WO2017028753A1 - 多重pcr引物及其应用 - Google Patents

多重pcr引物及其应用 Download PDF

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WO2017028753A1
WO2017028753A1 PCT/CN2016/094893 CN2016094893W WO2017028753A1 WO 2017028753 A1 WO2017028753 A1 WO 2017028753A1 CN 2016094893 W CN2016094893 W CN 2016094893W WO 2017028753 A1 WO2017028753 A1 WO 2017028753A1
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seq
primer
multiplex pcr
tcr
sequence
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French (fr)
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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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深圳市瀚海基因生物科技有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention belongs to the field of molecular biology, relates to multiplex PCR primers and applications thereof, and in particular to a multiplex PCR primer for amplifying human BCR and application thereof.
  • Leukemia is a malignant clonal disease of the blood system characterized by an increase in immature cells in bone marrow and/or peripheral blood.
  • the total number of leukemia cells in the patient is about 10 12 .
  • the total number of residual leukemia cells is less than 10 9 .
  • This morphological method is difficult to detect and in vivo.
  • the state in which a small amount of leukemia cells remain remains called minimal residual disease (MRD).
  • MRD minimal residual disease
  • Residual MRD in the body has a >50% recurrence rate in patients with acute T lymphocytic leukemia. Therefore, it is more important to design an individualized treatment plan to regularly detect MRD.
  • V and J gene segments at the T cell locus will produce various rearrangements in the synthesis of receptors. Nucleotides between VJ, VD and DJ conjugates are independent of template insertion or deletion, and high frequency variation. Similarly, this further increases the potential diversity of the receptor. This potential diversity of receptors is difficult to randomly generate the same CDR3 sequence, making each CDR3 sequence effectively a unique tag for a T cell clone. Therefore, sequencing the sequence composition of the CDR3 region of the T lymphocyte IGH gene can well reflect the composition and response status of the TCR immune pool.
  • mpFC multiparametric flow cytometry
  • mpFC has a sensitivity of 10 -4 for recurrent disease
  • complex multidimensional data relies on the analysis of experimental personnel, and human factors have a large impact, which is not conducive to clinical standardized testing.
  • the expression level of leukemia antigen after chemotherapy has an interference effect on mpFC detection of MRD.
  • Dependence on molecular means can improve the sensitivity of detecting MRD, which can reach 10 -5 ; however, real-time quantitative PCR needs to expand the diversity of rearranged sequences according to the special primers designed by patients, which is expensive to detect, labor intensive, and very It is difficult to form a standardized experimental process.
  • the present invention provides a multiplex PCR primer for amplifying human TCR and its use.
  • the present invention provides a multiplex PCR primer comprising an upstream primer and a downstream primer, the upstream primer being one-to-one corresponding to the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: a sequence consisting of the sequence of the upstream primer having 0 to 3 nucleotides more or less than the corresponding sequence in SEQ ID NO: 1 to SEQ ID NO: 25, and the downstream primer consisting of SEQ ID NO: 26
  • the nucleotide sequence shown in SEQ ID NO: 38 is composed of a set of sequences corresponding one to one, and the sequence in the downstream primer is more or less 0 to 3 than the corresponding sequence in SEQ ID NO: 26 to SEQ ID NO: 38. Nucleotides.
  • a "base” can represent a nucleotide, for example, when counting, 1 bp is used to represent 1 nucleotide.
  • more or less 0 to 3 nucleotides is preferably 0 to 3 nucleotides more or less at the 3' end of the corresponding primer.
  • the present invention sets at least 25 upstream primer sequences for the variable region V region of the human TCR, and at least 13 downstream primer sequences for the joining region J of the TCR, and the target strand is amplified by multiplex PCR.
  • the PCR product was used to obtain a high throughput sequencing library.
  • the present invention employs at least 13 downstream primer sequences and the at least 25 upstream primer sequences to randomly combine to form a paired primer, and then performs a multiplex PCR reaction to amplify the TCR CDR3 region.
  • the upstream primer is an upstream primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 25, and the downstream primers are SEQ ID NO: 26 to SEQ ID NO A downstream primer set consisting of the nucleotide sequence shown in :38.
  • the 1-3 extra bases which are more than the corresponding nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 38 are bases complementary to the TCR of interest.
  • a set of sequences one-to-one corresponding to the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 25 means that the set of sequences also contains 25 sequences, and the 25 sequences Each of the sequences is more or less 0 to 3 nucleotides than the corresponding sequence in SEQ ID NO: 1 to SEQ ID NO: 25.
  • the sequence of the set has a sequence of 0 to 3 nucleotides more or less than the sequence shown in SEQ ID NO: 1, and the other sequence of the set has a sequence other than the sequence shown in SEQ ID NO: 2. More or less sequences of 0 to 3 nucleotides...
  • the sequence of the group also has a sequence of 0 to 3 nucleotides more or less than the sequence shown in SEQ ID NO: 25, and the sequence consisting of 25 sequences
  • the group formed the upstream primer in the present invention.
  • the meaning of "a set of sequences one-to-one corresponding to the nucleotide sequences shown in SEQ ID NO: 26 to SEQ ID NO: 38" is similar to the above.
  • nucleotides are represented by SEQ ID NO: 1 to SEQ ID NO: 25 or SEQ ID NO: 26 to SEQ ID NO: 38. More or less 0 to 3 nucleotides in sequence", based on the corresponding nucleotide sequences shown in "SEQ ID NO: 1 to SEQ ID NO: 38", when a PCR primer is designed by those skilled in the art, Appropriate extension or truncation of the length of the PCR primer is obtained (the extension is still complementary to the corresponding fragment of interest).
  • the extension or truncation may be contiguous nucleotides or non-contiguous nucleotides, either extended or truncated at the ends or extended or truncated in the middle.
  • the ends are elongated or truncated.
  • the extension or truncation of nucleotides in the middle of the corresponding sequence can be continuously less 0-3 nucleotides, and may also be 0-3 nucleotides in succession.
  • multiplexed multiplex PCR primer set (“nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 38") obtains a better amplification effect, in a predictable range Further, a multiplex PCR primer set obtained by prolonging or truncating each primer by 0 to 3 bases can also obtain a preferable multiplex PCR amplification effect.
  • the 5' end of the downstream primer and/or the 5' end of the upstream primer respectively comprise a tag sequence
  • the tag sequence is a sequence bar code composed of 6-8 nucleotide sequences, wherein At least one nucleotide difference between the sequence barcodes is described.
  • the primer sequence-providing primer provided by the invention can add a tag sequence to each RNA molecule or DNA molecule in the sample to be tested, the tag sequence is randomly combined by four basic bases of ATCG, and the tag sequence is different from each other, e.g., the number of the nucleotide sequence of the tag is eight (example 8N tag sequence as represented embodiment of the present invention), may be obtained 109 different combinations of tag sequence; number seven nucleotide sequence tag At this time, 10 8 different combinations of tag sequences can be obtained; when the number of bases of the tag sequence is six, 10 7 different combinations of tag sequences can be obtained, and the number of bases of the tag sequence is eight. Or seven, or six.
  • the upstream primer further comprises an upstream primer set consisting of the nucleotide sequence shown in SEQ ID NO: 41 to SEQ ID NO: 65, and/or
  • the downstream primer further includes a downstream primer set consisting of the nucleotide sequences set forth in SEQ ID NO: 66 to SEQ ID NO: 78.
  • the present invention provides a method of obtaining a TCR, comprising the steps of:
  • the nucleic acid of the sample to be tested is taken; the nucleic acid is amplified by a multiplex PCR reaction to obtain a multiplex PCR product, wherein the multiplex PCR reaction is carried out using the multiplex PCR primer of the first aspect of the invention.
  • the sample to be tested is DNA and/or RNA.
  • the amount of the nucleic acid is not less than DNA or RNA contained in 0.5 cells.
  • the system for performing multiplex PCR is configured with reference to a common PCR system; when the nucleic acid of the sample to be tested is RNA, the cDNA is first synthesized by reverse transcription, and then synthesized into a second. Chain DNA, in this case, the step of reverse transcription synthesis of cDNA is equivalent to one cycle of multiplex PCR (using only the upstream primer set or the downstream primer set), and the step of synthesizing the second strand DNA is equivalent to one cycle of multiplex PCR (only downstream) Primer set or upstream primer set).
  • the sample to be tested is human peripheral blood mononuclear cells.
  • the RNA sample to be tested is a total RNA obtained by extracting human peripheral blood mononuclear cells (preferably using an RNA kit).
  • the sample to be tested is derived from a small residual lesion of human leukemia.
  • the multiplex PCR reaction is used to amplify the nucleic acid of the sample to be tested, and the step of obtaining the multiplex PCR product is: first, countering the downstream primer set The transcription primers are used to synthesize cDNA; then, using the synthesized cDNA as a template, an upstream primer set is added, and multiplex PCR is performed to amplify the cDNA to obtain a multiplex PCR product.
  • the multiplex PCR reaction is used to amplify the nucleic acid of the sample to be tested, and the step of obtaining the multiplex PCR product is: first, the upstream primer set The cDNA was synthesized as a reverse transcription primer; then the downstream primer set was added using the synthesized cDNA as a template, and multiplex PCR was performed to amplify the cDNA to obtain a multiplex PCR product.
  • each upstream primer is equimolar mixed; in the downstream primer set composed of 4 downstream primers, each downstream primer is equimolar mixed.
  • the 5' end of the downstream primer and the 5' end of the upstream primer respectively comprise a tag sequence, the tag sequence being a sequence bar code consisting of 6-8 nucleotide sequences, wherein the sequence bar code is between At least one nucleotide is different.
  • the amount of the template in the system of the multiplex PCR reaction, is 1-3 ug/50 ul system.
  • the procedure of the multiplex PCR reaction is:
  • the above procedure is specifically: pre-denaturation at 95 ° C for 15 min, denaturation at 94 ° C for 15 s, annealing at 65 ° C for 90 s, extension at 72 ° C for 30 s, cycle 25 to 30 times, and finally extension at 72 ° C for 10 min.
  • gel extraction recovers a DNA fragment having a fragment length of 100-150 bp.
  • the present invention provides a method of obtaining a TCR, comprising the steps of:
  • the multiplex PCR amplification product was subjected to sequencing library construction to obtain a TCR sequencing library.
  • the length and number of TCR sequences obtainable by the TCR high-throughput sequencing library provided by the invention are favorable for the polymorphism analysis of the TCR sequence and the distribution analysis of the length polymorphism of the TCR high clone CDR3 region.
  • the resulting multiplex PCR product is engineered for high throughput sequencing and high throughput sequencing results are analyzed by bioinformatics.
  • the present invention provides a method for sequencing a TCR library of a minimal residual disease of leukemia, comprising the steps of:
  • the TCR sequencing library was sequenced.
  • the present invention provides an analysis method for TCR diversity, comprising:
  • the sequencing results were analyzed to obtain analysis results of BCR diversity.
  • the sequencing is high throughput sequencing.
  • the invention provides a kit comprising a multiplex PCR primer according to the first aspect of the invention.
  • the kit can be used to detect TCR diversity, for example, to detect TCR diversity in minimal residual disease of leukemia.
  • the present invention provides a multiplex PCR primer according to the first aspect of the present invention or a method for obtaining a TCR according to the second aspect, for use in detecting BCR diversity, for example, in detecting a small residual lesion TCR of leukemia The application of diversity.
  • the multiplex PCR primers provided by the present invention and their applications have the following beneficial effects: 1) obtaining human TCR sequences; 2) obtaining human-specific TCR CDR3 sequences, especially increasing the detection rate of low copy number T cell clones.
  • FIG. 1 is an electrophoresis diagram of an agarose gel according to an embodiment of the present invention, wherein FIG. 1-a is genomic DNA; FIG. 1-b is a PCR product obtained by multiplex PCR;
  • the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
  • the nucleotide sequence shown by SEQ ID NO: 1 to SEQ ID NO: 38 in the present invention is a designed primer sequence.
  • the nucleotide sequences shown in SEQ ID NO: 39 to SEQ ID NO: 40 are the linker sequences used in the construction of the Chinese library of the examples; the nucleotide sequences shown in SEQ ID NO: 41 to SEQ ID NO: 78 are Primer sequences used in the inventive examples.
  • SEQ ID NO: 1 is ATTTCACTCTGAAGATCCGGTCC.
  • SEQ ID NO: 2 is CCTGACTTGCACTCTGAACTAAAC.
  • SEQ ID NO: 3 is GGAGGGACGTATTCTACTCTGAA.
  • SEQ ID NO: 4 is TTCTTGACATCCGCTCACCAG.
  • SEQ ID NO: 5 is TCCTTTCCTCTCACTGTGACATCG.
  • SEQ ID NO: 6 is AAACAGTTCCAAATCGMTTCTC.
  • SEQ ID NO: 7 is CAAGTCGCTTCTCACCTGAAT.
  • SEQ ID NO: 8 is GCCAGTTCTCTAACTCTCGCTC.
  • SEQ ID NO: 9 is TCAGGTCGCCAGTTCCCTAAYTA.
  • SEQ ID NO: 10 is CAATGGCTACAATGTCTCYAGA.
  • SEQ ID NO: 11 is TGATGGTTATAGTGTCTCCAGA.
  • SEQ ID NO: 12 is CGATGGCTACAATGTATCCAGAT.
  • SEQ ID NO: 13 is GGGATCCGTCTCCACTCTGAMG.
  • SEQ ID NO: 14 is GGGATCCGTCTCTACTCTGAAGA.
  • SEQ ID NO: 15 is GGGATCTTTCTCCACCTTGGAG.
  • SEQ ID NO: 16 is CCTCACTCTGGAGTCTGCT.
  • SEQ ID NO: 17 is CCTCACTCTGGAGTCMGCTA.
  • SEQ ID NO: 18 is GCAGAGAGGCTCAAAGGAGTAG.
  • SEQ ID NO: 19 is ATCGATTCTCAGCTAAGATGC.
  • SEQ ID NO: 20 is ATCGATTCTCAGCAGAGATGCCT.
  • SEQ ID NO: 21 is CTGTAGCCTTGAGATCCAGGCTAC.
  • SEQ ID NO: 22 is CAGATCAGCTCTGAGGTGC.
  • SEQ ID NO: 23 is GGAGATGTTCCTGARGGGTACA.
  • SEQ ID NO: 24 is GCCCTCACATACCTCTCAGTACC.
  • SEQ ID NO: 25 is CTCCCTGTCCCTAGAGTCTGCCAT.
  • SEQ ID NO:26 is CTTACCTACAACTGTGAGTCTGGTG.
  • SEQ ID NO:27 is CTTACCTACAACGGTTAACCTGGTC.
  • SEQ ID NO: 28 is CTTACCTACAACAGTGAGCCAACTT.
  • SEQ ID NO: 29 is CACATCCAAGACAGAGAGCTGGGTT.
  • SEQ ID NO: 30 is CTTACCTAGGATGGAGAGTCGAGTC.
  • SEQ ID NO: 31 is CAACTCTGTCACAGTGAGCCTG.
  • SEQ ID NO: 32 is CCTTCTTACCTAGCACGGTGA.
  • SEQ ID NO: 33 is CTTACCCAGTACGGTCAGC.
  • SEQ ID NO: 34 is CCGCTTACCGAGCACTGTC.
  • SEQ ID NO: 35 is CCAGCTTACCCAGCACTGA.
  • SEQ ID NO: 36 is CGAGCACCAGGAGCCGCGT.
  • SEQ ID NO: 37 is CTCGCCCAGCACGGTCAGCCT.
  • SEQ ID NO: 38 is CTTACCTGTGACCGTGAGC.
  • SEQ ID NO: 39 is CAGACGTGTGCTCTTCCGATCTAG .
  • SEQ ID NO: 40 is CTACACGACGCTCTTCCGATCT .
  • primers of the present invention are as follows (the underlined portion is the sequencing company linker sequence):
  • 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.
  • Primers were designed for the alignment of all V and J genes in TCR. Oligo 7.0 and MFEprimer-2.0 were used to analyze primer dimers and stem-loop mismatches, and were set upstream of the CDR3 region of the TCR (ie, FR3 region). The upstream primer, designed a reverse primer for the downstream of the J gene, amplifies the CDR3 region sequence.
  • the primer set provided in this example covers most of the VDJ recombination fragments. Since the small sequence changes will lead to a significant decrease in the amplification effect of the primers, the inventors designed two sets of multiplex PCR primer sets for different segments of the TCR region for different purposes. After two sets of pre-experiment screening, the present invention selects the expansion. The primer set with the best effect is shown in the above table.
  • Embodiment 1 of the present invention provides a method for preparing a T lymphocyte receptor (TCR) DNA sample, comprising the following steps:
  • the genomic DNA of the cells obtained in the step (1) was extracted using a PureLink Genomic DNA Mini Kit (Life Technology, Cat. No: K1820-00) kit, and the concentration and purity of the DNA were measured using Nanodrop 2000 (Thermo), and then the genomic DNA was preserved. .
  • the results of DNA extraction electrophoresis are shown in Figure 1-a (for genomic DNA fragments, see lanes 1-2; M is DNA Marker).
  • Embodiment 2 of the present invention provides a method for constructing a TCR high-throughput sequencing library of a minimal residual disease of leukemia using a multiplex PCR primer of a TCR library of a minimal residual disease of leukemia, comprising the following steps:
  • the TCR primer was taken, and the multiplex PCR system was configured according to the kit instructions using QIAGEN Multiplex PCR Kit (Cat. No. 206143), wherein the TCR primer included an upstream primer and a downstream primer.
  • the upstream primer is an upstream primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 25, and the downstream primer is a nucleoside represented by SEQ ID NO: 26 to SEQ ID NO: A downstream primer set consisting of an acid sequence.
  • Each of the upstream primers was equimolarly mixed, the total concentration of the primers was 10 micromoles, and the respective downstream primers were mixed in an equimolar amount, and the total concentration of the primers was 10 micromoles, and the amount of the template was adjusted, and 3 ug was used in this example.
  • the sequencing primer is added to the upstream primer and the downstream primer respectively, specifically: the upstream primer of the illumina sequencing company is respectively connected to the 5' end of the upstream primer.
  • a linker sequence such as the nucleotide sequence set forth in SEQ ID NO: 39
  • a downstream primer linker sequence of Illumina Sequencing Co., Ltd. at the 5' end of the downstream primer such as the nucleotide sequence set forth in SEQ ID NO: 40
  • the specific steps refer to the illumina high-throughput sequencing library construction specification;
  • the PCR instrument program is set up according to the following multiplex PCR conditions to perform multiplex PCR:
  • the PCR product was stored at 4 ° C and detected by electrophoresis.
  • the target fragment of about 250 bp was cut under ultraviolet light (see the lanes 1-4 for the target fragment; M is DNA Marker, the horizontal line is In order to make the target fragment more prominently added).
  • the library fragment with a fragment length of about 250 bp was selected, and the purified CDR3 fragment was obtained by tapping.
  • the gel recovery step was carried out by QIAGEN QIAquick gel purification kit according to routine laboratory procedures; Nanodrop 2000 was tested for DNA concentration and sent to the company for high concentration. Flux sequencing (sequencing with Illumina hiseq 2000, 2*100 pair-end).
  • CDR3ID CDR3 Sequence(nt) CDR3 Sequence(aa) Reads unique Ratio >C34_uniquecdr3nt_1 GCCAGCAGCGGTAACAGGGTAGGCACCGGGGAGCTGTTT ASSGNRVGTGELF 9933 4.95% >C34_uniquecdr3nt_2 GCCAGCAGCTTAACTAGCGGTTCGCAAGAGACCCAGTAC ASSLTSGSQETQY 3074 1.52% >C34_uniquecdr3nt_3 GCCACCAACGACAGGGGGACAGGGTCCGGGGAGCTGTTT ATNDRGTGSGELF 2985 1.48% >C34_uniquecdr3nt_4 GCCAGCAGTGAGCCTGGGATTAGCAATCAGCCCCAGCAT ASSEPGISNQPQH 1974 0.98% >C34_uniquecdr3nt_5 AGTGCTAGTGACCCCGGACTAGCGGGAGAGGGGAATGAGCAGTTC SASDPGLAGEGNEQF
  • Total reads number total sample comparison reads
  • Immune sequences numebr compares the number of reads to the target area
  • Unknown sequences numebr failed to compare the number of reads in the database
  • product sequencesnumber identification Number of reads that are efficiently translated for the TCR ⁇ chain
  • Non_productive sequences number the number of reads identified as TCR ⁇ chains but not efficiently translated
  • In-frame sequences number the number of reads identified as TCR ⁇ chains and still in the normal reading frame
  • -of_frame sequences number number of reads identified as TCR ⁇ chain but with frameshift mutation
  • Total CDR3sequences number total number of all sequences capable of detecting CDR3
  • Unique CDR3nt sequences number sequence of all CDR3 deduplicated base sequences Number of species
  • Unique CDR3aa sequences number The number of amino acid sequence species in which all CDR3 sequences are deduplicated.
  • the sequence information, amino acid information, number of fragments and proportion of each CDR3 sequence can be accurately known.
  • the present invention obtained statistical analysis results of representative clones of CDR3 of high-throughput sequencing sequence, and the results are shown in FIG. 2, and FIG. 2 shows the combined use of VJ in the TCR CD3 region.
  • the unique CDR3 sequences are more than 10 4 .
  • the TCR library of minimal residual disease of leukemia can be constructed by the method of the present invention to cover the diversity information of the CDR3 gene and improve the detection rate of low copy number T cell clones.

Abstract

提供一种多重PCR引物，包括上游引物和下游引物，所述上游引物由与SEQ ID NO：1～SEQ ID NO：25所示的核苷酸序列一一对应的一组序列组成，所述上游引物中的序列比SEQ ID NO：1～SEQ ID NO：25中的相应序列多或少0～3个核苷酸，所述下游引物由与SEQ ID NO：26～SEQ ID NO：38所示的核苷酸序列一一对应的一组序列组成，所述下游引物中的序列比SEQ ID NO：26～SEQ ID NO：38中的相应序列多或少0～3个核苷酸。

Description

多重PCR引物及其应用
本申请要求了2015年08月14日提交中国专利局的，申请号201510501106.4，发明名称为“一种基于高通量测序构建白血病微小残留病灶TCR文库的多重PCR引物和方法”的中国专利申请的优先权，上述在先申请的内容以引入的方式并入本申请中。
技术领域
本发明属于分子生物学领域，涉及多重PCR引物及其应用，特别地，涉及一种扩增人BCR的多重PCR引物及其应用。
背景技术
白血病(leukemia)是以骨髓和/或外周血中幼稚细胞增多为特征的血液系统恶性克隆性疾病。在初诊时，病人体内白血病细胞总数约为1012，经化疗取得形态学的完全缓解(Complete Remission，CR)后，残留的白血病细胞总数低于109，这种形态学方法难以检测、且体内仍残存着少量白血病细胞的状态称为白血病微小残留病(minimal residual disease，MRD)。体内残留的MRD使急性T淋巴白血病患者有>50％复发率。因此要定期检测MRD，对设计个体化的治疗方案显得更为重要。
T细胞基因座上大量V、J基因片段在受体的合成中会产生各种多样重排，在V-J，V-D和D-J接合体之间的核苷酸不依赖模板插入或删除，与高频变异类似，这进一步增加了受体潜在的多样性。受体的这种潜在多样性很难随机产生相同的CDR3序列，从而使每个CDR3序列有效地成为一个T细胞克隆的唯一标签。因此对于T淋巴细胞IGH基因CDR3区域的序列组成进行测序可以很好的反映TCR免疫组库的组成及应答状况。
目前临床上MRD的主要检测方法是：多参数流式细胞术(multiparametric flow cytometry，mpFC)和实时定量PCR技术。虽然mpFC对于复发疾病的检测灵敏度为10-4，但是复杂的多维数据依赖于实验人员分析，人为因素影响大，不利于临床标准化检测。此外，在化疗后白血病抗原的表达水平对MRD的mpFC检测具有干扰作用。依赖于分子手段可以提高检测MRD的灵敏度，可以达到10-5；然而，实时定量PCR需根据患者设计特殊的引物将多样性丰富的重排序列扩增出来，其检测费用昂贵、劳动力密集、很难形成标准化实验流程。
目前，人的TCR或获得或分析方法仍有待改进。
发明内容
有鉴于此，本发明提供了一种扩增人TCR的多重PCR引物及其应用。
第一方面，本发明提供了一种多重PCR引物，包括上游引物和下游引物，所述上游引物由与SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列一一对应的一组序列组成，所述上游引物中的序列比SEQ ID NO:1～SEQ ID NO:25中的相应序列多或少0～3个核苷酸，所述下游引物由与SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列一一对应的一组序列组成，所述下游引物中的序列比SEQ ID NO:26～SEQ ID NO:38中的相应序列多或少0～3个核苷酸。
如本发明所述的，“碱基”可代表核苷酸，比如，计数时，用1bp代表1个核苷酸。
如本发明所述的，“多或少0～3个核苷酸”，优选为在对应引物的3’端多或少0～3个核苷酸。
本发明通过针对人TCR的可变区V区(variable)设置至少25条上游引物序列，针对TCR的连接区(joining)J区设置至少13条下游引物序列，通过多重PCR扩增出目的链的PCR产物，获得高通量测序文库。
本发明采用至少13条下游引物序列与所述至少25条上游引物序列随机组合成配对引物，然后进行多重PCR反应，扩增TCR CDR3区。
根据本发明一实施例，所述上游引物为SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列组成的上游引物组，所述下游引物为SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列组成的下游引物组。
如本发明所述的，所述比SEQ ID NO:1～SEQ ID NO:38所示的相应核苷酸序列多出的1～3个碱基为与目的TCR互补的碱基。
本发明中，“由与SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列一一对应的一组序列”，是指该组序列也包含25条序列，而且该25条序列中的每条序列比SEQ ID NO:1～SEQ ID NO:25中的相应序列多或少0～3个核苷酸。例如，该组序列中有一条序列比SEQ ID NO:1所示的序列多或少0～3个核苷酸的序列，该组序列中有另一条序列比SEQ ID NO:2所示的序列多或少0～3个核苷酸的序列…该组序列还有一条序列比SEQ ID NO:25所示的序列多或少0～3个核苷酸的序列，这25个序列构成的序列组形成了本发明中的上游引物。同样地，“由与SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列一一对应的一组序列”所代表的含义与上述类似。
如本发明所述的，本领域技术人员可以理解的，所述的“比SEQ ID NO:1～SEQ ID NO:25或SEQ ID NO:26～SEQ ID NO:38所示的相应核苷酸序列多或少0～3个核苷酸”为本领域技术人员在设计PCR引物时，在如“SEQ ID NO:1～SEQ ID NO:38所示的相应核苷酸序列”的基础上，适当的延长或截短PCR引物长度即可获得(延长部分仍然与相应的目的片段互补)。所述延长或截短可以是连续核苷酸的，也可以是不连续核苷酸的，可以在末端延长或截短也可以在中间延长或截短。根据本发明的一个实施例，在末端延长或截短。根据本发明的另一个实施例，在相应序列的中间进行延长或截短核苷酸，可以连续少 0-3个核苷酸，也可以连续多0-3个核苷酸。
本领域技术人员可以理解的，若摸索的多重PCR引物组(“SEQ ID NO:1～SEQ ID NO:38所示的核苷酸序列”)获得了较佳的扩增效果，在可预测范围内，适当的将各条引物延长或截短0～3个碱基后所得多重PCR引物组，也可以获得较佳的多重PCR扩增效果。
本发明实施方式中，所述下游引物的5’端和/或上游引物的5’端分别包括标签序列，所述标签序列为由6～8个核苷酸序列组成的序列条码，其中，所述序列条码之间至少有一个核苷酸不同。
本发明提供的带标签序列的引物可以给待测样品中的每个RNA分子或DNA分子都加上了一个标签序列，所述标签序列由ATCG四种基本碱基随机组合，且所述标签序列互不相同，例如，标签序列的碱基个数是八个时(本发明实施例中表示为8N标签序列)，可以获得109个不同的标签序列组合；标签序列的碱基个数是七个时，可以获得108个不同的标签序列组合；标签序列的碱基个数是六个时，可以获得107个不同的标签序列组合，所述标签序列的碱基个数是八个，或者七个，或者六个。
