CN106755410A - A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence - Google Patents
A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence Download PDFInfo
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Abstract
The invention belongs to molecular Biological Detection field, more particularly to a kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence, including acquisition human blood sample 10mL carries out PMBC in EDTA anticoagulant tubes, using lymphocyte separation medium Ficoll 1077（PBMC）Separation, the total serum IgE of PBMC is extracted using the method for Trizol, agents useful for same be RNAzol RT, RNA reverse transcriptions into cDNA, and in the ends of cDNA 5 ' addition joint, PCR1, PCR2 and purifying and carry out the steps such as high-flux sequence simultaneously.By the anti-sense primer of the sense primer from joint to C areas, TCR in lymphocyte is obtained（Alpha chains or Beta chains）And BCR（Heavy chains or Light chains）Gene order total length information.
Description
Technical field
It is more particularly to a kind of to detect that T is thin simultaneously based on high-flux sequence the invention belongs to molecular Biological Detection field
Born of the same parents and the method in B cell immune group storehouse.
Background technology
Substantial amounts of V (variable region), D (variable region), J (bonding pad) genetic fragments are received in T, B cell on T, B cell locus
Each species diversity can be produced to recombinate in the formation of body.It is unique that the restructuring of this V-D-J genes imparts each T, B cell oneself
T, B-cell receptor (TCR, BCR) so that the sequence of each TCR and BCR can effectively turn into T, a B cell gram
Grand unique biomarker.
Because the maximum feature of TCR and BCR genes is the random restructuring of V, D, J genetic fragment, so, for unknown gene
Sequence, it is difficult to a sense primer is designed for recognizing 5 ' terminal sequences of TCR, BCR gene, so cannot also utilize round pcr
Amplification TCR, BCR gene and sequencing.
And another TCR sequence measurement, the method for Multiplex PCR, the partial sequence letter in tcr gene can only be sequenced
Breath, so that sequencing gene information is imperfect.In addition, the primer of Multiplex PCR method is according to known V, the design of J genes
, this sequencing result is confined to the known of wild type.But in cancer patient, the gene mutation of cancer cell is very normal
See, if the BCR or TCR of leukaemia cancer cell generate mutation, then the primer of known array is possible to None- identified
Gene after mutation, for testing result, is easy for causing false negative.Also, the immune group storehouse sequencing side of Multiplex PCR
Method, is only sequenced to TCR or BCR, has just needed the primer of tens pairs, huge if detection TCR and BCR simultaneously
Primer quantity can allow the efficiency and specificity of whole PCR amplification to become very poor.
So, TCR and BCR can be simultaneously detected in an experimental implementation there is presently no a technology, using us
Method (single pair of primer method) can simultaneously detect the TCR and BCR of multiple samples, save experimental period and reagent, cost of labor.
The technology can be applied in the research of immunogene group, can not only detect the minimal residual after lymphoid malignancy treatment
Disease, additionally it is possible to judge immunologic reconstitution, food hypersenstivity, detection of immunity disease after HSC transplanting etc..
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of high-flux sequence that is based on builds φt cell receptor and B simultaneously
The cDNA joints and single pair of primer of cell receptor library construction, and library preparation method, by from the sense primer of joint to C
The anti-sense primer in area, while obtaining the total length information of TCR and BCR gene orders.
Solve a kind of side for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence of above technical problem
Method, it is characterised in that：Comprise the following steps：
(1) human blood sample 10mL is obtained in EDTA anticoagulant tubes；
(2) to carry out peripheral blood mononuclear using lymphocyte separation medium Ficoll-1077 (Sigma Co., USA #10771) thin
The separation of born of the same parents (PBMC)；
The effect of lymphocyte separation medium is that lymphocyte can be separated from whole blood, because T cell is detected for us
Object, T cell belongs to one kind of lymphocyte, and the RNA that the cell mass after separation is obtained is to eliminate red blood cell, blood platelet etc.
The RNA's of cell.So building the total serum IgE that storehouse uses, to include T cell RNA template purity higher.
