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Primer and probe for detecting ZNF384-related fusion gene by multiple fluorescence PCR technology, and application

A technology of EP300-ZNF384 and ARID1B-ZNF384, which is applied in the fields of life science and biology, can solve problems such as insufficient sensitivity, high cost of FISH reagents, and easy contamination of the process, and achieve the effects of less sample demand, improved efficiency, and high specificity

Inactive Publication Date: 2021-03-19
济南艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the ZNF384 gene and some fusion partner chromosomal regions (TCF3, EWSR1, EP300, ARID1B) are located near the terminal telomeres of their respective chromosome arms, it greatly increases the difficulty of conventional G-band karyotype analysis; FISH reagents are expensive and less sensitive than PCR; The method of PCR combined with electrophoresis takes a long time, the process is easy to be polluted, the result is difficult to judge, and the cost of the reaction system is relatively high, so it is not suitable for clinical mass screening

Method used

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  • Primer and probe for detecting ZNF384-related fusion gene by multiple fluorescence PCR technology, and application
  • Primer and probe for detecting ZNF384-related fusion gene by multiple fluorescence PCR technology, and application
  • Primer and probe for detecting ZNF384-related fusion gene by multiple fluorescence PCR technology, and application

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Embodiment 1

[0047] Embodiment 1: The amplification primer probes of ZNF384-related fusion genes of the present invention are shown in the following table:

[0048]

Embodiment 2

[0050] Extraction of blood RNA:

[0051] Add 0.5ml of whole blood sample to 1ml of erythrocyte lysate, mix by inverting, leave at room temperature for 5min, then centrifuge at 4000rpm for 3min; discard the supernatant; resuspend in erythrocyte lysate, repeat washing twice to observe the volume of cell clumps, reaching 10-30ul. Add 1 ml Trizol Reagent and pipette repeatedly until there are no cell clumps in the lysate, and let stand at room temperature for 5 minutes. Add 200 μl of chloroform to the above Trizol solution, vortex and mix until fully emulsified, let stand on ice for 10 minutes, and then centrifuge at 14,000 rpm for 10 minutes. Take 450 μl of the supernatant and transfer it to a centrifuge tube filled with pre-cooled isopropanol, mix up and down, put it on ice at -30°C for 10 minutes, then centrifuge at 14,000 rpm for 10 minutes, discard the supernatant, add 1 ml of 75% ethanol to wash the precipitate, and then 14,000 rpm Centrifuge for 5 min; discard the supernat...

Embodiment 3

[0054] Multiplex fluorescent PCR amplification:

[0055] Configure the reverse transcription system according to the following reagents and reagent volume, Primer mix 1ul, 5*RT buffer 4ul, Enzyme Mix 1ul (Toyobo (Shanghai) Biotechnology Co., Ltd.); add deionized water 10ul; finally add sample RNA template 4ul. The reverse transcription program is: 37°C for 60 minutes, 98°C for 5 minutes. Sample cDNA was prepared.

[0056] Configure the PCR amplification system according to the following reagents and reagent volumes, each primer of the primary screening system is 0.1ul, and each probe is 0.05ul; THUNDERBIRD Probe qPCR Mix 12.5ul, 50*ROX 0.5ul (Toyobo (Shanghai) Biotechnology Co., Ltd.); Add 8 ul of deionized water; finally add 2 ul of cDNA template; the amplification program is as follows: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 sec, extension at 58°C for 35 sec, 40 cycles, and fluorescence signal collection set at 58°C for extension.

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Abstract

The invention relates to a primer and a probe for detecting a ZNF384-related fusion gene by a multiple fluorescence PCR technology, and application in a kit. The ZNF384 fusion genes related to an ALLdisease are integrated together to carry out screening, and diagnosis, treatment and prognosis monitoring of the ALL disease can be more comprehensively assisted. Compared with past methods, includingFISH and the like, the method disclosed by the invention has higher accuracy and is convenient to interpret a result. In addition, the primer and the probe are reasonably matched and optimized, a detection condition is optimal, and therefore, efficiency is greatly improved. In addition, the multiple fluorescence PCR technology is high in sensitiveness and specificity, has a low sample requirementand a low cost and is suitable for screening clinical samples on a large scale.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to primers and probes for detecting ZNF384-related fusion genes using multiple fluorescent PCR technology and their application in kits. The ZNF384-related fusion genes screened in the present invention include: EP300-ZNF384, ARID1B-ZNF384, CREBBP-ZNF384, TCF3-ZNF384, TAF15-ZNF384, BMP2K-ZNF384, a total of 6 types. Background technique [0002] Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and the leading cause of death in children. Genetic abnormalities (gene fusion, aneuploidy, etc.) are the main cause of ALL, and molecular typing based on this is of great significance for clinical diagnosis, risk classification and targeted therapy. There are still 20% to 30% of ALL with unknown genetic characteristics. With the development of genomics technology, transcription factor zinc-finger protein 384 (zinc-finger protein 384, ZNF384) fusion positiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/16C12Q2600/166C12Q2600/118C12Q2563/107C12Q2561/101C12Q2537/143C12Q2531/113
Inventor 董越邹媛杜翠张辰
Owner 济南艾迪康医学检验中心有限公司
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