Nucleic acid sequence for EGFR gene exon 18 G719X mutation detection, test kit and application of test kit

Abstract

The invention discloses a nucleic acid sequence for EGFR gene exon 18 G719X gene mutation detection, a test kit and application of the test kit, and belongs to the technical field of molecular diagnosis. A primer and probe for detecting the EGFR gene exon 18 G719X mutation, provided by the invention, can be used for specifically amplifying and detecting the G719X mutation of the exon 18 of the EGFR gene. The invention further discloses a detection test kit for detecting the EGFR gene exon 18 G719X gene mutation based on a microdroplet type digital PCR system. The G719X mutation of the EGFR gene exon 18 can be rapidly, sensitively and accurately detected. The kit has the advantages of simplicity and convenience in operation, strong specificity, high sensitivity, accurate result, low cost, high throughput and the like, can be used for rapidly detecting the exon 18 G719X mutation of the EGFR gene clinically, and provides reference basis for clinical diagnosis and treatment of the exon 18G719X mutation of the EGFR gene.

Description

technical field

[0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleotide sequence, a kit and an application thereof for detecting the mutation of the G719X gene in exon 18 of the EGFR gene. Background technique

[0002] The epidermal growth factor receptor gene is located on the short arm of human chromosome 7 and consists of 188,307 bases, including 28 exons. Its tyrosine kinase domain is encoded by exons 18-24. Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene C-erbB-1 (HER-1), located on the cell membrane. EGFR mainly transmits signals to the nucleus through two pathways, one is Ras→Raf→MAPK pathway; the other is PI3K→PKC→IKK pathway. When the signal is transmitted to the nucleus, it causes an increase in the transcription level of genes in the nucleus, resulting in cell proliferation and transformation. Abnormalities in EGFR signaling are responsible for the development of v...

Examples

Embodiment 1

[0037] Example 1: The primers and probes for detecting exon 18 of the EGFR gene G719X were synthesized by Shanghai Bailig Biotechnology Co., Ltd., and the sequence is as follows:

[0038] Primers for amplifying exon 719 of EGFR gene 18:

[0039] F1 (SEQ ID NO: 1): 5'-ggatcttgaaggaaactgaa-3';

[0040] R1 (SEQ ID NO: 2): 5'-tatacaccgtgccgaac-3';

[0041] Probes for detecting the wild type of EGFR gene 18 exon 719 site:

[0042] P-719-WT (SEQ ID NO: 3): 5'-tgct+g+g+gctcc-3';

[0043] Among them, the 5' end of the EGFR gene exon 719 wild-type probe P-719-WT (SEQ ID NO: 3) sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group. In addition, the probe The 5th, 6th, and 7th nucleotides in the sequence are modified by LNA (indicated by + sign).

[0044] Probes for detecting G719X mutant gene in exon 18 of EGFR gene:

[0045] P-G719S-mut (SEQ ID NO: 4): 5'-tgct+g+a+gct+cc-3'

[0046] P-G719C-mut (SEQ ID NO: 5): 5'-tgct+g+t+gctc+cg-3...

Embodiment 2

[0055] Example 2: Preparation method of EGFR gene exon 18 G719X mutation detection kit.

[0056] (1) PCR reaction solution: 2*supermix (purchased from Bio-rad Company in the United States, item number 186-3026), which is a 2*PCR reaction master mix, which contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP and other components, stored at -20°C;

[0057] (2) Primer-probe premix: a mixture of nucleotide sequences shown in SEQ ID NO: 1 to 3, wherein the concentration of each primer is 100 μM, the concentration of the probe is 10 μM, and the concentration of each nucleic acid sequence in 1 to 3 The mother solution is mixed at a volume ratio of 9:9:25; the mixed solution of the nucleotide sequences shown in SEQ ID NO: 4 to 8, wherein the concentration of each probe is 25 μM, and the mother solution of each nucleic acid sequence is 1: Mix at a volume ratio of 1:1:1:1; the concentration of the final primer / probe in the reaction system is 900 / 250nM; store ...

Embodiment 3

[0060] Embodiment 3: detection method.

