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

An immunological library and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as inconvenience and directness, and achieve the effect of low cost and good sequencing time.

Active Publication Date: 2015-01-07
武汉希望组生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

Most researchers have focused on Illumina's Genome Analyzer sequencer or HiSeq2000, which can generate read leng

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

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Embodiment Construction

[0022] The whole blood immune repertoire detection method based on high-throughput sequencing technology provided by the present invention comprises the following steps:

[0023] (1) RNA is directly extracted from whole blood samples;

[0024] (2) Generate single-stranded cDNA from RNA reverse transcription, connect the oligonucleotide linker sequence to the 3' end of the single-stranded cDNA, use general primers and special targeting primer P-VDJ whose sequence is GAC CTC GGG TGG GAA CAC The special primer pair for PCR reaction and the single-stranded cDNA connected with the oligonucleotide linker sequence are used as PCR templates for PCR reaction to amplify the immune receptor genome;

[0025] (3) High-throughput sequencing.

[0026] The method provided by the present invention will be further described below in conjunction with examples.

[0027] Using previously collected whole blood samples from two malignant tumor patients and two benign patients, which were stored at...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2525/191C12Q2535/122
Inventor 王凯高帆
Owner 武汉希望组生物科技有限公司
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