根据本发明一实施例，所述上游引物还包括SEQ ID NO:41～SEQ ID NO:65所示的核苷酸序列组成的上游引物组，和/或，
所述下游引物还包括SEQ ID NO:66～SEQ ID NO:78所示的核苷酸序列组成的下游引物组。
第二方面，本发明提供了一种获得TCR的方法，包括如下步骤：
取待测样品的核酸；采用多重PCR反应对所述核酸进行扩增，以获得多重PCR产物，其中，所述多重PCR反应利用本发明第一方面所述的多重PCR引物来进行。
本发明实施方式中，所述待测样品为DNA和/或RNA。
本发明实施方式中，所述核酸的量不少于0.5个细胞中包含的DNA或RNA。
当所述取待测样品的核酸为DNA时，进行多重PCR的体系参照普通PCR体系进行配置；当所述取待测样品的核酸为RNA时，要先进行逆转录合成cDNA，再合成第二链DNA，此时，逆转录合成cDNA的步骤相当于一个循环的多重PCR(只采用上游引物组或下游引物组)，合成第二链DNA的步骤也相当于一个循环的多重PCR(只采用下游引物组或上游引物组)。
根据本发明一实施例，所述待测样本为人外周血单个核细胞。
本发明实施方式中，所述待测RNA样品为(优选采用RNA试剂盒)提取人外周血单个核细胞获得的总RNA。
根据本发明一实施例，所述待测样本来自人白血病微小残留病灶。
在本发明一实施方式中，当待测样品的核酸为RNA，所述的采用多重PCR反应，扩增待测样品的核酸，获得多重PCR扩增产物的步骤为：先以下游引物组为反转录引物合成cDNA；然后以合成的cDNA为模板，加入上游引物组，进行多重PCR，扩增cDNA，获得多重PCR扩增产物。
在本发明另一实施方式中，当待测样品的核酸为RNA品，所述的采用多重PCR反应，扩增待测样品的核酸，获得多重PCR扩增产物的步骤为：先以上游引物组为反转录引物合成cDNA；然后以合成的cDNA为模板，加入下游引物组，进行多重PCR，扩增cDNA，获得多重PCR扩增产物。
优选地，所述多重PCR反应的体系中，13条上游引物组成的上游引物组中，各上游引物等摩尔混合；4条下游引物组成的下游引物组中，各下游引物等摩尔混合。
优选地，所述下游引物的5’端和上游引物的5’端分别包括标签序列，所述标签序列为由6～8个核苷酸序列组成的序列条码，其中，所述序列条码之间至少有一个核苷酸不同。
根据本发明一实施例，所述多重PCR反应的体系中，模板量为1～3ug/50ul体系。
根据本发明一实施例，所述多重PCR反应的程序为：
Figure PCTCN2016094893-appb-000001
上述程序具体为：95℃预变性15min，94℃变性15s，65℃退火90s，72℃延伸30s，循环25～30次，最后72℃后延伸10min。
优选地，所述多重PCR反应结束后，电泳，割胶回收片段长度为100-150bp的DNA片段。
第三方面，本发明提供了获得TCR的方法，包括如下步骤：
利用如本发明第二方面所述的获得TCR的方法获得多重PCR扩增产物；
对所述多重PCR扩增产物进行测序文库构建，以获得TCR测序文库。
本发明提供的TCR高通量测序文库能获得的长度和数量丰富的TCR序列，有利于TCR序列的多态性程度分析及TCR高克隆CDR3区长度多态性分布分析。
优选地，将所得多重PCR产物进行建库后进行高通量测序，并通过生物信息学分析高通量测序结果。
第四方面，本发明提供了对白血病微小残留病灶TCR文库进行测序的方法，包括如下步骤：
利用本发明第三方面所述的获得TCR测序文库的方法获得TCR测序文库；
对所述TCR测序文库进行测序。
第五方面，本发明提供了一种TCR多样性的分析方法，包括：
利用本发明第四方面所述的测序方法获得测序结果；
对所述测序结果进行分析，以获得BCR多样性的分析结果。
优选地，所述测序为高通量测序。
在高通量测序平台的基础上，通过对人TCR基因CDR3区域进行全面的生物信息学分析，获得了TCR在VDJ重组时的基因偏好性(usage patterns)，基因组合、连接多样性信息，以及CDR3序列中氨基酸的偏好性(usage patterns)、CDR3氨基酸序列的长度多样性以及连接处N端碱基的特性。正是这些因素形成数量庞大且种类多样的TCR受体库。
第六方面，本发明提供了一种试剂盒，包括如本发明第一方面所述的多重PCR引物。
所述试剂盒可应用于检测TCR多样性，例如用于检测白血病微小残留病灶TCR多样性。
第七方面，本发明提供了一种如本发明第一方面所述的多重PCR引物或第二方面所述的获得TCR的方法在检测BCR多样性中的应用，例如在检测白血病微小残留病灶TCR多样性中的应用。
本发明提供的多重PCR引物及其应用的有益效果为：1)获得了人TCR序列；2)获得了人特异性的TCR CDR3序列，尤其提高了低拷贝数T细胞克隆的检出率。
附图说明
图1为本发明实施例提供的琼脂糖凝胶电泳图，其中，图1-a为基因组DNA；图1-b为多重PCR所得的PCR产物；
图2为本发明实施例所得序列的VDJ重组分析结果。
具体实施方式
材料及试剂说明：
T淋巴细胞相关白血病患者：来源于深圳市人民医院，患者知情同意。非特殊说明，本发明实施例采用的试剂均为市售商品，本发明实施例采用的数据库均为公开的在线数据库。
本发明中SEQ ID NO:1～SEQ ID NO:38所示的核苷酸序列为设计的引物序列。SEQ ID NO:39～SEQ ID NO:40所示的核苷酸序列为实施例中文库构建时使用的接头序列；SEQ ID NO:41～SEQ ID NO:78所示的核苷酸序列为本发明实施例中使用的引物序列。
其中，SEQ ID NO：1为ATTTCACTCTGAAGATCCGGTCC。SEQ ID NO：2为CCTGACTTGCACTCTGAACTAAAC。SEQ ID NO：3为GGAGGGACGTATTCTACTCTGAA。
SEQ ID NO：4为TTCTTGACATCCGCTCACCAG。SEQ ID NO：5为TCCTTTCCTCTCACTGTGACATCG。SEQ ID NO：6为AAACAGTTCCAAATCGMTTCTC。
SEQ ID NO：7为CAAGTCGCTTCTCACCTGAAT。SEQ ID NO：8为GCCAGTTCTCTAACTCTCGCTC。SEQ ID NO：9为TCAGGTCGCCAGTTCCCTAAYTA。
SEQ ID NO：10为CAATGGCTACAATGTCTCYAGA。SEQ ID NO：11为TGATGGTTATAGTGTCTCCAGA。SEQ ID NO：12为CGATGGCTACAATGTATCCAGAT。
SEQ ID NO：13为GGGATCCGTCTCCACTCTGAMG。SEQ ID NO：14为GGGATCCGTCTCTACTCTGAAGA。SEQ ID NO：15为GGGATCTTTCTCCACCTTGGAG。
SEQ ID NO：16为CCTCACTCTGGAGTCTGCT。SEQ ID NO：17为CCTCACTCTGGAGTCMGCTA。
SEQ ID NO：18为GCAGAGAGGCTCAAAGGAGTAG。SEQ ID NO：19为ATCGATTCTCAGCTAAGATGC。SEQ ID NO：20为ATCGATTCTCAGCAGAGATGCCT。
SEQ ID NO：21为CTGTAGCCTTGAGATCCAGGCTAC。SEQ ID NO：22为CAGATCAGCTCTGAGGTGC。SEQ ID NO：23为GGAGATGTTCCTGARGGGTACA。
