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Method and kit for quantitative detection of BCR-ABL fusion genes

A technology of fusion gene and quantitative detection, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of insufficient accuracy and poor sensitivity.

Inactive Publication Date: 2019-04-02
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But it is highly dependent on the Ct value, the accuracy is not enough, and the sensitivity is not good

Method used

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  • Method and kit for quantitative detection of BCR-ABL fusion genes
  • Method and kit for quantitative detection of BCR-ABL fusion genes
  • Method and kit for quantitative detection of BCR-ABL fusion genes

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0094] Step 2, preparation of dPCR system

[0095] Preparation before dPCR system preparation: Take out the specific primers and probes, RT-buffer, RNase, Fluorescein sodium salt, 7-deaza-dGTP, RNase-free water, etc. in the kit, melt at room temperature, and vortex to mix well Centrifuge for 10 seconds to prepare a dPCR system; the composition of the dPCR system is shown in Table 5:

[0096] Table 5 dPCR system

[0097] Specific Primers and Probes

1μL

RT-buffer

5μL

RNase

3μL

Fluorescein sodium salt

2.5μL

7-deaza-dGTP

1μL

RNase-free water

Make up to 20 μL

[0098] The nucleotide sequence information of the primer probe is as follows:

[0099] Nucleotide sequence information of primer probes for detection of BCR-ABL fusion gene:

[0100] Table 6

[0101]

[0102] Step 3, add sample

[0103] Take 5 μL of the sample DNA template prepared in step 1 and 5 μL of each control sample in the kit, add the sa...

Embodiment 1

[0115] Embodiment 1: Kit

[0116] The composition, packaging and quantity (48 reactions / box) of a test kit for the quantitative detection of BCR-ABL fusion gene, as shown in Table 8,

[0117] Table 8 The composition, packaging and quantity of the kit

[0118]

[0119]

Embodiment 2

[0120] Embodiment 2: Sensitivity detection and minimum detection rate experiment

[0121] The gDNA control sample is the nucleic acid containing the wild-type BCR-ABL fusion gene derived from Caco2 cell line DNA; the sensitivity reference product consists of two kinds of BCR-ABL P210 fusion gene and BCR-ABL P190 fusion gene template DNA and ABL internal reference gene template DNA (internal reference Gene copy number ≥ 10 4 ) were mixed in a certain proportion, and the IS% of the mixture was 0.1%, 0.01%, and 0.0032% respectively; wherein, the template DNA of BCR-ABL P210 and BCR-ABL P190 was derived from artificially synthesized large fragments 1 and 2; the negative control was RNase -free water;

[0122] Take 5 μL each of the negative control, gDNA control, and sensitivity reference, and add the sample to the eight-tube tube of the dPCR reaction system prepared in step 2, so that the total volume of each tube of dPCR reaction solution is 25 μL; tightly cover the tube cap of ...

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PUM

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Abstract

The invention provides a method and kit for quantitative detection of BCR-ABL fusion genes. The specific technical scheme comprises that two BCR-ABL fusion genetypes are detected only by two differentupstream primers, a common downstream primer, a fluorescent labeled probe and a set of ABL internal reference gene detection system. With use of the specific primers and probes provided by the invention, the two fusion types of BCR-ABL can be detected only by one tube of a material, and the kit has the advantages of high specificity, good sensitivity and high accuracy.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular, the invention relates to a method and kit for quantitative detection of BCR-ABL fusion gene. Background technique [0002] Leukemia, also known as blood cancer, is a malignant clonal disease of hematopoietic stem cells. Leukemia cells are produced in the bone marrow and then spread to the blood and throughout the body. According to foreign statistics, leukemia accounts for about 3% of the total incidence of tumors, and is the most common malignant tumor in children and young people. The incidence rate of leukemia is 4.8-7.1 / 100,000 in men and 3.2-4.6 / 100,000 in women; among countries in the world, the incidence rate in Europe and North America is the highest, and its death rate is 3.2-7.4 / 100,000 population. The incidence rate in Asia and South America is relatively low, and the mortality rate is 2.8-4.5 / 100,000 population. [0003] Chronic myeloid leukemia (CML) malignant c...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2545/114C12Q2563/159
Inventor 蒋析文朱小亚颜尔聪
Owner DAAN GENE CO LTD
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