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Reaction system for detecting EML4-ALK fusion gene based on digital PCR and applications of reaction system

A technology of fusion gene and reaction system, applied in the field of molecular biology detection, can solve problems such as reducing the probability of false positives

Pending Publication Date: 2021-09-10
远辰生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a reaction system based on digital PCR detection of EML4-ALK fusion gene, which improves the fusion mutation by optimizing the primer probe specificity of EML4-ALK fusion gene. The detection rate is high, and the special RNA sample pretreatment process is assisted to further reduce the probability of false positives. Combining the above characteristics and advantages, the detection system of the present invention can realize three kinds of EML4-ALK fusion mutation sites in one reaction The synchronous detection has great application value for the detection of micro samples, which makes up for the difficulties in clinical sampling and small sample volume.

Method used

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  • Reaction system for detecting EML4-ALK fusion gene based on digital PCR and applications of reaction system
  • Reaction system for detecting EML4-ALK fusion gene based on digital PCR and applications of reaction system
  • Reaction system for detecting EML4-ALK fusion gene based on digital PCR and applications of reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Design and establishment of a digital PCR detection system for EML4-ALK gene fusion mutation

[0054] 1. Design of primer-probe combination

[0055] First, according to the subtypes of EML4-ALK gene fusion variants reported in the literature, a variety of subtypes were analyzed, and three main types of fusion variants were selected (accounting for more than 85% of EML4-ALK gene fusion variant subtypes) . Multiple sets of primers and probes were designed across the fusion breakpoint region for specific screening.

[0056] Primer design principles: The primer pair used to detect the fusion variation of the EML4-ALK gene in the present invention is designed by Primer5 and NCBI Blast software; the length of the primers is between 18-25 nucleotides, and the GC content of the primers is between 40%- Between 60%, the Tm value of the primer is ≥60°C. The Tm values ​​of each primer are roughly close to ensure that the primer pair can be amplified efficiently at the ...

Embodiment 2

[0074] Example 2 Detection of tumor tissue samples

[0075] Two cases of tumor tissue samples were selected, and the detection system of the present invention was used to detect their RNA, and the specific operation steps were as follows:

[0076] 1. RNA extraction from tumor tissue samples

[0077] The RNeasy FFPE Kit from QIAGEN was used to extract RNA from 2 tumor tissue samples, and Qubit was used to detect the RNA concentration and quality for later use.

[0078] 2. Perform reverse transcription reaction on RNA to obtain cDNA.

[0079] 2.1 RNA samples were denatured at 72°C for 3 minutes, then cooled in an ice bath;

[0080] 2.2 Reverse transcription reaction: 48°C, 60 minutes; 50°C, 2 minutes, 48°C, 2 minutes, 10 cycles; 70°C enzyme inactivation, 15 minutes; 4°C incubation, the reaction is over.

[0081] 3. PCR amplification

[0082] 3.1 dPCR amplification system preparation

[0083] Prepare the amplification reaction according to the digital PCR reaction system: dP...

Embodiment 3

[0090] Example 3 Detection of plasma free nucleic acid samples

[0091] The plasma samples of 2 patients with lung cancer were selected, and the detection system of the present invention was used to detect their plasma cfRNA, and the specific operation steps were as follows:

[0092] 1. Plasma cfRNA Extraction

[0093] The QIAamp ccfDNA / RNA Kit from QIAGEN was used to extract RNA from 2 plasma samples, and Qubit was used to detect the RNA concentration and quality for later use.

[0094] 2. Perform reverse transcription reaction on RNA to obtain cDNA.

[0095] 2.1 RNA samples were denatured at 72°C for 3 minutes, then cooled in an ice bath;

[0096] 2.2 Reverse transcription reaction: 48°C, 60 minutes; 50°C, 2 minutes, 48°C, 2 minutes, 10 cycles; 70°C enzyme inactivation, 15 minutes; 4°C incubation, the reaction is over.

[0097] 3. PCR amplification

[0098] 3.1 dPCR amplification system preparation

[0099]Prepare the amplification reaction according to the digital PCR ...

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Abstract

The invention provides a reaction system for detecting an EML4-ALK fusion gene based on digital PCR and applications of the reaction system. A detection system comprises a specific primer and a probe of the EML4-ALK fusion gene. By screening and optimizing primer and probe sequences and concentration proportion combination in a PCR amplification system, the detection rate of fusion mutation is increased, to assist a special RNA sample pretreatment process, so as to further reduce false positive probability. In addition, a digital PCR platform is used. The detection rate of various types of samples is remarkably improved. The detection on trace samples, such as free nucleic acid and exosome RNA, shows higher sensitivity and detection success rate.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a reaction system for detecting EML4-ALK fusion gene based on digital PCR and its application. Background technique [0002] With the advancement of bioanalysis technology and the development of information technology, as well as the in-depth understanding of gene-based medicine, the concept of precision medicine (Precision Medicine) has been extracted from the concepts of system medicine and translation medicine, and has become a highly comprehensive and detailed medical science. The new model of medicine is expected to improve the level of disease prevention and disease treatment. As many major countries in the world put forward the strategy of precision medicine, precision medicine has become the development direction of future medicine. As the first year of precision medicine, 2015 has important historical significance. Today, precision medicine has become...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2521/107C12Q2531/113C12Q2563/159C12Q2563/107
Inventor 徐梅波张建
Owner 远辰生物科技(苏州)有限公司
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