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Multiplex detection kit for EGFR gene mutation

A detection kit and multiple detection technology, applied in the field of biotechnology and tumor diagnosis, can solve the problems of low sensitivity, long time, and accuracy of small fragment deletion.

Pending Publication Date: 2020-03-24
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] ①Sanger sequencing method: The detection of gene mutations requires the amplification, purification, sequencing, and sequence analysis of the samples to be tested. Detect mutations with an abundance of more than 20%, with many false negatives
[0009] ④High-throughput sequencing method: High-throughput sequencing method is expensive, and the read length is about 30-250bp, which is shorter than Sanger sequencing, which imposes a burden on sequence splicing. The test results still need to be verified by Sanger sequencing, and for the detection of exon and endon Insufficient accuracy of small fragment deletions containing subjunction regions
The high-throughput sequencing method is cumbersome to operate, which limits the sequencing speed

Method used

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  • Multiplex detection kit for EGFR gene mutation
  • Multiplex detection kit for EGFR gene mutation
  • Multiplex detection kit for EGFR gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] The invention provides a method for detecting multiple gene mutations in lung cancer, primers, probes and a kit. The specific implementation steps are as follows:

[0135] (1) Extraction of nucleic acid templates from samples to be tested

[0136] Nucleic acid extraction or purification reagents from Sun Yat-Sen University Daan Gene Co., Ltd. (Tissue samples can use Yuesui Machinery Equipment No. 20170666; ascites and plasma samples can use Yuesui Machinery Equipment Number 20180628), and nucleic acid extraction is performed according to the kit instructions. The nucleic acid after tissue sample extraction is diluted to a concentration of 2-100ng / μL (the concentration of nucleic acid extracted from pleural fluid and plasma is not less than 2ng / μL), and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequent experiments or stored at -80°C for future use, avoiding repeated freezing and thawing.

[0137] (2...

Embodiment 2

[0156] Embodiment 2 Sensitivity and accuracy detection

[0157] The sensitivity reference product with a nucleic acid concentration of 2 ng / μL is composed of detection gene locus plasmid DNA or cell line, mixed with wild-type cell line nucleic acid in a certain proportion, and the gene mutation rate of the mixture is 1%.

[0158] (1) Sample addition

[0159] Take 3 μL of the DNase system and add it to the PCR reaction tube. Centrifuge briefly for 15 s.

[0160] Take 5 μL of negative quality control substance, sensitivity reference substance (with a concentration of 2 ng / μL mutation rate of 1%), and positive quality control substance, and add them to the reaction tubes in sequence according to Table 5. Close the cap of the PCR reaction tube tightly, mix well, and centrifuge briefly for 10 s.

[0161] For each sensitivity reference product, 5 repeated experiments.

[0162] (2) Real-time fluorescent PCR amplification:

[0163] Set the real-time fluorescent PCR amplification ...

Embodiment 3

[0171] Embodiment 3 clinical sample detection

[0172] Extraction of test sample nucleic acid:

[0173] (1) Extraction of nucleic acid templates from clinical samples to be tested

[0174] 100 negative clinical samples were collected, and 1 case each of the 28 EGFR gene mutation clinical samples confirmed by conventional methods was randomly mixed in after numbering. Nucleic acid extraction or purification reagents from Sun Yat-Sen University Daan Gene Co., Ltd. (Tissue samples can be used in Guangdong Sui Ji Bei No. 20170666; ascites and plasma samples can use Yue Sui Ji Bei No. 20180628), and nucleic acid extraction was carried out according to the kit instructions, and the nucleic acid after tissue sample extraction was diluted to a concentration of 2-100 ng / μL (ascites and plasma extraction The concentration of nucleic acid is not less than 2ng / μL), and the purity should meet A 260 / A 280 The ratio ranges between 1.7-2.3. The template can be used directly for subsequen...

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Abstract

The invention provides a multiplex detection kit for EGFR gene mutation. Specifically, the invention discloses a primer for multiplex detection of EGFR gene mutation, a probe and a kit containing theprimer and probe mixed solution. According to the invention, 28 mutation types of the lung cancer EGFR gene can be detected simultaneously. The method and the kit of the invention have advantages of simple detection process, many detectable mutation types, high sensitivity, small sample dependence and the like.

Description

technical field [0001] The invention belongs to the fields of biotechnology and tumor diagnosis, in particular, the invention relates to a multiple detection kit for EGFR gene mutation. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality, among which non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer patients, and the 5-year survival rate of advanced non-small cell lung cancer patients is about 15%. With the development of technology, more and more attention has been paid to gene mutations in tumors. Non-small cell lung cancer has a high degree of heterogeneity, resulting in extremely diverse clinical manifestations and therapeutic effects. [0003] Epidermal growth factor receptor (EGFR) is a transmembrane protein widely distributed on the cell membrane and is a member of the receptor tyrosine kinase family. The combination of EGFR and epidermal growth factor (EGF) can activate related ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 蒋析文朱小亚邓洁黄志文钟灵秀
Owner DAAN GENE CO LTD
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