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Primer probe combination and kit for detecting EGFR gene mutation

A technology of primer probes and kits, which is applied in the fields of biotechnology and tumor diagnosis, and can solve problems such as low sensitivity, time-consuming, and accuracy of small fragment deletions

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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  • Primer probe combination and kit for detecting EGFR gene mutation
  • Primer probe combination and kit for detecting EGFR gene mutation
  • Primer probe combination and kit for detecting EGFR gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] 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:

[0113] (1) Extraction of nucleic acid templates from samples to be tested

[0114] 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.

[0115] (2...

Embodiment 2

[0133] Embodiment 2 Sensitivity and accuracy detection

[0134] The sensitivity reference product with a nucleic acid concentration of 2ng / μL is composed of the detection gene locus plasmid DNA or cell line, and the wild-type cell line nucleic acid is mixed in a certain proportion, and the gene mutation rate of the mixture is 1%.

[0135] (1) Sample addition

[0136] Take 3 μL of the DNase system and add it to the PCR reaction tube. Centrifuge briefly for 15s.

[0137] 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 tube in sequence according to the order in Table 5. Close the cap of the PCR reaction tube tightly, mix well, and centrifuge briefly for 10s.

[0138] For each sensitivity reference product, 5 repeated experiments.

[0139] (2) Real-time fluorescent PCR amplification:

[0140] Set the real-time fluorescent...

Embodiment 3

[0148] Embodiment 3 clinical sample detection

[0149] Extraction of test sample nucleic acid:

[0150] (1) Extraction of nucleic acid templates from clinical samples to be tested

[0151] 100 negative clinical samples were collected, and 1 clinical sample of 18 EGFR gene exon 19 gene deletions confirmed by conventional methods was randomly mixed in after numbering, using nucleic acid extraction or purification reagents ( Tissue samples can use Yuesui Machinery No. 20170666; pleural fluid and plasma samples can use Yuesui Machinery No. 20180628), and nucleic acid extraction is carried out according to the kit instructions. The nucleic acid after tissue sample extraction is diluted to a concentration of 2-100ng / μL (chest The concentration of nucleic acid extracted from ascites and plasma should not be 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 fo...

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Abstract

The invention provides a primer probe combination and a kit for detecting EGFR gene mutation. Specifically, the invention discloses a primer for multiplex detection of EGFR gene mutation, a probe anda kit including a primer and probe mixed solution. According to the invention, 18 gene deletion mutations of exon 19 of the lung cancer EGFR gene can be simultaneously detected. The method and the kitof the invention have the advantages of simple detection process, multiple detectable mutation types, high sensitivity, small sample dependence and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and tumor diagnosis, in particular, the invention relates to a combination of primers and probes and a kit for detecting 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 fact...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 蒋析文朱小亚邓洁黄志文钟灵秀
Owner DAAN GENE CO LTD
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