(3) total serum IgE of PBMC is extracted using the method for Trizol, agents useful for same is RNAzol RT (MRC companies of U.S. #
RN190)；
The method of Trizol extracts RNA, and step is as follows：
Harvesting, is transferred in 1.5ml centrifuge tubes, adds 1ml Trizol, mixes, and is stored at room temperature 5min.
0.2ml chloroforms are added, 15s is vibrated, 2min is stood.
0.5ml isopropanols are added, liquid in pipe is gently mixed, be stored at room temperature 10min.
4 DEG C of centrifugations, 12000g × 10min abandons supernatant.
The ethanol of 1ml 75% is added, gently washing precipitation.4 DEG C of centrifugations, 7500g × 5min abandons supernatant.
Natural air drying, adds the DEPC H2O dissolvings of 50ul, obtains lymphocyte total serum IgE.
(4) into cDNA, and simultaneously in the ends of cDNA 5 ' addition joint, 5 ' ends are drawn when being expanded with PCR later for RNA reverse transcriptions
Thing is combined；
In reverse transcription, while adding joint, loss of the RNA during multistep reaction can be minimized.RNA is in operation
Middle stability extreme difference, is very easy to degraded, and a small amount of step can to the full extent reduce degraded, while also save can be used for
The cDNA preparation times of amplification.
Joint is the nucleic acid linker at cDNA 5 ' ends, the Oligo joints of XCR 5 '.
(5)PCR1:Expanded by way of 1 sense primer, the anti-sense primer of 2 or more than 2 restructuring TCR and
BCRcDNA；
(6) PCR2 and purifying：For PCR1 products (TCR the and BCR sequences after amplification) add Illumina high-flux sequences
The upper machine joint and label of instrument, while being expanded again to increase more upper machine gene dosage；After PCR reactions terminate, using magnetic bead
Carry out DNA purifying.
PCR primer typically all contains excessive primer, Taq DNA enzymatics and dNTP.The presence of these compositions will be directly affected
To processes such as follow-up library quality inspection, sequencing reactions, purifying can remove these influences the accessory substance of subsequent experimentals.Meanwhile, it is pure
The process of change is also a process for clip size screening, and the DNA fragmentation in the present invention is in 700bp or so, using difference
The magnetic bead of volume mixes with PCR primer, and magnetic bead/DNA different volume ratios can adsorb different size of fragment, using above-mentioned
Magnetic bead volume, can successfully remove mistake (error) fragment and primer dimer when PCR is expanded, machine text in the sequencing of let us
There was only our sequencing target DNA fragments in storehouse so that sequencing result is more accurate, reduce error.
As shown in figure 3, being only found that a peak for fragment by library quality inspection.
(7) high-flux sequence is carried out：The cDNA library of gained is sequenced by Illumina MiSeq platforms, is sequenced
Pattern is PE300, and library denaturant concentration is 2nM, and upper machine concentration is 20pM, and by bioinformatic analysis high-flux sequence knot
Really；
Up to a million obtained by the end connectors of XCR 5 ' of the invention, PCR primer and sequencing library preparation method
TCR and BCR sequences, the technology can be applied in the research of immunogene group, can not only detect that lymphoid malignancy is controlled
Minimal residual disease after treatment, additionally it is possible to judge immunologic reconstitution, food hypersenstivity, detection of immunity disease after HSC transplanting etc..
Step (4) center tap is the Oligo joints of XCR 5 ', and its base sequence is：5’ATGCATCGGATCTTCAGCA
TGAACTTrGrGrG 3’。
1 sense primer and 2 anti-sense primers and primer sequence are in the step (5)：
Mix each RNA sample according to following ratio in heretofore described step (4)：
Reagent volume 1X (μ L),
RNA 8,
The Oligo of XCR 3 ' (dT) primer (10 μM) 1,
Hatch in 72 DEG C 3 minutes, then 4 DEG C 1 minute.
PCR reaction buffers are prepared according to following ratio
Mixing PCR reactions caching liquid and RNA samples, cDNA reverse transcriptions are started according to following response procedures
42 DEG C 60 minutes
70 DEG C 10 minutes
4 DEG C permanent
After reaction terminates, so that it may obtain and with the addition of total cDNA (such as Fig. 1) of joint at 5 ' ends.
