Kit for simultaneously detecting 45 mutation sites of human EGFR (Epidermal Growth Factor Receptor) gene

A technology for detecting human mutation sites, applied in the fields of biotechnology and medicine, can solve the problem of lack of relevant reports on mutation site detection, and achieve the effect of convenient standardized operation, fewer components, and standardized operation.

Active Publication Date: 2018-05-04
BEIJING ACCB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In addition, the current primers, probes, and kits for detecting human EGFR gene mutations can only detect several or more than 20 deletion mutations, and there are no related reports on t

Method used

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  • Kit for simultaneously detecting 45 mutation sites of human EGFR (Epidermal Growth Factor Receptor) gene
  • Kit for simultaneously detecting 45 mutation sites of human EGFR (Epidermal Growth Factor Receptor) gene
  • Kit for simultaneously detecting 45 mutation sites of human EGFR (Epidermal Growth Factor Receptor) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Taking 10 samples as an example to illustrate the use of this kit:

[0084] (1) Take out 12 8-tube tubes from the kit, place them on a 96-well plate, melt the detection reagent I and the positive quality control product PC, centrifuge briefly, and place them on ice;

[0085] (2) Take 12 single PCR tubes, mark 1-10 and NC, PC respectively, add 3 μl Taq DNA polymerase to each tube, add to the bottom of the tube, and place on ice; Add 27 μl of 10 DNA samples to be tested, negative quality control product NC and positive quality control product PC, pipette to mix, close the cap tightly and centrifuge briefly (Note: Do not use a vortex shaker when mixing; follow-up);

[0086] The positive quality control product PC is the TE solution (10mM Tris-HCl, 1mM EDTA, pH8.0) of the internal reference gene IR and human EGFR mutant gene plasmids (Table 1, * mark), wherein the final concentration of each plasmid is 5 ×10 -4 ng / μL.

[0087] Negative quality control product NC is a bu...

Embodiment 2

[0090] The detection limit experiment of EGFR gene mutation in the human EGFR wild-type genomic DNA of embodiment 2

[0091] Get human EGFR wild-type genomic DNA, human EGFR mutant gene plasmids (Table 1, * mark) and dilution buffer, prepare human EGFR wild-type genomic DNA containing 20ng / μl at the same time, and the content is respectively wild-type 10%, 5% %, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% of the human EGFR mutant gene plasmids of the detection limit reference; preparations containing 10ng / μl of human EGFR wild-type genomic DNA and the content were respectively Wild-type 10%, 5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% detection limit reference substance of human EGFR mutant gene plasmids; preparation of human EGFR wild type containing 5ng / μl at the same time Genomic DNA and its contents are wild-type 10%, 5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, and 0.5% of the detection limit reference products of human EGFR mutant gene plasmids, and the positive detection rate is recorded (The result...

Embodiment 3

[0093] Example 3 Negative reference product coincidence rate experiment

[0094] 10 cases of negative reference samples of 15ng / μl human EGFR wild-type genomic DNA were detected once each, and the negative detection status was recorded. It was found that the test results of all EGFR wild-type nucleic acid samples were negative, and the negative coincidence rate was 100%.

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Abstract

The invention discloses a primer, probe, detection system and kit for detecting 45 types of mutation of 18 to 21 exons of a human EGFR (Epidermal Growth Factor Receptor) gene. According to the kit disclosed by the invention, an IR-IC double-quality-control reaction system is established based on an oligonucleotide probe technology and 45 types of gene mutation of an epidermal growth factor receptor EGFR can be simultaneously detected. The primer, probe and kit, disclosed by the invention, have high selectivity and strong specificity and are convenient to operate; standardized operation of a laboratory is convenient to realize and is convenient and rapid; a detection result can be referred when a doctor selects people who can orally take anti-EGFR target treatment medicines from patients with non-small-cell lung cancer.

Description

technical field [0001] The invention relates to a kit, in particular to a kit for simultaneously detecting 45 mutation sites of exons 18-21 of human EGFR gene, belonging to the fields of biotechnology and medicine. Background technique [0002] The human epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) gene is located on the short arm of human chromosome 7 (7p12), is about 118kb in length, and consists of 28 exons. The transcribed mRNA is about 5.6kb in length and encodes a transmembrane glycoprotein with a molecular weight of 170kD. The intracellular region has tyrosine kinase (tyrosine kinase, TK) activity and is responsible for transmitting extracellular signals into the cell. Abnormal EGFR activation can promote tumor cell proliferation, migration, differentiation, angiogenesis, and can inhibit tumor cell apoptosis. EGFR forms a homodimer after binding to its ligand, and can also form a heterodimer with other TK receptors, leading to the activat...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/166
Inventor 莫敏俐李晖陈钊
Owner BEIJING ACCB BIOTECH
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