Specific primer for rapidly screening EGFR gene exon 18-21 mutation and its application

An exon-specific technology, applied in the field of genetic engineering, can solve the problems of drug resistance induced by gefitinib and erlotinib, and achieve the effect of simple and fast operation, low detection cost and fast speed

Inactive Publication Date: 2017-12-01
北京青航基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This mutation is only found in patients who relapse after drug treatment, and the mutation confers resistance to gefitinib and erlotinib in patients with non-small cell lung cancer

Method used

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  • Specific primer for rapidly screening EGFR gene exon 18-21 mutation and its application
  • Specific primer for rapidly screening EGFR gene exon 18-21 mutation and its application
  • Specific primer for rapidly screening EGFR gene exon 18-21 mutation and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] An in vitro detection method for rapid screening of EGFR gene exon 18-21 mutation, comprising the following steps:

[0030] (1) Extraction of Dried Blood Spot DNA

[0031] The total DNA extraction was carried out according to the instructions of the Magnetic Bead Method Dried Blood Spot Genomic DNA Extraction Kit (Tiangen), as follows:

[0032] 1. Take a new 96-well deep-well plate, and add three 3*3mm dried blood spot samples to each well. Add 200 µL of buffer GA to a 96 deep-well plate.

[0033] 2. Add 20 μL of proteinase K solution, vortex for 10 seconds to mix well, put it into a constant temperature oscillator preheated to 56 degrees, and lyse at 900 rpm for 30 minutes.

[0034] 3. During the sample lysis in the previous step, add 10 μL magnetic bead suspension G, 200 μL buffer GB and 200 μL absolute ethanol to a new 96-well deep-well plate, and mix by whipping or shaking for 10 seconds.

[0035] 4. After the sample is lysed, briefly centrifuge the deep-well pla...

Embodiment 2

[0055] Embodiment 2: clinical sample detection

[0056] The method of Example 1 was used to detect exons 18-21 of the EGFR gene in the blood of multiple cases of physical examination populations, and the detection results were as follows:

[0057] Exons 18, 19, 20, and 21 of the EGFR gene in the blood of 81 cases of colorectal cancer, 69 cases of lung cancer, 79 cases of liver cancer, 100 cases of breast cancer, and 45 cases of esophageal cancer were detected, and 19 cases of colorectal cancer were successfully detected. Cancer patients, 18 lung cancer subjects, 6 liver cancer patients, 24 breast cancer patients, and 8 esophageal cancer patients had mutations in the EGFR gene in blood DNA, and then the PCR product was detected by next-generation sequencing and compared with the human genome sequence. After comparison, it was confirmed that these 75 samples did have changes in DNA sequences.

[0058] Blood samples from 81 patients with colorectal cancer, of which 19 cases (No....

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Abstract

The invention provides a specific primer for rapidly screening EGFR gene exon 18-21 mutation and its application method. The specific primer is prepared by (1), extracting peripheral blood sample gene group DNA; (2), designing and compounding totally four pairs of specific primers for amplifying EGFR gene exon 18-21; (3), applying the primers to perform high-resolution melting curve analysis on the sample gene group DNA to unite sole PCR; (4), the sample with positive result in the curve analysis is the one with EGFR gene mutation. The detecting method is not limited by the mutation alkali locus and type, and a sequence specificity probe is not in need; after finishing PCR, the high-resolution melting can be directly operated, thus the analysis on the sample mutation is completed. The operation is simple and fast; the using cost is low, and the result is exact; the real pipe closing operation can be realized; meanwhile, specific to the rapid screening of people, the specific primer is featured by high throughput, fast speed, reagent boxing, low detecting cost, and others.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. In particular, it relates to a specific primer for rapid screening of EGFR gene exon 18-21 mutation and its application. Background technique [0002] In recent years, the incidence and mortality of malignant tumors in my country have shown a clear upward trend, and have leapt to the top of the cause of death among Chinese residents. However, there is no breakthrough research on the early detection, early diagnosis and prevention of malignant tumors, and some patients are already in the middle and late stages when they see a doctor, and the curative effect is poor. Therefore, improving the early diagnosis level of malignant tumors is an effective way to improve the efficacy of malignant tumor diagnosis and treatment. At present, in clinical practice, serological detection methods are mostly used to monitor tumor markers in serum or other body fluids, and to dynamically make judgment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/106C12Q2600/156C12Q2527/107
Inventor 曾瀚其他发明人请求不公开姓名
Owner 北京青航基因科技有限公司
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