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Kit and device for BCR/TCR gene rearrangement detection

A gene rearrangement and kit technology, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of non-direct application, non-coverage, false negative, etc., and achieves low difficulty in operation steps. , avoid false negatives, improve the effect of sensitivity

Active Publication Date: 2022-06-24
SHANGHAI ORIGIMED CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The existing BCR / TCR clonal rearrangement detection method designed 3 VH-JH, 2 DH-JH, 2 IGK, 1 IGL, 3 TRB, 2 TRG, 1 TCRD, 3 BCL1-IGH, 1 BCL2-IGH has a total of 18 multiplex PCR reactions, involving 107 different primers, but the primers do not cover all the V and J gene fragments, which is prone to multiple PCR primer bias, which is not directly applicable to high-throughput sequencing, and cannot be used for PCR and sequencing Amplified mismatch repair, prone to false negative test results

Method used

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  • Kit and device for BCR/TCR gene rearrangement detection
  • Kit and device for BCR/TCR gene rearrangement detection
  • Kit and device for BCR/TCR gene rearrangement detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0197] Example 1 Obtaining the sample genome

[0198] According to before and after treatment and sample specificity, it can be divided into:

[0199] Human pre-treatment bone marrow / blood samples 200 microliters in EDTA anticoagulant tubes, human pre-treatment tissue samples.

[0200] Human post-treatment bone marrow / blood samples 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml in EDTA anticoagulant tubes.

[0201] Further, the nucleic acid extraction reagent in the kit of the present invention can be used to extract genomic DNA, or a commercially available nucleic acid extraction kit can be used to extract genomic DNA. QIAamp DNA Mini kit (Cat. No. 51304), QIAGEN’s QIAampDNA Midi Kit (Cat. No. 51185) reagent instructions to extract DNA.

[0202] Specifically, the components of FFPE sample DNA extraction reagent (QIAGEN, 56404) include: ATL lysate, AL lysate, AW1 purification eluate, AW2 purification eluate, ATE elua...

Embodiment 2

[0205] Example 2 Multiplex PCR amplification and library construction

[0206] Using the library construction kit, add multiple pairs of primers for V gene fragments and J gene fragments to the multiplex PCR reaction system, and add internal reference gene primers and samples for specific PCR amplification at the same time. The PCR amplification reagents come from QIAGEN Multiplex PCR of QIAGEN plus Kit (Cat. No. 206152). Specifically, the components of the PCR amplification reagent (QIAGEN, 206152) include: 2x multiplex PCR amplification enzyme, 5x amplification buffer, nuclease-free water, and 10x electrophoretic dye.

[0207] BCR / TCR clonal rearrangement detection is based on NGS detection after two rounds of multiplex PCR. The first round of PCR uses the multiplex PCR primers of each gene to carry out reactions in separate tubes or in the same tube to amplify the sequence of complementarity determining region 3 (CDR3) that determines the diversity of BCR or TCR. The secon...

Embodiment 3

[0273] Example 3 Fitting of B / T cell proportion standard curve

[0274] Taking the standard curve of B cell proportion as an example, as shown in Table 12, B cells are mixed into T cells at a ratio of 0.05, 0.1, 0.2, 0.5, and 1. The total input amount is 100 ng, for example, the B cell content is 0.05 , then mix 5 nanograms of B cells and 95 nanograms of T cells, do 2 repetitions for each mixing ratio, and then perform multiple PCR library construction according to the method of the above-mentioned Example 2, and then perform sequencing, the samples of group A and the samples of group B That is, two replicate samples under the same mixing ratio, determine the number of ACTB internal parameter reads in group A and the total reads in group A according to the sequencing data of group A samples, and determine the internal parameter reads number of group B ACTB and group B according to the sequencing data of group B samples The total number of reads. According to the sequencing da...

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Abstract

The invention relates to a primer combination product for BCR / TCR gene rearrangement detection. The primer combination product comprises a primer for detecting BCR gene rearrangement and / or a primer for detecting TCR gene rearrangement, the primer for detecting the BCR gene rearrangement comprises a primer composition for detecting IGH-VDJ rearrangement, IGH-DJ rearrangement, IGK-VJ rearrangement, IGK-V-KDE rearrangement, IntroRSS-KDE rearrangement, IGL-VJ rearrangement, BCL1-IGH rearrangement and BCL2-IGH rearrangement, and the primer composition is used for detecting the IGH-VDJ rearrangement, the IGH-DJ rearrangement, the IGK-VJ rearrangement, the IGK-V-KDE rearrangement, the IntroRSS-KDE rearrangement, the IGL-VJ rearrangement and the BCL2-IGH rearrangement. The primer for detecting the TCR gene rearrangement comprises a primer composition for detecting TRB-VDJ rearrangement and TRG-VJ rearrangement. The kit disclosed by the invention can be used for performing rearrangement detection on nine different types of genes through one-tube multiple PCR, and has the advantages of low difficulty in operation steps and high standardization degree, so that false negative detection is reduced.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit and device for BCR / TCR gene rearrangement detection. Background technique [0002] The immune response of immune cells to complex invading pathogens (antigens) depends on their highly diverse antigen recognition receptors B cell surface receptors (B cell receptor, BCR) and T cell surface receptors (T cell receptor, TCR ). [0003] B cell surface receptor (BCR) is an immunoglobulin molecule IG that grows on the surface of B lymphocytes and consists of two heavy chains (H) and two light chains ( , ) are connected by disulfide bonds, the genes encoding the heavy chain and the light chain are not on the same chromosome, the gene IGH encoding the heavy chain H is located on the long arm of chromosome 14, and the gene encoding the light chain The gene IGK is located on the short arm of chromosome 2 and encodes the light chain The gene IGL is located on the long arm...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C40B50/06C12N15/11G16B30/00
CPCC12Q1/6886C12Q1/686C40B50/06G16B30/00C12Q2600/156C12Q2600/166C12Q2537/143
Inventor 王维锋蔡佳程张科武海燕郑新李锋
Owner SHANGHAI ORIGIMED CO LTD
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