Method for detecting BCR-ABL fused gene ABL kinase domain mutation

A technology that fuses genes and kinase regions, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc.

Inactive Publication Date: 2019-05-21
武汉康圣达医学检验所有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned Sanger sequencing method has low sensitivity, and the HRM method can only detect specific mutation sites in samples. If multiple sites are to be detected, th

Method used

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  • Method for detecting BCR-ABL fused gene ABL kinase domain mutation
  • Method for detecting BCR-ABL fused gene ABL kinase domain mutation
  • Method for detecting BCR-ABL fused gene ABL kinase domain mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 COLD-PCR combined with next-generation sequencing method to detect mutations in the ABL kinase region of the BCR / ABL fusion gene in peripheral blood

[0033] COLD-PCR combined with next-generation sequencing method to detect BCR-ABL fusion gene in peripheral blood Simultaneous detection of ABL kinase region drug resistance mutation detection kit in two types of M-bcr and m-bcr, including: RNA extraction solution: Red blood cell lysate, Trizol, chloroform, absolute ethanol; reverse transcription reagent: SuperScript II Reverse Transcriptase (ThermoFisher Scientific); detection system PCR reaction solution; library construction reaction solution; Miseq platform sequencing reaction solution; positive control, negative control and blank Control substance.

[0034] Detection system PCR reaction solution includes: 10×LA PCR Buffer II (Mg 2+Plus); dNTP Mixture (10mMeach); LA Taq enzyme; primers for amplifying BCR / ABL fusion gene M-bcr and m-bcr spliced ​​BCR-ABL fus...

Embodiment 2

[0081] Example 2 COLD-PCR combined with next-generation sequencing method to detect mutations in the ABL kinase region of the BCR / ABL fusion gene in bone marrow

[0082] COLD-PCR combined with next-generation sequencing method to detect BCR-ABL fusion gene in bone marrow Simultaneous detection of ABL kinase region drug resistance mutation detection kit in two shear types M-bcr and m-bcr, including: RNA extraction solution: red blood cells Lysis solution, Trizol, chloroform, absolute ethanol; reverse transcription reagent: SuperScript II Reverse Transcriptase (ThermoFisher Scientific); detection system PCR reaction solution; library construction reaction solution; Miseq platform sequencing reaction solution; positive control, negative control and blank Control substance.

[0083] Detection system PCR reaction solution includes: 10×LA PCR Buffer II (Mg 2+ Plus); dNTP Mixture (10mMeach); LATaq enzyme; amplification of BCR / ABL fusion gene M-bcr and m-bcr spliced ​​BCR-ABL fusion ...

Embodiment 3

[0130] Example 3 Comparison of the method of COLD-PCR combined with next-generation sequencing and the method of traditional PCR+next-generation sequencing to detect mutations in the ABL kinase region of the BCR-ABL fusion gene

[0131] 1. T-A cloning method to construct the T315I mutant plasmid, the steps are as follows:

[0132] 1.1 Take bone marrow samples from CML patients with T315I mutation, extract RNA, and reverse transcribe cDNA;

[0133] 1.2 Using cDNA as a template and using the sequences shown in SEQ ID NO.1 and SEQ ID NO.3 as primers, amplify the M-bcr type BCR-ABL fusion gene;

[0134] 1.2.1 BCR-ABL fusion gene amplification reaction system:

[0135] Table 21

[0136] Element

Amount added (μl)

LATaq enzyme

0.2

10×LAPCR Buffer II (Mg 2+ Plus)

2

dNTP Mixture

0.8

primer mix

2

cDNA

5

wxya 2 o

10

total capacity

20

[0137] 1.2.2 BCR-ABL fusion gene amplification procedure: ...

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Abstract

The invention provides a method for detecting the BCR-ABL fused gene ABL kinase domain mutation. In combination with COLD-PCR and a second-generation sequencing method, the method detects the ABL kinase domain mutation in two shearing types M-bcr and m-bcr of a BCR-ABL fused gene in peripheral blood or marrow, that is, the BCR-ABL fused gene of M-bcr and m-bcr is amplified in the same system, thenan ABL kinase domain is amplified through COLD-PCR, a wild type template is sealed according to the COLD-PCR principle, a small number of mutant genes are enriched at the temperature Tc, and the detection sensitivity is improved through the second-generation sequencing method. The method has the advantages of being easy to operate, high in repeatability, high in flux during specimen detection, short in reporting time and the like; the method is particularly suitable for detecting a small number of mutant cloning plants. By means of the method, the mutation detecting sensitivity and the compound mutation detecting capacity are remarkably improved.

Description

technical field [0001] The invention relates to the technical field of genome sequencing, in particular to a method and a kit for detecting mutations in the ABL kinase region of the BCR-ABL fusion gene. Background technique [0002] Chronic myelocytic leukemia (CML) is a hematopoietic stem cell clonal proliferative disease characterized by myeloid hyperplasia, peripheral blood leukocytosis and splenomegaly. For CML, the expansion of mutant clones may be a gradual process. The proportion of mutant clones is low at the beginning, but once it appears, it will increase rapidly and lead to disease progression. Drug resistance mutations often appear before or during the blast phase of CML. Therefore, regular monitoring of mutations is necessary to detect mutations early and adjust treatment regimens accordingly. For some mutations with poor prognosis, early detection at low levels is very meaningful. Detection results of drug-resistant mutations in the ABL kinase region of the B...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11C12Q1/6886
Inventor 尤隽丹郝玮李小青
Owner 武汉康圣达医学检验所有限公司
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