WO2019061196A1 - 一种用于检测的pcr引物及其应用 - Google Patents

一种用于检测的pcr引物及其应用 Download PDF

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WO2019061196A1
WO2019061196A1 PCT/CN2017/104105 CN2017104105W WO2019061196A1 WO 2019061196 A1 WO2019061196 A1 WO 2019061196A1 CN 2017104105 W CN2017104105 W CN 2017104105W WO 2019061196 A1 WO2019061196 A1 WO 2019061196A1
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primer
pcr
sequence
primers
round
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PCT/CN2017/104105
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French (fr)
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周清
王飞
王磊
尹悦露
吴靓
赵正琦
李贵波
杨乃波
侯勇
李波
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深圳华大生命科学研究院
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Priority to PCT/CN2017/104105 priority Critical patent/WO2019061196A1/zh
Priority to CN201780095491.4A priority patent/CN111148836A/zh
Publication of WO2019061196A1 publication Critical patent/WO2019061196A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • the invention belongs to the field of molecular biology, relates to a PCR primer for detection and application thereof, and particularly relates to a PCR primer for detecting a target fragment of a single cell, a method for detecting a target fragment and an application thereof.
  • Cells in the same tissue are often considered to be functional units with the same state, and traditional assays analyze the average response of the cell population. However, it is necessary to understand the dynamic process of the population more accurately, and it is necessary to analyze individual cells.
  • sequencing the DNA or RNA of a single cell it is shown that the function at the tissue system level is composed of heterogeneous cells.
  • Single-cell sequencing in a single cell unit through whole genome or transcriptome amplification, high-throughput sequencing, can solidify the genetic structure and gene expression status of individual cells, reflect intercellular heterogeneity, in tumors, developmental biology Microbiology, neuroscience and other fields play an important role and are becoming the focus of life science research.
  • the DNA or RNA in a cell is only at the picograms level, at a level of 10 pg, so much that it is far from the minimum loading requirements of existing sequencers. Therefore, it is necessary to amplify a small amount of nucleic acid molecules in a single cell.
  • the SMART-seq method is a better method for full-length RNA-seq, and the principle is as follows: (1) The reverse transcription is initiated by "primer amplification primers with PCR (poly) with poly(T)". This ensures that the reverse transcription starts from the poly(A) position at the 3' end of the template, and the reverse transcription product is ligated with a downstream primer sequence that can be PCR amplified; (2) reversed with SMARTscribe RT enzyme. Transcription, this enzyme has a characteristic: it will extend several C bases beyond the template at the end of transcription; (3) using "primers with 3 g-PCR upstream amplification sequences" and reverse transcription The first strand is bonded, and the three g bases here are RNA bases, not DNA bases.
  • PCR-amplified upstream primer sequence is also introduced into the reverse transcription product; (4) after the PCR primer sequence is introduced both upstream and downstream, PCR Can be carried out efficiently; (5) PCR products are built and further sequenced.
  • the library can be subjected to subsequent sequencing or the entire transcript data. This method is currently the most commonly used method for studying single-cell gene expression profiles, and the entire transcript information can be obtained. However, in some scientific research, the expression level of some genes of some cells is often only concerned, and it is not necessary to obtain complete transcript information. At this time, the cost of the method is too high.
  • CEL-Seq Cell Expression by Linear amplification and Sequencing
  • This technique introduces a unique barcode sequence during reverse transcription.
  • cDNA can be used as a whole pool. Because it contains barcode information, it is convenient to distinguish a single cDNA molecule from the pool.
  • Subsequent in vitro linearization of cDNA pooling was performed and sequenced. This method is also the reverse transcription of the entire total RNA, and finally the information obtained by the DNA barcode is also the information of the entire transcript.
  • the main advantage of linear amplification is error The rate is relatively low, but both linear amplification and PCR have sequence-dependent preferences. It is also not suitable for studying the expression levels of several individual genes in a single cell.
  • SCRB-Seq single-cell RNA barcoding and sequening technology. Single cells are dispensed into the microwells using PCR amplification combined with flow cytometry (FACS) or other cell sorting methods. SCRB-seq is similar to Smart-seq, except that SCRB-seq integrates specific cell barcodes to resolve the source of amplified molecules and quantify transcripts more accurately. Furthermore, SCRB-Seq does not generate full-length cDNA, but enriches the 3' end of RNA like CEL-seq.
  • the DROP-seq technology a two-drip-based DROP-seq, uses a microfluidic device to place tiny beads with cells in tiny droplets, creating a fast, inexpensive, high-throughput single cell.
  • RNA-seq method This technique isolates cells in tiny droplets and mounts barcode primers for amplification, thereby detecting thousands of cells.
  • the researchers found that the number of genes that Drop-seq detected in a single cell was less than half that of Smart-Seq/C1, CEL-seq, and SCRB-seq.
  • high-throughput Drop-seq and SCRB-seq are relatively cost-effective when studying differential expression at a statistical level.
  • the present invention provides a PCR primer for detection and an application thereof, and the present invention can avoid the whole transcriptome, only study the expression of several genes in a single cell, or obtain a complex rearranged gene.
  • the genetic sequence is not limited to the PCR primer for detection and an application thereof.
  • the invention provides a PCR primer pair comprising two primers, wherein one primer comprises SEQ ID NO:1, the sequence 1 is a complementary sequence designed according to a linker sequence, and the other primer comprises a sequence 2, the sequence 2 A complementary sequence designed according to a fragment of interest, and the two primers are upstream and downstream primers.
  • the method can be used for amplification of DNA or amplification of RNA
  • the PCR primer pair of the present invention is directed to amplification of DNA, which can be introduced into the 5' end of one of the strands of the DNA strand, and the sequence 2 is designed according to the 5' end of the complementary strand.
