CN105063032A - Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing - Google Patents

Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing Download PDF

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CN105063032A
CN105063032A CN201510500391.8A CN201510500391A CN105063032A CN 105063032 A CN105063032 A CN 105063032A CN 201510500391 A CN201510500391 A CN 201510500391A CN 105063032 A CN105063032 A CN 105063032A
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primer
seqidno
sequence
leukemia
multiple pcr
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葛良进
刘松
林群婷
刘丽春
曾立董
黄莎莎
黄亮
李改玲
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SHENZHEN HANHAI GENE BIOTECHNOLOGY CO Ltd
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Priority to CN201710085553.5A priority patent/CN106834472A/en
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Priority to PCT/CN2016/094892 priority patent/WO2017028752A1/en
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Abstract

The invention provides multiple PCR primers for constructing a leukemia minimal residual disease BCR library based on high-flux sequencing. The multiple PCR primers comprise upstream primers and downstream primers, wherein the upstream primers are an upstream primer group composed of nucleotide sequences which are 0-3 basic groups more or less than the nucleotide sequences shown in the SEQ ID NO: 1-SEQ ID NO: 13, and the downstream primers are composed of nucleotide sequences which are 0-3 basic groups more or less than the nucleotide sequences shown in the SEQ ID NO: 14-SEQ ID NO: 17. The provided multiple PCR primer group can efficiently construct the B lymphocyte receptor high-flux sequencing library of a leukemia patient, so that rich leukemia minimal residual disease BCR rearrangement information can be obtained.

Description

A kind of multiple PCR primer and method building Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence
Technical field
The invention belongs to biology field, particularly relate to a kind of multiple PCR primer and the method that build Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence.
Background technology
Leukemia (leukemia) increases blood system malignant clone disease for feature with juvenile cell in marrow and/or peripheral blood.When first visit, in patient body, leukemia total cellular score is about 10 12, after chemotherapy obtains morphologic complete incidence graph (CompleteRemission, CR), residual leukemia cell's sum is lower than 10 9, this morphological method be difficult to detect and in body still the state of remaining a small amount of leukemia cell be called Minimal Residual Disease of Leukemia (minimalresidualdisease, MRD).MRD residual in body makes acute T leukemic lymphoblastoid patient have >50% recurrence rate.Therefore want periodic detection MRD, the treatment plan of design individuation is seemed even more important.
On B cell locus, a large amount of V, J gene fragment can produce various various rearrangement in the synthesis of acceptor, at V-J, Nucleotide between V-D and D-J conjugant does not rely on template and inserts or delete, similar with high frequency closedown, which in turns increases the diversity that acceptor is potential.This potential diversity of acceptor is difficult to the identical CDR3 sequence of random generation, thus makes each CDR3 sequence effectively become the unique tags of a B cell clone.Therefore the sequence for bone-marrow-derived lymphocyte IGH gene C DR3 region forms composition and the response situation of carrying out order-checking and can well reflect BCR immune group storehouse.
The main detection method of MRD is clinically at present: multi-parameter Flow Cytometry (multiparametricflowcytometry, mpFC) and Real-time quantitative PCR.Although mpFC is 10 for the detection sensitivity of recurrent disease -4but the multidimensional data of complexity depends on experimenter and analyzes, and human factor impact is large, is unfavorable for that clinical criteriaization detects.In addition, after chemotherapy, the mpFC detection of expression level to MRD of leukemia antigen has interference effect.Depend on Molecular tools and can improve the sensitivity detecting MRD, can 10 be reached -5; But real-time quantitative PCR need design special primer according to patient and increase out by the retracing sequence that diversity is abundant, its testing cost costliness, labour intensive, very difficult formation standardized assay flow process.
Have benefited from fast development and the widespread use of high throughput sequencing technologies of future generation, DNA and RNA order-checking platform plays outstanding pushing effect in all respects of genomics.Therefore, a kind of multiple PCR primer and the method that build Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence is provided.
Summary of the invention
Given this, the invention provides a kind of multiple PCR primer and the method that build Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence.
