CN103571945A - Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types - Google Patents
Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types Download PDFInfo
- Publication number
- CN103571945A CN103571945A CN201310450028.0A CN201310450028A CN103571945A CN 103571945 A CN103571945 A CN 103571945A CN 201310450028 A CN201310450028 A CN 201310450028A CN 103571945 A CN103571945 A CN 103571945A
- Authority
- CN
- China
- Prior art keywords
- probe
- primer
- abl
- tamra
- fam
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000523 sample Substances 0.000 title claims abstract description 120
- 230000004927 fusion Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012216 screening Methods 0.000 title abstract description 10
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical compound NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 title description 16
- 210000004369 blood Anatomy 0.000 claims abstract description 48
- 239000008280 blood Substances 0.000 claims abstract description 48
- 108010056708 bcr-abl Fusion Proteins Proteins 0.000 claims abstract description 36
- 239000002773 nucleotide Substances 0.000 claims description 38
- 125000003729 nucleotide group Chemical group 0.000 claims description 38
- 239000002299 complementary DNA Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 238000011156 evaluation Methods 0.000 claims description 11
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000007500 overflow downdraw method Methods 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000010839 reverse transcription Methods 0.000 claims description 5
- 108700039887 Essential Genes Proteins 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000003149 assay kit Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 28
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000003753 real-time PCR Methods 0.000 abstract description 3
- 238000003752 polymerase chain reaction Methods 0.000 abstract 1
- 238000013461 design Methods 0.000 description 16
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 238000002123 RNA extraction Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 210000000349 chromosome Anatomy 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 5
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 5
- 108700025690 abl Genes Proteins 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000017105 transposition Effects 0.000 description 5
- 238000010200 validation analysis Methods 0.000 description 5
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 4
- 101150049556 Bcr gene Proteins 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 102100026008 Breakpoint cluster region protein Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101000933320 Homo sapiens Breakpoint cluster region protein Proteins 0.000 description 2
- 206010020880 Hypertrophy Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004992 fission Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 238000007499 fusion processing Methods 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000004214 philadelphia chromosome Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
- C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
- C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method, primer, probe and kit for detecting six unusual fusion types of BCR-ABL fusion genes such as e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3. The method comprises the following steps: performing primary screening on blood samples by adopting two tubes of fluorescent quantitative polymerase chain reaction (PCR), and quantitatively indentifying specific types of the blood samples belonging to the a2 or a3 group through primary screening judgment. Therefore, the screening cost is reduced, and the detection throughput is also improved. According to the detection method, primer, probe and kit, high sensitivity, high specificity and high detection throughput can be realized, and the method is a rapid and accurate type screening and identifying method.
Description
Technical field
The invention belongs to life science and biological technical field, be particularly related to the non-common pattern of fusion method for distinguishing of a kind of Clinical screening BCR-ABL fusion gene, primer, probe and test kit, adopt Real-Time Fluorescent Quantitative PCR Technique, BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3, these 6 kinds non-common types of e14a3 in human blood sample are carried out to preliminary examination and type evaluation.
Background technology
BCR-ABL fusion gene is long-armed upper by No. 9 long-armed transposition to 22 karyomit(e)s of karyomit(e), causes ABL proto-oncogene and the BCR gene to occur to merge and generation.T (9; 22) (q34; Q11) No. 22 karyomit(e)s after transposition are called again Philadelphia chromosome (Ph karyomit(e)).Ph karyomit(e) and BCR-ABL fusion gene are the molecular basises that chronic myelocytic leukemia (Chronic Myeloid Leukemia, CML) produces.In addition, BCR-ABL fusion gene is also shown in acute lymphoblastic leukemia (AcuteLymphoblastic Leukemia, ALL), rare in acute myeloid leukemia (Acute Myeloblastic Leukemia, AML).
When No. 9 karyomit(e)s and No. 22 karyomit(e) generation transpositions, the fracture position on No. 9 karyomit(e)s is usually located on the 1st intron of abl gene, is secondly intron 2; Fracture position on No. 22 karyomit(e)s can be positioned on a plurality of sections of BCR gene, therefore can form multiple fusion type.In various fusion types, have three kinds to be modal, be respectively: e13a2(is called not only b2a2), e14a2(but be called b3a2) and e1a2; It is exactly the BCR-ABL P210 often saying that first two merges type, and the latter is often called BCR-ABL P190.In addition, report that more non-common type has: e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3.In CML patient, two kinds of pattern of fusion of BCR-ABL e13a2 and e14a2 account for altogether～and 98%; But not common fusion type at least accounts for 1-2%, even more.In adult and children ALL patient, BCR-ABL positive rate is respectively～and 25% and～3%; This wherein e13a2, e14a2 and e1a2 account for altogether～99%; But not common fusion type also at least accounts for 1-2%.
