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Primer combination and kit for detecting minimal residual diseases of T-cell leukemia by high-throughput sequencing

A minimal residual disease and primer combination technology, applied in the field of molecular biology detection, can solve the problems of unrecognized genes, false negatives, and difficulty in designing upstream primers to identify the 5' end sequence of TCR genes, etc., to achieve high construction efficiency and stable efficiency , The effect of simple and convenient use method

Active Publication Date: 2017-07-18
杭州艾沐蒽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since the biggest feature of the TCR gene is the random recombination of the V, D, and J gene segments, it is difficult to design an upstream primer to identify the 5' end sequence of the TCR gene for the unknown gene sequence, so PCR technology cannot be used to amplify TCR genes and sequencing
[0008] Another TCR sequencing method, that is, the method of multiple primer PCR, can only sequence part of the sequence information in the TCR gene, which makes the sequenced gene information incomplete.
In addition, the primers of the multiple primer PCR method are designed according to the known V and J genes, but in cancer patients, gene mutations in cancer cells are very common. If the TCR of leukemia cancer cells has a mutation, then the known sequence The primers may not be able to recognize the mutated gene, and it is easy to cause false negatives for the test results

Method used

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  • Primer combination and kit for detecting minimal residual diseases of T-cell leukemia by high-throughput sequencing
  • Primer combination and kit for detecting minimal residual diseases of T-cell leukemia by high-throughput sequencing
  • Primer combination and kit for detecting minimal residual diseases of T-cell leukemia by high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] The primer combination and kit used in the present invention to perform high-throughput sequencing to detect T-cell leukemia minimal residual disease includes the following steps:

[0104] (1) Obtain 10mL of human blood samples in EDTA anticoagulant tubes;

[0105] (2) Peripheral blood mononuclear cells (PBMCs) were separated using the lymphocyte separation medium Ficoll-1077 (Sigma, USA #10771);

[0106] (3) Utilize the method of Trizol to extract the total RNA of PBMC, the reagent used is RNAzol RT (US MRC company #RN190);

[0107] (4) RNA is reverse-transcribed into cDNA, and an adapter is added to the 5' end of the cDNA for the 5' end primer binding during subsequent PCR amplification. The specific steps are as follows,

[0108] Reagents used:

[0109]·TCR 3'Oligo(dT) primer (10μM)

[0110] 5X reverse transcription buffer (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl 2 )

[0111] Dithiothreitol, DTT (20mM) US Thermo Scientific #R0861

[0112] · dNTP Mix (10mM...

Embodiment 2

[0184] Embodiment 1 of the present invention provides a method for preparing a T lymphocyte receptor (TCR) RNA sample, comprising the following steps:

[0185] 10 milliliters (mL) of fresh peripheral blood samples were collected and operated according to the instructions of Ficoll-1077 (Sigma Company #10771, USA) to obtain relatively pure peripheral blood mononuclear cells (PBMC);

[0186] Adopt the method of Trizol to extract the total RNA of PBMC, reagent used is RNAzol RT (US MRC company #RN190), the total RNA obtained, utilize 2.0 Fluorometer (#Q32866 from Thermo Fisher Scientific, USA), with RNA HS Assay Kit kit (Thermo Fisher Scientific, USA #Q32852) was used to measure the RNA concentration, and then the RNA was reverse transcribed;

Embodiment 3

[0188] Embodiment 2 of the present invention provides a method for constructing a TCR high-throughput sequencing library for minimal residual disease of leukemia using a single pair of primers of the TCR library for minimal residual disease of leukemia, including the following steps:

[0189] Using the RNA obtained in Example 1 as a template for reverse transcription, the cDNA to which the TCR 5' end adapter was added was obtained according to the reagents and steps in the fourth part of the above "second aspect". Purify the products (libraries) of PCR 1, PCR 2 and PCR 2 according to the reagents and steps in the fifth, sixth and seventh parts of the above "second aspect".

[0190] After the library was purified, use Agilent 2100Bioanalyzer (US Agilent #G2939AA) to detect the purity and size of the library. The kit used was Agilent DNA 1000Kit (US Agilent #5067-1504), and the test results were as follows: image 3 As shown, the size of the library is 686bp, and the purity of th...

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Abstract

The invention belongs to the field of molecular biology and in particular relates to a primer combination and kit for detecting minimal residual diseases of T-cell leukemia by high-throughput sequencing. The primer combination is characterized by comprising a TCR (T Cell Receptor) 5'Oligo connector, a TCR 3'Oligo (dT) primer, a TCR C-region primer and upstream and downstream primers of a label; and the kit comprises the primer combination in claim 1 or 2. Full-length information of a TCR gene sequence is obtained from the upstream primer of the connector to a downstream primer of a C region.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a primer combination and a kit for high-throughput sequencing detection of T-cell leukemia minimal residual disease. Background technique [0002] Leukemia is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, impaired differentiation, and apoptosis, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function. T-cell leukemia is a malignant disease of T cells. Clonal growth. [0003] With the continuous improvement of chemotherapy, specific targeted therapy, hematopoietic stem cell transplantation (HSCT) and other technologies, the treatment effect of leukemia is improving day by day. However, recurrence is still a difficulty in the cure of leukemia. (MRD) related. MRD ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/122C12Q2525/191
Inventor 孙涛刘潇
Owner 杭州艾沐蒽生物科技有限公司
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