Probe, primer and kit for detecting ALK (anaplastic lymphoma kinase) gene expression

A detection kit and a technology for detecting differences in gene expression, applied in the biological field, can solve problems such as difficult interpretation, long detection cycle, and inconspicuous separation signals, and achieve the effects of wide clinical application range, fast detection speed, and wide detection range

Active Publication Date: 2013-09-11
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The previous RT-PCR method for ALK fusion gene detection was designed and detected for the reported ALK fusion types, and the detection spectrum can only cover known ALK fusion types, therefore, it does not have unknown/unreported ALK fusions It is difficult to avoid false positives caused by amplification; the IHC method can detect the expression of tumor-specific antigens without losing the ability to distinguish normal tissues from pathological tissues However, the IHC method is aimed at the expression of ALK protein, and there may be false positives (increased expression of ALK protein may be due to the fusion of ALK gene in tumor cells, and may Due to the abnormal expression of ALK

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  • Probe, primer and kit for detecting ALK (anaplastic lymphoma kinase) gene expression
  • Probe, primer and kit for detecting ALK (anaplastic lymphoma kinase) gene expression
  • Probe, primer and kit for detecting ALK (anaplastic lymphoma kinase) gene expression

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Embodiment 1

[0075] Using the system of the present invention to detect plasmids, plasmid templates for experiments (containing ALK expression exon fragments), and using the above-mentioned fluorescent PCR to detect ALK expression are as follows:

[0076] (1) Plasmid treatment and extraction:

[0077] Plasmids were extracted using a plasmid extraction kit from TIANGEN (HighPure Plasmid Kit, DP116), and the specific extraction steps were performed according to the instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mmol / L, PH8.0), the quality of the extraction was detected by a UV spectrophotometer, and the concentration was determined, and then the DNA was adjusted with Tris-HCl (10mmol / L, PH8.0) solution. Concentration to use as a PCR template, take 5 μ L for PCR reaction amplification.

[0078] (2) Establish PCR reaction system

[0079] The cDNA template obtained above was used as a template for real-time fluorescent PCR amplification, and PCR amplification was perf...

Embodiment 2

[0100] A total of 3845 cases of paraffin-embedded tissue samples of clinical lung cancer were detected by using the present invention, and the steps of detecting ALK gene expression differences of 3845 cases of clinical samples by using the specific primer and probe fluorescent PCR system of the present invention were as follows:

[0101] (1) Sample processing and RNA extraction:

[0102] (a) Take each of the above lung cancer samples, add 1ml of xylene, mix well, centrifuge at 14000RPM for 2min at room temperature, discard the supernatant, add 1ml of absolute ethanol to the pellet, shake and mix (remove xylene), 14000RPM at room temperature Centrifuge for 2 minutes to discard the supernatant, open the cap of the centrifuge tube, and dry at 37°C;

[0103] (b) Add 150 μl Buffer PKD and 10 μl proteinase K to the centrifuge tube, shake and mix, incubate at 56°C for 15 minutes, and incubate at 80°C for 15 minutes. After cooling down to room temperature, add 16ul DNase Booster Buff...

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Abstract

The invention discloses primer, a probe and a kit for detecting ALK (anaplastic lymphoma kinase) gene expression difference. The primer mainly comprises the following sequences: SEQ ID NO1-SEQ ID NO12. The primer and the probe disclosed by the invention can specifically detect the ALK gene expression difference, so as to determine whether ALK gene fusion exists. A real-time fluorescent PCR (polymerase chain reaction) system for detecting the ALK gene expression difference is built; qualitative evaluation of an ALK gene expression difference exon is provided by detecting the difference of 5'-end and 3'-end gene expression quantities; and the primer and the probe are suitable for a patient with a non-small cell lung cancer before entering personalized targeted therapy. The method has the advantages that (1) sensitivity is high, wherein five copies of positive plasmids can be detected; (2) operation is simple, detection is cheap, and clinical application range is wide; (3) sample detection range is wide, wherein the sample can be used as a fresh pathological tissue, a paraffin-embedded tissue (slice) or a frozen pathological slice; (4) fast detection speed is fast, wherein the detection process can be finished just by 160 minutes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting differential expression of ALK gene. Background technique [0002] Receptor-type tyrosine kinase Anaplastic lymphoma kinase (Anaplastic lymphoma kinase, ALK) was first discovered in anaplastic large cell lymphoma (Anaplastic large cell lymphoma, ALCL), which is formed by the translocation of chromosomes 2 and 5 The fusion protein contains the 3' end intracellular domain of ALK and the 5' end domain of nucleophosmin (NPM). Subsequent studies have found that normal ALK is exclusively expressed in the nervous system, such as the brain and nerve cord, especially in the brain of newborns. The ALK gene is located at chromosome 2p23. Under normal circumstances, human ALK can be transcribed to produce a 6222bp mRNA, which consists of 29 exons and encodes a 1620 amino acid sequence of 200KDa type I transmembrane protein ALK. The protein is a receptor ty...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 施伟杰林清华张鹏程陈昌盛宋庆涛阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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