At present, it is clinical when doubtful CML, ALL and AML patient are done to the examination of BCR-ABL fusion gene, conventionally only for e13a2, e14a2 and tri-kinds of types of e1a2, make detection, other non-common types are to chance on during by chromosome karyotype analysis or Fluorescence in situ hybridization (FISH) technical inspection.There is following shortcoming in this examination mode judging by visual inspection: (1) exists undetected situation to the examination of the non-common type of BCR-ABL fusion gene.First, the prerequisite of chromosome karyotype analysis is to cultivate the karyocyte in sample, has nuclear fission phase; If cell cultures failure, without nuclear fission phase, cannot analyze.Secondly, in cell cultures breeding, certain class cell meeting hypertrophy, if normal cell hypertrophy can " flood sick cell " to fall, causes the normal illusion of karyotype.And cannot bone marrow extraction in the situation that, extract peripheral blood and do chromosome karyotype analysis, also have false negative and occur.Moreover the chromosomal transposition of some case is extremely small, there is invisible Ph karyomit(e), this is cannot find in the chromosome examination of analyzing by visual inspection.And FISH method is when detecting BCR-ABL and merging, although without culturing cell, also can detect invisible Ph karyomit(e), its inspection charge is too high, and most of patient can not select the method to detect, therefore do not have the effect of real examination.(2) cannot distinguish the concrete type of BCR-ABL, thereby just cannot carry out targetedly the monitoring of MRD (Minimal Residua Disease, MRD).Chromosome karyotype analysis is by naked eyes, chromosomal transposition to be identified; Equally, FISH is by fluorescent signal, observes the fusion of coloured differently body, and submicroscopic variation cannot be observed.(3) be detected as power and sensitivity is low.
At present, even if targetedly the non-common type of BCR-ABL fusion gene is identified, also be first by carrying out multiplex PCR (m μ ltiplex PCR) or nest-type PRC (nested PCR) method, to increase, then in conjunction with agarose gel electrophoresis or order-checking.The shortcoming that multiplex PCR carries out examination in conjunction with agarose gel electrophoresis or order-checking is as follows: (1) exists non-specific amplification, there will be many PCR product bands during electrophoresis, is prone to false positive; (2) while analyzing by electrophoresis there is subjectivity in result judgement, and particularly, when the monitoring to MRD, critical result cannot be carried out clear and definite analysis judgement, cause cost to increase, and greatly extends and can arrive by order-checking the time cycle; (3) sensitivity is lower than nest-type PRC and fluorescent PCR; (4) can only carry out qualitative detection; (5) length consuming time, if identify that by electrophoresis PCR product needs 5-6 hour; If identify by order-checking, need 10-12 hour.And nest-type PRC carries out examination in conjunction with agarose gel electrophoresis or order-checking and also has following shortcoming: (1) process is loaded down with trivial details, easily pollutes, and when electrophoresis is identified, the judgement of critical result is more subjective; (2) PCR reaction system is many, and cost is high, is not suitable for high-throughout pattern detection; (3) can only carry out qualitative detection; (4) length consuming time, if identify that by electrophoresis PCR product needs 5-6 hour; If identify by order-checking, need 10-12 hour.Therefore be no matter first by carrying out multiplex PCR (m μ ltiplex PCR) or nest-type PRC (nested PCR) method increases, the non-common type of BCR-ABL fusion gene is identified in recycling agarose gel electrophoresis or order-checking, all can not meet the highly sensitive and good advantage of specificity simultaneously.
Summary of the invention
Deficiency in view of the non-common type method of current examination BCR-ABL fusion gene, the present invention designs these 6 kinds non-common pattern of fusion method for distinguishing, primer, probe and test kits for detection of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3, thus can be in high sensitivity, with high specificity, high-throughput ground and examination rapidly and accurately and identify the non-common type of BCR-ABL fusion gene.