First aspect, the invention provides a kind of multiple PCR primer building Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence, comprise upstream primer and downstream primer, the upstream primer group of the nucleotide sequence composition that described upstream primer is more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:13 or few 0 ~ 3 base;
The nucleotide sequence composition that described downstream primer is more than the nucleotide sequence shown in SEQIDNO:14 ~ SEQIDNO:17 or few 0 ~ 3 base.
As described in the present invention, " base " can represent Nucleotide, such as, during counting, represents 1 Nucleotide with 1bp.
As described in the present invention, " many or few 0 ~ 3 base ", is preferably in many or few 0 ~ 3 bases of 3 ' end of corresponding primer.
The present invention arranges at least 13 upstream primer sequences by the V district, variable region (variable) for people BCR, joining region (joining) J district for BCR arranges at least 4 downstream primer sequences, by multiplexed PCR amplification object fragment, obtain multiple PCR products and build BCR high-throughput sequencing library.
Preferably, described upstream primer is the upstream primer group of the nucleotide sequence composition shown in SEQIDNO:1 ~ SEQIDNO:13, and described downstream primer is the downstream primer group of the nucleotide sequence composition shown in SEQIDNO:14 ~ SEQIDNO:17.
As described in the present invention, described 1 ~ 3 base had more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:17 is the base with object BCR complementation.
As described in the present invention, it will be appreciated by those skilled in the art that, described " nucleotide sequence of more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:13 or SEQIDNO:14 ~ SEQIDNO:17 or few 0 ~ 3 base " is for those skilled in the art are when designing PCR primer, on the basis of such as " nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:17 ", suitable prolongation or brachymemma PCR primer length can obtain (prolongation still with corresponding object fragment complementation);
It will be appreciated by those skilled in the art that, if the multiple PCR primer group of groping (" nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:17 ") obtains preferably expanding effect, in predictable range, suitable to gained multiple PCR primer group after each bar primer extension or brachymemma 0 ~ 3 base, also can obtain preferably the Efficiency of Mutiplex PCR.
Preferably, 5 ' end of described downstream primer and 5 ' end of upstream primer comprise sequence label respectively, and described sequence label is the sequence bar code be made up of 6 ~ 8 nucleotide sequences, wherein, has a Nucleotide difference between described sequence bar code at least.
The primer of tape label sequence provided by the invention adds a sequence label can to each RNA molecule in testing sample or DNA molecular, described sequence label is by the basic base random combine of ATCG tetra-kinds, and described sequence label is different, such as, (in the embodiment of the present invention, be expressed as 8N sequence label) when the base number of sequence label is eight, can 10 be obtained 9individual different sequence labels combination; When the base number of sequence label is seven, can 10 be obtained 8individual different sequence labels combination; When the base number of sequence label is six, can 10 be obtained 7individual different sequence labels combination, the base number of described sequence label is eight, or seven, or six.
Second aspect, the invention provides a kind of multiple PCR method building Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence, comprises the steps:
Get testing sample;
Multi-PRC reaction amplification testing sample is adopted to obtain multiple PCR products, wherein, the primer sets that multi-PRC reaction adopts comprises upstream primer and downstream primer, and described upstream primer is the upstream primer group of the nucleotide sequence composition of more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:13 or few 0 ~ 3 base of 3 ' end;
The nucleotide sequence composition that described downstream primer is more than the nucleotide sequence shown in SEQIDNO:14 ~ SEQIDNO:17 or few 0 ~ 3 base of 3 ' end.
Preferably, described testing sample is DNA sample or RNA sample.
When described to get testing sample be DNA sample time, the system of carrying out multiplex PCR is configured with reference to regular-PCR system; When described to get testing sample be RNA sample time, first to carry out reverse transcription synthesis cDNA, resynthesis second chain DNA, now, the step of reverse transcription synthesis cDNA is equivalent to the multiplex PCR (only adopting upstream primer group or downstream primer group) of a circulation, and the step of synthesizing the second chain DNA is also equivalent to the multiplex PCR of a circulation (only adopting downstream primer group or upstream primer group).
