Method for simultaneously detecting multiple trace sample TCR (T cell receptor) repertoires

A technology in samples and samples, which is applied in the fields of biotechnology and clinical immunology, can solve the problems of easy operation errors, time-consuming and labor-intensive, frequency distribution deviation, etc., and achieve the effect of reducing sample demand, reducing sequencing costs and accurate detection

Active Publication Date: 2016-01-27
THE FIRST PEOPLES HOSPITAL OF FOSHAN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

②Due to the design of different primers for each family for RT-PCR amplification, the difference in the amplification efficiency of each TCRVα and Vβ gene family itself will cause the deviation of the frequency distribution, which cannot objectively reflect the frequenc

Method used

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  • Method for simultaneously detecting multiple trace sample TCR (T cell receptor) repertoires
  • Method for simultaneously detecting multiple trace sample TCR (T cell receptor) repertoires
  • Method for simultaneously detecting multiple trace sample TCR (T cell receptor) repertoires

Examples

Experimental program
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Embodiment 1

[0102] The 3 samples adopted in this embodiment are respectively:

[0103] Sample 1: healthy human PBMC cells;

[0104] Sample 2: PBMC cells from tuberculosis patients;

[0105] Sample 3: Jurket cell line.

[0106] Among them, the separation of PBMC cells adopts the density gradient method, and the specific steps are as follows:

[0107] 1) Add an appropriate amount of Ficoll lymphocyte separation medium into a 15ml graduated sterile centrifuge tube;

[0108] 2) Take heparin anticoagulated peripheral venous blood and mix and dilute it with an equal amount of RPMI-1640, absorb 2 times the volume of anticoagulated blood and slowly superimpose it on the lymphocyte separation solution along the tube wall, keeping the interface intact;

[0109] 3) 18~20℃, 1800~2000rpm / min horizontal centrifugation for 20~30min;

[0110] 4) After centrifugation, the liquid in the tube is divided into four layers, the upper layer is plasma and diluent, and the bottom of the tube is mainly red blo...

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Abstract

The invention discloses a method for simultaneously detecting multiple trace sample TCR (T cell receptor) repertoires. The method includes: taking a sample RNA (ribonucleic acid) as a template to synthesize a first-strand cDNA (complementary deoxyribonucleic acid), performing 5'-terminal RACE PCR, designing specific 5' RACE nested PCR primers and TCR alpha-beta gene specific primers, performing RT-PCR amplification to obtain alpha-beta gene amplification products of each sample TCR, mixing the multiple sample PCR products for sequencing, distinguishing different samples according to joining regions of the specific primers, and analyzing frequency distribution of V and J gene segments in each sample. The method for simultaneously detecting the multiple trace sample TCR repertoires has the advantages that on the premise that distinguishing of different samples is guaranteed, sequencing cost of the TCR repertoires is greatly reduced, and authenticity in frequency distribution of each family is guaranteed; establishment of disease entity RCR repertoires requiring a great quantity of samples is promoted.

Description

technical field [0001] The invention belongs to the fields of biotechnology and clinical immunology, and in particular relates to a method for detecting T cell receptor pools (Tcell receptor, TCR) of multiple trace samples. Background technique [0002] T cells are the main component of lymphocytes, which have a variety of biological functions, such as directly killing target cells, assisting or inhibiting B cells to produce antibodies, responding to specific antigens and mitogens, and producing cytokines, etc. The body's heroic fighter against infection and tumor formation. T cells recognize pathogens and their toxins in the external environment and tumor antigens produced by gene mutations in the internal environment through their surface receptor (Tcell receptor, TCR) molecules. However, the antigens in the environment are ever-changing. How do they recognize the ever-changing antigens in nature? The classic clonal selection theory (clonal selection theory) believes that...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 罗微金亚彬
Owner THE FIRST PEOPLES HOSPITAL OF FOSHAN
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