The object of the present invention is to provide for examination BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-other primers of common pattern of fusion of e14a3 and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, described primer and probe also comprise that its nucleotide sequence is as follows for increasing as primer and the probe of the house-keeping gene ABL of internal reference:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
The present invention also provides for the identification of these 6 kinds non-other primers of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 and probe, it is characterized in that,
Identify that the primer of e6a2 type and the nucleotide sequence of probe are as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e8a2 type and the nucleotide sequence of probe are as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e19a2 type and the nucleotide sequence of probe are as follows:
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e1a3 type and the nucleotide sequence of probe are as follows:
E1F：5’-CTCGCAGAACTCGCAACAG-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e13a3 type and the nucleotide sequence of probe are as follows:
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e14a3 type and the nucleotide sequence of probe are as follows:
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, identify that be selected from the primer of any type and the nucleotide sequence of probe in e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 type also comprises that amplification is as primer and the probe of the house-keeping gene ABL of internal reference, its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’
The present invention also provides a kind of examination and has identified BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-common pattern of fusion method for distinguishing of e14a3, it is characterized in that, said method comprising the steps of:
(1) extract the RNA in blood sample;
(2) by the RNA reverse transcription cDNA in step (1);
(3) utilize the described cDNA of fluorescent PCR amplification step (2), carry out described other preliminary examination of non-common pattern of fusion;
(4) if by the preliminary examination of step (3), determine that described blood sample is not that described non-common pattern of fusion is other, detect and finish; If by the preliminary examination of step (3), determine that described blood sample is that described non-common pattern of fusion is other, is increased and is carried out concrete type Quantitative measurement by fluorescent PCR again.
Further, utilize the preliminary examination of described step (3) to judge that described blood preparation belongs to a2 group, a3 group or be not that described non-common pattern of fusion is other.
Further, for the amplimer of described step (3) and the nucleotides sequence of probe, classify as:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, according to the judged result in step (3), if a2 group is carried out e6a2, e8a2, e19a2 type is identified; If a3 group, carries out e1a3, e13a3, e14a3 type is identified.
Further, in described step (4), for carrying out the primer of concrete type Quantitative measurement by the amplification of described fluorescent PCR and the nucleotides sequence of probe is classified as:
Identify that the primer of e6a2 type and the nucleotide sequence of probe are as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e8a2 type and the nucleotide sequence of probe are as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e19a2 type and the nucleotide sequence of probe are as follows:
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
Identify that the primer of e1a3 type and the nucleotide sequence of probe are as follows:
E1F：5’-CTCGCAGAACTCGCAACAG-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e13a3 type and the nucleotide sequence of probe are as follows:
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e14a3 type and the nucleotide sequence of probe are as follows:
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
The present invention also provides a kind of examination and has identified BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-other test kits of common pattern of fusion of e14a3, comprise blood rna extracting solution, reverse transcription reagent, pcr amplification reaction system, described pcr amplification reaction system comprises primer and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
Because these 6 kinds non-other incidences of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 are low, therefore the present invention is by the design optimization of primer probe, take two pipe PCR reactions first by the method for multi-primers combined with fluorescent PCR, to carry out blood sample primary dcreening operation, to belonging to the blood preparation of a2 group or a3 group, carry out again the method for concrete type Quantitative measurement, reduce like this cost of examination, improved again the flux detecting.
Being grouped as follows of the design of primer probe of the present invention and examination system: according to BCR-ABL in fusion process, the relatively-stationary feature of fracture position on abl gene, examination system is divided into two groups: a2 group (fracture position is positioned at abl gene the 1st intron), by e6a2, e8a2, e19a2 type forms; A3 group (fracture position is positioned at abl gene intron 2), by e1a3, e13a3, e14a3 type forms.Meanwhile, on the exon 2 of abl gene, design downstream primer and the fluorescent probe of a2 group; On the 3rd exon, design downstream primer and the fluorescent probe of a3 group.Subsequently, according to downstream primer design with it suitable carry out PCR reaction upstream primer.The upstream primer of a2 group lays respectively at the 6th exon, the 8th exon and the 19th exon of BCR gene; Just in time can detect e6a2, e8a2, tri-kinds of types of e19a2 with downstream primer combination like this.In like manner, the upstream primer of a3 group lays respectively at the 1st exon, the 13rd exon and the 14th exon of BCR gene, with downstream primer combine detection e1a3, e13a3 and tri-kinds of types of e14a3.Three upstream primers of this design share the matching method of a downstream primer and probe, reduced in a tube reaction system owing to there are many primer probes and mutually suppressed situation about disturbing, not only saved cost but also increased sensitivity and the specificity of detection system.No matter be in a2 group or in a3 group simultaneously, because downstream primer in every group shares, so should follow first design total downstream primer on the same group, design again the principle of interior different other upstream primers of pattern of fusion on the same group, if not so other upstream primer of pattern of fusion in design on the same group in advance designs its downstream primer again, then other upstream primer of another pattern of fusion in design on the same group designs its downstream primer again, the very possible inconsistent phenomenon of downstream primer that produces twice design, and cause downstream primer to redesign, thereby bring great time waste.
