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Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology

A technology that combines genes and compositions, applied in the fields of life sciences and biology, can solve problems such as subjectivity, multiple PCR reaction systems, and complex chromosomal translocations

Active Publication Date: 2016-09-28
WUHAN ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, in some cases, the chromosomal translocations are complex and tiny, which cannot be analyzed by naked eyes
Furthermore, the sensitivity of karyotype analysis cannot meet the needs of MRD (Minimal Residua Disease, minimal residual disease) detection
However, the method of multiple nested PCR combined with electrophoresis takes a long time, the process is cumbersome, easy to pollute, and the judgment of the result is more subjective.
Moreover, there are many PCR reaction systems and the cost is high, so it is not suitable for high-throughput sample detection
The disadvantage of only qualitative detection can not meet the needs of high sensitivity and specificity at the same time

Method used

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  • Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology
  • Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology
  • Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0285] Extraction of RNA from whole blood: Add 1ml of 1× red blood cell lysate to a 1.5ml centrifuge tube, take 0.5ml of the whole blood sample to be tested, and mix by inverting. Centrifuge at 4000rpm for 3min, discard the supernatant, add erythrocyte lysate and wash once to get the desired cells; add 1 ml Total RNA Isolation Reagent, blow and pipe repeatedly until no obvious cell clumps, add 200μl chloroform, vortex mix for 30s, place on ice Let stand for 10min. Centrifuge at 14,000rpm at 4°C for 10min. Use a pipette to draw 450 μl of the supernatant and transfer it to another centrifuge tube, add an equal volume of pre-cooled isopropanol, invert and mix well, and then let stand on ice for 10 minutes. Centrifuge at 14,000rpm at 4°C for 10min. Then wash and centrifuge once with 75% ethanol and absolute ethanol respectively. Dry at room temperature for 5 min, add 50 μl DEPC-H2O to dissolve.

Embodiment 2

[0287] Reverse transcription: Take 4ul of the RNA solution in Example 1 (concentration about 200ng / ul), add 1ul Primer mix (ReverTra Ace qPCR RT Kit) and 3ul DEPC-H2O and mix well, pre-denature at 70°C for 5min; quench on ice for 1min, then add 5 *RT buffer 4ul (ReverTra Ace qPCR RT Kit), Enzyme Mix 1ul (ReverTra Ace qPCR RT Kit), and add DEPC-H20 7ul to a total volume of 20ul. After reacting at 37°C for 60 minutes, it was inactivated at 98°C for 5 minutes, and the cDNA of the sample to be tested was obtained.

Embodiment 3

[0289] Multiplex fluorescent PCR screening: Configure screening reagents according to the materials and dosages shown in the table below. The screening reagent contains a total of 5 reaction tubes: one tube for each of the first group to the fifth group; ABL is the internal reference test in the first group, which is used to judge whether the quality of RNA extraction meets the requirements. 2ul of each cDNA obtained in Example 2 was added. Detection was performed according to the following procedure: pre-denaturation at 95°C for 60s; 10s at 95°C and 50s at 58°C, a total of 40 cycles; fluorescence was collected at 58°C. The result is as image 3 - Shown in A: Screening shows positive for the second group.

[0290]

[0291]

[0292]

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Abstract

The invention relates to a primer, a probe, a composition and a method for screening and identifying MLL rearrangement correlated fusion genes by utilizing a multi-fluorescent polymerase chain reaction (PCR) technology. The screened MLL rearrangement correlated fusion genes comprise thirteen relatively common fusion genes, i.e., MLL-AF9, MLL-AF6, MLL-AF4, MLL-AF1P, MLL-AF1Q, MLL-AF10, MLL-AF17, MLL-AFX1, MLL-ELL, MLL-ENL, MLL-SEPT6, MLL-CBP and dup MLL. The primer, the probe, the testing combination way and the detection method are convenient, economical, fast, good in specificity, high in sensitivity and large in flux, thus being suitable for clinical test of large-batch samples.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer, a probe, a composition and a method for screening MLL rearrangement-related fusion genes by using multiple fluorescent PCR technology. Background technique [0002] Abnormal MLL rearrangements are seen in approximately 5-10% of acute leukemia (AL) patients. Among them, except for the MLL-AF9 fusion gene, which is an intermediate prognostic indicator, the rest of the abnormalities associated with MLL rearrangement are associated with poor prognosis. In the clinical treatment of acute leukemia, especially for the treatment of AML, a prognostic evaluation stratification will be carried out, and different treatment options will be selected according to different prognostic stratifications. At the same time, it is also necessary to monitor the expression level of the fusion gene during the treatment to predict the curative effect and the possibility ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/16C12Q2563/107C12Q2537/143
Inventor 邹媛杜翠陈红梅夏成青
Owner WUHAN ADICON CLINICAL LAB
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