  • the PCR primer pair of the present invention is directed to amplification of RNA introduced into the 3' end of the first strand cDNA, which is a primer designed according to the 5' end of the fragment of interest.
  • the primer comprising sequence 1 is an upstream primer and the primer comprising sequence 2 is a downstream primer.
  • a SMART template conversion technique is combined with a gene-specific primer, and a reverse IS sequence (linker sequence) is introduced into the 3' end of the first strand cDNA of the transcript during reverse transcription.
  • the subsequent PCR design is based on the IS sequence to obtain the upstream primer, and at the same time, the gene-specific primers, such as PD-1, GAPDH, TCR, BCR, etc., different genes have different downstream specific primers, and the single cells are amplified. Certain genes.
  • primers can be designed according to the conserved region, and a 5'-end primer phosphorylation modification or restriction endonuclease can be introduced during PCR.
  • the PCR product is self-ligated and cyclized into double-stranded circular DNA, and the full-length sequence of the target gene can be obtained by sequencing.
  • the designed primers follow general primer design principles, such as GC content 40-60%, no secondary hairpin structure, no primer dimer, etc., and primer design should be designed as far as possible without base polymorphism. .
  • the linker sequence is introduced into the 3' end of the cDNA by a translation template method during RNA reverse transcription.
  • the upstream primer is 18-25 nt in length, for example 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt or 25 nt, preferably 23 nt.
  • the linker sequence is introduced by a template method using a linker primer, which is designed according to the linker sequence, wherein the linker primer can be any fixed length sequence, those skilled in the art.
  • the nucleotide sequence of the adaptor primer of the present invention is shown in SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 is as follows:
  • nucleotide sequence of the upstream primer of the present invention is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the downstream primer of the present invention is preferably a nested primer, and the downstream primer includes an external primer, an intermediate primer or an internal primer. Any one or a combination of at least two of the primers, the combination may be, for example, a combination of an external primer and an intermediate primer, a combination of an intermediate primer and an internal primer, a combination of an external primer, an intermediate primer, and an internal primer, preferably an external primer. , an intermediate primer or a combination of internal primers.
  • a three-round nested primer is selected, and the inventors have found that not only the sample with a small initial amount in the single cell of the present application, but also the mechanism for the TCR is so complicated, the result after amplification is very accurate.
  • nucleotide sequence of the downstream primer is as follows:
  • SEQ ID NO. 3 GCAGACAGACTTGTCACTGG;
  • SEQ ID NO. 4 TGGTCGGGGAAGAAGCCTGTG;
  • SEQ ID NO. 5 TGGATTTAGAGTCTCTCAGCTGGTACACG;
  • SEQ ID NO. 6 TCTGCTTCTGATGGCTCAAACACAGC;
  • SEQ ID NO. 7 GGTACACGGCAGGGTCAGGGTTC;
  • SEQ ID NO. 8 TTCTGATGGCTCAAACACAGCGA;
  • the TCR is a heterodimer composed of two different subunits
  • 95% of the T cell receptors are composed of an ⁇ subunit and a ⁇ subunit, and each subunit contains two cells.
  • the external primer SEQ ID NO. 4, the intermediate primer SEQ ID NO. 6 and the internal primer SEQ ID NO. 8 were used for amplification of the ⁇ subunit of the TCR.
  • nucleotide sequence of the downstream primer is as follows:
  • SEQ ID NO. 9 AGGCTCTCTTTGATCTGCGCCT;
  • SEQ ID NO. 10 GGTAGGTGCCGCTGACATTGCG;
  • SEQ ID NO. 11 AGTTGTGTGACACGGAAGCGGC;
  • nucleotide sequence of the downstream primer is as follows:
  • SEQ ID NO. 12 CCTTTTGGCTCCCCCCTGCAAA;
  • SEQ ID NO. 13 CTCCACGACGTACTCAGCGCCA;
  • SEQ ID NO. 14 TCTCAGCCTTGACGGTGCCATG.
  • the present invention also provides a method for detecting a target segment, comprising the following steps:
  • the PCR-amplified target fragment of the step (1) is derived from a single cell derived from peripheral blood mononuclear cells of peripheral blood.
  • the isolated single cells described in the step (1) are conventional techniques in the art, and are not particularly limited herein.
  • the present invention employs a method comprising the following specific steps: peripheral blood mononuclear cells are stained with antibodies, and flow-through The CD8+ T cells were sorted by a cytometer, single cells were picked up by a micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate, immediately placed on dry ice, and the samples were stored at -80 ° C before amplification or In liquid nitrogen.
  • the lysate formulation is as follows:
  • the lysed single cells are preferably subjected to the following steps: a PCR tube containing a single cell is placed in a PCR machine, and the hot lid temperature is set to 75 °C. The single cells were incubated at 72 ° C for 3-5 min, immediately after the completion of the lysis, and placed on ice for 1 min; centrifuged at 10,000 rpm for 4 s for 30 s, and immediately transferred to ice.
  • step (1) the step of reverse transcription of the lysed mRNA to obtain the first strand cDNA is further included.
  • the mRNA reverse transcription system comprises TSO.
  • the final concentration of the TSO is 0.5-2 ⁇ M, and may be, for example, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.8 ⁇ M or 2 ⁇ M, preferably 0.1 ⁇ M.
  • the reverse transcription system is as follows:
  • the reverse transcription conditions of the mRNA are: 42 ° C for 90 min 1 cycle; 50 ° C for 2 min, 42 ° C for 2 min, 10 cycles; 70 ° C for 15 min 1 cycle; stored at 4 ° C.
  • the downstream primer described in the step (1) is a nested primer, and the downstream nested primer is used for amplification.
  • the amplification is nested PCR amplification, and the number of the nested PCR amplifications is 2-3 times, preferably 3 times.