According to statistics, these 6 kinds non-common pattern of fusion not in, the incidence of any type is probably at 1-2%, thereby easily out in the cold and undetected in clinical detection.If adopt, utilize multiple fluorescence PCR to carry out examination in single tube, the fluorophor adopting for the probe of each type must be different, and this not only can increase testing cost, and sensitivity, specificity can not meet clinical requirement, and result reliability is also poor.If each type is identified separately, need to carry out six pipe detections, simultaneously also in view of these 6 kinds non-common pattern of fusion not in, the incidence of any type is only 1-2% left and right, this just means in 1000 parts of blood samples, at least by 980 duplicate samples, does not belong to any in not of these 6 kinds non-common pattern of fusion, if this at least 980 duplicate samples is all carried out to six pipe detections, so not only to consume the more blood sample sample of patient, also greatly increase reagent and human cost simultaneously.The present invention utilizes the array mode of corresponding many upstream primers of a downstream primer probe, first carry out carrying out quantitative typing after qualitatively screening, the reaction system of primary dcreening operation inspection is tapered to two pipes, so just can above-mentioned at least 980 duplicate samples be excluded in the primary dcreening operation stage, just need not carry out again follow-up detection by quantitative and somatotype, therefore this has not only saved cost, has increased again the flux of a batch of detection, for high-throughout blood sample screening facilitates.Moreover, for the probe of each type, can share identical fluorophor, also help and reduce reagent cost of development, for ease of the exploitation of product.In addition, this array mode is combined with fluorescent quantitative PCR technique, avoid the reaction of uncapping of nest-type PRC, improved the accuracy of result, avoid the generation of polluting; Also improved the easy judgement of result, made the more objective readability of detected result.Finally, when identifying, introduces concrete type ABL typical curve, both carried out somatotype, carried out again quantitatively, be conducive to clinically generation development and prognosis in conjunction with result comprehensive assessment diseases such as bone marrow examination, chromosome karyotype analysis, immunophenotypings, carry out targetedly MRD monitoring, predicting recurrence risk simultaneously.
Accompanying drawing explanation
Fig. 1 is primer of the present invention and probe design site plan
Fig. 2 is used primer of the present invention, probe and detection method a blood sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 3 is used primer of the present invention, probe and detection method second blood sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 4 is used primer of the present invention, probe and detection method the 3rd blood sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 5 is used primer of the present invention, probe and detection method the 4th blood sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 6 is used primer of the present invention, probe and detection method the 5th blood sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 7 is used primer of the present invention, probe and detection method the 6th sample to be checked to be carried out to the amplified fluorescence graphic representation of examination and type evaluation
Fig. 8 is the amplified fluorescence graphic representation that uses primer of the present invention, probe and detection method to carry out examination to the 7th blood sample to be checked
Embodiment
Embodiment 1:
The extraction of blood rna: add 1ml1 * erythrocyte cracked liquid in 1.5ml centrifuge tube, get blood sample 0.5ml to be checked, put upside down and mix.Centrifugal 4000rpm, 3min, inhales and abandons supernatant, adds erythrocyte cracked liquid washing once, obtains required cell; Add l ml TotalRNA Isolation Reagent (Shanghai Pu Fei Bioisystech Co., Ltd), pressure-vaccum is until the obvious cell mass of nothing adds chloroform 200 μ l repeatedly, and whirlpool mixes 30s, on ice standing 10min.Then, 4 ℃ centrifugal 14,000rpm, 10min.With pipettor, draw supernatant liquor 450 μ l and be transferred in another centrifuge tube, add isopyknic pre-cold isopropanol, put upside down mix after at standing 10min on ice.4 ℃ centrifugal 14,000rpm, 10min.Then with 75% ethanol and dehydrated alcohol, wash respectively once centrifugal.Drying at room temperature 5min, adds 50 μ l DEPC-H2O to dissolve, and obtains RNA extracting solution.
10 * erythrocyte splitting liquid formula is: NH
4cl82g, NaHCO
38.4g, EDTA-Na
23.72g, adds ddH
2o is settled to 1000ml.
Embodiment 2:
Reverse transcription: get the about 200ng/ μ of the RNA extracting solution 4 μ l(concentration l in embodiment 1) add 1 μ l Primer mix(ReverTra AceqPCR RT Kit, purchased from Japan, spin (Shanghai) bio tech ltd) and 3 μ l DEPC-H2O mix, 70 ℃ of denaturation 5min; After quenching 1min, add 4 μ l5*RT buffer(ReverTra Ace qPCR RT Kit on ice), 1 μ l Enzyme Mix(ReverTra Ace qPCR RT Kit), and add 7 μ l DEPC-H
20 to cumulative volume be 20 μ l.37 ℃ of 60min react rear 98 ℃ of 5min deactivations, and gained is the cDNA of blood sample to be checked.
