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Nucleic acid composition and kit for detecting EGFR gene mutation and detection method of EGFR gene mutation

A technology of nucleic acid composition and kit, which is applied in the field of genetic engineering and can solve problems such as low detection sensitivity and failure to meet actual needs

Pending Publication Date: 2021-02-05
SUZHOU ZHONGKE ADVANCED TECH RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection sensitivity of these methods is low and cannot meet the actual needs

Method used

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  • Nucleic acid composition and kit for detecting EGFR gene mutation and detection method of EGFR gene mutation
  • Nucleic acid composition and kit for detecting EGFR gene mutation and detection method of EGFR gene mutation
  • Nucleic acid composition and kit for detecting EGFR gene mutation and detection method of EGFR gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] A kit for detecting EGFR gene mutation is provided, and the composition of the kit is shown in Table 1.

[0115] The kit includes nucleic acid compositions for detecting EGFR gene mutation (as shown in Table 2 and Table 3), dNTPs, PCR reaction solution, enzyme mixture, positive control substance and blank control substance.

[0116] Wherein, the enzyme mixed solution includes the enzyme I mixed solution. Enzyme I mixture is a mixture of Taq enzymes and antibodies without 5' to 3' exonuclease activity. Enzyme II mixture is a mixture of exonuclease activity and antibody from 5' end to 3' end. Among them, the Taq enzyme was purchased from TAKARA Biological Company and Guangzhou Baiwang Biotechnology Co., Ltd., and the antibody was purchased from TOYOBO Company.

[0117] Mutation detection PCR reaction solution includes: 2mM MgCl2, 50mM Tris, pH8.3, 500mg / L BSA, 100μM dNTPs, 0.4μL enzyme I mixture, 0.1μM forward primer, 0.5μM reverse primer , 0.5 μM probe.

[0118] The ...

Embodiment 2

[0131] Adopt the detection limit of the test kit of the detection EGFR gene mutation of embodiment 1

[0132] Take human EGFR wild-type genome, human EGFR mutant gene plasmids and TE buffer buffer, and prepare human EGFR wild-type DNA containing 5ng / μL and human EGFR whose content is 10%, 1% and 0.1% of wild-type respectively For each mutant gene plasmid, record the positive detection rate.

[0133] The specific implementation steps are as follows:

[0134] 1. Extraction of samples to be tested

[0135] Tissue samples were extracted using QIAamp DNA FFPE Tissue Kit from QIAGEN, and plasma samples were extracted using QIAamp Circulating Nucleic Acid Kit from QIAGEN. The extraction process should be carried out in strict accordance with the instructions. The nucleic acid after tissue sample extraction should be diluted to a concentration of 2-100 ng / μL, the concentration of nucleic acid after plasma extraction should not be less than 2 ng / μL, and the purity should satisfy the...

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Abstract

The invention relates to a nucleic acid composition and kit for detecting EGFR gene mutation and a detection method of EGFR gene mutation. The nucleic acid composition for detecting EGFR gene mutationcomprises a first primer pair with sequences shown in SEQ ID No. 1 and SEQ ID No. 2, a first probe with the sequence shown in SEQ ID No. 3, a second primer pair with the sequences shown in SEQ ID No.4 and SEQ ID No. 5 and a second probe with the sequence shown in SEQ ID No. 6. The nucleic acid composition has higher specificity for EGFR gene mutation, can detect EGFR gene mutation, and has higher detection sensitivity.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a nucleic acid composition and a kit for detecting EGFR gene mutations and a detection method for EGFR gene mutations. Background technique [0002] EGFR (Epidermal Growth Factor Receptor) is the epidermal growth factor receptor, which is the expression product of the proto-oncogene C-erbB-1 (HER1), located on the cell membrane, and is a kind of tumor cell proliferation, growth, repair and survival. Membrane proteins that play an important role. [0003] At present, the main methods for EGFR gene mutation detection are as follows: 1) Sanger sequencing method; direct sequencing method has always been the gold standard for gene mutation detection, but the sequencing method has many disadvantages, such as time-consuming, subsequent PCR products need to be processed , the procedure is complex and prone to contamination leading to inaccurate results. In addition, the sensitivity o...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 张炳为辜嘉周树民
Owner SUZHOU ZHONGKE ADVANCED TECH RES INST CO LTD
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