Comprised the following steps that in the step (5)：Reaction system is prepared according to following ratio：
Reaction system is prepared according to above-mentioned reaction system, mixes above-mentioned 5 sample reagent in PCR test tubes, mixed, number is centrifuged
Second, the liquid residue on tube wall is centrifuged in ttom of pipe.Holding test tubes start fortune in PCR amplification instrument according to above-mentioned response procedures
OK.
The response procedures of PCR 1 are in the step (5):
95 DEG C 3 minutes；95 DEG C 30 seconds；65 DEG C 1 minute, 25 circulation；72 DEG C 1 minute；4 DEG C permanent.
Comprised the following steps that in the step (6)：
Reaction system is prepared according to following ratio：
The response procedures of PCR 2 are in the step (6):
94 DEG C 3 minutes；94 DEG C 30 seconds；55 DEG C 30 seconds, 18 circulation；72 DEG C 20 minutes, 72 DEG C 1 minute, 4 DEG C are permanent.
Specific purification step is as follows in the step (6)：
(1) 80 μ L AMPure XP Beads of addition enter PCR2 product, mix.
(2) in incubation at room temperature 10 minutes.
(3) magnetic bead-PCR2 product mixtures test tube is placed on magnetic frame, and all magnetic beads of wait are adsorbed on magnetic frame
Afterwards, all supernatants are sucked with pipettor, is abandoned.
(4) the addition ethanol of 150 μ L 70% is hatched 30 seconds on magnetic bead, and all supernatants are sucked with pipettor, is abandoned.
(5) the 4th step is repeated 2 times.
(6) test tube cap is opened, is waited 5 minutes, treat that magnetic bead is air-dried, there is no any ethanol to residue in test tube.
(7) test tube is removed from magnetic frame, and adds 50 μ L and remove nuclease water, suspension magnetic bead is blown and beaten using pipettor.
(8) tube back magnetic frame, after waiting all magnetic beads to be all adsorbed in magnetic frame, transfer supernatant is in new test tube
In.The PCR2 products after purifying are just contained in supernatant.
The present invention is based on high throughput sequencing technologies, and TCR, BCR are sequenced the library constructing method of single pair of primer, by obtaining
After blood PBMC RNA, while RNA to cDNA reverse transcriptions are carried out, to 5 ' one joint of end addition of cDNA, so as to pass through
The joint design amplification sense primer of this known array, then coordinate tcr gene and BCR genes 3 ' to hold C areas' gene (constant region)
Design anti-sense primer, the purpose of whole TCR and BCR sequences gene is expanded so as to reach.
Therefore it provides a kind of φt cell receptor and B-cell receptor detection means of being built based on high throughput sequencing technologies is simultaneously
It is very significant, it is effective to save experimental period and reagent, cost of labor.
The present invention provides the beneficial effect of the joint for building TCR and BCR libraries simultaneously based on high-flux sequence, primer and method
It is really：Obtain people TCR (Alpha chains or Beta chains) and BCR (Heavy chains or Light chains) complete genome sequence simultaneously；
The present invention on the basis of high-flux sequence platform, by simultaneously people's TCR and BCR gene sequencing result is carried out entirely
The bioinformatic analysis in face, obtain the gene Preference of TCR and BCR when VDJ is recombinated, and VDJ assortment of genes information is immunized
Group clone's kind of information, immune group diversity information, the nucleotide sequence and amino acid of all CDR1, CDR2, CDR3 of immune group
Sequence information, the abrupt information on gene, etc..Exactly these factor quantity of formation are huge and immune group storehouse of wide variety.
Brief description of the drawings
RNA reverse transcription cDNA schematic diagrames in Fig. 1 present invention
Fig. 2 is sequenced upper machine joint schematic diagram to be expanded TCR and BCR cDNA and being added using twice PCR in the present invention
Fig. 3 is sequencing library quality inspection result in the present invention.