Embodiment 3:
Fluorescent PCR primary dcreening operation: according to each material as shown in table 1 and consumption configuration preliminary screening agent.Preliminary screening agent is altogether containing 3 reaction tubess: each pipe of a2 group, a3 group and ABL; Wherein ABL is internal reference detector tube, for judging that RNA extracts quality and whether meets the requirements.Add each 2 μ l of gained cDNA in embodiment 2.By following program, detect: 95 ℃ of denaturation 30s; 95 ℃ of 10s, 58 ℃ of 35s, totally 40 circulations; In the time of 58 ℃, gather fluorescence.HUNDERBIRD Probe qPCR Mix spins (Shanghai) bio tech ltd purchased from Japan.
Table 1
Embodiment 4:
First blood sample to be checked is carried out to RNA extraction by the method described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, primary dcreening operation shows that first blood sample to be checked belongs to a2 group.Acquired results is as shown in Fig. 2-A.Then each type of a2 group is identified respectively and quantitatively.Reagent configuration is as shown in table 2, and adds each 2 μ l of obtained cDNA.Trace routine is with embodiment 3.Through identifying that first blood sample to be checked is BCR-ABL fusion gene e8a2 type, acquired results, as shown in Fig. 2-B, show that according to detecting computed in software its relative quantification result (BCR-ABL e8a2/ABL) is 161.81%.Finally this sample is carried out to sequence verification, be defined as BCR-ABL fusion gene e8a2 type.
Table 2
Embodiment 5:
Second blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 3-A: primary dcreening operation shows that second blood sample to be checked belongs to a3 group.Again each type of a3 group is identified respectively and quantitatively.Reagent configuration is as shown in table 3, and adds each 2 μ l of obtained cDNA.Trace routine is with embodiment 3.Acquired results is as shown in Fig. 3-B: through identifying that first blood sample to be checked is BCR-ABL fusion gene e1a3 type, according to detecting computed in software, show that its relative quantification result (BCR-ABL e1a3/ABL) is 50.97%.By this sample detection validation that checks order, be defined as BCR-ABL fusion gene e1a3 type.
Table 3
Embodiment 6:
The 3rd blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 4-A: primary dcreening operation shows that the 3rd blood sample to be checked belongs to a3 group.Again each type of a3 group is identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 5.Acquired results is as shown in Fig. 4-B: through identifying that the 3rd blood sample to be checked is BCR-ABL fusion gene e13a3 type, according to detecting computed in software, show that its relative quantification result (BCR-ABL e13a3/ABL) is 384.62%.By this sample detection validation that checks order, be defined as BCR-ABL fusion gene e13a3 type.
Embodiment 7:
The 4th blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 5-A: primary dcreening operation shows that the 4th blood sample to be checked belongs to a2 group.Again each type of a2 group is identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 4.Acquired results is as shown in Fig. 5-B: through identifying that the 4th blood sample to be checked is BCR-ABL fusion gene e19a2 type, according to detecting computed in software, show that its relative quantification result (BCR-ABL e19a2/ABL) is 70.88%.By this sample detection validation that checks order, be defined as BCR-ABL fusion gene e19a2 type.
Embodiment 8:
The 5th blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 6-A: primary dcreening operation shows that the 5th blood sample to be checked belongs to a2 group.Again each type of a2 group is identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 4.Acquired results is as shown in Fig. 6-B: through identifying that the 5th blood sample to be checked is BCR-ABL fusion gene e6a2 type, according to detecting computed in software, show that its relative quantification result (BCR-ABL e6a2/ABL) is 70.95%.By this sample detection validation that checks order, be defined as BCR-ABL fusion gene e6a2 type.
Embodiment 9:
The 6th blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 7-A: primary dcreening operation shows that the 6th blood sample to be checked belongs to a3 group.Again each type of a3 group is identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 5.Acquired results is as shown in Fig. 7-B: through identifying that the 6th blood sample to be checked is BCR-ABL fusion gene e14a3 type, according to detecting computed in software, show that its relative quantification result (BCR-ABL e14a3/ABL) is 96.29%.By this sample detection validation that checks order, be defined as BCR-ABL fusion gene e14a3 type.
