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Ewing sarcoma related fusion gene detection probe composition, kit and application thereof

A fusion gene and detection probe technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of subjective error, cumbersome operation, time-consuming result interpretation, etc., to improve detection The effect of improving the output rate, improving the accuracy and reducing the missed detection rate

Active Publication Date: 2021-06-29
SHANGHAI BIOTECAN PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH detection probes are costly and cumbersome to operate, time-consuming to interpret results, and subjective errors exist. At the same time, EWS-FLI1 fusion only occurs in about 85% of Ewing sarcoma patients, which may lead to missed detection.
[0006] CN108559780A discloses primer pairs, probes, kits and two-step detection methods for the detection of tumor fusion gene EWS-FLI1, which belong to the field of genetic engineering technology and can solve the existing cumbersome detection operations for fusion gene EWS-FLI1, Interpretation of results is time-consuming and has technical problems such as subjective errors
However, the primer pair, probe, and kit can only detect two sites of the fusion gene EWS-FLI1, and cannot completely cover the gene mutation region that causes Ewing sarcoma

Method used

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  • Ewing sarcoma related fusion gene detection probe composition, kit and application thereof
  • Ewing sarcoma related fusion gene detection probe composition, kit and application thereof
  • Ewing sarcoma related fusion gene detection probe composition, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Nucleic acid extraction and reverse transcription

[0060] In this embodiment, nucleic acid is first extracted from the sample, and Qubit 3.0 is used for quality control, and then a strand of cDNA is synthesized using the extracted nucleic acid as a template;

[0061] Prepare a one-strand cDNA synthesis reaction system on ice according to Table 1, use a pipette to gently blow and mix well, and then centrifuge briefly; place the reaction system in a PCR instrument, and perform one-strand cDNA synthesis according to the procedures in Table 2;

[0062] Table 1

[0063] Element Volume (μL) DNA / RNA 8.5 RNA Fragmentation Mix 8.5 1st Strand Buffer (1st Strand Buffer) 7 1st Strand Enzyme Mix 1

[0064] Table 2

[0065] temperature(°C) time 25 10min 42 15min 70 15min 4 Keep

[0066] Using one-strand cDNA as a template, prepare a second-strand cDNA synthesis reaction system on ice accordi...

Embodiment 2

[0073] Example 2 Pre-library Construction

[0074] In this example, the DNA sample extracted / reverse transcribed in Example 1 was used for pre-library construction, and the steps are as follows:

[0075] Mix 5 μL DNA Fragmentation-Add A Tailing Buffer (DNA Frag-A Tailing Buffer) and 2.5 μL DNA Fragmentation-Add A Tailing Enhancer (DNA Frag-A Tailing Enhancer) by gentle pipetting, then add DNA sample, 10 μL DNA Fragmentation enzyme (DNA Frag Enzyme) and 1.2 μL of A tailing enzyme (End Repair-A Tailing Enzyme Mix), gently pipette to mix;

[0076] Briefly centrifuge the prepared system to the bottom of the tube and immediately transfer it to a 4°C pre-cooled PCR instrument (hot lid temperature is 70°C), and perform the reaction according to the procedures in Table 5;

[0077] table 5

[0078] temperature(°C) time 32 22min 65 30min 4 Keep

[0079] Prepare the reaction system according to Table 6, gently pipette and mix thoroughly, place in a PC...

Embodiment 3

[0091] Example 3 Target Fragment Capture

[0092] (1) Transfer 200-500ng of the quantified pre-library to a new centrifuge tube, concentrate using a concentrator-dryer (temperature not exceeding 45°C), then add 5 μL of enzyme-free water and 7.7 μL of Universal Blocker Mix (IL)), fully oscillated and mixed, placed on the PCR instrument (with hot lid mode) to execute the program according to Table 10;

[0093] Table 10

[0094] temperature(°C) time 95 5min 65 Keep

[0095] (2) Prepare a hybridization mixture in a 1.5mL EP tube according to Table 11, mix well and then centrifuge quickly, transfer all to the above-mentioned pre-library mixture (always kept on the PCR instrument), and then hybridize at 65°C for 16 hours after gently pipetting ;

[0096] Table 11

[0097]

[0098] (3) After the hybridization of the pre-library and the probe is completed, add 200 μL of magnetic beads, pipette to mix, rotate and incubate at room temperature for 30 mi...

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Abstract

The invention provides an Ewing sarcoma related fusion gene detection probe composition, kit and application thereof. Target genes of the probe composition comprise NUTM2B whole exons, FUS whole exons, YWHAE whole exons, CIC whole exons, EWSR1 whole exons, EWSR1 introns 7-8, EWSR1 introns 8-9 and BCOR whole exons. Aiming at common gene fusion mutation forms of Ewing sarcoma, the probe composition fully covering fusion gene mutation sites is designed, the detection rate of different fusion phenomena is remarkably improved, and the omission ratio is reduced.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to a detection probe composition, kit and application of Ewing sarcoma-related fusion genes. Background technique [0002] Ewing sarcoma (ES) is a rare small round cell malignant tumor belonging to the Ewing sarcoma family of tumors (ESFT). Although the disease is rare, ES occurring in bone is the second most common primary malignant bone tumor affecting children and adolescents. This type of tumor is highly malignant, prone to recurrence, and poor prognosis. [0003] Ewing sarcoma is characterized by the fusion of the EWS gene (EWSR1) with several genes of the ETS gene family (FLI1, ERG, ETV1, ETV4, FEV) on chromosome 22q12. In 85% of Ewing's sarcoma patients, the fusion of EWS and FLI1 gene on chromosome 11 and the transcription of EWS-FLI1 fusion gene caused by the corresponding t(11;22)(q24;q12) chromosomal translocation occurred. In 5% to 10% of cases, EWS is fused with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6827C12N15/11
CPCC12Q1/6886C12Q1/6827C12Q2600/118C12Q2600/112C12Q2600/156C12Q2525/173C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 林灵侯群星魏利然刘静仪张朔王永攀武晓会文明楼敬伟
Owner SHANGHAI BIOTECAN PHARMA
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