CN107267629A - A kind of method of the B cell minimal residual detection of leukaemia - Google Patents
A kind of method of the B cell minimal residual detection of leukaemia Download PDFInfo
- Publication number
- CN107267629A CN107267629A CN201710570750.6A CN201710570750A CN107267629A CN 107267629 A CN107267629 A CN 107267629A CN 201710570750 A CN201710570750 A CN 201710570750A CN 107267629 A CN107267629 A CN 107267629A
- Authority
- CN
- China
- Prior art keywords
- primer
- sequence
- pcr
- minimal residual
- residual detection
- 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.)
- Pending
Links
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
- 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
- C12Q1/6869—Methods for sequencing
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)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to technical field of biological, and in particular to a kind of method of the B cell minimal residual detection of leukaemia.The present invention designs 10 upstream primer sequences for people BCR variable region V areas (variable), for BCR A, D, M, G genes constant region (constant) respectively designs 1 downstream primer sequence, E genes constant region for BCR designs 2 downstream primer sequences, and aim sequence is gone out by multiplexed PCR amplification, aim sequence adds base A in the end of Product Sequence 3 ' after purification and is connected with joint sequence, enter performing PCR amplification to the connection product sequencing primer containing joint, obtain amplified production, it is purified, sequencing, sequencing result is compared with cancer cell RNA amplification Product Sequence.The method sensitivity is up to 10‑6, broad covered area is detected, almost all of B-cell receptor is can detect.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of side of the B cell minimal residual detection of leukaemia
Method.
Background technology
ALL (ALL) is one of most common adult acute leukemia, and it is that a class is drenched with former children
Malignant hematologic disease with the characteristics of bar undesired cell proliferation, accumulation, infiltration.Multinomial research report uses systematic treating scheme, and ALL suffers from
Person's complete remission rate (CR) up to 70%~90%, 3-5 disease-free survivals rate be 60%.Although the first of adult ALL controls CR rates oneself has
Significantly improve, but high recurrence rate, long-term survival rate is still relatively low.Equally, children acute myelocytic leukemia (acute
Myeloidleukemia, AML) 20% or so of acute leukemia (acute leukemia, AML) is accounted for, but account for children AL
More than the 50% of number of dying of illness.It can obtain patient using newtype drug and the advanced therapeutic scheme of HSCT at present
Long-term disease-free survival (disease free survival, DFS) close to 70% and 5 years Event-free survival rates more than 50%
(event free survival, EFS), but recurrence rate and case fatality rate are equally higher.Occurs the master of similar above Recurrent death
It is exactly that minimal residual disease (MRD) is caused to want reason.ALL is in morbidity about 10 in body12Left and right
Leukaemia, but by the use in conjunction of Treated with Chemotherapeutic Drugs thing, when up to complete incidence graph, still residue I0 in vivo8-1010Left and right
Leukaemia, but by existing morphologic detection, oneself can not make accurate test and appraisal, and this to these residual leukernic cells
A little remaining leukaemias are the root of leukemia relapse, therefore still remaining leukaemia in vivo after this leukaemia complete incidence graph
The state of cell is referred to as microresidual disease (minimal residualdisease, MRD).
Current MRD detection techniques are mainly flow cytometry (flowcytometry, FCM) and polymerase chain reaction
(polymerase chain reaction, PCR) technology.Although PCR sensitivity is high, the fusion that can be used for PCR to monitor
It is less, use narrow range；Although gene mutation, proto-oncogene are overexpressed, coverage rate is high, and sensitivity is low；And FCM is applied to
Most of patient, as a result accurately, quickly, expense is low, sensitivity is high, up to 10-3~10-4.Although mpFC is for recurrent disease
Detection sensitivity is 104, but complicated multidimensional data is analyzed dependent on experimenter, human factor influence is big, is unfavorable for facing
Bed standardization detection.In addition, mpFC detection of the expression of leukemia antigen to MRD has interference effect after chemotherapy.According to
Detection MRD sensitivity can be improved in Molecular tools by relying, and can reach 105；However, real-time quantitative PCR need to be set according to patient
The special primer of meter amplifies the retracing sequence that diversity is enriched to come, and its testing cost costliness, labour intensive, is hardly formed
Standardized assay flow.