Embodiment 10:
The 7th blood sample to be checked carried out to RNA extraction by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Figure 8: the 7th blood sample to be checked do not belong to a2 group, do not belong to a3 group yet, product everyone to get rid of the 7th blood sample to be checked be e6a2, e8a2, e19a2, e1a3, e13a3, the possibility of e14a3 type.Dyed body karyotyping results verification is that caryogram is normal.
Embodiment 11:
According to the BCR-ABL fusion gene type result of embodiment 4 to embodiment 10 gained, summarize.Result is as shown in table 4.
Table 4
As shown in Table 4, the present invention designs for detection of the other e6a2 of the non-common pattern of fusion of BCR-ABL fusion gene, e8a2, e19a2, e1a3, e13a3 and e14a3 method, primer, probe and test kit can detect this six kinds of types.The result detecting is all consistent with the result that order-checking detects or chromosome karyotype analysis obtains.
SEQUENCE LISTING
<110> Shenyang company limited of Ai Dikang medical test institute
<120> examination and the non-common pattern of fusion method for distinguishing of evaluation BCR-ABL, primer, probe and test kit
<130>
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
gagccagaag caacaaagat g 21
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
ttcctgtcca gcatcaatga g 21
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<400> 3
<210> 4
<211> 18
<212> DNA
<213> artificial sequence
<400> 4
<210> 5
<211> 26
<212> DNA
<213> artificial sequence
<400> 5
cccttcagcg gccagtagca tctgac 26
<210> 6
<211> 19
<212> DNA
<213> artificial sequence
<400> 6
<210> 7
<211> 21
<212> DNA
<213> artificial sequence
<400> 7
gatgctgacc aactcgtgtg t 21
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
<210> 9
<211> 20
<212> DNA
<213> artificial sequence
<400> 9
<210> 10
<211> 24
<212> DNA
<213> artificial sequence
<400> 10
ctccgggtct taggctataa tcac 24
<210> 11
<211> 22
<212> DNA
<213> artificial sequence
<400> 11
gatacgaagg gagggtgtac ca 22
<210> 12
<211> 18
<212> DNA
<213> artificial sequence
<400> 12
<210> 13
<211> 29
<212> DNA
<213> artificial sequence
<400> 13
tgcttctgat ggcaagctct acgtctcct 29
Claims (10)
1. for examination BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-other primers of common pattern of fusion of e14a3 and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’- TGGAGGAGGTGGGCATCTA -3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
E1F：5’- CTCGCAGAACTCGCAACAG -3’
E13F：5’- GATGCTGACCAACTCGTGTGT -3’
E14F：5’- GTCATCGTCCACTCAGCCAC -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
2. primer as claimed in claim 1 and probe, is characterized in that, described primer and probe also comprise that its nucleotide sequence is as follows for increasing as primer and the probe of the house-keeping gene ABL of internal reference:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT - TAMRA-3’。
3. for the identification of these 6 kinds non-other primers of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 and probe, it is characterized in that,
Identify that the primer of e6a2 type and the nucleotide sequence of probe are as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e8a2 type and the nucleotide sequence of probe are as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e19a2 type and the nucleotide sequence of probe are as follows:
E19F：5’- TGGAGGAGGTGGGCATCTA -3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e1a3 type and the nucleotide sequence of probe are as follows:
E1F：5’- CTCGCAGAACTCGCAACAG -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e13a3 type and the nucleotide sequence of probe are as follows:
E13F：5’- GATGCTGACCAACTCGTGTGT -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e14a3 type and the nucleotide sequence of probe are as follows:
E14F：5’- GTCATCGTCCACTCAGCCAC -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
4. primer as claimed in claim 3 and probe, it is characterized in that, evaluation is selected from the primer of any type and the nucleotide sequence of probe in e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 type and also comprises that amplification is as primer and the probe of the house-keeping gene ABL of internal reference, and its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT - TAMRA-3’。
5. examination and evaluation BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-common pattern of fusion method for distinguishing of e14a3, is characterized in that, said method comprising the steps of:
(1) extract the RNA in blood sample;
(2) by the RNA reverse transcription cDNA in step (1);
(3) utilize the described cDNA of fluorescent PCR amplification step (2), carry out described other preliminary examination of non-common pattern of fusion;
(4) if by the preliminary examination of step (3), determine that described blood sample is not that described non-common pattern of fusion is other, detect and finish; If by the preliminary examination of step (3), determine that described blood sample is that described non-common pattern of fusion is other, is increased and is carried out concrete type Quantitative measurement by fluorescent PCR again.
6. method as claimed in claim 5, is characterized in that, utilizes the preliminary examination of described step (3) to judge that described blood preparation is to belong to a2 group, a3 group or is not that described non-common pattern of fusion is other.