SEQ ID NO：24为GCCCTCACATACCTCTCAGTACC。SEQ ID NO：25为CTCCCTGTCCCTAGAGTCTGCCAT。SEQ ID NO：26为CTTACCTACAACTGTGAGTCTGGTG。
SEQ ID NO：27为CTTACCTACAACGGTTAACCTGGTC。SEQ ID NO：28为CTTACCTACAACAGTGAGCCAACTT。SEQ ID NO：29为CATACCCAAGACAGAGAGCTGGGTT。
SEQ ID NO：30为CTTACCTAGGATGGAGAGTCGAGTC。SEQ ID NO：31为CATACCTGTCACAGTGAGCCTG。SEQ ID NO：32为CCTTCTTACCTAGCACGGTGA。
SEQ ID NO：33为CTTACCCAGTACGGTCAGC。SEQ ID NO：34为CCGCTTACCGAGCACTGTC。
SEQ ID NO：35为CCAGCTTACCCAGCACTGA。SEQ ID NO：36为CGAGCACCAGGAGCCGCGT。
SEQ ID NO：37为CTCGCCCAGCACGGTCAGCCT。SEQ ID NO：38为CTTACCTGTGACCGTGAGC。SEQ ID NO：39为CAGACGTGTGCTCTTCCGATCTAG。SEQ ID NO：40为CTACACGACGCTCTTCCGATCT。
具体地，本发明引物如下(下划线部分为测序公司接头序列)：
表1.多重PCR引物序列
Figure PCTCN2016094893-appb-000002
Figure PCTCN2016094893-appb-000003
Figure PCTCN2016094893-appb-000004
注，本领域技术人员可以理解的是，核苷酸缩写如下：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。
设计引物：针对TCR所有的V和J基因进行了比对分析，采用Oligo 7.0和MFEprimer-2.0对引物二聚体以及茎环错配进行分析，在TCR的CDR3区上游(即FR3区)设置了上游引物，针对J基因下游设计反向引物，扩增CDR3区域序列。
本实施例提供的引物组覆盖了大部分VDJ重组片段。由于很小的序列变化将导致引物扩增效果显著降低，发明人分别针对不同目的TCR区域的不同区段设计了2组多重PCR引物组，在经过2组预实验筛选后，本发明选取了扩增效果最佳的引物组，如上表所示。
实施例1
本发明实施例1提供了一种T淋巴细胞受体(TCR)DNA样品的制备方法，包括如下步骤：
(1)收集的新鲜外周血样本各10毫升(ml)，按LymphoPrep试剂盒(Axis-shield,Cat.No.AS1114544UK)说明书操作，获得相对较纯的PBMC；
(2)采用PureLink Genomic DNA Mini Kit(Life Technology,Cat.No:K1820-00)试剂盒提取步骤(1)所得细胞的基因组DNA，并用Nanodrop2000(Thermo)测定DNA的浓度及纯度，然后保存基因组DNA。 DNA提取电泳结果如图1-a所示(基因组DNA片段参见泳道1-2；M是DNA Marker)。
实施例2
本发明实施例2提供了一种采用白血病微小残留病灶TCR文库的多重PCR引物构建白血病微小残留病灶TCR高通量测序文库的方法，包括如下步骤：
以实施例1所得基因组DNA为扩增模板，取TCR引物，再采用QIAGEN公司Multiplex PCR试剂盒(货号：206143)，按试剂盒说明书配置多重PCR体系，其中，TCR引物包括上游引物和下游引物，所述上游引物为SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列组成的上游引物组，所述下游引物为SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列组成的下游引物组。
各上游引物等摩尔混合，引物总浓度是10微摩尔，各下游引物等摩尔混合，引物总浓度是10微摩尔，模板量可以调整，本实施例中采用3ug。
为方便送测，若无特别说明，本发明实施例进行多重PCR时，分别在上游引物和下游引物加上测序接头，具体为：在上游引物的5’端分别接上illumina测序公司的上游引物接头序列(如SEQ ID NO:39所示的核苷酸序列)，在下游引物的5’端分别接上illumina测序公司的下游引物接头序列(如SEQ ID NO:40所示的核苷酸序列)，具体步骤参照illumina高通量测序文库构建说明书；
再按下述多重PCR的条件设置PCR仪器程序，进行多重PCR：
Figure PCTCN2016094893-appb-000005
PCR结束后，4℃保存PCR产物并电泳检测，结果如图1-b所示，在紫外下切下约250bp左右的目的片段(目的片段参见泳道1-4；M是DNA Marker，横线是本发明为了让目的片段更突显添加的)。
挑选片段长度约为250bp的文库片段，割胶回收，得到纯化后的CDR3片段，胶回收步骤采用QIAGEN公司QIAquick胶纯化试剂盒，按常规实验室操作进行；Nanodrop 2000测试DNA浓度，并送公司进行高通量测序(采用Illumina hiseq2000测序，2*100pair-end)。
采用本发明的引物组以及多重PCR构库后，高通量测序得到大约百万条序列。将测序结果进行生物信息学统计分析(生物信息学分析采用南方科技大学在线软件Immune Repertoire Analysis Pipeline(iRAP，http://www.sustc-genome.org.cn/irap/)，部分比对结果如下表2所示。其中，表2为CDR3unique克隆数量及分布，包括表2-1和表2-2。
表2-1.CDR3unique克隆数量及分布
Total reads number 632578 1446220 1290247 819400
immune sequences number 530520 1372065 1202415 744817
Unknown sequences numebr 102058 74155 87832 74583
productive sequences number 407301 1028722 853018 513292
Non_productive sequences number 123219 343343 349397 231525
In-frame sequences number 428029 1078737 928999 541143
Out-of_frame sequences number 100555 287854 269823 200430
Total CDR3 sequences number 398774 1003918 829923 493541
Unique cdr3 nt sequences number 19841 59699 77170 48323
Unique cdr3 aa sequences number 17896 50188 66465 41578
表2-2.CDR3unique克隆数量及分布
CDR3ID CDR3 Sequence(nt) CDR3 Sequence(aa) Reads unique Ratio
＞C34_uniquecdr3nt_1 GCCAGCAGCGGTAACAGGGTAGGCACCGGGGAGCTGTTT ASSGNRVGTGELF 9933 4.95％