Fig. 4 for the present invention in TCR sequencing results carry out bioinformatic analysis comparison, find out every information (portion of sequence
Point)
Fig. 5 for the present invention in BCR sequencing results carry out bioinformatic analysis comparison, find out every information (portion of sequence
Point)
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail:
Embodiment 1
A kind of method that utilization high-flux sequence detects φt cell receptor and B-cell receptor simultaneously, it is characterised in that：Including
Following steps：
(1) human blood sample 10mL is obtained in EDTA anticoagulant tubes；
(2) peripheral blood mononuclear is carried out using lymphocyte separation medium Ficoll-1077 (Sigma Co., USA #10771)
The separation of cell (PBMC)；
(3) total serum IgE of PBMC is extracted using the method for Trizol, agents useful for same is RNAzol RT (MRC companies of U.S. #
RN190)；
(4) utilize2.0Fluorometer (U.S. Thermo Fisher Scientific company #
Q32866), coordinateRNA HS Assay Kit kits (U.S. Thermo Fisher Scientific company #
Q32852 RNA concentration) is determined, reverse transcription RNA is subsequently used for；
(5) RNA reverse transcriptions are into cDNA, and 5 ' the end primer knots when the ends of cDNA 5 ' addition joint is expanded with PCR later
Close, comprise the following steps that,
The reagent for using：
The Oligo of XCR 3 ' (dT) primer (10 μM)
5X reverse transcription buffers (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl2)
Dithiothreitol (DTT), DTT (20mM) U.S. Thermo Scientific#R0861
DNTP Mix (10mM) U.S. Invitrogen#18427088
RNAse Out (40U/ μ L) U.S. Invitrogen#10777019
The Oligo of XCR 5 ' joints (10 μM)
Superscript II RT (200U/ μ L) U.S. Invitrogen#18064022
Mix each RNA sample according to following ratio：
Hatch in 72 DEG C 3 minutes, then 4 DEG C 1 minute.
PCR reaction buffers are prepared according to following ratio
| Reagent | Volume 1X (μ L) |
| 5X reverse transcription buffers | 3.5 |
| DTT(20mM) | 1 |
| dNTP(10mM) | 1 |
| RNAse Out | 1 |
| The Oligo of XCR 5 ' joints (10 μM) | 1 |
| Superscript II RT | 1 |
Mixing PCR reactions caching liquid and RNA samples, RNA reverse transcriptions are started according to following response procedures
42 DEG C 60 minutes
70 DEG C 10 minutes
4 DEG C permanent
After reaction terminates, so that it may obtain and with the addition of total cDNA (such as Fig. 1) of joint at 5 ' ends
(6) PCR1." single pair of " or more than 1 pair by way of primer, or by 1 sense primer, 2 or more than 2
Anti-sense primer expands restructuring TCR and BCR cDNA simultaneously, comprises the following steps that：
The reagent for using：
Q5 High-Fidelity 2X Master Mix (U.S. NEB#M0492L)
The end connectors of XCR 5 ' primer (sense primer)
TCR C areas primer (anti-sense primer)
BCR C areas primer (anti-sense primer)
Remove nuclease water (U.S. Thermo Scientific AM9914G)
Reaction system is prepared according to following ratio：
The response procedures of PCR 1 are:
(7) PCR2 (label PCR).It is PCR1 products (TCR and BCR sequences after amplification) addition Illumina high fluxs
The upper machine joint and label of sequenator, while being expanded again to increase more upper machine gene dosage.Comprise the following steps that：
The reagent for using：
Q5 High-Fidelity 2X Master Mix (U.S. NEB#M0492L)
Label sense primer
Label TCR anti-sense primers
Label B CR anti-sense primers
Remove nuclease water (U.S. Thermo Scientific AM9914G)
Reaction system is prepared according to following ratio：
The response procedures of PCR 2 are:
(8) PCR2 product purifications.After above-mentioned PCR reactions terminate, DNA purifying is carried out using magnetic bead, comprised the following steps that：
The reagent for using：
Agencourt AMPure XP Beads (U.S. Beckman#A63882)
Specific purification step is as follows：
1. 80 μ L AMPure XP Beads of addition enter PCR2 product, mix.
2. in incubation at room temperature 10 minutes.