Preferably, described RNA sample to be measured is the total serum IgE that (preferably adopting RNA test kit) extracts human peripheral blood single nucleus cell acquisition.
Preferably, when testing sample is RNA sample, described employing multi-PRC reaction, amplification testing sample, obtaining the step of multiple PCR products is: first with downstream primer sets for reverse transcription primer synthesizes cDNA; Then with synthesis cDNA for template, add upstream primer group, carry out multiplex PCR, amplification cDNA, obtain multiple PCR products.
Preferably, when testing sample is RNA sample, described employing multi-PRC reaction, amplification testing sample, obtaining the step of multiple PCR products is: first with upstream primer sets for reverse transcription primer synthesizes cDNA; Then with synthesis cDNA for template, add downstream primer group, carry out multiplex PCR, amplification cDNA, obtain multiple PCR products.
Preferably, described upstream primer is the upstream primer group of the nucleotide sequence composition shown in SEQIDNO:1 ~ SEQIDNO:13, and described downstream primer is the downstream primer group of the nucleotide sequence composition shown in SEQIDNO:14 ~ SEQIDNO:17.
Preferably, in the system of described multi-PRC reaction, in the upstream primer group of 13 upstream primer compositions, mole mixing such as each upstream primer; Article 4, in the downstream primer group that downstream primer forms, mole mixing such as each downstream primer.
Preferably, 5 ' end of described downstream primer and 5 ' end of upstream primer comprise sequence label respectively, and described sequence label is the sequence bar code be made up of 6 ~ 8 nucleotide sequences, wherein, has a Nucleotide difference between described sequence bar code at least.
Preferably, in the system of described multi-PRC reaction, template amount is 1 ~ 3ug/50ul system.
Preferably, the program of described multi-PRC reaction is:
Said procedure is specially: 95 DEG C of denaturation 15min, 94 DEG C of sex change 15s, and 65 DEG C of annealing 90s, 72 DEG C extend 30s, circulate 25 ~ 30 times, extend 10min after last 72 DEG C.
Preferably, after described multi-PRC reaction terminates, electrophoresis, it is the DNA fragmentation of 100-150bp that fragment length is reclaimed in rubber tapping.
The BCR sequence that the obtainable length of BCR high-throughput sequencing library provided by the invention and quantity are enriched, is conducive to polymorphism degree analyzing and the distributional analysis of BCR height clone CDR3 section length polymorphism of BCR sequence.
Preferably, after gained multiple PCR products being carried out build storehouse, carry out high-flux sequence, and by bioinformatic analysis high-flux sequence result.
On the basis of high-flux sequence platform, by carrying out comprehensive bioinformatic analysis to people BCR gene C DR3 region, obtain the gene Preference (usagepatterns) of BCR when VDJ recombinates, the assortment of genes, junctional diversity information, and in CDR3 sequence, the length diversity of amino acid whose Preference (usagepatterns), CDR3 aminoacid sequence and junction N hold the characteristic of base.Huge and the BCR receptoire of wide variety of these factor quantity of formations just.
The third aspect, the invention provides the multifarious test kit of a kind of detection Minimal Residual Disease of Leukemia stove BCR, comprises the multiple PCR primer building Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence as described in relation to the first aspect.
Fourth aspect, the invention provides a kind of multiple PCR method building Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence built described in the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library or second aspect based on high-flux sequence as described in relation to the first aspect and is detecting the application in Minimal Residual Disease of Leukemia stove BCR diversity.
Provided by the inventionly build the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library and the beneficial effect of method based on high-flux sequence and be: 1) obtain people BCR sequence; 2) obtain the BCRCDR3 sequence of human specific, especially improve the recall rate of low copy number B cell clone.
Accompanying drawing explanation
The agarose gel electrophoresis figure of the genome that Fig. 1 provides for the embodiment of the present invention and PCR primer;
Fig. 2 is the VDJ recombination analysis result of embodiment of the present invention gained sequence.
Embodiment
Material and reagent illustrate:
Bone-marrow-derived lymphocyte related leukemia patient: derive from Shenzhen people's hospital, patient's informed consent.No special illustrates, the reagent that the embodiment of the present invention adopts is commercial goods, and the database that the embodiment of the present invention adopts is disclosed online database.
