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Method for screening eosinophilic granulocyte increase related fusion genes by using multiplex fluorescence PCR method technology, primer, probe and composition

An eosinophil and multiple fluorescence technology, which is applied in the field of screening fusion genes related to eosinophilia, can solve the problems of economic convenience, wide detection range and accurate results, and achieve high accuracy, improved efficiency, and high precision Effect

Inactive Publication Date: 2021-04-06
WUHAN ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of the fact that the current screening of eosinophilia-related fusion genes cannot meet the requirements of economic convenience, wide detection range and accurate results, etc.

Method used

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  • Method for screening eosinophilic granulocyte increase related fusion genes by using multiplex fluorescence PCR method technology, primer, probe and composition
  • Method for screening eosinophilic granulocyte increase related fusion genes by using multiplex fluorescence PCR method technology, primer, probe and composition
  • Method for screening eosinophilic granulocyte increase related fusion genes by using multiplex fluorescence PCR method technology, primer, probe and composition

Examples

Experimental program
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Effect test

Embodiment 1

[0069] The amplification primers designed by the present invention are shown in the table below:

[0070]

Embodiment 2

[0072] Extraction of blood RNA:

[0073] Add 1ml of 10× red blood cell lysate into a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated whole blood after mixing, invert and mix.

[0074] Place at room temperature for 5 minutes, and centrifuge at 4000 rpm for 3 minutes. Discard the supernatant, and the resulting mass is the sample leukocyte. Add 1 ml Trizol Reagent to the cell pellet. Blow and suck repeatedly with a pipette gun until there are no obvious cell clumps in the lysate, and let stand at room temperature for 5 minutes. Add 200 μl of chloroform (1 / 5 volume of Trizol Reagent) to the above-mentioned Trizol preservation solution, close the cap of the centrifuge tube tightly, and vortex for 30 seconds. After being fully emulsified to pink (no phase separation), let stand on ice for 10 minutes. Centrifuge at 14,000rpm at 4°C for 10min. Carefully remove the centrifuge tube from the centrifuge, pipette vertically and transfer 450 μl of the supernatant to another n...

Embodiment 3

[0077] Reverse transcription amplification: Configure the PCR amplification system according to the following reagents and reagent volumes, among which primer mix 2.5μl, add deionized water 7.5μl, add RNA template 6μl, pre-denature at 70°C for 5 minutes and place on ice; add 5*RT buffer 4 μl, 1 μl Enzyme Mix (Toyobo (Shanghai) Biotechnology Co., Ltd.), 7 μl deionized water. The amplification program is: 37°C 10min; 98°C 5min

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Abstract

The invention relates to a method for screening eosinophilic granulocyte increase related fusion genes by using a multiplex fluorescence PCR method technology, a primer, a probe and a composition. The screened eosinophilic granulocyte increase related fusion genes comprise six types, namely, FIP1L1-PDGFRa, ETV6-PDGFRbeta, ETV6-ABL, PCM1-JAK2, BCR-FGFR1 and ZNF198-FGFR1. The real-time fluorescence PCR technology is combined with the Tapman probe, eosinophilic granulocyte increase related fusion genes are integrated together so as to be screened, and therefore, diagnosis, treatment and prognosis monitoring of myeloproliferative diseases can be assisted more comprehensively. The method, the primer, the probe and the composition are economical, high in accuracy, high in specificity and suitable for clinical detection of large-batch samples.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a method, primers, probes and composition for screening fusion genes related to eosinophilia by using multiple fluorescent PCR technology. The eosinophilia-related fusion genes screened in the present invention include: FIP1L1-PDGFRa, ETV6-PDGFRβ, ETV6-ABL, PCM1-JAK2, BCR-FGFR1, ZNF198-FGFR1. [0002] technical background [0003] Eosinophils are an important type of cells in the human body, accounting for about 0.5% to 3.0% of the total number of white blood cells. Clinically, eosinophilia is more common, and there are many reasons for inducing it, which vary from person to person. Hypereosinophilic syndrome (HES) is a variant of myeloproliferative disease characterized by increased eosinophils with target organ damage. It manifests as unexplained persistent increase of blood and / or bone marrow eosinophils (eosinopilcell, EC). The revised World Health Or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/16C12Q2600/166C12Q2600/118C12Q2537/143C12Q2563/107C12Q2561/101
Inventor 张辰邹媛杜翠董越
Owner WUHAN ADICON CLINICAL LAB
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