[0061] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0062] 1. Preparation of EGFR gene exon 18 G719X test sample, positive control, and negative control DNA template: use QIAamp Circulating Nucleic Acid Kit (Catalog No. 55114) from QIAGEN Company, and operate according to the kit instructions.

[0063] 2. Use 1 pair of specific primers and 6 specific probes to detect the G719X mutation gene in exon 18 of the EGFR gene, specifically including the following steps;

[0064] (1) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, thaw at room temperature, and put them on an ice box for later use. Within 10 minutes before a...

technical field

[0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleotide sequence, a kit and an application thereof for detecting the mutation of the G719X gene in exon 18 of the EGFR gene. Background technique

[0002] The epidermal growth factor receptor gene is located on the short arm of human chromosome 7 and consists of 188,307 bases, including 28 exons. Its tyrosine kinase domain is encoded by exons 18-24. Epidermal growth factor receptor (EGFR) is the expression product of proto-oncogene C-erbB-1 (HER-1), located on the cell membrane. EGFR mainly transmits signals to the nucleus through two pathways, one is Ras→Raf→MAPK pathway; the other is PI3K→PKC→IKK pathway. When the signal is transmitted to the nucleus, it causes an increase in the transcription level of genes in the nucleus, resulting in cell proliferation and transformation. Abnormalities in EGFR signaling are responsible for the development of v...

Examples

Embodiment 1

[0037] Example 1: The primers and probes for detecting exon 18 of the EGFR gene G719X were synthesized by Shanghai Bailig Biotechnology Co., Ltd., and the sequence is as follows:

[0038] Primers for amplifying exon 719 of EGFR gene 18:

[0039] F1 (SEQ ID NO: 1): 5'-ggatcttgaaggaaactgaa-3';

[0040] R1 (SEQ ID NO: 2): 5'-tatacaccgtgccgaac-3';

[0041] Probes for detecting the wild type of EGFR gene 18 exon 719 site:

[0042] P-719-WT (SEQ ID NO: 3): 5'-tgct+g+g+gctcc-3';

[0043] Among them, the 5' end of the EGFR gene exon 719 wild-type probe P-719-WT (SEQ ID NO: 3) sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group. In addition, the probe The 5th, 6th, and 7th nucleotides in the sequence are modified by LNA (indicated by + sign).

[0044] Probes for detecting G719X mutant gene in exon 18 of EGFR gene:

[0045] P-G719S-mut (SEQ ID NO: 4): 5'-tgct+g+a+gct+cc-3'

[0046] P-G719C-mut (SEQ ID NO: 5): 5'-tgct+g+t+gctc+cg-3...

Embodiment 2

[0055] Example 2: Preparation method of EGFR gene exon 18 G719X mutation detection kit.

[0056] (1) PCR reaction solution: 2*supermix (purchased from Bio-rad Company in the United States, item number 186-3026), which is a 2*PCR reaction master mix, which contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP and other components, stored at -20°C;

[0057] (2) Primer-probe premix: a mixture of nucleotide sequences shown in SEQ ID NO: 1 to 3, wherein the concentration of each primer is 100 μM, the concentration of the probe is 10 μM, and the concentration of each nucleic acid sequence in 1 to 3 The mother solution is mixed at a volume ratio of 9:9:25; the mixed solution of the nucleotide sequences shown in SEQ ID NO: 4 to 8, wherein the concentration of each probe is 25 μM, and the mother solution of each nucleic acid sequence is 1: Mix at a volume ratio of 1:1:1:1; the concentration of the final primer / probe in the reaction system is 900 / 250nM; store ...

Embodiment 3

[0060] Embodiment 3: detection method.

[0061] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0062] 1. Preparation of EGFR gene exon 18 G719X test sample, positive control, and negative control DNA template: use QIAamp Circulating Nucleic Acid Kit (Catalog No. 55114) from QIAGEN Company, and operate according to the kit instructions.

[0063] 2. Use 1 pair of specific primers and 6 specific probes to detect the G719X mutation gene in exon 18 of the EGFR gene, specifically including the following steps;

[0064] (1) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, thaw at room temperature, and put them on an ice box for later use. Within 10 minutes before a...

Images