3. magnetic bead-PCR2 product mixtures test tube is placed on magnetic frame, after waiting all magnetic beads to be adsorbed on magnetic frame,
All supernatants are sucked with pipettor, is abandoned.
4. the addition ethanol of 150 μ L 75% is hatched 30 seconds on magnetic bead, and all supernatants are sucked with pipettor, is abandoned.
5. the 4th step is repeated 2 times.
6. test tube cap is opened, is waited 5 minutes, treat that magnetic bead is air-dried, there is no any ethanol to residue in test tube.
7. test tube is removed from magnetic frame, and add 50 μ L and remove nuclease water, suspension magnetic bead is blown and beaten using pipettor.
8., tube back magnetic frame, after waiting all magnetic beads to be all adsorbed in magnetic frame, transfer supernatant is in new test tube
In.The PCR2 products after purifying are just contained in supernatant.
(9) after library purifying terminates, using the Bioanalyzer of Agilent 2100 (Agilent companies of U.S. #
G2939AA the purity and size in library) are detected, the kit for using is that (U.S. Agilent is public for the Kit of Agilent DNA 1000
Department #5067-1504), testing result as shown in figure 3, library size 768bp or so scope, and library purity is at a relatively high,
And have not seen other non-specific amplification sequences.
(10) utilize2.0Fluorometer (U.S. Thermo Fisher Scientific company #
Q32866), coordinateDsDNA HS Assay Kit kits (U.S. Thermo Fisher Scientific company #
Q32851 DNA library concentration) is determined, and send the company to carry out high-flux sequence.The cDNA library of gained is passed through into IlluminaPlatform (Illumina companies of the U.S.) is sequenced, and sequencing pattern is PE300, and library denaturant concentration is 2nM, and upper machine is dense
It is 20pM to spend, and by bioinformatic analysis high-flux sequence result.
Material and reagent explanation
Healthy volunteer's informed consent.No special illustrates that the reagent that the present invention is used is commercial goods, the present invention
The database that embodiment is used is disclosed online database.
Specifically, the termination of reverse transcription 5 ' header sequence of the present invention, primer sequence are following (5 ' -3 ')：
Reverse transcription step
The Oligo of XCR 3 ' (dT) primer：
5’TTTTTTTTTTTTTTTTTTTTGA 3’
The Oligo joints of XCR 5 '：
5’ATGCATCGGATCTTCAGCATGAACTTrGrGrG 3’
PCR1 steps
The end connector primers of XCR 5 '：
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’
TCR-alpha chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’
TCR-beta chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’
BCR-Heavy chain C areas primer：
IgA：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’
IgG：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’
IgM：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’
BCR-Light chain C areas primer：
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’
Selected as needed for anti-sense primer, such as to do the primer that TCR-beta chains just select TCR-Beta, if
BCR-light (kappa) is, this primer is just selected.TCR sequencings are generally done, BCR sequencings, are one, a chains
What chain was surveyed, determined according to the chain that purpose is detected, TCR with BCR respectively select one together with survey, can also all be placed on one in the present invention
Rise and survey, make use of has primer and 7 anti-sense primers on 1, TB cell receptors whole chain can be surveyed.
PCR2 steps
Label sense primer：
5’CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’
Label TCR anti-sense primers：
5’AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC 3’
Label B CR anti-sense primers：
5’AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT 3’
RG=RNA nucleotides
In Tag primerUnderscorePart is sequenced sequence label for Illumina, and [] interior sequence is replaced by following table sequence,
During for detecting multiple samples simultaneously, using different index1-index2 or index1-index3 combinations and biological information
Learn φt cell receptor (TCR) and B-cell receptor (BCR) sequencing result that algorithm distinguishes multiple samples.
Design of primers：Joint sequence and TCR during for RNA reverse transcriptions in 5 ' end additions are divided in BCR C areas gene
Analysis, is analyzed using Oligo 7.36 and Primer Premier 6.0 to primer dimer and stem ring mispairing, at 5 ' ends
Manual splice is provided with sense primer, and reverse primer is designed for C downstream of gene, and amplification TCR transcribes subregion sequence with BCR total lengths
Row, wherein the FR1 of TCR and BCR immune groups storehouse is contained, CDR1, FR2, CDR2, FR3, CDR3, FR4 region.