Particularly, primer of the present invention following (underscore part is order-checking company joint sequence):
Table 1. multiple PCR primer sequence
Note, it will be appreciated by persons skilled in the art that Nucleotide abbreviation is as follows:
R＝A/G，Y＝C/T，M＝A/C，K＝G/T，S＝C/G，W＝A/T，H＝A/C/T，B＝C/G/T，V＝A/C/G，D＝A/G/T，N＝A/C/G/T。
Design primer: V and the J gene all for BCR has carried out compare of analysis, Oligo7.0 and MFEprimer-2.0 is adopted to analyze primer dimer and the mispairing of stem ring, upstream primer is provided with in the upstream, CDR3 district (i.e. FR3 district) of BCR, for J downstream of gene design reverse primer, amplification CDR3 regional sequence.
The primer sets that the present embodiment provides covers most of VDJ recombinant fragment.Because very little sequence variation will cause primer amplification Be very effective to reduce, contriver is respectively for different section designs 3 groups of multiple PCR primer groups in different object BCR region, after 3 groups of preliminary experiment screenings, the present invention have chosen the primer sets of expanding effect the best, as shown above.
Embodiment 1
The embodiment of the present invention 1 provides the preparation method of a kind of b lymphocyte receptor (BCR) DNA sample, comprises the steps:
(1) the fresh peripheral blood sample of collecting each 10 milliliters (ml), by the operation of LymphoPrep test kit (Axis-shield, Cat.No.AS1114544UK) specification sheets, obtains relatively pure PBMC;
(2) PureLinkGenomicDNAMiniKit (LifeTechnology is adopted, Cat.No:K1820-00) genomic dna of test kit extraction step (1) gained cell, and by concentration and purity that Nanodrop2000 (Thermo) measures DNA, then preserve genomic dna.
(genomic DNA fragment is see swimming lane 1-2 as shown in Fig. 1-a for DNA extraction electrophoresis result; M is DNAMarker).
Embodiment 2
The embodiment of the present invention 2 provides a kind of method adopting the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library to build Minimal Residual Disease of Leukemia stove BCR high-throughput sequencing library, comprises the steps:
With embodiment 1 gained genomic dna for amplification template, get BCR primer, adopt QIAGEN company MultiplexPCR test kit (article No.: 206143) again, by test kit specification sheets configuration multiplex PCR system, wherein, BCR primer comprises upstream primer and downstream primer, described upstream primer is the upstream primer group of the nucleotide sequence composition shown in SEQIDNO:1 ~ SEQIDNO:13, and described downstream primer is the downstream primer group of the nucleotide sequence composition shown in SEQIDNO:14 ~ SEQIDNO:17.
Mole mixing such as each upstream primer, total primer concentration is 10 micromoles, and mole mixing such as each downstream primer, total primer concentration is 10 micromoles, and template amount can adjust, and adopts 3ug in the present embodiment.
Send survey for convenience, unless otherwise noted, when the embodiment of the present invention carries out multiplex PCR, sequence measuring joints is added respectively at upstream primer and downstream primer, be specially: the upstream primer joint sequence (nucleotide sequence as shown in SEQIDNO:18) connecting illumina order-checking company at 5 ' end of upstream primer respectively, connect the downstream primer joint sequence (nucleotide sequence as shown in SEQIDNO:19) of illumina order-checking company respectively at 5 ' end of downstream primer, concrete steps build specification sheets with reference to illumina high-throughput sequencing library;
Again by the condition setting PCR instrument device program of following multiplex PCR, carry out multiplex PCR:
After PCR terminates, preserve PCR primer electrophoresis detection for 4 DEG C, result is as shown in Fig. 1-b, and under ultraviolet, cutting the object fragment of about about 250bp, (object fragment is see swimming lane 1-3; M is DNAMarker, and horizontal line is that the present invention more highlights interpolation to allow object fragment).
