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A method for detecting bcr and tcr immune repertoire in plasma cfdna

An immune library, plasma technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of not being able to fully and comprehensively display the disease progress of patients, and achieve the elimination of randomly introduced errors, The effect of improving accuracy

Active Publication Date: 2018-08-03
BEIJING GENEPLUS TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are limitations in the analysis and research of individual specific clones, which cannot fully and comprehensively display the disease progression of patients

Method used

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  • A method for detecting bcr and tcr immune repertoire in plasma cfdna
  • A method for detecting bcr and tcr immune repertoire in plasma cfdna
  • A method for detecting bcr and tcr immune repertoire in plasma cfdna

Examples

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Embodiment 1

[0065] Example 1 Detects the design of primers for BCR and TCR in plasma cfDNA

[0066] The immunological polymorphism of BCR is based on the recombination arrangement of V(D)J region fragments. The TCR CDR3 region just covers the TRBV-Junction-TRBD-Junction-TRBJ region, which directly determines the antigen specificity of the TCR due to the largest variation. Primers were designed for the BCR and TCR gene sequences, aiming to amplify the full-length BCR H chain and TCR Beta chain CDR3 region.

[0067] The primer structure of BCR / TCR-PCR1: from the 5' end to the 3' end, it contains the linker sequence, the specific tag sequence and the upstream and downstream primers for BCR / TCR amplification.

[0068] Design of upstream and downstream primers for BCR amplification: based on the homology analysis of the 7 major family ORFs of BCR V genes (FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4), designed with the leader region of the V gene Upstream primers (22 in total, see the B-PCR1 upst...

Embodiment 2

[0080] Example 2 Establishment of method for detecting BCR and TCR immune repertoire in plasma cfDNA

[0081] 1. Plasma cfDNA extraction:

[0082] 1.1 Plasma Extraction Take 2 tubes (5mL / tube) of peripheral blood from the subject, put them in EDTA anticoagulant tubes, gently turn them upside down (to prevent cell rupture), mix thoroughly 6-8 times, and perform the following within 4-6 hours on the day of blood collection Processing: Centrifuge at 1600g for 10 minutes at 4°C, and divide the supernatant (plasma) into multiple 1.5mL / 2mL centrifuge tubes, and the middle layer of white blood cells cannot be absorbed during the suction process; 16000g at 4°C Centrifuge for 10 minutes to remove residual cells, and transfer the supernatant (plasma) to a new 1.5mL / 2mL centrifuge tube. If the white blood cells cannot be sucked to the bottom of the tube, the required plasma after separation is obtained.

[0083]1.2 After the plasma sample is processed, the separated plasma and remaining...

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Abstract

The invention provides a method for detecting BCR and TCR immune repertoire in blood plasma cfDNA. The method includes: blood plasma cfDNA extraction, BCR H chain overall length multiple PCR amplification, TCR beta chain CDR3 multiple PCR amplification, PCR amplification after the products of the two previous amplifications are mixed in an equal-volume manner, high-throughput sequencing, and immune repertoire precise information analysis. The primers of the three amplifications are respectively shown in SEQ ID No. 1-28, SEQ ID No. 29-77 and SEQ ID No. 78-79. The invention further provides a kit, which contains the primers, for detecting the BCR and TCR in the blood plasma cfDNA. By the method and the kit which are promising in application prospect in the field of non-invasive detection, the BCR and TCR in the blood plasma cfDNA can be detected at the same time, precise information analysis and precise quantification of immune repertoire subcloning are achieved.

Description

technical field [0001] The invention belongs to the technical field of immune repertoire sequencing, in particular to a method for detecting BCR and TCR immune repertoires in plasma cfDNA. Background technique [0002] Thanks to the rapid development and wide application of next-generation high-throughput sequencing technologies, DNA and RNA sequencing platforms play a prominent role in promoting various aspects of genomics. At the same time, this emerging technical field has also been applied to the sequencing of immune repertoires of T cells and B cell receptors. [0003] Conventional immune repertoire sequencing of T cells and B cell receptors is mainly based on two major strategies, first: based on cell DNA through TCR / BCR gene rearrangement rules, in TCR (alpha / beta / gamma / delta chain) and BCR The variable region (variable) gene of (heavy / light chain) is set with upstream primers, and the downstream primers are set at the joining region (joining) J gene or constant regi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/686C12Q1/6869C12Q2537/143C12Q2535/122
Inventor 易鑫吕小星赵美茹管彦芳刘涛杨玲
Owner BEIJING GENEPLUS TECH
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