Primer combination for simultaneously detecting T cell and B cell immune repertoire for high-throughput sequencing and kit

Abstract

The invention belongs to the field of molecular biological detection and especially relates to a primer combination for simultaneously detecting T cell (TCR) and B (BCR) cell immune repertoire for high-throughput sequencing and a kit. The primer combination comprises an XCR 3' Oligo (dT) primer, an XCR 5' Oligo joint, an XCR 5' terminal joint primer, a TCR-alpha chain C zone primer, a TCR-beta chain C zone primer, IgA, IgG and IgM in a BCR-Heavy chain C zone primer, Kappa and Lambda in IgM, BCR-Light chain C zone primer and upstream and downstream label primers. The kit comprises a primer combination according to the claim 1 or 2. The overall length information of a gene sequence of TCR (Alpha chain or Beta chain) and BCR (Heavy chain or Light chain) in lymphocyte is acquired.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer combination and a kit for simultaneous detection of T cell and B cell immune repertoires for 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 am...

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.

Images