Have benefited from the fast development and extensive use of high throughput sequencing technologies of future generation, DNA and RNA microarray datasets are in gene
The various aspects that group is learned play prominent impetus.Therefore, this research provides a kind of for B cell leukemia minimal residual
The method of disease detection.
The content of the invention
There is provided the white blood that a kind of energy high flux detects minimal residual in order to overcome the above-mentioned deficiency of prior art by the present invention
The method of sick cell, the method sensitivity is up to 10-6, broad covered area is detected, almost all of B-cell receptor is can detect.This hair
Bright purpose is achieved through the following technical solutions：
A kind of method of the B cell minimal residual detection of leukaemia, comprises the following steps：
(1) sample rna and cancer cell RNA are collected, is preserved after determining its concentration through detected through gel electrophoresis, Nanodrop；
(2) multiple PCR products are obtained by the method for one-step RT-PCR to sample rna and cancer cell RNA respectively；
(3) difference magnetic beads for purifying multiple PCR products, remove unnecessary primer；
(4) 3 ' the ends addition base A of multiple PCR products after purification, obtains the PCR with 3 ' cohesive end A and produces
Thing；
(5) PCR primer that will have 3 ' cohesive end A is connected with joint sequence, obtains the connection product containing joint；
(6) enter performing PCR to the connection product sequencing primer containing joint to expand, acquisition amplified production, purified, sequencing,
Sample rna amplified production sequence is compared with cancer cell RNA amplification Product Sequence.
Further, in the system that one-step RT-PCR reacts in the step (2), template amount is 50~500ng/20 μ l.
Further, one-step RT-PCR primer includes 10 designed for people BCR variable V areas in the step (2)
1 anti-sense primer and the E for BCR that sense primer, A, D, M, G gene constant region (constant) for BCR are respectively designed
2 anti-sense primers of gene constant region design, the sequence of 10 sense primers is SEQ ID N0:1~SEQ ID N0:10,6
The sequence of anti-sense primer is SEQ ID N0:11~SEQ ID:16.
Further, each sense primer equimolar mixing in the one-step RT-PCR, total primer concentration is 10 μm of ol, downstream
Primer integral molar quantity is identical with sense primer integral molar quantity.
Further, magnetic beads for purifying PCR primer described in the step (3) is concretely comprised the following steps：
A) take out magnetic bead vibration and mix 5min, the amount for taking out 45 μ l/ samples is put in room temperature 10min；
B) RT-PCR product respectively takes out 20 μ l, and 30 μ lH are added into each sample2O, is added according to 45 μ l/ samples
Magnetic bead, is inhaled with rifle and blows about 10 mixings, be put in room temperature 2min；
C) uncap and be put in 1min or so on magnetic frame, liquid becomes clarification can sucking liquid, 125 μ l85% of addition second
Wash out and discard after alcohol, 1min, magnetic bead is put in room temperature 8min (5-10min) and dried；
D) 30 μ l ddH are added2O back dissolvings.
Further, 3 ' the ends addition base A of the multiple PCR products in the step (4) after purification reaction system
For：
A) multiple PCR products of 34 μ l purifying；
B) 5 μ l Klenow buffer solutions；
c)10μl 1mM dATP；
D) 1 μ l Klenow fragments.
Further, jointing reaction system is in the step (5)：
A) DNA of 11 μ l steps 3；
B) 15 μ l DNA connection buffer solutions；
c)1μl 1:The linker oligonucleotides of 20 dilutions；
D) 3 μ l DNA ligases.
Further, the linker oligonucleotides center tap sequence is：
- the 3' of normal chain 5 '：ACACTCTTTCCCTACACGACGCTCTTCCGATCT
- the 3' of minus strand 5 ':ATCTCGTATGCCGTCTTCTGCTTG
Further, the sequencing primer in the step (6) is：
First sequencing primer：5"AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG
ATCT
Second sequencing primer：5"CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
Further, the pcr amplification product in the step (6) is using Ago-Gel separation, cut above and below 400bp
What the fragment at edge was purified
Beneficial effects of the present invention：By above technical scheme, it is used for high flux the invention provides one kind and detects white blood
The method of the B cell minimal residual of disease, sensitivity is up to 10-6, broad covered area is detected, almost all of B-cell receptor is can detect.
