Method for accurately detecting T cell immune repertoire based on high-throughput sequencing and primer system thereof

A cellular immunity, high-throughput technology, applied in the field of molecular biology detection, can solve the problems of repeatability and specificity not as good as DNA detection results, complex processing of RNA samples, etc., to reduce bias and amplification errors, comprehensive and accurate immunization Assessment of status, effect of elimination of purification steps

Active Publication Date: 2021-07-16
武汉弘康医学检验实验室股份有限公司
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Problems solved by technology

This method can accurately quantify and detect the TCR immune repertoire, but due to the complexity of RNA sample processing and the fluctuation of RNA expression, its repeatability and specificity are not as good as DNA detection results, so it is not an ideal raw material

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  • Method for accurately detecting T cell immune repertoire based on high-throughput sequencing and primer system thereof
  • Method for accurately detecting T cell immune repertoire based on high-throughput sequencing and primer system thereof
  • Method for accurately detecting T cell immune repertoire based on high-throughput sequencing and primer system thereof

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Embodiment Construction

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the implementation manners in the present invention, all other implementation manners obtained by persons of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0043] 1. DNA extraction and fragmentation

[0044] Take 1 peripheral blood sample, follow the QIAamp DNABLoodMini Kit (Qiagen) extraction kit instructions, carry out peripheral blood DNA extraction, and then use Qubit to quantify, requiring blood DNA to be greater than 600ng at least.

[0045] 2. Amplification of multiplex PCR primers specific to the CDR3 region of the TCRBeta chain with molecular tags

[0046] TCR Beta chain CDR3 region-specific...

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Abstract

The invention provides a method for accurately detecting a T cell immune repertoire based on high-throughput sequencing and a primer system thereof, the primer system comprises a PCR1 primer and a PCR2 primer, the PCR1 primer sequence and the PCR2 primer sequence comprise an upstream V primer and a downstream J primer, and a sequencing linker sequence and a molecular tag sequence are respectively inserted into target areas of the upstream primer and the downstream primer of the PCR1; the PCR2 primer takes a PCR1 product as a template, and a sequencing primer sequence and an index tag are introduced. The primer system and the method for accurately detecting a T cell immune repertoire based on high-throughput sequencing have the characteristics of simplicity and convenience in sample treatment, rapidness in experimental operation and accurate quantification of TCR cloning clusters.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a method for accurately detecting T cell immune repertoire based on high-throughput sequencing and a primer system thereof. Background technique [0002] T cell receptor (T cell receptor, TCR) is a molecule on the surface of T cells that specifically recognizes antigens and mediates immune responses. It is one of the most polymorphic regions in the human genome and determines how the human immune system adapts to the environment. Variety. The diversity of T cell receptor library (including gene recombination and selective expression) directly reflects the state of the body's immune response. During the development of T cells, CDR1, 2 and FR regions are relatively conserved, and the CDR3 region is rearranged by V, D and J to form a functional TCR coding gene (T cell clone), because V (65-100 species), D (2 species) and J (13 species) gene fragments themselves have dive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/6876C12N15/11G16B30/10
CPCC12Q1/6869C12Q1/6876G16B30/10C12Q2600/16C12Q2525/191C12Q2531/113C12Q2535/122C12Q2537/143C12Q2563/179Y02A50/30
Inventor 吕小星张剑袁萌伍炜康叶丽曼吕星星
Owner 武汉弘康医学检验实验室股份有限公司
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