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Primers, method, and kit used for detecting BCR/ABL fusion gene ABL kinase domain drug resistance mutation sites

A technology of drug resistance mutation sites and fusion genes, which can be applied in the fields of life science and biology and can solve problems such as aerosol pollution

Active Publication Date: 2015-03-18
SHANGHAI ADICON CLINICAL LAB LNC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the method used to detect mutations in the ABL kinase region of the BCR / ABL fusion gene is mainly the traditional two-tube nested PCR method. For a long time, nested PCR has been widely used because it can improve the specificity of the product and expand the yield. However, the drawbacks of the two-tube nested PCR method, which is prone to aerosol pollution, have been difficult to overcome.
Therefore, the application of this method in some laboratories with high experiment density and many projects, such as clinical inspection centers, is limited.

Method used

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  • Primers, method, and kit used for detecting BCR/ABL fusion gene ABL kinase domain drug resistance mutation sites
  • Primers, method, and kit used for detecting BCR/ABL fusion gene ABL kinase domain drug resistance mutation sites
  • Primers, method, and kit used for detecting BCR/ABL fusion gene ABL kinase domain drug resistance mutation sites

Examples

Experimental program
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Effect test

Embodiment 1

[0119] A kit for detecting drug-resistant mutation sites in the ABL kinase region in the two shear types M-bcr and m-bcr of the BCR / ABL fusion gene, including: RNA extraction solution: erythrocyte lysate, Trizol, chloroform, absolute ethanol; Reagents for reverse transcription: ReverTra Ace qPCR RT Kit (TOYOBO); detection system PCR reaction solution; sequencing system reaction solution; positive control, negative control and blank control.

[0120] Detection system PCR reaction solution includes: 2×PCR Buffer; dNTPs (2mM); KOD FX DNA Polymerase (1U / μl); M-bcr shear for amplifying the ABL kinase region in the spliced ​​M-bcr of BCR / ABL fusion gene Cut-type outer upstream and downstream primers M-bcr-Outer-F (10 μM), M-bcr-Outer-R (10 μM); amplify the M-bcr of the ABL kinase region in the cut-type M-bcr of the BCR / ABL fusion gene M-bcr-Inner-F (20μM) and M-bcr-Inner-R (20μM) for the shear type inner upstream and downstream primers; M for the ABL kinase region in the shear type ...

Embodiment 2

[0137] The operation process of blood RNA extraction, reverse transcription, amplification and sequencing is as follows:

[0138](1) Extract RNA from blood: Add 1ml of erythrocyte lysate to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Let stand at room temperature for 10 minutes; centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml Trizol to the cells, and pipette repeatedly until sedimentation Dissolve completely, let stand at room temperature for 5 minutes; add 0.2ml chloroform, shake evenly; centrifuge at 14000rpm at 4°C for 10 minutes, absorb the supernatant layer and transfer to another new centrifuge tube; add an equal volume of isopropanol, mix well up and down, and let stand at room temperature Centrifuge at 14000rpm at 4°C for 10min, discard the supernata...

Embodiment 3

[0166] The nucleic acid detection kit of the present invention is used to detect clinical blood samples.

[0167] 20 samples of anticoagulated blood from patients with chronic myeloid leukemia (CML) were taken for examination, and blood RNA was extracted, reagents were prepared and tested according to the method described in Example 2.

[0168] For each sample, 2 μl of cDNA produced by reverse transcription was added to the PCR reaction solution of the detection system, and positive, negative, and blank control experiments were performed at the same time. A 96-well ordinary PCR machine can detect 46 samples at the same time, each sample has 2 repetitions, a positive control, a negative control and a blank control. The detection time is 160 minutes.

[0169] After each sample was sequenced twice, it was compared with the wild-type reference sequence of the ABL kinase region in the two spliced ​​types M-bcr and m-bcr of the BCR / ABL fusion gene (Genbank accn: NM_005157.4 / NM_0215...

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Abstract

The invention discloses primers, a method, and a kit used for detecting ABL kinase domain drug resistance mutation sites in two shear-type M-bcr and m-bcr in BCR / ABL fusion gene. The kit comprises; (i) inner and outer primers used for amplifying M-bcr shear-type and m-bcr shear-type fusion gene ABL kinase domains; (ii) an antisense primer complementary with the outer primer; and (iii) a pair of sequencing primers covering all mutation hotspots in the BCR / ABL fusion gene ABL kinase domains. According to the invention, with a tube nested PCR technology, the kit and the method can be used for rapidly detecting the mutation of drug resistance sites in BCR / ABL fusion gene ABL kinase domains in chronic myelogenous leukemia patients.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to primers, methods and kits for detecting drug-resistant mutation sites in the ABL kinase region of the BCR / ABL fusion gene, which can be used for rapid detection of chronic myelogenous leukemia by adopting single-tube nested PCR technology Mutations of drug resistance sites in the ABL kinase region of the BCR / ABL fusion gene in patients. Background technique [0002] Chronic myeloid leukemia (CML) is a malignant clonal proliferative disease of the blood system that occurs in hematopoietic stem cells. Abnormal white blood cells in 95 percent of CML patients contain a chromosome called "Philadelphia" because a segment of chromosome 22 is misplaced on chromosome 9, and a segment of chromosome 9 is misplaced on chromosome 22 On the chromosome, chromosome 22 is shortened, and an oncogenic fusion gene BCR / ABL is formed on it. The BCR / ABL fusion gene leads to th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6848C12Q1/686C12Q1/6886C12Q2547/101C12Q2549/119C12Q2600/106C12Q2600/156C12Q2531/113
Inventor 周晓犊陈奕磊李文静王淑一
Owner SHANGHAI ADICON CLINICAL LAB LNC
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