  • the nested PCR specifically includes:
  • the first round of nested PCR conditions described in step (1') are: 95 ° C 3 min, 1 cycle; 98 ° C 20 s, 55 ° C 15 s, 72 ° C 2 min, 25 cycles ; 72 ° C 5 min, 1 cycle; stored at 4 ° C;
  • the second round of nested PCR conditions described in step (2') are: 95 ° C 3 min, 1 cycle; 98 ° C 20 s, 60 ° C 15 s, 72 ° C 2 min, 25 cycles ; 72 ° C 5 min, 1 cycle; stored at 4 ° C;
  • the third round of nested PCR conditions described in step (3') are: 95 ° C 3 min, 1 cycle; 98 ° C 20 s, 60 ° C 15 s, 72 ° C 2 min, 35 cycles ; 72 ° C 5 min, 1 cycle; stored at 4 ° C.
  • the sequencing described in step (2) is sanger sequencing and/or Miseq sequencing.
  • the method for detecting a TCR includes the following steps:
  • the invention provides a kit for detecting a fragment of interest, the kit comprising the PCR primer pair of the first aspect.
  • the present invention provides the PCR primer pair according to the first aspect, the detection method according to the second aspect or The kit of the third aspect is for detecting the use of a fragment of interest of a single cell.
  • the fragment of interest is selected from, but not limited to, TCR (T cell receptor), BCR (B cell receptor), PD-1 (programmed death receptor 1) or GAPDH (glyceraldehyde-3-phosphate Any one or a combination of at least two of hydrogenases.
  • the invention provides the use of a PCR primer pair according to the first aspect or a kit according to the third aspect for the preparation of a medicament for immunological diagnosis and/or prognosis monitoring of a disease.
  • the present invention has the following beneficial effects:
  • the primers and methods of the present invention are effective for different fragments of interest, and it is not necessary to design specific upstream primers for a single different target fragment, and transcription of the mRNA strand is performed by SMART template conversion technique by using a linker primer in reverse transcription.
  • the 3' end of the first strand cDNA introduces a fixed IS sequence, and the subsequent PCR uses the upstream primer designed according to the linker sequence, and the specific downstream primer is used to successfully amplify the target fragment;
  • the invention adopts three-round nested PCR, which is suitable for samples with template as low as 10pg total RNA, and can simultaneously acquire the sequence of a single cell target fragment, which has high amplification sensitivity, complete coverage and relatively low cost;
  • the method of the present invention explores a new immunological mechanism, constructs a large-scale full-length target fragment immune group library, facilitates disease diagnosis and health management, and develops neo TCR-T tumor immune cell therapy in combination with tumor new antigen, and performs tumor immunotherapy, cancer. Or prognosis monitoring of autoimmune diseases can help guide doctors in medication and scientific research.
  • Figure 1 is a technical schematic diagram of the method of the present invention
  • Example 2 is a diagram showing electrophoresis results in Example 2 of the present invention, wherein the marker is a 100 bp marker, and the bottom is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp (brightest band), 600 bp, 700 bp, 800 bp, 900 bp, 1000bp and 1500bp.
  • the marker is a 100 bp marker
  • the bottom is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp (brightest band), 600 bp, 700 bp, 800 bp, 900 bp, 1000bp and 1500bp.
  • the linker sequence is introduced into the 3' end of the first strand of cDNA by a template method using a linker primer as shown in SEQ ID NO. 1, SEQ ID NO.
  • the nucleotide sequences shown are as follows:
  • the upstream primer is designed according to the adaptor sequence, which is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the downstream primer is designed according to the C region of the TCR gene, and the complete sequence of the C region of the T cell ⁇ and ⁇ chain is as follows:
  • TCRA C Region SEQ ID NO. 15:
  • the primers of the Miseq sequencing platform and the Sanger sequencing platform are designed separately.
  • the PE300 sequencing strategy requires that the library Insert length be no more than 600bp, and the TCR ⁇ chain 5' UTR sequence +V+D+J region sequence is just within 600bp, so we The downstream primer is placed 20 to 30 bp near the 5' end of the C region as follows:
  • the method for detecting a TCR includes the following steps:
  • Peripheral blood mononuclear cells were stained with antibodies, and CD8+ T cells were sorted by flow cytometry. Single cells were picked up by micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate and immediately placed in dry ice. Above, the sample is stored at -80 ° C or liquid nitrogen before amplification;
  • the first strand cDNA of all mRNAs is synthesized
  • the upstream primer is SEQ ID NO. 2, and the nucleotide sequence shown by SEQ ID NO. 2 is: 5'-AAGCAGTGGTATCAACGCAGAGT-3'
  • the downstream primers are as follows:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • electrophoresis was performed using a 2% agarose gel, taking 15 ⁇ L of the product, and adding 3 ul of Loading buffer. Mix well and electrophorese at 130V for 45min. The target strip was recovered and the results were as shown in Fig. 2, and the intact ⁇ -chain of TCR was obtained.
  • sequencing can directly obtain the full length of V(D)J, and at the same time, the ⁇ and ⁇ pairing sequence information of T cells can be obtained.
  • the method for detecting PD-1 includes the following steps:
  • Peripheral blood mononuclear cells were stained with antibodies, and CD8+ T cells were sorted by flow cytometry. Single cells were picked up by micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate and immediately placed in dry ice. Above, the sample is stored at -80 ° C or liquid nitrogen before amplification;
  • the first strand cDNA of all mRNAs is synthesized
  • the upstream primer is SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is: 5'-AAGCAGTGGTATCAACGCAGAGT-3'
  • the downstream primers are as follows:
  • SEQ ID NO. 9 AGGCTCTCTTTGATCTGCGCCT;
  • SEQ ID NO. 10 GGTAGGTGCCGCTGACATTGCG;
  • SEQ ID NO. 11 AGTTGTGTGACACGGAAGCGGC;
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • the electrophoresis was detected. Using a 2% agarose gel, 15 ⁇ L of the product was added, 3 ul of Loading buffer was added, and the mixture was electrophoresed at 130 V for 45 min.
