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Lung cancer related gene mutation detection method, primer and reagent

A detection method and technology for lung cancer, which are applied in biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of large sample size, high false negative rate, and complicated operation of multiple mutation sites.

Inactive Publication Date: 2017-06-13
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the Sanger sequencing method, the operation is cumbersome, the sensitivity is low, and the false negative rate is high; in the fluorescent quantitative PCR method, each pair of primers can only detect one mutation, and the detection of multiple mutation sites is cumbersome and requires a large sample size

Method used

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  • Lung cancer related gene mutation detection method, primer and reagent
  • Lung cancer related gene mutation detection method, primer and reagent
  • Lung cancer related gene mutation detection method, primer and reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Detection method of lung cancer related gene mutation based on Ion Torrent sequencing platform

[0109] This embodiment adopts the following reagents: 5×Ion Ampliseq TM HiFi Master Mix (Life Company), 2 x Ion Ampliseq TM Primer Pool (Life Corporation), FuPa Reagent (Life Corporation), Ion P1Adapter and IonXpress TM Barcode X (Life Corporation), Switch Solution (Life Corporation), DNA Ligase (Life Corporation), XP Reagent (Beckman Corporation), Platium TMPCR SuperMix High Fidelity (Life Company), Library Amplification Primer Mix (Life Company), Nuclease-FreeWater, absolute ethanol.

[0110] Before the experiment of this method, a reagent needs to be freshly prepared: 75% ethanol.

[0111] Instruments and equipment needed for this experiment: centrifuge, magnetic stand, pipette, PCR instrument, oscillator, fluorometer Qubit3.0.

[0112] Main experimental steps:

[0113] (1) gDNA extraction of samples to be tested

[0114] In this example, the gDNA sa...

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Abstract

The invention discloses a lung cancer related gene mutation detection method, primer and reagent based on a high-flux sequencing platform, which are used for detecting 50 mutant sites of four lung cancer related genes of EGFR, KRAS, BRAF and PIK3CA. The detection method comprises the steps of sample DNA (deoxyribonucleic acid) extraction, multiplex PCR (polymerase chain reaction) target segment enrichment, primer elimination, linker connection, library amplification, library detection, high-flux sequencing and the like. According to the invention, the library building cost is lowered, and the possibility of mutation introduction from PCR amplification is reduced, so that the sequencing result is more accurate, and the detection flux and detection sensitivity are improved; and meanwhile, reference can be provided for molecular diagnosis and individualized medication of lung cancer.

Description

technical field [0001] The present invention designs the field of gene sequencing, especially the field of high-throughput sequencing, specifically a method for detecting mutations in lung cancer-related genes based on the Ion Torrent sequencing platform, a set of primer pairs and reagents. Background technique [0002] Lung cancer is the cancer with the highest morbidity and mortality in my country, and non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for about 80% of all lung cancers. Chemotherapy is currently the main method for the treatment of advanced lung cancer, and it is also one of the preoperative and postoperative adjuvant treatments for patients. Clinically, some patients have poor chemotherapy effect and chemotherapy failure. The main reason is that the tumor cells are not sensitive or resistant to anticancer drugs. Therefore, it is particularly important to understand the factors related to the sensitivity of patients to chemotherapy dr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2535/122C12Q2537/143
Inventor 杨学习吴英松李明王庆曹治家
Owner GUANGZHOU DARUI BIOTECH
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