Whole blood immune repertoire detection method based on high-flux sequencing technology

Abstract

The invention provides a whole blood immune repertoire detection method based on high-flux sequencing technology. The method comprises the following steps: (1) directly extracting RNA (ribonucleic acid) from a whole blood sample; (2) carrying out reverse transcription on the RNA to generate single chain cDNA (complementary deoxyribonucleic acid), connecting an oligonucleotide joint sequence to the tail end of the single chain cDNA, carrying out PCR (polymerase chain reaction) reaction on the single chain cDNA subjected to oligonucleotide joint sequence connection by using a universal primer and a special targeted primer P-VDJ (of which the sequence is GAC CTC GGG TGG GAA CAC) as a special primer pair for PCR reaction, and amplifying the immune receptor genome; and (3) carrying out high-flux sequencing. The method of directly extracting the RNA from the whole blood sample is more suitable for clinic; the single target specific primer is utilized to uniformly amplify the variable region of the subtype T cell receptor gene; and a Hi-SEQ 2500 of the Illumina corporation and a bar code are utilized for sequencing, thereby ensuring the lower cost, the sequencing time of less than 48 hours and favorable sequencing depth.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a whole blood immune repertoire detection method based on high-throughput sequencing technology. Background technique [0002] The importance of monitoring TCRs in human health and disease has been increasingly recognized. Recent studies have shown that the T cell receptor repertoire has been found to affect a wide range of disease categories, including malignancies, autoimmune diseases, and infectious diseases, and, if one considers that the immune system broadly affects almost all human health and diseases, immune The system's reach is expected to be larger. Commonly used recent methods such as multiparameter flow cytometry, profiling, or custom-designed real-time PCR assays, while providing some resolution, are labor-intensive and do not provide deeper resolution to Determine the patient's V(D)J recombination pattern. [0003] With the development of massively paral...

AI Technical Summary

Problems solved by technology

Most researchers have focused on Illumina's Genome Analyzer sequencer or HiSeq2000, which can generate read lengths from 50 bases to 100 bases, but infer long sequences from short reads, and thus cannot Convenient and direct

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