Detection range is narrow, sensitivity is relatively low, cost is high when the method solves detection Minimal Residual Disease of Leukemia in the prior art asks
Topic.
Brief description of the drawings
Fig. 1 be the embodiment of the present invention 1 in adjunction head after screen purpose fragment agarose gel electrophoresis figure；
Fig. 2 is gained PCR primer after Illumina PCR amplifications in the embodiment of the present invention 1；
The flow chart that Fig. 3 detects for the present invention for B cell leukemia minimal residual disease.
Embodiment
Show that example illustrates certain embodiments of the present invention, and should not be construed as the model of the limitation present invention
Enclose.Present disclosure can be improved from material, method and reaction condition simultaneously, it is all these to improve, all should
Within the spirit and scope for falling into the present invention.No special illustrates that the reagent that the embodiment of the present invention is used is commercial goods, this
The database that inventive embodiments are used is disclosed online database.
Embodiment 1：
1.RNA is extracted：
The embodiment of the present invention 1 provides a kind of preparation method of b lymphocyte receptor (BCR) RNA sample, including following step
Suddenly：
(1) collect each 10 milliliters of fresh peripheral blood sample, by LymphoPrep kits (Axis-shield,
Cat.No.AS1114544UK) specification is operated, and obtains relatively pure PBMC；
(2) adopt the PBMC of gained in step (1), using Trizol Reage, article No. 15596-018, specification 200ml,
Producer's INVITROGEN kits are extracted to patient RNA.Step is as follows：
A) often pipe PBMC adds 1ml Trizol, is mixed, 5min is placed on ice；
B) chloroform 0.2ml/ pipes are added, 15s is shaken.15-30 DEG C of incubation 2-3min, 4 DEG C, 12000g centrifuges 15min；
C) upper strata colourless liquid is drawn to be transferred in new EP pipes；
D) isometric isopropanol is added, is mixed, 15-30 DEG C of incubation 10-30min, 4 DEG C, 12000g centrifuges 10min；
E) supernatant is removed, 75% ethanol 1ml, vortex oscillation 30s is added, 4 DEG C, 7500g centrifuges 5min；
F) exhausted supernatant, and air blast in super-clean bench is deposited in pipe and stands 3-5min；
G) RNA concentration and purity is determined with Nanodrop2000 (Thermo)；Add the dissolving of 20ulDEPC water, -80 DEG C
Refrigerator is preserved.
The method that the B cell minimal residual for leukaemia of the present invention is detected, can be in Patient leukemic's morbidity
When by above RNA extraction method extract cancer cell RNA, after treatment again pass through above RNA extraction method extract sample rna.
2. One step RT-PCR is inverted and expanded to RNA
Multiple PCR products are obtained by the method for one-step RT-PCR to sample rna and cancer cell RNA respectively, by for
People BCR variable region V areas (variable) design 10 upstream primer sequences, for BCR A, D, M, G gene constant region
(constant) 1 downstream primer sequence is respectively designed, the E genes constant region for BCR designs 2 downstream primer sequences, this hair
As shown in table 1, as shown in table 2, One step RT-PCR reaction condition is as shown in table 3 for its PCR reaction system for bright primer.
Multiplexed PCR amplification primer in the present invention of table 1
In the system of the multi-PRC reaction, in the sense primer group of 10 sense primer compositions, each sense primer etc. rubs
You mix, and total primer concentration is 10 μm of ol；In the anti-sense primer group of 6 anti-sense primer compositions, anti-sense primer mole and upstream
Primer mole is identical.
The PCR reaction systems of table 2 (20 μ l)
| 5XBuffer | 4μl |
| Mix | 1.5μl |
| Primers | 1μl |
| RNA | 500ng(N) |
| H2O | 13.5-N |
The One step RT-PCR reaction condition of table 3
Said procedure is specially：42 DEG C of reverse transcriptions 50min, 95 DEG C of pre-degenerations 15min, 94 DEG C of denaturation 30s, 55 DEG C of annealing
2min, 72 DEG C of extension 30s, 10min is extended after circulation 16 times, last 72 DEG C.