Select the CDR3 fragment that fragment length is about 250bp, rubber tapping is reclaimed, and obtains the CDR3 fragment after purifying, and glue recycling step adopts QIAGEN company QIAquick gel purification kit, and laboratory operation is carried out routinely; Nanodrop2000 test dna concentration, and send company to carry out high-flux sequence (adopting Illuminahiseq2000 order-checking, 2*100pair-end).
After adopting primer sets of the present invention and multiplex PCR structure storehouse, high-flux sequence obtains about 1,000,000 sequences.(bioinformatic analysis adopts the online software I mmuneRepertoireAnalysisPipeline (iRAP of southern University of Science and Technology sequencing result to be carried out information biology statistical study, http://www.sustc-genome.org.cn/irap/), part comparison result is as shown in table 2 below.Wherein, table 2 is CDR3unique clone's quantity and distribution, comprises table 2-1 and table 2-2.
Table 2-1.CDR3unique clones quantity and distribution
Total reads number 2031308 1987751 1438302 783456 960837 1073258
immune sequences number 1997429 1895548 1373941 757634 921853 1027270
Unkncwn sequences numebr 33879 92203 64361 25822 38984 45988
productive sequences number 1665974 1485444 1069650 617752 683431 630666
Kon_prcductive sequences number 331455 410104 304291 139882 238422 396604
In-frame sequences number 1742745 1554969 1130464 648423 751370 823125
Out-of_frame sequences number 249753 336743 240804 107677 168555 201943
Total CDR3 sequences number 1247543 1458146 1048736 607143 668171 615392
Unique cdr3 nt sequences number 97346 68127 103347 56940 76489 60181
Unique cdr3 aa sequences number 88832 56874 90119 50791 67745 52124
Table 2-2.CDR3unique clones quantity and distribution
CDR3 ID CDR3 Sequence(nt) CDR3 Sequence(aa) Reads urique Ratio
＞CG2_uniquecdr3nt_1 AGCGTGAGAGCGGTACAAGAGACCCAGTAC SVRAVQETQY 11553 6.54％
＞CG2_uniquecdr3nt_2 GCCACCAGTGATTTGCAGGGGGATGCCGGGAGCTGTTT ATSDLQGDAGELF 6669 3.78％
＞CG2_uniquecdr3nt_3 GCCAGCACTGGTCACCTAAATGAAAAACTGTTT ASTGHLNEKLF 6439 3.64％
＞CG2_uniquecdr3nt_4 GCCAGCAGCTTAGAGGCGCATGCGAACACCGGGGAGCTGTTT ASSLEAHANTGELF 5730 3.24％
＞CG2_uniquecdr3nt_5 GCTAGTGGTGCAGGGTTACTCTGGACTGAAGCTTTC ASGAGLLWTEAF 5673 3.21％
＞CG2_uniquecdr3nt_6 GCCACCAGCACACATACGTTGGCGGGGGGCCGGGGGGATACGCAGTAT ATSRDTLAGGRGDTQY 1907 1.08％
＞CG2_uniquecdr3nt_7 GCCAGCAGCTACACCTCGAACACCGGGGAGCTGTTT ASSYTSNTGELF 1743 0.99％
＞CG2_uniquecdr3nt_8 GCCAGCAGCTTATCCCAGGGCCAGGACACTGAAGCTTTC ASSLSQGQDTEAF 1154 0.65％
＞CG2_uniquecdr3nt_9 GCCACCAGTTGGGGCACCACCACCTACAATGAGCAGTTC ATSWGTTTYNEQF 1035 0.59％
＞CG2_uniquecdr3nt_10 GCCAGCAGCGAGCTGACAGGGGGGGGGCTCGAGCAGTAC ASSELTGGGLEQY 934 0.53％
By comparison and bioinformatic analysis, the sequence information of every bar CDR3 sequence can be known accurately, amino acid information, number and proportion.Through BCR compare of analysis, present invention obtains the statistic analysis result of high-flux sequence sequence C DR3 representativeness clone, as shown in Figure 2, Fig. 2 is that BCRCD3 district V-J combinationally uses situation to result.As shown in Figure 2, obtained by primer of the present invention in the BCR sequence of closely white ten thousand, the CDR3 sequence of unique is greater than 10 4bar.
