Kit used for detecting relative expression of AML1-ETO fusion gene

A technology of relative expression and fusion genes, applied in the field of gene detection kits, can solve the problems of high cost and poor specificity, and achieve the effects of simple operation, reduced detection cost, and good specificity.

Active Publication Date: 2012-10-17
北京艾迪康医学检验实验室有限公司
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  • Abstract
  • Description
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Problems solved by technology

Among them, since 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; while the cost of the double-probe hybridization method is relatively expensive

Method used

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  • Kit used for detecting relative expression of AML1-ETO fusion gene
  • Kit used for detecting relative expression of AML1-ETO fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The kit for detecting the relative expression of AML1-ETO fusion gene of the present invention comprises:

[0025] Red blood cell lysate;

[0026] TRIzol;

[0027] Chloroform;

[0028] Anhydrous ethanol;

[0029] ReverTra Ace qPCR RT Kit (TOYOBO);

[0030] Detection system PCR reaction solution: THUNDERBIRD Probe qPCR Mix (2×), upstream and downstream primers for detection of target genes AML1-ETO-F, AML1-ETO-R each 0.8uM, AML1-ETO-Probe (probe) 0.4uM; detection The internal reference gene Abl uses the upstream and downstream primers abl-F, abl-R each 0.8uM, abl-probe (probe) 0.4uM; where:

[0031]AML1-ETO-F: GTCTTTCACAAACCCACCGCAAG

[0032] AML1-ETO-R: GTCAGCCTAGATTGCGTCTTCA

[0033] AML1-ETO-Probe: FAM-TGAGAAGCACTCCACAATGCCAGACTCACC-TAMRA

[0034] abl-F: GCCGTGAAGACCTTGAAGGAG

[0035] abl-R: ATGATATAGAACGGGGGCTC

[0036] abl-Probe: FAM-ACCTGGTGCAGCTCCTTGGG-TAMRA.

[0037] Positive control substance: a solution containing the AML1-ETO gene;

[0038] Negative...

Embodiment 2

[0040] The using method of kit of the present invention:

[0041] (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

[0051] Using the nucleic acid detection kit of the present invention to detect clinical specimens

[0052] A total of 80 cases of anticoagulant blood samples from patients with acute promyelocytic leukemia (APL) submitted for inspection were extracted, and genomic RNA was extracted, reagents were prepared and tested according to the method described in Example 2.

[0053] 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.

[0054] The experimental results are compared with the results reported by the special inspection laboratory to determine the accuracy of the sample detection. Some positive results are as follows:

[0055] ...

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Abstract

The invention discloses a kit used for detecting relative expression of the AML1-ETO fusion gene. The kit comprises erythrocyte lysate, TRIzol, chloroform, anhydrous ethanol, a ReverTra Ace qPCR RT kit, a detection system PCR reaction solution, a positive control and a negative control, and is characterized in that the detection system PCR reaction solution comprises THUNDERBIRD qPCR MIX, the upstream and downstream primers of AML1-ETO-F and AML1-ETO-R and the probe of AML1-ETO-Probe used for detecting target genes, and the primers of ab1-F and ab1-R and the probe of ab1-Probe used for detecting the internal control gene Ab1. The kit provided in the invention can detect the expression level of the AML1-ETO fusion gene in patients with acute myeloid leukemia (AML) and can effectively save detection time and improve detection precision.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a gene detection kit, which can express the AML1-ETO fusion gene in human patients with acute myeloid leukemia (AML) by using real-time fluorescence quantitative PCR technology of probes. Level detection can effectively save detection time and improve detection accuracy. Background technique [0002] Chromosomal translocations are common in acute myeloid leukemia (AML), some of which are specifically associated with subtypes of AML. In AML, t(8;21)(q22;q22) is one of the most common chromosomal abnormalities, which can be found in nearly 40% of AML-M2 subtype patients and 8% to 20% of primary AML patients. This translocation leads to the fusion of the AML1 gene (acutemyeloblastic leukemia one gene) on chromosome 21 and the ETO gene (eight twenty-one gene, also known as MTG8 gene) on chromosome 8, forming AML1-ETO and ETO-AML1 fusion genes. Since the latter cannot b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/686C12Q2561/101
Inventor 周晓犊方国伟王淑一徐建成
Owner 北京艾迪康医学检验实验室有限公司
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