After using the end connectors of PBMC RNA 5 ' of the invention, primer and library construction, high-flux sequence obtains about several
Million TCR and BCR sequences.
Sequencing result carries out bioinformatic analysis, and (analysis of biological information uses the aligner of Bowtie 2
(Ver.2.1.0), TCR and BCR derive from international immunogene information system according to storehouse matching www.imgt.org),
Shown in partial analysis comparison result below figure 4,5.
By bioinformatic analysis, every information of TCR and BCR sequences, amino acid information, bar can be accurately known
Number and proportion etc..By TCR and BCR comparative analyses, present invention obtains high-flux sequence sequence TCR and BCR sequence
The statistic analysis result of information, as a result as shown in Fig. 4 (TCR) and 5 (BCR).
The above results show that building TCR and BCR libraries simultaneously using the method for the present invention can cover TCR and BCR genes
Diversity information, improve the recall rate of low copy number immune cell clones and save time, reagent and cost of labor.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Sequence table SEQ UENCE LISTING
<110>Sun Tao
<120>A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence
<160> 34
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>It is artificial synthesized
<220>
<223>The Oligo of XCR 3 ' (dT) primer
<400> 1
ttttttttttttttttttttga 22
<210> 2
<211> 32
<212> DNA
<213>It is artificial synthesized
<220>
<223>The Oligo joints of XCR 5 '
<400> 2
atgcatcggatcttcagcatgaacttrgrgrg 32
<210> 3
<211> 57
<212> DNA
<213>It is artificial synthesized
<220>
<223>The end connector primers of XCR 5 '
<400> 3
gtctcgtgggctgggcgatgtgtatgagagacagcatgcatcggatcttcagcatga 57
<210> 4
<211> 62
<212> DNA
<213>It is artificial synthesized
<220>
<223>TCR-alpha chain C areas primer
<400> 4
tcgtcgccagcgtcggaagtgtataagagacagtctcagctggtacatatcgatgtcagggt 62
<210> 5
<211> 62
<212> DNA
<213>It is artificial synthesized
<220>
<223>TCR-beta chain C areas primer
<400> 5
tcgtcgccagcgtcggaagtgtataagagacagtcgcagcgtcagatgtgtataagagacag 62
<210> 6
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223>BCR-Heavy chain C areas primers-IgA
<400> 6
tacgcgatatcgctctgcgctaactagtcgtactaggctcctgggttccgaagcc 55
<210> 7
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223> IgG
<400> 7
tacgcgatatcgctctgcgctaactagtcgtactagcgcctgagaaggacgacac 55
<210> 8
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223> IgM
<400> 8
tacgcgatatcgctctgcgctaactagtcgtactaggggaattcttctgggagac 55
<210> 9
<211> 57
<212> DNA
<213>It is artificial synthesized
<220>
<223>BCR-Light chain C areas primers-Kappa
<400> 9
tacgcgatatcgctctgcgctaactagtcgtactaccttccactctatattggcctc 57
<210> 10
<211> 56
<212> DNA
<213>It is artificial synthesized
<220>
<223> Lambda
<400> 10
tacgcgatatcgctctgcgctaactagtcgtactagccactgtatccgctcccggg 56
<210> 11
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 11
caagcagaagacggcatacgagatatctatcggtctcgtgggctgg 46
<210> 12
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 12
caagcagaagacggcatacgagattcaggtgagtctcgtgggctgg 46
<210> 13
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 13
caagcagaagacggcatacgagatcactagttgtctcgtgggctgg 46
<210> 14
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 14
caagcagaagacggcatacgagatgaattgccgtctcgtgggctgg 46
<210> 15
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 15
caagcagaagacggcatacgagat atgtacaagtctcgtgggctgg 46
<210> 16
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 16
caagcagaagacggcatacgagatgattcagtgtctcgtgggctgg 46
<210> 17
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 17
caagcagaagacggcatacgagatctgttcgtgtctcgtgggctgg 46
<210> 18