7. method as claimed in claim 5, is characterized in that, for the amplimer of described step (3) and the nucleotides sequence of probe, classifies as:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’- TGGAGGAGGTGGGCATCTA -3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
E1F：5’- CTCGCAGAACTCGCAACAG -3’
E13F：5’- GATGCTGACCAACTCGTGTGT -3’
E14F：5’- GTCATCGTCCACTCAGCCAC -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
8. method as claimed in claim 6, is characterized in that, according to the judged result in step (3), if a2 group is carried out e6a2, and e8a2, e19a2 type is identified; If a3 group, carries out e1a3, e13a3, e14a3 type is identified.
9. method as claimed in claim 5, is characterized in that, in described step (4), for carrying out the primer of concrete type Quantitative measurement by the amplification of described fluorescent PCR and the nucleotides sequence of probe is classified as:
Identify that the primer of e6a2 type and the nucleotide sequence of probe are as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e8a2 type and the nucleotide sequence of probe are as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e19a2 type and the nucleotide sequence of probe are as follows:
E19F：5’- TGGAGGAGGTGGGCATCTA -3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
Identify that the primer of e1a3 type and the nucleotide sequence of probe are as follows:
E1F：5’- CTCGCAGAACTCGCAACAG -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e13a3 type and the nucleotide sequence of probe are as follows:
E13F：5’- GATGCTGACCAACTCGTGTGT -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
Identify that the primer of e14a3 type and the nucleotide sequence of probe are as follows:
E14F：5’- GTCATCGTCCACTCAGCCAC -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
10. an examination and identify BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and these 6 kinds non-other test kits of common pattern of fusion of e14a3, comprise blood rna extracting solution, reverse transcription reagent, pcr amplification reaction system, described pcr amplification reaction system comprises primer and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’- TGGAGGAGGTGGGCATCTA -3’
A2R：5’- AGTTCCAACGAGCGGCTT -3’
A2P：5’-FAM- CCCTTCAGCGGCCAGTAGCATCTGAC -TAMRA-3’
E1F：5’- CTCGCAGAACTCGCAACAG -3’
E13F：5’- GATGCTGACCAACTCGTGTGT -3’
E14F：5’- GTCATCGTCCACTCAGCCAC -3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT - TAMRA-3’。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310450028.0A CN103571945B (en) | 2013-09-27 | 2013-09-27 | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310450028.0A CN103571945B (en) | 2013-09-27 | 2013-09-27 | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103571945A true CN103571945A (en) | 2014-02-12 |
| CN103571945B CN103571945B (en) | 2015-05-06 |
Family
ID=50044653
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310450028.0A Active CN103571945B (en) | 2013-09-27 | 2013-09-27 | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103571945B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838792A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia |
| CN106399462A (en) * | 2015-07-27 | 2017-02-15 | 上海睿玻生物科技有限公司 | BCR-ABL fusion gene amplification kit and BCR-ABL fusion gene detection kit |
| CN107674914A (en) * | 2016-08-02 | 2018-02-09 | 熊术道 | One step single tube standard measure RT PCR detect the expression quantity of chronic myelogenous leukemia BCR ABL1 genes |
| CN111808959A (en) * | 2020-07-17 | 2020-10-23 | 杭州艾迪康医学检验中心有限公司 | Primer and method for detecting TCF3-HLF fusion gene |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
| CN102758006A (en) * | 2012-04-25 | 2012-10-31 | 武汉艾迪康医学检验所有限公司 | Kit for detecting relative expression of leukemia BCR/ABL (b3a2, b2a2) fusion gene |
-
2013
- 2013-09-27 CN CN201310450028.0A patent/CN103571945B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
| CN102758006A (en) * | 2012-04-25 | 2012-10-31 | 武汉艾迪康医学检验所有限公司 | Kit for detecting relative expression of leukemia BCR/ABL (b3a2, b2a2) fusion gene |
Non-Patent Citations (1)
| Title |
|---|
| THOMAS BURMEISTER等: "A multiplex PCR for improved detection of typical and atypical BCR–ABL fusion transcripts", 《LEUKEMIA RESEARCH》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399462A (en) * | 2015-07-27 | 2017-02-15 | 上海睿玻生物科技有限公司 | BCR-ABL fusion gene amplification kit and BCR-ABL fusion gene detection kit |
| CN105838792A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia |
| CN105838792B (en) * | 2016-04-22 | 2019-07-12 | 上海荻硕贝肯生物科技有限公司 | For the primer of qualitative detection leukemia fusion gene, probe, kit and method |
