A method for simultaneous detection of T-cell and B-cell immune repertoires based on high-throughput sequencing

Abstract

The invention belongs to the field of molecular biological detection and in particular relates to a method for simultaneously detecting immune repertoires of a T cell and a B cell on the basis of high-throughput sequencing. The method for simultaneously detecting the immune repertoires of the T cell and the B cell on the basis of high-throughput sequencing comprises the steps of obtaining 10 mL human blood sample; putting the 10 mL human blood sample in an EDTA anticoagulant tube; separating peripheral blood monouclear cells (PBMC) by using a lymphocyte separation medium Ficoll-1077; extracting total RNA of the PBMC by using a Trizol method, wherein a reagent used is cDNA reversely transcribed from RNAzolRT and RNA; adding a linker, PCR1 and PCR2 to 5' end of the cDNA; carrying out purification and high-throughput sequencing on the cDNA and the like. Total length information of gene sequences of TCR (Alpha chain or Beta chain) and BCR (Heavy chain or Light chain) in lymphocyte are obtained from an upstream primer of the linker and a downstream primer of a C-region.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a method for simultaneously detecting T cell and B cell immune repertoires based on high-throughput sequencing. Background technique [0002] A large number of V (variable region), D (variable region), and J (junction region) gene fragments on T and B cell loci will produce various diverse recombinations in the formation of T and B cell receptors. This V-D-J gene recombination endows each T and B cell with its own unique T and B cell receptors (TCR, BCR), so that the sequence of each TCR and BCR can effectively become a T and B cell clone unique biomarker. [0003] Since the biggest feature of TCR and BCR genes is the random recombination of V, D, and J gene segments, it is difficult to design an upstream primer for identifying the 5' end sequences of TCR and BCR genes for unknown gene sequences, so it is also impossible to use PCR technology to amplify TCR and BCR ge...

AI Technical Summary

Problems solved by technology

[0003] Since the biggest feature of TCR and BCR genes is the random recombination of V, D, and J gene segments, it is difficult to design an upstream primer for identifying the 5' end sequences of TCR and BCR genes for unknown gene sequences, so it is also impossible to use Amplification of TCR and BCR genes and sequencing by PCR technology

[0004] Another TCR sequencing method, the method of multiple primer PCR, can only sequence part of the sequence information in the TCR gene, which makes the sequenced gene information incomplete

In addition, the primers of the multiple primer PCR method are designed according to the known V and J genes, and the sequencing results are limited to the known genes of the wild type

However, in cancer patients, gene mutations in cancer cells are very common. If there is a mutation in the BCR or TCR of leukemia cancer cells, the primers with known sequences may not be able to identify the mutated gene. For the test results, prone to false negatives

Moreover, the immune library sequencing method of multiple primer PCR requires dozens of pairs of primers only for TCR or BCR sequencing. If TCR and BCR are detected at the same time, the huge number of primers will increase the efficiency and specificity of the entire PCR amplification. sex becomes bad

[0005] Therefore, there is currently no technology that can simultaneously detect TCR and BCR in one experimental operation. Our method (single-pair primer method) can simultaneously detect TCR and BCR in multiple samples, saving experimental time, reagents, and labor costs.

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