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Primer for detecting low-frequency mutation of epidermal growth factor receptor gene T790M and application of primer

A technology of epidermal growth factor and T790M, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of low accuracy of low-frequency mutation detection results, save treatment time and improve positive results The detection rate, the effect of reducing the false positive or false negative rate

Inactive Publication Date: 2017-06-09
HANGZHOU D A GENETIC ENG
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a detection kit for low-frequency mutation of epidermal growth factor receptor (EGFR) gene T790M, to solve the low accuracy rate of detection results of low-frequency mutation of epidermal growth factor receptor (EGFR) gene T790M in the prior art Shortcomings

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  • Primer for detecting low-frequency mutation of epidermal growth factor receptor gene T790M and application of primer
  • Primer for detecting low-frequency mutation of epidermal growth factor receptor gene T790M and application of primer
  • Primer for detecting low-frequency mutation of epidermal growth factor receptor gene T790M and application of primer

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Embodiment 1

[0038] Example 1: Reagents.

[0039] (1) DNA extraction reagents:

[0040] Purchased from QIAGEN Company.

[0041] (2) Reaction solution:

[0042] PCR Buffer: purchased from Fermentas, USA;

[0043] Primers SEQ ID NO: 1-4, synthesized by Shanghai Yingjun Biotechnology Co., Ltd.;

[0044] MgCl 2 : purchased from the U.S. Fermentas company;

[0045] 0.2mM dNTPs: purchased from Fermentas, USA;

[0046] 2U / μL Taq DNA polymerase: purchased from Fermentas, USA.

[0047](3) Reagent for single-strand purification:

[0048] 75% (v / v) ethanol solution: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.;

[0049] 0.2M NaOH: purchased from Shanghai Shisi Hewei Chemical Co., Ltd.;

[0050] 10mM Tris-Acetate (pH 7.6): Tris-base was purchased from Sigma, USA, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.;

[0051] Binding buffer: 10mM Tris-HCl (Tris-base was purchased from Sigma, USA; hydrochloric acid was purchased from Hangzhou Chemic...

Embodiment 2

[0058] Embodiment 2: detection method.

[0059] Instruments: Bio-Rad S1000 PCR instrument, Beckman Microfuge 22R desktop micro-refrigerated centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGEN PyroMark Q96ID sequencer.

[0060] (1) Extract tissue DNA from paraffin specimens, the specific steps are as follows: place the paraffin specimens in xylene to remove paraffin; add lysis buffer and proteinase K, digest the tissue under denaturing conditions, and lyse the cells; incubate at 90°C, reverse Formalin cross-linking; pass the lysate through the silica gel membrane to adsorb DNA to the silica gel membrane, add rinsing solution to wash the impurities, and finally elute the high-purity and concentrated DNA from the silica gel membrane to obtain the genomic DNA collection solution.

[0061] (2) using the DNA obtained in step (1) as a template, and using EGFR-specific primers to perform PCR amplification;

[0062] Wherein, th...

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Abstract

The invention discloses a primer for detecting low-frequency mutation of an epidermal growth factor receptor gene T790M and an application of the primer, and belongs to the field of molecular pathological diagnosis. The primer comprises amplification primers shown by SEQ ID NO:1-2, a sequencing primer shown by SEQ ID NO:3, and an amplification refractory primer shown by SEQ ID NO:4, wherein the last two bases of a 5' terminal of the amplification refractory primer are same as the first two bases of a 3' terminal of an upstream primer of the amplification primers; the 5' terminal of the SEQ ID NO:2 is used for marking a biotin; the 3' terminal of the SEQ ID NO:4 is used for performing phosphorylation treatment; and the above nucleotide sequences are used together in one detection. The primer provided by the invention improves detection sensitivity, can be used for detecting mutation of less than 0.1%, and ensures that results are intuitive, determination is simpler, more accurate and faster, the false negative rate of detection results is greatly reduced, and useless or useful clinical targeted medicine selection for patients is avoided, thereby saving precious treatment time for the patients, and improving the quality of life of the patients.

Description

technical field [0001] The invention belongs to the field of molecular pathological diagnosis, and specifically relates to a primer for accurately and qualitatively detecting the T790M low-frequency mutation of the epidermal growth factor receptor (EGFR) gene related to lung cancer and its application. Background technique [0002] Lung cancer is very harmful and has a high mortality rate. It is currently the number one cause of cancer death in the world. In 1995, 600,000 people died of lung cancer in the world, and the number is increasing every year. In 2003, the death rate announced by the World Health Organization (WHO) The incidence rate is 1.1 million per year, and the incidence rate is 1.2 million per year. 75% to 80% of lung cancers are non-small cell lung cancers. Treatment methods such as radiotherapy and chemotherapy for patients with advanced lung cancer not only reduce the quality of life, but also have poor curative effect. Iressa (Iressa, Gefitinib) is curren...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/122C12Q2565/301C12Q2531/113C12Q2523/308
Inventor 任绪义张锋虞闰六曹子豪周韵
Owner HANGZHOU D A GENETIC ENG
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