3.PCR reaction products are purified
By the amplified production magnetic beads for purifying in step 2, concretely comprise the following steps：
1) amount for taking out magnetic bead vibration mixing 5min 45 μ l/ samples of taking-up is put in room temperature 10min；
2) RT-PCR product respectively takes out 20 μ l, then each sample adds 30 μ lH2O, finally adds magnetic according to 45 μ l/
Pearl, is inhaled with rifle and blows about 10 mixings, be put in room temperature 2min；
3) uncap and be put in 1min or so on magnetic frame, liquid, which becomes clarification, can wash out liquid, add 125 μ l85% second
Wash out and discard after alcohol, 1min, magnetic bead is put in room temperature 8min (5-10min) and dried；
4) 30 μ l ddH are added2O back dissolvings.
4. 3 ' ends of multiple PCR products after purification attach base A
PCR primer after above-mentioned magnetic beads for purifying is carried out plus base A, detailed process is：Mixed in 1.5ml centrifuge tubes with
Lower composition (50 μ l)：
1) multiple PCR products (or moisturizing to 34 μ l) of 34 μ l purifying；
2) 5 μ l Klenow buffer solutions=NEB buffer 2；
3)10μl 1mM dATP；
4) 1 μ l Klenow fragments.
5. jointing reacts：
The PCR primer that will have 3 ' cohesive end A is connected with joint sequence, obtains the connection product containing joint, and connection connects
Head reaction system is that following component (30 μ l) is mixed in 1.5ml centrifuge tubes：
1) 11 μ l3 ' ends attach the DNA of base A step 3；
2) 15 μ l DNA connection buffer solutions；
3)1μl 1:The linker oligonucleotides of 20 dilutions；
4) 3 μ l DNA ligases.
When doing multiple repeat simultaneously, it is ensured that each parallel reaction adds different joints, marks clear.Joint connection is anti-
The joint sequence answered is：
- the 3' of normal chain 5 ':ACACTCTTTCCCTACACGACGCTCTTCCGATCT
- the 3' of minus strand 5 ':ATCTCGTATGCCGTCTTCTGCTTG
Before butt joint product enters performing PCR amplification unnecessary joint, concrete operations side are removed, it is necessary to be screened using gel
Method is：
The 6 μ l sample-loading buffers for diluting 10 times are added in the reaction solution of 30 μ l jointings reaction, loading is to two
In E-gel glue hole.The 50bp DNA ladder loadings for taking 20 μ l to dilute 10 times in addition, electrophoresis 20 minutes.
DNA (as shown in Figure 1) of the size between 150bp to 450bp is cut, it is pure with QIAquick gel purification kits
Change, the elution of 30 μ l elution buffers.
6. connection product Illumina PCR are expanded：
Illumina pcr amplification primers thing 1.1
5"AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
Illumina pcr amplification primers thing 2.1
5"CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
Illumina PCR primers 1.1 and 2.1 are carried out 1 with Gibco water：1 dilution, mixes following component in PCR pipe
(60ul)：
1) the product DNA of 30 μ l jointings reaction；
2) 28 μ l Phusion PCR premixed liquids；
3) primer 1.1 of 1 μ l dilutions；
4) the primer 2 .1 of 1 μ l dilutions.
PCR reaction conditions are as shown in table 4：
The PCR reaction conditions of table 4
Molecular weight screening, purifying are carried out on 7.2% Ago-Gel
The 1 μ l sample-loading buffers diluted are added in the PCR reaction solutions of previous step, loading is into three E-gel glue holes.
50bp the and 100bp DNA ladder loadings for taking 20 μ l to dilute 10 times respectively in addition.Electrophoresis 30 minutes.Size is cut in 400bp
The DNA (as shown in Figure 2) of left and right, DNA is purified with QIAquick gel purification kits, is eluted with 30 μ l elution buffers.
8. sequencing
Cancer cell RNA amplified production sequence and the amplified production sequence of sample rna are respectively obtained according to the method described above, are led to
The sequencing of Illumina HighSeq2500 platforms is crossed, and regard cancer cell RNA amplified production sequence as follow-up residue detection
Whether canonical sequence, the amplified production sequence of sample rna is compared therewith, determine thin containing remaining leukaemia in cell to be measured
Born of the same parents.