| CN107674914A (en) * | 2016-08-02 | 2018-02-09 | 熊术道 | One step single tube standard measure RT PCR detect the expression quantity of chronic myelogenous leukemia BCR ABL1 genes |
| CN111808959A (en) * | 2020-07-17 | 2020-10-23 | 杭州艾迪康医学检验中心有限公司 | Primer and method for detecting TCF3-HLF fusion gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103571945B (en) | 2015-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107406885A (en) | Use the size and number Distortion Detect cancer of plasma dna | |
| CN102146476B (en) | Fluorescent quantitative PCR kit for detecting alpha-globin gene deletion | |
| CN105483218A (en) | Seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof | |
| CN103571945B (en) | Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types | |
| CN108866244A (en) | Detect RPA primer and probe, kit and its method of prawn irido virus | |
| CN109504780A (en) | DNA methylation qPCR kit and application method for lung cancer detection | |
| CN105861641A (en) | Primer, kit and method for detecting CHO cell DNA residues | |
| CN108588226A (en) | Detect the miRNA combination of breast cancer patients with brain transfer and the kit containing the combination | |
| CN103614477A (en) | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for diagnosing human spinal muscular atrophy | |
| CN105969866A (en) | Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology | |
| CN109609632A (en) | Reagent, kit and the application of detection fusion gene | |
| CN105648088A (en) | AD or MCI detection marker and detection method thereof | |
| CN101955994B (en) | Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation | |
| CN106191311A (en) | A kind of quick detection Cavia porcellus LCMV, SV, PVM, Reo 3 virus multiple liquid phase method for gene chip and reagent | |
| CN108342478B (en) | Circulating tumor cell metabolism typing marker and application thereof | |
| CN103103287B (en) | Multigene-based influenza C virus real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection reagent | |
| CN103074438B (en) | Multi-gene detection kit for guiding administration of warfarin and detection method of multi-gene detection kit | |
| CN102925559B (en) | Kit for quantitatively detecting W515 site mutation of MPL genes | |
| CN102212621B (en) | Detection kit and detection method for hepatitis B virus genotyping | |
| CN106086226A (en) | A kind of blood plasma miRNA mark relevant to IgA nephropathy auxiliary diagnosis and application thereof | |
| CN102286625A (en) | Seminal plasma micro RNA combination relevant to male reproductive function disorder and application thereof | |
| CN110373457A (en) | A kind of mRNA marker and its application for ulcerative colitis diagnosis | |
| CN106755330B (en) | Cancer-related gene expression difference detection kit and application thereof | |
| CN104087671A (en) | Kit used for detecting number of human chromosomes 21 | |
| CN110358825A (en) | Detect the kit and its application method of people BCR-ABL fusion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20140212 Assignee: Nanchang Adicon ClinIcal Tersting Institute Co., Ltd. Assignor: SHENYANG ADICON CLINICAL LABORATORIES CO., LTD. Contract record no.: 2015990001017 Denomination of invention: Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types Granted publication date: 20150506 License type: Exclusive License Record date: 20151209 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: Nanchang Adicon ClinIcal Tersting Institute Co., Ltd. Assignor: SHENYANG ADICON CLINICAL LABORATORIES CO., LTD. Contract record no.: 2015990001017 Date of cancellation: 20160204 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160219 Address after: 330029, 1111, Qingshan Avenue, hi tech Development Zone, Jiangxi, Nanchang, 1 Patentee after: Nanchang Adicon ClinIcal Tersting Institute Co., Ltd. Address before: 110044 No. 252-9, Union Road, Dadong District, Liaoning, Shenyang Patentee before: SHENYANG ADICON CLINICAL LABORATORIES CO., LTD. |
|
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 330029, 1111, Qingshan Avenue, hi tech Development Zone, Nanchang, Jiangxi, 1, Nanchang Patentee after: Nanchang ADICON Clinical Institute Co Ltd Address before: 330029, 1111, Qingshan Avenue, hi tech Development Zone, Nanchang, Jiangxi, 1, Nanchang Patentee before: Nanchang Adicon ClinIcal Tersting Institute Co., Ltd. |
|
| CP03 | Change of name, title or address |
Address after: 330029 Nanchang high tech Development Zone, Jiangxi, Castle Peak Road, No. 1111 Patentee after: Nanchang ADICON clinical laboratories Ltd. Address before: 330029, 1111, Qingshan Avenue, hi tech Development Zone, Nanchang, Jiangxi, 1, Nanchang Patentee before: Nanchang ADICON Clinical Institute Co Ltd |
|
| CP03 | Change of name, title or address |