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EGFR gene mutation detection system and kit of detection system

A technology of EGFRT790M and system, applied in the direction of microbial measurement/inspection, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as unsatisfactory

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

However, due to the particularity of plasma cell-free DNA, its content in plasma and its tiny amount, a single sampling cannot meet the needs of experimental samples for individual detection of a single site

Method used

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  • EGFR gene mutation detection system and kit of detection system
  • EGFR gene mutation detection system and kit of detection system
  • EGFR gene mutation detection system and kit of detection system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Design and establishment of EGFR gene mutation detection system

[0063] 1. Design of primer-probe combination

[0064] First, refer to the GeneBank sequence number of the EGFR (epidermal growth factor receptor) gene reported in the literature, find the gene sequence corresponding to each site from the NCBI database, and design specificity upstream and downstream of the T790M, 19DEL, and L858R mutation sites confirmed by research. Amplify the primer pair, and simultaneously design wild-type and mutant-specific probes in the case of covering the mutation site nucleotides.

[0065] Primer design principles: The primer pairs used to detect EGFR gene mutation sites T790M, 19DEL, and L858R in the present invention are designed by Primer5 and NCBI Blast software; the length of the primers is between 18-26 nucleotides, and the GC content of the primers is Between 40% and 60%, the Tm value of the primer is ≥60°C. The Tm values ​​of each primer are roughly close to e...

Embodiment 2

[0087] Example 2 Detection of Patient Tissue Samples

[0088] Two cases of non-small cell lung cancer FFPE samples were selected, and the detection system of the present invention was used to detect the cell line DNA. The specific operation steps were as follows:

[0089] 1. DNA extraction from non-small cell lung cancer FFPE samples

[0090] Use the QIAamp DNA FFPE Tissue Kit from QIAGEN to extract DNA from non-small cell lung cancer FFPE samples, use Qubit to detect DNA concentration and quality, and finally dilute to a concentration of 10ng / μL for later use.

[0091] 2. PCR amplification

[0092] 2.1 dPCR amplification system preparation

[0093] Prepare the amplification reaction according to the system of the QIAcuity system digital PCR reaction system: 10 μL of 4×dPCR-enzyme reaction solution, 4.0 μL of 10× primer-probe mixture, 2.0 μL of FFPE DNA, and make up to 40 μL with deionized water.

[0094] 2.2 Add the reaction mixture to the sample well of the dPCR nano-micr...

Embodiment 3

[0105] Example 3 Detection of plasma free nucleic acid samples

[0106] The plasma samples of 3 patients with lung cancer were selected, and the detection system of the present invention was used to detect the plasma free nucleic acid (cfDNA). The specific operation steps are as follows:

[0107] 1. Plasma cell-free nucleic acid (cfDNA) extraction

[0108] The MagMAX™ Cell-Free DNA Isolation Kit from Applied Biosystems was used to extract cfDNA from the plasma samples of 3 lung cancer patients, and then the concentration and quality of cfDNA were detected using Qubit.

[0109] 2. PCR amplification

[0110] 2.1 dPCR amplification system preparation

[0111] Prepare the amplification reaction according to the system of the QIAcuity system digital PCR reaction system: 10 μL of 4×dPCR-enzyme reaction solution, 4.0 μL of 10× primer-probe mixture, 20.0 μL of cfDNA, and make up to 40 μL with deionized water.

[0112] 2.2 Add the reaction mixture to the sample well of the dPCR nano...

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Abstract

The invention provides an EGFR (epidermal growth factor receptor) gene mutation detection system and a kit of the detection system. The detection system can synchronously detect three sites T790M, 19DEL and L858R of an EGFR gene in a reaction system, so that the detection of a trace DNA (deoxyribonucleic acid) sample is realized, and meanwhile, based on a digital PCR (polymerase chain reaction) detection platform of a multicolor fluorescent channel, the sites with gene mutation can be clearly prompted, and an accurate and reliable reference basis is provided for subsequent clinical diagnosis and evaluation. According to the present invention, the EGFR gene mutation detection system is especially suitable for detection of plasma free DNA (cfDNA), a plurality of EGFR gene mutation sites can be detected through the same reaction system based on a digital PCR technology platform, the blank of clinical practical application is filled for trace sample patients and patients with tissue sample sampling difficulty, and the detection system has a wide application prospect and a huge market value.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to an EGFR gene mutation detection system and a kit thereof. Background technique [0002] The human epidermal growth factor receptor family (EGFR family) is a transmembrane tyrosine kinase receptor, and the activation of the receptor kinase domain is of great significance to the relative signal transmission of cancer cell proliferation and growth. Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene C-erbB-1 (HER-1), located on the cell membrane. EGFR mainly transmits signals to the nucleus through two pathways, one is Ras→Raf→MAPK pathway; the other is PI3K→PKC→IKK pathway. When the signal is transmitted to the nucleus, it will cause an increase in the transcription level of genes in the nucleus, resulting in cell proliferation and transformation. Abnormality of EGFR signaling is the cause of many tumors. [0003] Targeted drug...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2563/159C12Q2563/107C12Q2565/631
Inventor 徐梅波李玉玺
Owner 远辰生物科技(苏州)有限公司