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 18
caagcagaagacggcatacgagattatacggcgtctcgtgggctgg 46
<210> 19
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 19
aatgatacggcgaccaccgagatctacactagctacttcgtcgccagcgtc 51
<210> 20
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 20
aatgatacggcgaccaccgagatctacacattatagctcgtcgccagcgtc 51
<210> 21
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 21
aatgatacggcgaccaccgagatctacaccccgtacttcgtcgccagcgtc 51
<210> 22
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 22
aatgatacggcgaccaccgagatctacacgggtataatcgtcgccagcgtc 51
<210> 23
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 23
aatgatacggcgaccaccgagatctacacagcaggtgtcgtcgccagcgtc 51
<210> 24
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 24
aatgatacggcgaccaccgagatctacactatacgtatcgtcgccagcgtc 51
<210> 25
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 25
aatgatacggcgaccaccgagatctacaccacctagttcgtcgccagcgtc 51
<210> 26
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 26
aatgatacggcgaccaccgagatctacacgttgctactcgtcgccagcgtc 51
<210> 27
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 27
aatgatacggcgaccaccgagatctacaccacgatgttacgcgatatcgct 51
<210> 28
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 28
aatgatacggcgaccaccgagatctacactaatcgcttacgcgatatcgct 51
<210> 29
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 29
aatgatacggcgaccaccgagatctacacatattacctacgcgatatcgct 51
<210> 30
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 30
aatgatacggcgaccaccgagatctacaccctatacttacgcgatatcgct 51
<210> 31
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 31
aatgatacggcgaccaccgagatctacac ggatgaaatacgcgatatcgct 51
<210> 32
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 32
aatgatacggcgaccaccgagatctacacagtctttgtacgcgatatcgct 51
<210> 33
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 33
aatgatacggcgaccaccgagatctacactagtggtatacgcgatatcgct 51
<210> 34
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 34
aatgatacggcgaccaccgagatctacacgtatgaactacgcgatatcgct 51
Claims (10)
1. a kind of to be based on the method that high-flux sequence detects T cell (TCR) and B cell (BCR) immune group storehouse simultaneously, its feature exists
In：Comprise the following steps：
(1) human blood sample 10mL is obtained in EDTA anticoagulant tubes；
(2) separation of PMBC (PBMC) is carried out using lymphocyte separation medium Ficoll-1077；
(3) total serum IgE of PBMC is extracted using the method for Trizol, agents useful for same is RNAzol RT；
(4) RNA reverse transcriptions are into cDNA, and simultaneously in the ends of cDNA 5 ' addition joint；
(5)PCR1:Restructuring TCR and BCR is expanded by way of 1 sense primer, the anti-sense primer of 2 or more than 2
cDNA；
(6) PCR2 and purifying：The upper machine joint and label of Illumina high-flux sequence instrument are added for PCR1 products, while expanding
Increase, and DNA purifying is carried out using magnetic bead；
(7) high-flux sequence is carried out：The cDNA library of gained is sequenced by Illumina MiSeq platforms, pattern is sequenced
It is PE300, library denaturant concentration is 2nM, and upper machine concentration is 20pM, and by bioinformatic analysis high-flux sequence result.
2. it is according to claim 1 it is a kind of based on high-flux sequence detect T cell Minimal Residual Disease of Leukemia method, its feature
It is：Step (4) center tap is the Oligo joints of XCR 5 ', and its base sequence is：5’ATGCATCGGATCTTCAGCATGAACTTr
GrGrG 3’。
3. it is according to claim 1 it is a kind of based on high-flux sequence detect T cell Minimal Residual Disease of Leukemia method, its
It is characterised by：Single pair of primer and primer sequence are in the step (5).