＞C34_uniquecdr3nt_2 GCCAGCAGCTTAACTAGCGGTTCGCAAGAGACCCAGTAC ASSLTSGSQETQY 3074 1.52％
＞C34_uniquecdr3nt_3 GCCACCAACGACAGGGGGACAGGGTCCGGGGAGCTGTTT ATNDRGTGSGELF 2985 1.48％
＞C34_uniquecdr3nt_4 GCCAGCAGTGAGCCTGGGATTAGCAATCAGCCCCAGCAT ASSEPGISNQPQH 1974 0.98％
＞C34_uniquecdr3nt_5 AGTGCTAGTGACCCCGGACTAGCGGGAGAGGGGAATGAGCAGTTC SASDPGLAGEGNEQF 1946 0.96％
＞C34_uniquecdr3nt_6 AGTGCTAGAGATGCCAGACTAGCGGGAGCGGTCGGGGAGCTGTTT SARDARLAGAVGELF 1886 0.93％
＞C34_uniquecdr3nt_7 GCCAGTGGGTCGTATCTGGGGCAGTTC ASGSYLGQF 1822 0.90％
＞C34_uniquecdr3nt_8 GCCAGCAGTGGCAGCGGGAGTGGGGAGACCCAGTAC ASSGSGSGETQY 1512 0.75％
＞C34_uniquecdr3nt_9 GCCAGCAGTGGGACAGGGTCGGGAGAGACCCAGTAC ASSGTGSGETQY 1244 0.62％
＞C34_uniquecdr3nt_10 GCCAGCAGCCCCATGGGTGATGAAAAACTGTTT ASSPMGDEKLF 1226 0.61％
上表2-1中，Total reads number：样本总比对reads数；Immune sequences numebr：比对到目标区域的reads数；Unknown sequences numebr：未能在数据库成功比对的reads数；productive sequencesnumber：识别为TCRβ链并能有效翻译的reads数；Non_productive sequences number：识别为TCRβ链但并不能有效翻译的reads数；In-frame sequences number：识别为TCRβ链并且还在正常阅读框内的reads数；Out-of_frame sequences number：识别为TCRβ链但发生了移码突变的reads数；Total CDR3sequences number：所有能检测到CDR3的序列总数；Unique CDR3nt sequences number：所有的CDR3的序列去冗余后的碱基序列种类数；Unique CDR3aa sequences number：所有的CDR3的序列去冗余后的氨基酸序列种类数。
通过比对和生物信息学分析，可以准确地知道每条CDR3序列的序列信息，氨基酸信息，条数以及所占比例。经过TCR比对分析，本发明获得了高通量测序序列CDR3代表性克隆的统计分析结果，结果如图2所示，图2为TCR CD3区V-J组合使用情况。由图2可知，通过本发明的引物获得近白万条的TCR序列中，unique的CDR3序列大于104条。
上述结果表明，利用本发明的方法构建白血病微小残留病灶TCR文库能够覆盖CDR3基因的多样性信息，提高低拷贝数T细胞克隆的检出率。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (16)

  1. 一种多重PCR引物，其特征在于，包括上游引物和下游引物，所述上游引物由与SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列一一对应的一组序列组成，所述上游引物中的序列比SEQ ID NO:1～SEQ ID NO:25中的相应序列多或少0～3个核苷酸，
    所述下游引物由与SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列一一对应的一组序列组成，所述下游引物中的序列比SEQ ID NO:26～SEQ ID NO:38中的相应序列多或少0～3个核苷酸。
  2. 如权利要求1所述的多重PCR引物，其特征在于，所述下游引物的5’端和/或上游引物的5’端包括标签序列，所述标签序列为由6～8个核苷酸序列组成的序列条码，其中，所述序列条码之间至少有一个核苷酸不同。
  3. 如权利要求1所述的多重PCR引物，其特征在于，所述上游引物为SEQ ID NO:1～SEQ ID NO:25所示的核苷酸序列组成的上游引物组，所述下游引物为SEQ ID NO:26～SEQ ID NO:38所示的核苷酸序列组成的下游引物组。
  4. 如权利要求1-3任一所述的多重PCR引物，其特征在于，所述上游引物还包括SEQ ID NO:41～SEQ ID NO:65所示的核苷酸序列组成的上游引物组，和/或，
    所述下游引物还包括SEQ ID NO:66～SEQ ID NO:78所示的核苷酸序列组成的下游引物组。
  5. 一种获得TCR的方法，其特征在于，包括如下步骤：
    获得待测样品的核酸；
    采用多重PCR反应对所述核酸进行扩增，以获得多重PCR扩增产物，其中，所述多重PCR反应利用权利要求1-4任一项所述的多重PCR引物来进行。
  6. 如权利要求5所述的获得TCR的方法，其特征在于，所述待测样品的核酸为DNA和/或RNA。
  7. 如权利要求5所述的获得TCR的方法，其特征在于，所述核酸的量不少于0.5个细胞中包含的DNA和/或RNA。
  8. 如权利要求5所述的获得TCR的方法，其特征在于，所述待测样本为人外周血单个核细胞。
  9. 如权利要求5所述的获得BCR的方法，其特征在于，所述待测样本来自人白血病微小残留病灶。
  10. 如权利要求5所述的获得TCR的方法，其特征在于，当待测样品的核酸为RNA时，所述采用多重PCR反应对所述核酸进行扩增，以获得多重PCR扩增产物的步骤为：
    以上游引物或下游引物为反转录引物进行反转录，获得cDNA产物；
    以所述cDNA产物为模板，加入相应的下游引物或上游引物，进行多重PCR反应，获得所述多重PCR扩增产物。
  11. 一种获得TCR测序文库的方法，其特征在于，包括如下步骤：
    利用权利要求5-10任一项所述的方法获得多重PCR扩增产物；
    对所述多重PCR扩增产物进行测序文库构建，以获得TCR测序文库。
  12. 一种对TCR进行测序的方法，其特征在于，包括：
    利用权利要求11的方法获得TCR测序文库；
    对所述TCR测序文库进行测序。
  13. 一种TCR多样性的分析方法，其特征在于，包括：
    利用权利要求12的方法，获得测序结果；
    对所述测序结果进行分析，以获得TCR多样性的分析结果。
  14. 如权利要求1-4任一项所述的白血病微小残留病灶TCR文库的多重PCR引物在检测白血病微小残留病灶TCR多样性中的用途。
  15. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1-4任一项所述的多重PCR引物。
  16. 权利要求15的试剂盒在检测TCR多样性中的用途。
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