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Detection method for human EML4-ALK fusion gene

A technology of EML4E6-ALKE20 and EML4E13-ALKE20, which is applied in the field of human EML4-ALK fusion gene detection, can solve the problems of low differential expression level of ALK protein, high requirements for operation and interpretation technology, high cost of FISH detection, etc., and achieves convenient interpretation , low cost and high accuracy

Pending Publication Date: 2018-01-12
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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Problems solved by technology

Although FISH is currently the gold standard for detection of EML4-ALK fusion genes, FISH detection has high requirements for operation and interpretation techniques, and diagnostic physicians must undergo strict training in FISH operation and result interpretation. Reliable; current FISH assays are expensive and low throughput
IHC detects fusion by detecting the overexpression of ALK protein, but due to the low level of differential expression of ALK protein, its sensitivity and reproducibility are poor, and there is no standard operating procedure among different laboratories, the same detection results will be different explanation of

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  • Detection method for human EML4-ALK fusion gene
  • Detection method for human EML4-ALK fusion gene
  • Detection method for human EML4-ALK fusion gene

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Embodiment 1

[0073] Example 1: Fluorescence quantitative PCR rapid detection of human EML4-ALK fusion gene

[0074] Step 1: Extract sample RNA according to the above RNA extraction steps.

[0075] Step 2: Reverse transcribe the extracted RNA into cDNA according to the above reverse transcription steps.

[0076] Step 3: Fluorescent quantitative PCR reaction system configuration

[0077] Fluorescent quantitative PCR reaction system (20μL):

[0078]

[0079] The EML4-ALK fusion detection reaction and the internal reference RNase P reaction are divided into 2 tubes. These two reactions should be carried out for each sample, and a positive control and a negative control should be set up for each batch.

[0080] Step 4: Real-time quantitative PCR reaction is carried out

[0081] The reaction solution prepared in step 3 was reacted in the Stepone Plus fluorescent quantitative PCR instrument according to the following procedures: 37°C, 2min; 95°C pre-denaturation for 10min; 50cycles: 95°C, ...

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Abstract

The invention discloses a detection method for a human EML4-ALK fusion gene. According to the detection method, a primer is designed on each of fusion exons of EML4 terminals of EML4E2-ALK E20, EML4E3-ALK E20, EML4E6-ALK E20, EML4E13-ALK E20, EML4E14-ALK E20, EML4E15-ALK E20, EML4E17-ALK E20, EML4E18-ALK E20, and EML4E20-ALK E20, another common reverse primer is designed on each No.20 exon of ALK,and a Taqman-MGB probe is designed on each No.20 exon of ALK. Through one hybrid reaction and one internal reference reaction, the detection for 9 EML4-ALK fusion types is realized.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a human EML4-ALK fusion gene detection method. Background technique [0002] The EML4-ALK fusion gene is located on the short arm of chromosome 2 (2p21 and 2p23), its 5' end is a fragment of EML4, and its 3' end is a fragment of ALK, which is connected by the inverted EML4 gene fragment and the remaining ALK fragment . The EML4 gene can be broken at multiple sites, and the broken amino-terminal fragment replaces the extracellular domain and transmembrane domain of ALK, and directly connects with the intracellular catalytic domain located in the 20th exon of ALK, forming a variety of EML4- ALK fusion mutants. The signal transduction pathways of EML4-ALK are PI3-K / Akt, STAT3 / 5, Ras-MEK and PLC-γ / PIP2, etc. These pathways are closely related to cell survival, proliferation and migration. EML4-ALK gene fusion can promote the expression of oncogenic fusion protein caus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/686C12Q1/6886
Inventor 汪元涛朱月艳孙子奎
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD