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Primers, probes and methods for screening leukemia mll-sept6 fusion gene by real-time fluorescent PCR

A technology of real-time fluorescence and gene fusion, which is applied in the field of biotechnology detection, can solve the problems that karyotype analysis cannot satisfy MRD, sensitivity cannot be satisfied at the same time, and there are many PCR reaction systems, so as to avoid hematological recurrence, save detection time, and prevent The effect of clinical recurrence

Active Publication Date: 2021-02-23
北京艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Secondly, in some cases, the chromosomal translocations are complex and tiny, which cannot be analyzed by naked eyes
Furthermore, karyotype analysis cannot meet the needs of MRD (Minimal Residual Disease, minimal residual disease) detection
However, the method of multiple nested PCR combined with electrophoresis is time-consuming, cumbersome, easily polluted, the judgment of the result is more subjective, and there are many PCR reaction systems and high cost, so it is not suitable for high-throughput sample detection
It can only perform qualitative detection, and cannot meet the needs of high sensitivity and specificity at the same time

Method used

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  • Primers, probes and methods for screening leukemia mll-sept6 fusion gene by real-time fluorescent PCR
  • Primers, probes and methods for screening leukemia mll-sept6 fusion gene by real-time fluorescent PCR
  • Primers, probes and methods for screening leukemia mll-sept6 fusion gene by real-time fluorescent PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040]The method is used as an auxiliary indicator for early prevention and early diagnosis of human leukemia in clinic; it can also be used for relatively accurate screening of high-risk groups. Reagents include: erythrocyte lysate, TRIzol, chloroform, absolute ethanol, ReverTra Ace qPCR RT Kit (TOYOBO).

[0041] Detection system PCR reaction solution: ReverTra AceqPCR RT Kit (TOYOBO); THNDERBIRDProbe qPCR Mix (2×), MLL-SEPT6 upstream and downstream primers 0.32uM, MLL-SEPT6 probe 0.16uM; abl upstream and downstream primers 0.32uM , abl-probe (probe) 0.16uM; Wherein:

[0042] MLL(exon9)-F:GAACATCCTCAGCACTCTCTCC;

[0043] MLL(exon10)-F:TTGACTTCTGTTCCTATAACACCC;

[0044] MLL(exon11)-F:AAATTGGTGTTGTCGTCGTTG;

[0045] SEPT6(exon2)-R:CAAGCTGTCAAAACCCCACAT;

[0046] MLL-SEPT6-Probe:FAM-CCGAACTGTCCCCCCTGGCTG-TAMRA;

[0047] abl-F:GATACGAAGGGAGGGTGTACCA;

[0048] abl-R:CTCGGCCAGGGTGTTGAA;

[0049] abl-Probe:FAM-GCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA;

[0050] Positive control sub...

Embodiment 2

[0052] The operation flow of this method:

[0053] (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 the precipitation is complete Dissolve, 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 for 10 minutes Centrifuge at 14000rpm at 4°C for 10min, discard the supernatant, add 1ml of 75% ethanol, wash the tube wall upsi...

Embodiment 3

[0063] Clinical Sample Testing

[0064] Take 12 clinical samples to be tested, extract genomes, prepare reagents and detect according to the method described in Example 2.

[0065] Add 2ul of each sample to the detection system PCR reaction solution. At the same time, make positive, negative, and blank controls. Detect with a fluorescent PCR instrument for 100 minutes. The abl of all samples in the 12 screening samples had a line, but none of the samples of MLL exon9-SEPT6 exon2, MLL exon10-SEPT6 exon2 and MLL exon11-SEPT6 exon2 had a line.

[0066] The experimental results are as follows:

[0067]

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Abstract

The invention discloses a method for detecting the relative expression of MLL-SEPT6 fusion gene by using fluorescent quantitative PCR technology, including specific primers and probes, which can be used as a screening method for the trace residue of MLL-SEPT6 fusion gene in leukemia patients, so as to prevent timely intervention and treatment. Hematological recurrence, adjustment of treatment plan, evaluation of treatment effect, prediction of prognosis and prevention of clinical recurrence are all of great significance.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and particularly relates to a method for screening fusion genes, which uses probe Taqman real-time fluorescent PCR technology to detect the situation of MLL-SEPT6 fusion genes in human leukemia patients. 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. The etiology and pathogenesis of human leukemia are still not fully understood. The known causes include infectious factors, ionizing radiat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6848C12Q1/6886C12Q1/686C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 桑志高吴鹏飞王淑一
Owner 北京艾迪康医学检验实验室有限公司
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