The above results shows, utilizes method of the present invention structure Minimal Residual Disease of Leukemia stove BCR library can cover the diversity information of IGH gene.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. one kind builds the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence, it is characterized in that, comprise upstream primer and downstream primer, the upstream primer group of the nucleotide sequence composition that described upstream primer is more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:13 or few 0 ~ 3 base;
The nucleotide sequence composition that described downstream primer is more than the nucleotide sequence shown in SEQIDNO:14 ~ SEQIDNO:17 or few 0 ~ 3 base.
2. the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 1 based on high-flux sequence, it is characterized in that, 5 ' end of described downstream primer and 5 ' end of upstream primer comprise sequence label, described sequence label is the sequence bar code be made up of 6 ~ 8 nucleotide sequences, wherein, a Nucleotide difference is had at least between described sequence bar code.
3. the multiple PCR primer in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 1 based on high-flux sequence, it is characterized in that, described upstream primer is the upstream primer group of the nucleotide sequence composition shown in SEQIDNO:1 ~ SEQIDNO:13, and described downstream primer is the downstream primer group of the nucleotide sequence composition shown in SEQIDNO:14 ~ SEQIDNO:17.
4. build the multiple PCR method in Minimal Residual Disease of Leukemia stove BCR library based on high-flux sequence, it is characterized in that, comprise the steps:
Get testing sample;
Multi-PRC reaction amplification testing sample is adopted to obtain multiple PCR products, wherein, the primer sets that multi-PRC reaction adopts comprises upstream primer and downstream primer, and described upstream primer is the upstream primer group of the nucleotide sequence composition of more than the nucleotide sequence shown in SEQIDNO:1 ~ SEQIDNO:13 or few 0 ~ 3 base of 3 ' end;
The nucleotide sequence composition that described downstream primer is more than the nucleotide sequence shown in SEQIDNO:14 ~ SEQIDNO:17 or few 0 ~ 3 base of 3 ' end.
5. the multiple PCR method in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 4 based on high-flux sequence, it is characterized in that, 5 ' end of described downstream primer and 5 ' end of upstream primer comprise sequence label, described sequence label is the sequence bar code be made up of 6 ~ 8 nucleotide sequences, wherein, a Nucleotide difference is had at least between described sequence bar code.
6. the multiple PCR method in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 4 based on high-flux sequence, it is characterized in that, described upstream primer is the upstream primer group of the nucleotide sequence composition shown in SEQIDNO:1 ~ SEQIDNO:13, and described downstream primer is the downstream primer group of the nucleotide sequence composition shown in SEQIDNO:14 ~ SEQIDNO:17.
7. build the multiple PCR method in Minimal Residual Disease of Leukemia stove BCR library as claimed in claim 4 based on high-flux sequence, it is characterized in that, described testing sample is DNA sample or RNA sample.
8. the multiple PCR method in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 4 based on high-flux sequence, it is characterized in that, when testing sample is RNA sample, described employing multi-PRC reaction, amplification testing sample, the step obtaining multiple PCR products is: first above or downstream primer group is reverse transcription primer synthesis cDNA; Then with the cDNA of synthesis for template, add lower or upstream primer group, carry out multiplex PCR, amplification cDNA, obtains multiple PCR products.
9. detect the multifarious test kit of Minimal Residual Disease of Leukemia stove BCR, it is characterized in that, comprise the multiple PCR primer building Minimal Residual Disease of Leukemia stove BCR library as claimed in claim 1 based on high-flux sequence.
10. the application of multiple PCR primer in detection Minimal Residual Disease of Leukemia stove BCR diversity in Minimal Residual Disease of Leukemia stove BCR library is built as claimed in claim 1 based on high-flux sequence.
CN201510500391.8A 2015-08-14 2015-08-14 Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing Pending CN105063032A (en)

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RU2638800C2 (en) * 2015-12-29 2017-12-15 Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) Method for tumour b lymphoblasts availability markers determination
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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
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