SEQUENCE LISTING
<110>Wuhan Sai Yunbo bio tech ltd
<120>A kind of method of the B cell minimal residual detection of leukaemia
<130> 2017
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 52
<212> DNA
<213>Artificial sequence
<400> 1
acactctttc cctacacgac gctcttccga tctagcctac atggagctga gc 52
<210> 2
<211> 56
<212> DNA
<213>Artificial sequence
<400> 2
acactctttc cctacacgac gctcttccga tctaggtggt ccttacaatg accaac 56
<210> 3
<211> 53
<212> DNA
<213>Artificial sequence
<400> 3
acactctttc cctacacgac gctcttccga tcttctgcaa atgaacagcc tga 53
<210> 4
<211> 56
<212> DNA
<213>Artificial sequence
<400> 4
acactctttc cctacacgac gctcttccga tcttgttcaa atgagcagtc tgagag 56
<210> 5
<211> 53
<212> DNA
<213>Artificial sequence
<400> 5
acactctttc cctacacgac gctcttccga tcttctgcaa atgggcagcc tga 53
<210> 6
<211> 55
<212> DNA
<213>Artificial sequence
<400> 6
acactctttc cctacacgac gctcttccga tctttctccc tgaagctgaa ctctg 55
<210> 7
<211> 53
<212> DNA
<213>Artificial sequence
<400> 7
acactctttc cctacacgac gctcttccga tctgcctacc tgcagtggag cag 53
<210> 8
<211> 55
<212> DNA
<213>Artificial sequence
<400> 8
acactctttc cctacacgac gctcttccga tctttctccc tgcagctgaa ctctg 55
<210> 9
<211> 54
<212> DNA
<213>Artificial sequence
<400> 9
acactctttc cctacacgac gctcttccga tctgcatatc tgcagatcag cagc 54
<210> 10
<211> 53
<212> DNA
<213>Artificial sequence
<400> 10
acactctttc cctacacgac gctcttccga tctcagatca gcagcctaaa ggc 53
<210> 11
<211> 42
<212> DNA
<213>Artificial sequence
<400> 11
atctcgtatg ccgtcttctg cttgaagacc gatgggccct tg 42
<210> 12
<211> 42
<212> DNA
<213>Artificial sequence
<400> 12
atctcgtatg ccgtcttctg cttggaagac cttggggctg gt 42
<210> 13
<211> 44
<212> DNA
<213>Artificial sequence
<400> 13
atctcgtatg ccgtcttctg cttggggaat tctcacagga gacg 44
<210> 14
<211> 43
<212> DNA
<213>Artificial sequence
<400> 14
atctcgtatg ccgtcttctg cttggggtgt ctgcaccctg ata 43
<210> 15
<211> 43
<212> DNA
<213>Artificial sequence
<400> 15
atctcgtatg ccgtcttctg cttggaagac ggatgggctc tgt 43
<210> 16
<211> 44
<212> DNA
<213>Artificial sequence
<400> 16
atctcgtatg ccgtcttctg cttgttgcag cagcgggtca aggg 44
Claims (10)
1. the method for the B cell minimal residual detection of a kind of leukaemia, it is characterised in that comprise the following steps：
(1) sample rna and cancer cell RNA are collected, is preserved after determining its concentration through detected through gel electrophoresis, Nanodrop；
(2) multiple PCR products are obtained by the method for one-step RT-PCR to sample rna and cancer cell RNA respectively；
(3) difference magnetic beads for purifying multiple PCR products, remove unnecessary primer；
(4) 3 ' the ends addition base A of multiple PCR products after purification, obtains the PCR primer with 3 ' cohesive end A；
(5) PCR primer that will have 3 ' cohesive end A is connected with joint sequence, obtains the connection product containing joint；
(6) enter performing PCR amplification to the connection product sequencing primer containing joint, obtain amplified production, purified, sequencing, by sample
This RNA amplification Product Sequence is compared with cancer cell RNA amplification Product Sequence.
2. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
In the system that one-step RT-PCR reacts in step (2), template amount is 50~500ng/20 μ l.
3. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
In step (2) one-step RT-PCR primer include for people BCR variable V areas design 10 sense primers, the A for BCR, D,
2 downstreams of 1 anti-sense primer that M, G gene constant region (constant) are respectively designed and the E genes constant region design for BCR
Primer, the sequence of 10 sense primers is SEQ ID N0:1~SEQ ID N0:The sequence of 10,6 anti-sense primers is SEQ ID
N0:11~SEQ ID:16.
4. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 3, it is characterised in that described
Each sense primer equimolar mixing in one-step RT-PCR, total primer concentration is 10 μm of ol, the anti-sense primer integral molar quantity and institute
State sense primer integral molar quantity identical.
5. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
The magnetic beads for purifying multiple PCR products of step (3) are concretely comprised the following steps：
1) take out magnetic bead vibration and mix 5min, the amount for taking out 45 μ l/ samples is put in room temperature 10min；
2) RT-PCR product respectively takes out 20 μ l, and 30 μ l H are added into each sample2O, magnetic bead is added according to 45 μ l/ samples,
Inhaled with rifle and blow about 10 mixings, be put in room temperature 2min；
3) uncap and be put in 1min or so on magnetic frame, liquid become clarification can sucking liquid, add 125 μ l 85% ethanol,
Wash out and discard after 1min, magnetic bead is put in room temperature 8min (5-10min) and dried；
4) 30 μ l dd H are added2O back dissolvings.
6. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
3 ' the ends addition base A of multiple PCR products in step (4) after purification reaction system is：
1) multiple PCR products of 34 μ l purifying；
2) 5 μ l Klenow buffer solutions；
3)10μl 1mM dATP；
4) 1 μ l Klenow fragments.
7. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
Jointing reaction system is in step (5)：
1) DNA of 11 μ l steps 3；
2) 15 μ l DNA connection buffer solutions；
3)1μl 1:The linker oligonucleotides of 20 dilutions；
4) 3 μ l DNA ligases.
8. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 7, it is characterised in that described
Linker oligonucleotides center tap sequence is：
- the 3' of normal chain 5 '：ACACTCTTTCCCTACACGACGCTCTTCCGATCT
- the 3' of minus strand 5 ':ATCTCGTATGCCGTCTTCTGCTTG.
9. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
Sequencing primer described in step (6) is：
First sequencing primer：5"AATGATACGGCGACCACCGAGATCTACACTCTTT
CCCTACACGACGCTCTTCCGATCT
Second sequencing primer：5"CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT.
10. the method for the B cell minimal residual detection of a kind of leukaemia according to claim 1, it is characterised in that described
Pcr amplification product in step (6) is that the fragment for separating, cutting 400bp lower edges using Ago-Gel is purified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710570750.6A CN107267629A (en) | 2017-07-13 | 2017-07-13 | A kind of method of the B cell minimal residual detection of leukaemia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710570750.6A CN107267629A (en) | 2017-07-13 | 2017-07-13 | A kind of method of the B cell minimal residual detection of leukaemia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107267629A true CN107267629A (en) | 2017-10-20 |
Family
ID=60073435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710570750.6A Pending CN107267629A (en) | 2017-07-13 | 2017-07-13 | A kind of method of the B cell minimal residual detection of leukaemia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107267629A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680062A (en) * | 2018-12-18 | 2019-04-26 | 杭州艾沐蒽生物科技有限公司 | A method of detection minimal residual disease MRD |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710454A (en) * | 2013-12-31 | 2014-04-09 | 南方科技大学 | Method for carrying out high-throughput sequencing on TCR (T cell receptor) or BCR (B cell receptor) and method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing tag sequences |
| CN105063032A (en) * | 2015-08-14 | 2015-11-18 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
| CN106119251A (en) * | 2016-06-03 | 2016-11-16 | 刘鹏飞 | BCR heavy chain CDR3 leukemia is caused a disease sequence and screening technique and application |
| CN106755410A (en) * | 2016-12-23 | 2017-05-31 | 孙涛 | A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence |
| WO2017096239A1 (en) * | 2015-12-04 | 2017-06-08 | St. Jude Children's Research Hospital, Inc. | Cloning and expression system for t-cell receptors |
| CN106834450A (en) * | 2017-01-10 | 2017-06-13 | 武汉赛云博生物科技有限公司 | A kind of method for the detection of T cell Minimal Residual Disease of Leukemia |
-
2017
