?AF1Q

FG121R5B?ENL

FG122R6A?BCR

FG123R6B?BCR

FG124R6ABC?ABL

FG125R6C?TEL

FG126R6D?PDGFR

FG127R7A?DEK

FG128R7AB?CAN

FG129R7B?SET

FG130R7C?EV1

FG131R7C?EV1

FG132R8A?PLZF

FG133R8B?PML3

FG134R8C?PML3

FG135R8ABC?RARA

FG136R8DEF?NPM

FG137R8D?NPM

FG138R8F?MLF1

FG139ABCDEF?E2A

FG140ABCDEF?E2A

Third round PCR primer be second take turns primer pair answer ABCD configuration genes involved as: the corresponding primer of R1A is FG86, FG87, FG139, FG140, and the corresponding primer of R1B is FG86, FG88, FG139, FG140, obtains by that analogy relevant separated primer.

Reaction system is as follows:

Amplified reaction program is as follows:

A. first round pcr amplification reaction:

94 ℃, 10, min, carries out 1 circulation;

94 ℃, 30s → 59 ℃, 30s → 72 ℃, 45s; Carry out 25 circulations;

72 ℃, 8min; Carry out 1 circulation.

B. second take turns and third round pcr amplification reaction:

94 ℃, 10, min, carries out 1 circulation;

94 ℃, 30s → 60 ℃, 30s → 72 ℃, 60s; Carry out 20 circulations;

72 ℃, 8min; Carry out 1 circulation.

(4) PCR product is carried out to agarose gel electrophoresis detection, interpretation electrophoresis result.

For example understood embodiment of the present utility model above; what those skilled in the art should understand that is; do not departing under spirit and scope of the present utility model and can the details of technical solutions of the utility model and form modified or be replaced, but these modifications and replacement all fall in protection domain of the present utility model.

Claims

1. the test kit for 31 kinds of hemopathy fusion genes of examination, it is characterized in that: comprise box body (1), lid (2) and be arranged on the liner (3) in box body (1), described liner (3) is divided into upper and lower two portions by described box body (1), described liner (3) is provided with aperture, the Reagent Tube (4) of 32 reverse transcription Auele Specific Primers is housed, the Reagent Tube (5) of dNTP is housed, the Reagent Tube (6) of reversed transcriptive enzyme is housed, the Reagent Tube (7) of RNA enzyme inhibitors is housed and the Reagent Tube (8) of reverse transcription damping fluid is housed, the Reagent Tube (9) that PCR MIX reagent is housed is arranged in described aperture, the Reagent Tube that 110 pcr amplification primers are housed is respectively placed in the box body of described liner (3) below.

2. according to a kind of test kit for 31 kinds of hemopathy fusion genes of examination claimed in claim 1, it is characterized in that, described test kit also comprises: the Reagent Tube (10) that deionized water is housed.

3. according to a kind of test kit for 31 kinds of hemopathy fusion genes of examination claimed in claim 2, it is characterized in that: described liner (3) is provided with 7 apertures, described aperture is divided into two rows and is arranged in parallel on described liner (3).