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Method for detecting minimal residual disease in T cell leukemia based on high-throughput sequencing

A minimal residual disease, high-throughput technology, applied in the field of molecular biology detection, can solve problems such as false negatives, amplification of TCR genes and sequencing, and incomplete sequencing gene information

Inactive Publication Date: 2017-07-25
孙涛
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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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  • Method for detecting minimal residual disease in T cell leukemia based on high-throughput sequencing
  • Method for detecting minimal residual disease in T cell leukemia based on high-throughput sequencing
  • Method for detecting minimal residual disease in T cell leukemia based on high-throughput sequencing

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Experimental program
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Effect test

Embodiment 1

[0082] A method for detecting T-cell leukemia minimal residual disease by high-throughput sequencing, comprising the steps of:

[0083] (1) Obtain 10mL of human blood samples in EDTA anticoagulant tubes;

[0084] (2) Peripheral blood mononuclear cells (PBMCs) were separated using the lymphocyte separation medium Ficoll-1077 (Sigma, USA #10771);

[0085] (3) Utilize the method of Trizol to extract the total RNA of PBMC, the reagent used is RNAzol RT (US MRC company #RN190);

[0086] (4) The 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,

[0087] Specific steps are as follows,

[0088] Reagents used:

[0089] ·TCR 3'Oligo(dT) primer (10μM)

[0090] 5X reverse transcription buffer (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl 2 )

[0091] Dithiothreitol, DTT (20mM) US Thermo Scientific #R0861

[0092] · dNTP Mix (10mM) US Invitrogen#18427088

[0093] ·RNAse Out (40...

Embodiment 2

[0162] Embodiment 1 of the present invention provides a method for preparing a T lymphocyte receptor (TCR) RNA sample, comprising the following steps:

[0163] 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);

[0164] 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

[0166] 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:

[0167] 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".

[0168] 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 biological detection, and in particular relates to an RNA linker and a single pair of primers for constructing a high-throughput sequencing library for a T cell receptor (TCR) of T cell leukemia minimal residual disease based on high-throughput sequencing, and a preparation method of the DNA library. The method comprises the following steps: acquiring 10mL of a human blood sample, and placing the human blood sample in an EDTA anticoagulative tube; separating peripheral blood mononuclear cells (PBMC) by adopting lymphocyte separating liquid Ficoll-1077; extracting total RNA of PBMC by adopting a Trizol method, wherein the used reagent is RNAzol RT; carrying out inverse transcription on RNA to form cDNA, and meanwhile, adding a linker at the 5' terminal of cDNA; carrying out PCR1; carrying out PCR2 and purification; and carrying out high-throughput sequencing, and thus the full-length information of a TCR gene sequence is obtained from an upstream primer of the linker and a downstream primer of the C region.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a method for detecting T-cell leukemia minimal residual disease based on high-throughput sequencing. 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 refers to the sm...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q1/6886C12Q2600/158C12Q2535/122C12Q2525/191
Inventor 孙涛刘潇
Owner 孙涛
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