  • the method for detecting a TCR includes the following steps:
  • Peripheral blood mononuclear cells were stained with antibodies, and CD8+ T cells were sorted by flow cytometry. Single cells were picked up by micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate and immediately placed in dry ice. Above, the sample is stored at -80 ° C or liquid nitrogen before amplification;
  • the first strand cDNA of all mRNAs is synthesized
  • the upstream primer is SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is: 5'-AAGCAGTGGTATCAACGCAGAGT-3'
  • the downstream primers are as follows:
  • SEQ ID NO. 12 CCTTTTGGCTCCCCCCTGCAAA;
  • SEQ ID NO. 13 CTCCACGACGTACTCAGCGCCA;
  • SEQ ID NO. 14 TCTCAGCCTTGACGGTGCCATG.
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • electrophoresis was performed. Using a 2% agarose gel, 15 ⁇ L of the product was taken, and 3 ⁇ l of the loading buffer was added and mixed, and electrophoresed at 130 V for 45 minutes. The purpose of the strip is to recover the glue.
  • the primers and methods of the present invention are effective for different target fragments, and it is not necessary to design specific upstream primers for a single different target fragment, and the transcript of the mRNA chain is made by using SMART template conversion technology in reverse transcription.
  • the 5' end of the first strand cDNA introduces a fixed IS sequence, and the subsequent PCR uses the complementary sequence of the IS sequence as the upstream primer, and the specific downstream primer is used to successfully amplify the target fragment; otherwise, the present invention adopts three rounds.
  • Nested PCR which is suitable for samples with template as low as 10pg total RNA, can simultaneously acquire the sequence of a single cell target fragment with high amplification sensitivity, complete coverage and relatively low cost.

Abstract

本发明公开了一种用于检测的PCR引物及其应用，包括上游引物和下游引物；所述上游引物为根据一段接头序列设计的引物，所述接头序列引入到第一链cDNA的3'端；所述下游引物为根据检测的目的片段的3'端设计的下游引物序列。本发明引物和方法对不同目的片段均有效，不需要针对单一不同目的片段设计特异性的上游引物，通过在反转录过程中，采用SMART模板转换技术使得mRNA链的转录本第一链cDNA的3'端引入一段固定IS序列，后续PCR均以IS序列的互补序列为上游引物，同时配合特异性的下游引物，成功扩增出目的片段。

Description

一种用于检测的PCR引物及其应用 技术领域
本发明属于分子生物学领域，涉及一种用于检测的PCR引物及其应用，具体涉及一种用于检测单个细胞目的片段的PCR引物，检测目的片段的方法及其应用。
背景技术
同一组织中的细胞往往被认为是具有相同状态的功能单位，传统的检测分析的是细胞群体的平均反应。然而需要更精准的了解群体的动态变化过程，需要对单个细胞进行分析。通过对单个细胞的DNA或RNA进行测序，表明组织系统层面的功能是由异质性细胞构成的。单细胞测序以单个细胞为单位，通过全基因组或转录组扩增，进行高通量测序，能够结实单个细胞的基因结构和基因表达状态，反应细胞间的异质性，在肿瘤、发育生物学、微生物学、神经科学等领域发挥重要作用，正成为生命科学研究的焦点。
一个细胞里的DNA或RNA仅仅处在皮克(picograms)级，10pg的水平，这么少的量远远达不到现有测序仪最低的上样需求。因此必须对单细胞内的微量的核酸分子进行扩增。
现有技术中目前采用的具体实验技术有如下：
SMART-seq方法作为一种较好的全长RNA-seq的方法，其原理为：(1)用“带poly(T)的PCR下游扩增序列引物”启动反转录。这样保证了反转录是从模板最3’端的poly(A)位置起始，且反转录的产物上连上了一个可以PCR扩增的下游引物序列；(2)用SMARTscribe RT酶进行反转录，这个酶有个特点：会在转录的末端，延长出几个超过模板的C碱基；(3)用“带3个g-PCR上游扩增序列的引物”与反转录得到的第1链进行粘合，这里的3个g碱基是RNA碱基，而不是DNA碱基。这有利于引物与反转录酶相结合，再进行延伸，把PCR扩增的上游引物序列也引入到反转录产物中来；(4)在上、下游都引入了PCR引物序列之后，PCR可以高效进行；(5)PCR的产物进行建库，再深度测序。该文库可以进行后续的测序，或得整个转录本数据。该方法是目前研究单细胞基因表达谱最常用的方法，可以获得整个转录本信息。但是，在一些科学研究中，往往只关注一些细胞的某些基因的表达量，而不需要获得完整转录本信息，这时，该方法的成本就显得太高。
