Specific lymphocyte content analysis method and device based on tcr/bcr high-throughput sequencing

Abstract

The invention relates to a specific lymphocyte content analysis method and device based on TCR / BCR high-throughput sequencing, belonging to the technical field of gene detection. The analysis method includes the following steps: high-throughput sequencing: extract the genomic DNA of the biological sample to be tested, put in a predetermined amount of artificially synthesized TCR / BCR coding gene internal standard sequence, and perform high-throughput sequencing; data analysis: obtain the above-mentioned high-throughput Sequence data, analyze the obtained TCR / BCR gene sequence, and calculate the amplification factor according to the input internal standard sequence; compare the TCR / BCR gene sequence of the sample to be tested with the target lymphocyte antigen receptor gene sequence A, and obtain the target lymphocyte antigen receptor gene sequence A. The number of original antigen receptor gene sequences consistent with antigen receptor gene sequence A, the number of sequences with sequencing errors, the number of sequences with secondary rearrangements, and the number of sequences with mutations are used to obtain the total number of target lymphoid sequences; through the total number of target lymphoid sequences and The expansion factor is calculated to obtain the number of target lymphocytes, which can accurately determine the content of each T / B cell.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and device for analyzing the content of specific lymphocytes based on TCR / BCR high-throughput sequencing. Background technique [0002] Adaptive immunity is also called acquired immunity. Specific immune cells mainly include T lymphocytes involved in cellular immunity and B lymphocytes involved in humoral immunity. Adaptive immunity has the characteristics of antigen specificity and immune memory, and its antigen specificity means that different antigens need to be recognized and responded by different lymphocytes. In clinical practice, it is often necessary to determine the content of specific lymphocytes in biological samples (the composition of each component in biological samples is as follows: figure 1 shown), for example, the immune effect of the vaccine can be determined by detecting whether there are specific vaccine-specific immune cells; or the detectio...

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Problems solved by technology

[0005] However, by flow cytometry detection, the content of a type of cells with the same surface differentiation antigen is detected, and each cell cannot be accurately distinguished

Moreover, the detection sensitivity of the current common flow cytometry can only reach 1 / 10000. For samples with a content lower than 1 / 10000, false negative detection results will be given.

In flow cytometry, data interpretation depends on the operator and the laboratory, and the degree of standardization is low

Most importantly, for cancerous lymphocytes, the expression of their surface antigens will change at different times of cancer occurrence and treatment, which will seriously affect the accuracy of cancer cell detection

[0006] For detection by RQ-PCR, it is necessary to design specific primers and quantitative probes for the TCR / BCR genes of different target cells. If there is a mutation or secondary rearrangement in the binding region of the quantitative probe, a missed detection will occur

[0007] Accordingly, scientists also use high-throughput sequencing to detect the TCR / BCR genes of target cells. However, the current high-throughput sequencing for TCR / BCR often has detection bias due to gene mutations.

Moreover, the TCR / BCR research based on the multiplex PCR method will cause a large amplification bias due to the difference in the binding efficiency of the primers in the multiplex PCR primer system and the influence of the interaction between the primers. If the system is not optimized and corrected, it cannot accurately reflect The true level of sample clone

Moreover, the TCR / BCR research based on the RACE method is only applicable to RNA samples, and the conditions for sample collection and transportation are relatively strict

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