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Kit for detecting relative expression index of leukemia BCR/ABL (m-bcr) fusion gene

A technology of relative expression and fusion gene, which is applied in the field of kits for detecting the relative expression of BCR/ABL (m-bcr) fusion genes in leukemia, can solve the problems of high cost, inferior specificity, etc. The effect of reducing detection cost and simple operation

Active Publication Date: 2012-08-29
南京艾迪康医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Kit for detecting relative expression index of leukemia BCR/ABL (m-bcr) fusion gene
  • Kit for detecting relative expression index of leukemia BCR/ABL (m-bcr) fusion gene
  • Kit for detecting relative expression index of leukemia BCR/ABL (m-bcr) fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The kit for detecting the relative expression of leukemia BCR / ABL (m-bcr) fusion gene of the present invention comprises:

[0023] Red blood cell lysate;

[0024] TRIzol;

[0025] Chloroform;

[0026] Anhydrous ethanol;

[0027] ReverTra Ace qPCR RT Kit (TOYOBO);

[0028] Detection system PCR reaction solution: THUNDERBIRD Probe qPCR Mix (2×), BCR / ABL (m-bcr) upstream and downstream primers 0.8uM each, BCR / ABL (m-bcr) probe 0.4uM; abl upstream and downstream primers each 0.8uM, abl-probe (probe) 0.4uM; where:

[0029] m-bcr-F: GGCGCCTTCCATGGAGAC

[0030] m-bcr-R: TCCTTGGAGTTCCAACGA

[0031] m-bcr-Probe: TTTGAGCCTCAGGGTCTGAGTGAA

[0032] abl-F: GCCGTGAAGACCTTGAAGGAG

[0033] abl-R: ATGATATAGAACGGGGGCTC

[0034] abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA.

[0035] Positive control substance: respectively containing BCR / ABL (m-bcr) genome solution;

[0036] Negative control substance: no BCR / ABL (m-bcr) genome solution.

Embodiment 2

[0038] The using method of kit of the present invention:

[0039] (1) Extract tissue 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 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, stand at room temperature Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall upside down gently; ...

Embodiment 3

[0049] Using the nucleic acid detection kit of the present invention to detect clinical specimens

[0050] Acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) and a small number of chronic myeloid leukemia (CML) patients' anticoagulant blood samples sent for inspection were totally 50 cases, and genomic RNA was extracted and reagents were prepared according to the method described in Example 2 and detect.

[0051] Add 2ul of each sample to the detection system PCR reaction solution. At the same time, make a standard curve of positive, negative, blank control, and internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 38 samples at the same time, each sample has 2 repetitions, a positive control, a negative control and a blank control. The detection time is only 100 minutes.

[0052] The experimental results are compared with the results reported by the special inspection laboratory to determine the accuracy of the sample detection. ...

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Abstract

The invention discloses a kit for detecting a relative expression index of a leukemia BCR / ABL (m-bcr) fusion gene. The kit comprises a red blood cell lysis solution, TRIzol, chloroform, absolute ethanol, ReverTraAceqPCRRTKit, a detection system PCR (Polymerase Chain Reaction) reaction solution, a positive control sample and a negative control sample, and is characterized in that the detection system PCR reaction solution comprises THUNDERBIRDqPCRMIX, upstream and downstream primers m-bcr-F and m-bcr-R and a probe m-bcr-Probe for detecting target genes, and primers ab1-F and ab1-R and a probe ab1-Probe for detecting internal reference genes Ab1, wherein the m-bcr-F is GGCGCCTTCCATGGAGAC, the m-bcr-R is TCCTTGGAGTTCCAACGA, the m-bcr-Probe is TTTGAGCCTCAGGGTCTGAGTGAA, the ab1-F is GCCGTGAAGACCTTGAAGGAG, the ab1-R is ATGATATAGAACGGGGGCTC, and the ab1-Probe is FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a gene detection kit, which can detect human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and a small number of chronic myeloid Detection of the expression level of BCR / ABL (m-bcr) fusion gene in patients with cellular leukemia (CML) can effectively save detection time and improve detection accuracy. Background technique [0002] Leukemia is a kind of clonal malignant disease with abnormal hematopoietic stem cells. The leukemia cells in its clones lose the ability to further differentiate and mature and stagnate at different stages of cell development. In the bone marrow and other hematopoietic tissues, a large number of leukemia cells proliferate and accumulate and infiltrate other organs and tissues, and at the same time inhibit normal hematopoiesis. The clinical manifestations are anemia, bleeding, infection and infiltration of various organs....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 方国伟周晓犊王淑一徐建成
Owner 南京艾迪康医学检验所有限公司
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