CEL-Seq，即(Cell Expression by Linear amplification and Sequencing)技术，是一种采用线性扩增的常用测序方法。该技术是在反转录时引入唯一的barcode序列，此时cDNA可以作为一个pool整体，由于含有barcode信息，方便从pool中区分出单个cDNA分子。后续对cDNA pooling进行体外线性化转录，并进行测序。该方法同样是对整个total RNA进行反转录，最终通过DNA barcode辅助获得的也是整个转录本的信息。线性扩增的主要优势是错误 率比较低，不过线性扩增和PCR都存在序列依赖性偏好。同样对于研究单细胞中的某几个单独基因表达量并不适合。
SCRB-Seq，即(single-cell RNA barcoding and sequening)技术。是采用PCR扩增，结合流式细胞仪(FACS)或者其他细胞分选方法，把单细胞分配到微孔里去。SCRB-seq与Smart-seq比较相似，只不过SCRB-seq会整合特异性的细胞条码，以分辨扩增分子的来源，更准确的定量转录本。此外，SCRB-Seq并不生成全长cDNA，而是像CEL-seq一样富集RNA 3’端。
DROP-seq技术，以微滴为基础的两种DROP-seq，利用微流体装置将带有条码的微珠和细胞一起装入微小的液滴，建立了快速、廉价、高通量的单细胞RNA-seq方法。这种技术将细胞隔离在微小的液滴中，装上用于扩增的条码引物，由此检测数以千计的细胞。但研究者发现Drop-seq在单个细胞中检测的基因数还不到Smart-Seq/C1、CEL-seq和SCRB-seq的一半。不过，在统计学水平上研究差异性表达的时候，高通量Drop-seq和SCRB-seq相对来讲比较划算。
上述四种方法，都需要细胞数在96至上千，对于单细胞中的目的片段是无法扩增的。
现有技术中的其他方法就算能够对于单细胞进行扩增，都是对于单细胞整个转录组水平的扩增、测序。适用范围多为研究细胞转录组水平基因表达情况，各个平台成本和灵敏度不尽相同，但对于研究单个细胞部分基因表达的案例，成本太高，浪费过多的数据量。另外，对于单细胞转录组而言，由于后续测序平台的不同，很难通过生物信息学分析出复杂的重排基因的基因序列，如：TCR、BCR等基因。
发明内容
针对现有技术中的缺陷，本发明提供了一种用于检测的PCR引物及其应用，本发明可以避开全转录组，仅仅研究单细胞中几个基因的表达，或者获得复杂重排基因的基因序列。
第一方面，本发明提供了一种PCR引物对，包括两条引物，其中一条引物包含序列1，所述序列1为根据接头序列设计的互补序列，另一条引物包含序列2，所述序列2为根据目的片段设计的互补序列，且这两条引物互为上下游引物。
本发明中，所述方法可用于DNA的扩增或RNA的扩增；
本发明所述PCR引物对针对于DNA的扩增，所述接头序列可以引入DNA链的其中一条链的5’端，所述序列2则根据互补链的5’端进行设计。
本发明所述PCR引物对针对于RNA的扩增，所述接头序列引入第一链cDNA的3’端，所述序列2为根据目的片段的5’端设计的引物。
根据本发明，包含序列1的引物为上游引物，包含序列2的引物为下游引物。
本发明中，在反转录过程中，采用SMART模板转换技术与基因特异性引物相结合，在反转录过程中，转录本第一链cDNA的3’端引入一段固定IS序列(接头序列)，后续PCR均根据IS序列设计得到上游引物，同时配合基因特异性引物，如：PD-1、GAPDH、TCR、BCR等基因，不同的基因有不同的下游特异性引物，扩增出单细胞中的某些基因。
本发明中，对于序列较长且高度可变的基因，只要序列中含有保守区域，则可以根据保守区设计引物，通过在PCR过程中引入5’端引物磷酸化修饰或添加限制性内切酶位点等手段，实现PCR产物自连，环化成双链环状DNA，可以通过测序获得目的基因的全长序列
本发明中，所述设计引物遵循一般引物设计原则，例如GC含量40-60％、无二级发夹结构、无引物二聚体等，引物设计应该尽量设计在没有碱基多态性的位置。
根据本发明，所述接头序列是RNA反转录过程中通过转换模板法引入到cDNA的3’端。
根据本发明，所述上游引物的长度为18-25nt，例如可以是18nt、19nt、20nt、21nt、22nt、23nt、24nt或25nt，优选为23nt。
根据本发明，所述接头序列是利用接头引物通过转换模板法引入的，所述上游引物是根据所述接头序列设计的，其中所述接头引物可以是任意一段固定长度的序列，本领域技术人员可以根据需要进行设计，本发明的接头引物的核苷酸序列如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：
5’-AAGCAGTGGTATCAACGCAGAGTACGGG-3’；
本发明的上游引物的核苷酸序列如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：
5’-AAGCAGTGGTATCAACGCAGAGT-3’.
根据本发明，由于本发明的TCR检测为单细胞裂解后进行检测，起始样本浓度比较低，本发明所述下游引物优选为巢式引物，所述下游引物为包括外部引物、中间引物或内部引物中的任意一种或至少两种的组合，所述组合例如可以是外部引物和中间引物的组合，中间引物和内部引物的组合，外部引物、中间引物和内部引物的组合，优选为外部引物、中间引物或内部引物的组合。
本发明中，选用三轮巢式引物，发明人发现不仅能够针对本申请单细胞中的起始量小的样本，且其针对TCR如此复杂的机构，扩增后的结果非常准确。
对于TCR的检测，所述下游引物的核苷酸序列如下：
外部引物：
SEQ ID NO.3：GCAGACAGACTTGTCACTGG；
SEQ ID NO.4：TGGTCGGGGAAGAAGCCTGTG；
中间引物：
SEQ ID NO.5：TGGATTTAGAGTCTCTCAGCTGGTACACG；
SEQ ID NO.6：TCTGCTTCTGATGGCTCAAACACAGC；
内部引物：
SEQ ID NO.7：GGTACACGGCAGGGTCAGGGTTC；
SEQ ID NO.8：TTCTGATGGCTCAAACACAGCGA；
本发明中，由于TCR是异源二聚体，由两个不同的亚基所构成，95％的T细胞的受体由α亚基和β亚基构成，每一个亚基都含有两个细胞外的结构域：可变区与恒定区，所述外部引物SEQ ID NO.3，中间引物SEQ ID NO.5和内部引物SEQ ID NO.7用于TCR的α亚基的扩增，所述外部引物SEQ ID NO.4，中间引物SEQ ID NO.6和内部引物SEQ ID NO.8用于TCR的β亚基的扩增。
对于PD-1的检测，所述下游引物的核苷酸序列如下：
外部引物：
SEQ ID NO.9：AGGCTCTCTTTGATCTGCGCCT；
中间引物：
SEQ ID NO.10：GGTAGGTGCCGCTGACATTGCG；
内部引物：
SEQ ID NO.11：AGTTGTGTGACACGGAAGCGGC；
对于GAPDH的检测，所述下游引物的核苷酸序列如下：
外部引物：
SEQ ID NO.12：CCTTTTGGCTCCCCCCTGCAAA；
中间引物：
SEQ ID NO.13：CTCCACGACGTACTCAGCGCCA；
内部引物：
SEQ ID NO.14：TCTCAGCCTTGACGGTGCCATG.