- 2017-07-13 CN CN201710570750.6A patent/CN107267629A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710454A (en) * | 2013-12-31 | 2014-04-09 | 南方科技大学 | Method for carrying out high-throughput sequencing on TCR (T cell receptor) or BCR (B cell receptor) and method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing tag sequences |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
| CN105063032A (en) * | 2015-08-14 | 2015-11-18 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing |
| CN106834472A (en) * | 2015-08-14 | 2017-06-13 | 深圳市瀚海基因生物科技有限公司 | BCR diversity detection kit and application |
| WO2017096239A1 (en) * | 2015-12-04 | 2017-06-08 | St. Jude Children's Research Hospital, Inc. | Cloning and expression system for t-cell receptors |
| CN106119251A (en) * | 2016-06-03 | 2016-11-16 | 刘鹏飞 | BCR heavy chain CDR3 leukemia is caused a disease sequence and screening technique and application |
| CN106755410A (en) * | 2016-12-23 | 2017-05-31 | 孙涛 | A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence |
| CN106834450A (en) * | 2017-01-10 | 2017-06-13 | 武汉赛云博生物科技有限公司 | A kind of method for the detection of T cell Minimal Residual Disease of Leukemia |
Non-Patent Citations (1)
| Title |
|---|
| 石连霞: "急性淋巴细胞白血病微小残留病诊断研究进展", 《临床检验杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680062A (en) * | 2018-12-18 | 2019-04-26 | 杭州艾沐蒽生物科技有限公司 | A method of detection minimal residual disease MRD |
| CN109680062B (en) * | 2018-12-18 | 2022-12-02 | 杭州艾沐蒽生物科技有限公司 | Method for detecting minimal residual disease MRD |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103757106B (en) | Based on people's mthfr gene polymorphic detection test kit and the method for Taqman-MGB probe | |
| CN106701987A (en) | PCR (polymerase chain reaction) amplification system for genotyping of three SNP (single-nucleotide polymorphism) loci related to human folic acid metabolism and detection kit | |
| CN103352073B (en) | Primer system for detecting gene SNP related to genetic deafness, and use thereof | |
| CN105296621A (en) | Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene | |
| CN106119362B (en) | It is a kind of for detecting the primer sets and kit of HLA-B*1502 allele | |
| CN103276065A (en) | Primer system for detecting gene SNPs (single nucleotide polymorphisms) related to hereditary hearing loss and application of primer system | |
| CN108998505A (en) | A kind of gene polymorphism sites detection technique and its kit | |
| CN104946746A (en) | Folic acid heredity metabolism ability detection using mass spectrum | |
| CN109182501A (en) | A kind of folic acid metabolism genetic polymorphism detection primer and kit | |
| CN108977532B (en) | A kind of mankind's mthfr gene polymorphic detection kit and its preparation method and application | |
| CN106929582A (en) | A kind of method and kit for detecting EGFR genetic mutation | |
| CN104962621A (en) | Primer group used for detecting locus polymorphism of MTHFR genes and MTRR genes, method for detecting same and application of primer group | |
| CN107267629A (en) | A kind of method of the B cell minimal residual detection of leukaemia | |
| CN108949929A (en) | For detecting the product and its methods and applications of MTHFR and MTRR gene pleiomorphism simultaneously | |
| CN103911380A (en) | EPAS1 gene mutant and application thereof | |
| CN106834450A (en) | A kind of method for the detection of T cell Minimal Residual Disease of Leukemia | |
| CN103667267B (en) | For with the DNA probe storehouse of KRAS gene recombination and the method adopting its enrichment KRAS gene fragment | |
| CN110066868A (en) | A kind of primer sets, kit and the detection method of aspirin pharmaceutical relevant gene genetic polymorphism detection | |
| CN109306374A (en) | For detecting primer, detection method and the kit of mankind PIK3CA gene E545K mutation | |
| CN111235252A (en) | Method for distinguishing individual medication of nitrendipine by mass spectrometry through detecting product | |
| CN104946735A (en) | Kit and method for determining genotype of predetermined SNP site of DNA sample to be tested | |
| Duan et al. | Screening of T Cell‐Related Long Noncoding RNA‐MicroRNA‐mRNA Regulatory Networks in Non‐Small‐Cell Lung Cancer | |
| CN105296471A (en) | DNA label, PCR primer and application thereof | |
| CN103305600B (en) | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof | |
| CN110373467A (en) | A kind of detection reagent and detection method of human EGFR gene mutations site T790M |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171020 |
|
| RJ01 | Rejection of invention patent application after publication |