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

A lymphocyte, high-throughput technology, applied in the field of specific lymphocyte content analysis, can solve the problems of detection, time-consuming, affecting the accuracy of cancer cells, etc., and achieve the effect of accurate cloning and good performance.

Active Publication Date: 2021-07-30
深圳泛因医学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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

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Embodiment 1

[0053] Lymphohematopoietic tumor is a malignant disease that seriously endangers human health caused by malignant clonal proliferation of lymphocytes, including lymphocytic leukemia, myeloma and various lymphomas.

[0054] With the improvement of diagnosis and treatment technology, the diagnosis and treatment of lymphocytic tumors has achieved a qualitative leap. Minimal residual disease (MRD, Minimal residual disease) refers to a small amount of tumor cells remaining in the body after complete remission (CR, Complete Remission) after induction chemotherapy or after bone marrow transplantation. With the continuous improvement of chemotherapy, specific targeted therapy, and hematopoietic stem cell transplantation technology, the treatment effect of lymphocytic tumors is improving day by day. However, recurrence is still a problem that plagues the cure of this type of disease, and minimal residual disease is the main source of recurrence. Regular MRD detection for patients has i...

Embodiment 2

[0131] A specific lymphocyte content analysis kit based on TCR / BCR high-throughput sequencing, including: internal standard sequence, multiple PCR amplification primers and reagent components required for conventional high-throughput sequencing.

[0132] The internal standard sequence is the internal standard sequence of the coding gene of artificially synthesized TCR / BCR; the multiple PCR amplification primers are used for the amplification of the TCR / BCR gene, and the specific sequence is as SEQ ID NO.1-SEQ ID in Example 1 Shown in NO.56.

Embodiment 3

[0134] A specific lymphocyte content analysis device based on TCR / BCR high-throughput sequencing, including a high-throughput sequencing module and a data analysis module. Wherein, the high-throughput sequencing module uses the kit in Example 2, and is performed according to the high-throughput sequencing steps in Example 1; the data analysis module is performed according to the data analysis steps in Example 1.

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12M1/34C12M1/00
CPCC12Q1/6869C12Q2531/113C12Q2537/143C12Q2535/122C12Q2537/165C12Q2545/101
Inventor 王谢苏政
Owner 深圳泛因医学有限公司
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