第二方面，本发明还提供了一种检测目的片段的方法，包括如下步骤：
(1)以第一方面所述的上游引物和下游引物进行PCR扩增；
(2)测序验证，得到所述目的片段的序列。
根据本发明，步骤(1)所述PCR扩增的目的片段来源为单细胞，所述单细胞来源于外周血的外周血单个核细胞。
优选地，步骤(1)所述的分离单细胞为本领域的常规技术，在此不做特殊限定，本发明采用方法，包括如下具体步骤：外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中。
优选地，所述裂解液配方如下表所示：
Figure PCTCN2017104105-appb-000001
为了保证mRNAs都从单细胞中释放，所述裂解单细胞优选为如下步骤：将装有单细胞的PCR管置于PCR仪内，热盖温度设为75℃。将单细胞72℃孵育3-5min，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上。
优选地，步骤(1)之前还包括将裂解得到的mRNA进行反转录，得到第一链cDNA的步骤。
根据本发明，所述的mRNA反转录的体系包括TSO。
根据本发明，所述TSO的终浓度为0.5-2μM，例如可以是0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.2μM、1.3μM、1.5μM、1.6μM、1.8μM或2μM，优选为0.1μM。
优选地，所述的反转录体系如下表所示：
Figure PCTCN2017104105-appb-000002
根据本发明，所述的mRNA反转录的条件为：42℃ 90min 1循环；50℃ 2min，42℃ 2min，10循环；70℃ 15min 1循环；保存在4℃。
根据本发明，步骤(1)所述的下游引物为巢式引物，利用所述下游巢式引物进行扩增 时，所述扩增为巢式PCR扩增，所述巢式PCR扩增的次数为2-3次，优选为3次。
根据本发明，所述巢式PCR具体包括：
(1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
(2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
(3’)以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物。
根据本发明，针对TCR的扩增，步骤(1’)所述的第一轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
根据本发明，针对TCR的扩增，步骤(2’)所述的第二轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
根据本发明，针对TCR的扩增，步骤(3’)所述的第三轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，，72℃ 2min，35个循环；72℃ 5min，1循环；保存在4℃。
根据本发明，步骤(2)所述的测序为sanger测序和/或Miseq测序。
作为优选技术方案，所述检测TCR的方法包括如下步骤：
(1)分离外周血单个核细胞，裂解；
(2)将得到的mRNA进行反转录，得到第一链cDNA；
(3)以步骤(2)得到的第一链cDNA为模板，以权利要求1所述的上游引物和所述的巢式引物为下游引物进行PCR扩增，具体包括：
(1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
(2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
(3’)以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
(4)sanger和/或Miseq测序验证，得到所述T细胞受体的全长序列。
第三方面，本发明一种用于检测目的片段的试剂盒，所述试剂盒包含如第一方面所述的PCR引物对。
第四方面，本发明提供如第一方面所述的PCR引物对、第二方面所述的检测方法或如第 三方面所述的试剂盒用于检测单个细胞的目的片段的用途。
根据本发明，所述目的片段选自但不限于TCR(T细胞受体)、BCR(B细胞受体)、PD-1(程序性死亡受体1)或GAPDH(甘油醛-3-磷酸脱氢酶)中的任意一种或至少两种的组合。
第五方面，本发明提供如第一方面所述的PCR引物对或如第三方面所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
与现有技术相比，本发明具有如下有益效果：
(1)本发明引物和方法对不同目的片段均有效，不需要针对单一不同目的片段设计特异性的上游引物，通过在反转录过程中，采用接头引物通过SMART模板转换技术使得mRNA链的转录本第一链cDNA的3’端引入一段固定IS序列，后续PCR均以根据接头序列设计的上游引物，同时配合特异性的下游引物，成功扩增出目的片段；
(2)本发明采用三轮巢式PCR，适用于模板低至10pg总RNA的样品，能同时获取某一个单细胞的目的片段的序列，扩增灵敏度高，覆盖完整且成本相对较低；
(3)本发明方法探索免疫学新机制，构建大规模全长目的片段免疫组库，方便疾病诊断及健康管理，结合肿瘤新抗原开发neo TCR-T肿瘤免疫细胞疗法，进行肿瘤免疫治疗，癌症或自体免疫疾病预后监测，有助于指导医生用药和科学研究等。
附图说明
图1为本发明方法的技术原理图；
图2是本发明实施例2中的电泳结果图，其中，marker为100bp marker，从下到上为100bp、200bp、300bp、400bp、500bp(最亮条带)、600bp、700bp、800bp、900bp、1000bp和1500bp。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明。可以理解的是，此处所描述的具体实施例仅仅用于解释本发明，而非对本发明的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本发明相关的部分而非全部结构。
实施例1 TCR引物设计
引物设计的原理如图1所示，利用接头引物通过转换模板法在cDNA第一链的3’端引入接头序列，所述接头引物如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：
5’-AAGCAGTGGTATCAACGCAGAGTACGGG-3’；
根据接头序列设计上游引物，所述上游引物如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：
5’-AAGCAGTGGTATCAACGCAGAGT-3’；
所述下游引物根据TCR基因C区进行设计，所述T细胞α和β链的C区完整序列如下：
TCRA C区(SEQ ID NO.15)：
Figure PCTCN2017104105-appb-000003
TCRB C区(SEQ ID NO.16)：
Figure PCTCN2017104105-appb-000004
由于Miseq测序平台和Sanger测序平台的不同，所述Miseq测序平台和Sanger测序平台的引物分别设计。
Miseq测序
因Miseq目前最长的读长为300bp/reads，采用PE300的测序策略，要求文库Insert长度不超过600bp，TCR αβ链5’端UTR序列+V+D+J区序列正好在600bp以内，因此我们将下游引物设置在靠近C区的5’端20～30bp的位置，如下所示：
表1 人T细胞C区半长引物序列信息
Figure PCTCN2017104105-appb-000005
Figure PCTCN2017104105-appb-000006
实施例2 TCR在Miseq测序平台测序
所述检测TCR的方法，包括如下步骤：
(1)单细胞第一链cDNA合成：
a)配制裂解液，所述裂解液配方如下表所示：
Figure PCTCN2017104105-appb-000007
配制时，按样品数110％配制(如有10个细胞样品，则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净0.2ml离心管中，14000rpm，4℃离心离心30s(将液滴离心到管底并去除气泡)；冰盒放置，待后续加入细胞；
b)单细胞分离
外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中；
c)细胞裂解
将0.2ml PCR管置于PCR仪内，72℃，3min孵育(细胞mix样本增至5min)，热盖温度为75℃，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合；