4. a kind of side for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence according to claim 1
Method, it is characterised in that：In the step (4), comprise the following steps that：
Mix each RNA sample according to following ratio：
Reagent volume 1X (μ L),
RNA 8,
The Oligo of XCR 3 ' (dT) primer (10 μM) 1,
Hatch in 72 DEG C 3 minutes, then 4 DEG C 1 minute；
PCR reaction buffers are prepared according to following ratio：
5. it is according to claim 1 and 2 a kind of T cell and B cell immune group storehouse to be detected based on high-flux sequence simultaneously
Method, it is characterised in that：The mixing PCR reactions caching liquid and RNA samples, start cDNA and invert according to following response procedures
Record：
42 DEG C 60 minutes；70 DEG C 10 minutes；4 DEG C permanent.
6. a kind of side for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence according to claim 1
Method, it is characterised in that：In the step (5), reaction system is prepared according to following ratio：
7. the one kind according to claim 1 or 4 is based on high-flux sequence and detects T cell and B cell immune group storehouse simultaneously
Method, it is characterised in that：In the step (5), the response procedures of PCR 1 are:
95 DEG C 3 minutes；95 DEG C 30 seconds；65 DEG C 1 minute, 25 circulation；72 DEG C 1 minute；4 DEG C permanent.
8. a kind of side for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence according to claim 1
Method, it is characterised in that：In the step (6), reaction system is prepared according to following ratio：
9. a kind of according to claim 1 or 6 detects T cell and B cell immune group storehouse simultaneously based on high-flux sequence
Method, it is characterised in that：In the step (6), the response procedures of PCR 2 are:
94 DEG C 3 minutes；94 DEG C 30 seconds；55 DEG C 30 seconds, 18 circulation；72 DEG C 20 minutes, 72 DEG C 1 minute, 4 DEG C are permanent.
10. a kind of side for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence according to claim 1
Method, it is characterised in that：In the step (6), specific purification step is as follows：
(1) 80 μ L AMPure XP Beads of addition enter PCR2 product, mix；
(2) in incubation at room temperature 10 minutes；
(3) magnetic bead-PCR2 product mixtures test tube is placed after on magnetic frame, waiting all magnetic beads to be adsorbed on magnetic frame, is used
Pipettor sucks all supernatants, abandons；
(4) the addition ethanol of 150 μ L 70% is hatched 30 seconds on magnetic bead, and all supernatants are sucked with pipettor, is abandoned；
(5) the 4th step is repeated 2 times；
(6) test tube cap is opened, is waited 5 minutes, treat that magnetic bead is air-dried, there is no any ethanol to residue in test tube；
(7) test tube is removed from magnetic frame, and adds 50 μ L and remove nuclease water, suspension magnetic bead is blown and beaten using pipettor；
(8) tube back magnetic frame, after waiting all magnetic beads to be all adsorbed in magnetic frame, transfer supernatant in new test tube,
The PCR2 products after purifying are just contained in supernatant.
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| CN107893068A (en) * | 2017-10-20 | 2018-04-10 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRbetaCDR3 areas library |
| CN108410856A (en) * | 2018-03-29 | 2018-08-17 | 武汉光谷创赢生物技术开发有限公司 | A kind of structure of full-length cDNA synthetic method and its sequencing library |
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| CN107893068A (en) * | 2017-10-20 | 2018-04-10 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRbetaCDR3 areas library |
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| CN109161592A (en) * | 2018-08-02 | 2019-01-08 | 深圳市人民医院 | A kind of kit and the application in IgA nephrosis |
| 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 |
| CN111363783A (en) * | 2018-12-26 | 2020-07-03 | 武汉康测科技有限公司 | T cell receptor library high-throughput sequencing library construction and sequencing data analysis method based on unique recognition sequence |
| CN111363783B (en) * | 2018-12-26 | 2024-01-02 | 武汉康测科技有限公司 | T cell receptor library high-throughput sequencing library construction and sequencing data analysis method based on specific recognition sequence |
| CN110734958A (en) * | 2019-10-13 | 2020-01-31 | 湖南大地同年生物科技有限公司 | Construction method of high-throughput sequencing library of monomolecular label immune repertoire |
| CN117126921A (en) * | 2023-10-26 | 2023-11-28 | 立凌生物制药(苏州)有限公司 | Library construction method for detecting T cell and B cell immune repertoire, primer and kit thereof |
| CN117126921B (en) * | 2023-10-26 | 2024-01-26 | 立凌生物制药(苏州)有限公司 | Library construction method for detecting T cell and B cell immune repertoire, primer and kit thereof |
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