d)mRNA反转录
配制反转录体系如下：
Figure PCTCN2017104105-appb-000008
配制时，按样品数+0.5个配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次加入到上步离心管中；
吹打混匀、瞬时离心后，按如下条件进行反转录反应(75℃热盖)：
Figure PCTCN2017104105-appb-000009
所有mRNAs的第一链cDNA合成完毕；
(2)巢式PCR
采用实施例1中的引物，上游引物为SEQ ID NO.2，SEQ ID NO.2所示的核苷酸序列为：5’-AAGCAGTGGTATCAACGCAGAGT-3’
下游引物如下：
Figure PCTCN2017104105-appb-000010
a)第一轮PCR
以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000011
Figure PCTCN2017104105-appb-000012
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取15ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000013
b)第二轮PCR
以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000014
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000015
Figure PCTCN2017104105-appb-000016
c)第三轮PCR
以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000017
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000018
(3)电泳检测
巢式PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3ul Loading buffer 混匀，130V电泳45min。目的条带切胶回收，结果如图2所示，得到了完整的TCR的αβ链。
(4)送到公司去完成TA克隆和测序，测序结果如下：
α链序列
α1(SEQ ID NO.17)：V12+J21
Figure PCTCN2017104105-appb-000019
α2(SEQ ID NO.18)：V5+J36
Figure PCTCN2017104105-appb-000020
β链序列(SEQ ID NO.19)：：V27+J2
Figure PCTCN2017104105-appb-000021
Figure PCTCN2017104105-appb-000022
所述电泳产物测序结果统计如下2所示：
表2
Figure PCTCN2017104105-appb-000023
从表2可以看出，采用Miseq测序平台，测序可以直接得到V(D)J全长，且同时可以获得T细胞的α与β配对序列信息。
实施例3 PD-1测序
所述检测PD-1的方法，包括如下步骤：
(1)单细胞第一链cDNA合成：
a)配制裂解液，所述裂解液配方如下表所示：
Figure PCTCN2017104105-appb-000024
配制时，按样品数110％配制(如有10个细胞样品，则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净0.2ml离心管中，14000rpm，4℃离心离心30s(将液滴离心到管底并去除气泡)；冰盒放置，待后续加入细胞；
b)单细胞分离
外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中；
c)细胞裂解
将0.2ml PCR管置于PCR仪内，72℃，3min孵育(细胞mix样本增至5min)，热盖温度为75℃，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合；
d)mRNA反转录
配制反转录体系如下：
Figure PCTCN2017104105-appb-000025
配制时，按样品数+0.5个配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次加入到上步离心管中；
吹打混匀、瞬时离心后，按如下条件进行反转录反应(75℃热盖)：
Figure PCTCN2017104105-appb-000026
所有mRNAs的第一链cDNA合成完毕；
(2)巢式PCR
采用实施例1中的引物，
上游引物为SEQ ID NO.2，SEQ ID NO.2所示的核苷酸序列为：5’-AAGCAGTGGTATCAACGCAGAGT-3’
下游引物如下：
外部引物：
SEQ ID NO.9：AGGCTCTCTTTGATCTGCGCCT；
中间引物：
SEQ ID NO.10：GGTAGGTGCCGCTGACATTGCG；
内部引物：
SEQ ID NO.11：AGTTGTGTGACACGGAAGCGGC；
a)第一轮PCR
以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000027
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取15ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000028
b)第二轮PCR
以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000029
Figure PCTCN2017104105-appb-000030
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000031
c)第三轮PCR
以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000032
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000033
(3)电泳检测
巢式PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3ul Loading buffer混匀，130V电泳45min，目的条带切胶回收。
(4)送到公司去完成TA克隆和测序，测序结果如下：
实施例4 GAPDH的测序
所述检测TCR的方法，包括如下步骤：
(1)单细胞第一链cDNA合成：
a)配制裂解液，所述裂解液配方如下表所示：
Figure PCTCN2017104105-appb-000034
配制时，按样品数110％配制(如有10个细胞样品，则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净0.2ml离心管中，14000rpm，4℃离心离心30s(将液滴离心到管底并去除气泡)；冰盒放置，待后续加入细胞；
b)单细胞分离
外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中；
c)细胞裂解
将0.2ml PCR管置于PCR仪内，72℃，3min孵育(细胞mix样本增至5min)，热盖温度 为75℃，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合；
d)mRNA反转录
配制反转录体系如下：
Figure PCTCN2017104105-appb-000035
配制时，按样品数+0.5个配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次加入到上步离心管中；
吹打混匀、瞬时离心后，按如下条件进行反转录反应(75℃热盖)：
Figure PCTCN2017104105-appb-000036
所有mRNAs的第一链cDNA合成完毕；
(2)巢式PCR
采用实施例1中的引物，
上游引物为SEQ ID NO.2，SEQ ID NO.2所示的核苷酸序列为：5’-AAGCAGTGGTATCAACGCAGAGT-3’
下游引物如下：
外部引物：
SEQ ID NO.12：CCTTTTGGCTCCCCCCTGCAAA；
中间引物：
SEQ ID NO.13：CTCCACGACGTACTCAGCGCCA；
内部引物：
SEQ ID NO.14：TCTCAGCCTTGACGGTGCCATG.
a)第一轮PCR
以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000037
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取15ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000038
b)第二轮PCR
以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000039
Figure PCTCN2017104105-appb-000040
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000041
c)第三轮PCR
以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104105-appb-000042
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24ul加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104105-appb-000043
(3)电泳检测
巢式PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3μl上样缓冲液混匀，130V电泳45min。目的条带切胶回收。
(4)送到公司去完成TA克隆和测序，测序结果如下：
综上所述，本发明引物和方法对不同目的片段均有效，不需要针对单一不同目的片段设计特异性的上游引物，通过在反转录过程中，采用SMART模板转换技术使得mRNA链的转录本第一链cDNA的5’端引入一段固定IS序列，后续PCR均以IS序列的互补序列为上游引物，同时配合特异性的下游引物，成功扩增出目的片段；不仅如此，本发明采用三轮巢式PCR，适用于模板低至10pg总RNA的样品，能同时获取某一个单细胞的目的片段的序列，扩增灵敏度高，覆盖完整且成本相对较低。
上述仅为本发明的较佳实施例及所运用技术原理。本领域技术人员会理解，本发明不限于这里所述的特定实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本发明的保护范围。因此，虽然通过以上实施例对本发明进行了较为详细的说明，但是本发明不仅仅限于以上实施例，在不脱离本发明构思的情况下，还可以包括更多其他等效实施例，而本发明的范围由所附的权利要求范围决定。

Claims (14)

  1. 一种PCR引物对，其特征在于，包括两条引物，其中一条引物包含序列1，所述序列1为根据接头序列设计的互补序列，另一条引物包含序列2，所述序列2为根据目的片段设计的互补序列，且这两条引物互为上下游引物。
  2. 根据权利要求1所述的PCR引物对，其特征在于，所述接头序列是RNA反转录过程中通过转换模板法引入到cDNA的3’端。
  3. 根据权利要求1或2所述的PCR引物对，其特征在于，所述接头序列的长度为18-25nt。
  4. 根据权利要求3所述的PCR引物对，其特征在于，所述接头序列的长度为23nt。
  5. 根据权利要求1或2所述的PCR引物对，其特征在于，所述接头的核苷酸序列如SEQ ID NO.1所示。
  6. 根据权利要求1或2所述的PCR引物对，其特征在于，包含序列1的引物为上游引物，包含序列2的引物为下游引物；
    优选地，所述上游引物的核苷酸序列如SEQ ID NO.2所示；
    优选地，所述下游引物为巢式引物；
    优选地，所述下游引物包括外部引物、中间引物或内部引物中的任意一种或至少两种的组合，优选为外部引物、中间引物或内部引物的组合。
  7. 一种检测目的片段的方法，其特征在于，包括如下步骤：
    (1)以权利要求1或2所述的PCR引物对中的上游引物和下游引物进行PCR扩增；
    (2)测序验证，得到所述目的片段的序列。
  8. 根据权利要求7所述的方法，其特征在于，步骤(1)所述PCR扩增的目的片段来源为单细胞；
    优选地，所述单细胞来源于外周血的外周血单个核细胞；
    优选地，步骤(1)之前还包括将mRNA进行反转录，得到第一链cDNA的步骤；
    优选地，所述的mRNA反转录的体系包括接头序列；
    优选地，所述接头序列的终浓度为0.5-2μM，优选为0.1μM。
  9. 根据权利要求7或8所述的方法，其特征在于，步骤(1)所述下游引物为巢式引物，利用所述下游巢式引物进行扩增时，所述扩增的次数为2-3次，优选为3次；
    优选地，所述巢式PCR具体包括：
    (1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巣式PCR，得到第一轮扩增产物；
    (2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为 模板，进行第二轮巣式PCR，得到第二轮扩增产物；
    (3’)以权所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巣式PCR，得到第三轮扩增产物。
  10. 根据权利要求7-9中任一项所述的方法，其特征在于，步骤(2)所述的测序为sanger测序和/或Miseq测序。
  11. 一种用于检测目的片段的试剂盒，其特征在于，所述试剂盒包含如权利要求1-6任一项所述的PCR引物对。
  12. 一种如权利要求1-6任一项所述的PCR引物对、如权利要求7-10中任一项所述的检测方法或如权利要求11所述的试剂盒用于检测单个细胞的目的片段的用途。
  13. 根据权利要求12所述的用途，其特征在于，所述目的片段为T细胞受体、B细胞受体、程序性死亡受体1或甘油醛-3-磷酸脱氢酶中的任意一种或至少两种的组合。
  14. 一种如权利要求1-6任一项所述的PCR引物对或如权利要求11所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
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