Primers, probes and kit for detecting human EGFR gene mutations

Abstract

The invention belongs to the field of molecular biology and particularly relates to primers, probes and a kit for detecting human EGFR gene mutations. The invention provides 7 groups of primers and probes, which can accurately detect 29 types of common human EGFR gene mutations. The kit adopting the primers and the probes has high sensitivity, can detect 1% of micro mutation templates under the background of 20 ng of wild human genomes and reduces false-negative results. Besides, the kit is simple and convenient to operate, has high controllability, can be used for detecting mass samples and is favorable for clinical operation.

Description

technical field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a primer, a probe and a kit for detecting human EGFR gene mutation. Background technique

[0002] The human epidermal growth factor receptor (EGFR) gene is located in the 7p12-14 region of the short arm of human chromosome 7 and consists of 28 exons. The protein belongs to the receptor tyrosine kinase (TKI) family and exists in all normal epithelial and some cells of mesenchymal origin. EGFR can activate tyrosine kinases, thereby opening downstream signaling pathways and ultimately mediating a series of processes such as cell differentiation, survival, migration, invasion, adhesion, and cell damage repair, playing an important role in the malignant growth of tumors. Therefore, EGFR has become an important target for tumor targeted therapy research.

[0003] At present, clinical EGFR targeted therapy is mainly based on two mechanisms: small molecule tyrosine kinase i...

Examples

Embodiment 1

[0061] The clinical application of embodiment 1 detection kit of the present invention

[0062] 1. Construction of plasmid standard products: The present invention constructed plasmid standard products of 29 mutant types and wild types of EGFR genes as positive quality control and used for the construction of the early stage PCR system.

[0063] 2. Prepare PCR reaction system:

[0064] The content and final concentration of each reaction tube component are as follows:

[0065] 7 mutation detection tubes:

[0066] components

Volume (μL)

Final concentration / L

ARMS upstream primer

0.4

0.2μM

downstream primer

0.4

0.2μM

TaqMan probes

0.25

0.125μM

internal control primer F

0.4

0.2μM

internal control primer R

0.4

0.2μM

Internal control TaqMan probe

0.25

0.125μM

2*SuperReal PreMix(Probe)

10

1*

h 2 o

5.9

[0067] 1 quality control tube: ...

Embodiment 2

[0106] Extract DNA from the peripheral blood, frozen sections, and fresh tissues of 41 patients in Example 1 respectively, and use it to detect template DNA. The template DNA extracted from fresh tissues and paraffin sections can be detected, and the results are the same as those in Example 1, with little difference.

Embodiment 3

[0108]19 deletion mutants and wild-type plasmid standard items in the EGFR gene exon 19 prepared in Example 1 were mixed with the wild-type plasmid according to the copy number percentage of 1% and 10% of the mutant plasmid (total plasmid amount) For 2.4*10-5ng) prepare sample, adopt the detection method of embodiment 1 to detect, specific result is as follows:

[0109]

[0110]

[0111] Table 1

[0112]

[0113] Table 2

[0114]

[0115]

technical field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a primer, a probe and a kit for detecting human EGFR gene mutation. Background technique

[0002] The human epidermal growth factor receptor (EGFR) gene is located in the 7p12-14 region of the short arm of human chromosome 7 and consists of 28 exons. The protein belongs to the receptor tyrosine kinase (TKI) family and exists in all normal epithelial and some cells of mesenchymal origin. EGFR can activate tyrosine kinases, thereby opening downstream signaling pathways and ultimately mediating a series of processes such as cell differentiation, survival, migration, invasion, adhesion, and cell damage repair, playing an important role in the malignant growth of tumors. Therefore, EGFR has become an important target for tumor targeted therapy research.

[0003] At present, clinical EGFR targeted therapy is mainly based on two mechanisms: small molecule tyrosine kinase i...

Examples

Embodiment 1

[0061] The clinical application of embodiment 1 detection kit of the present invention

[0062] 1. Construction of plasmid standard products: The present invention constructed plasmid standard products of 29 mutant types and wild types of EGFR genes as positive quality control and used for the construction of the early stage PCR system.

[0063] 2. Prepare PCR reaction system:

[0064] The content and final concentration of each reaction tube component are as follows:

[0065] 7 mutation detection tubes:

[0066] components

Volume (μL)

Final concentration / L

ARMS upstream primer

0.4

0.2μM

downstream primer

0.4

0.2μM

TaqMan probes

0.25

0.125μM

internal control primer F

0.4

0.2μM

internal control primer R

0.4

0.2μM

Internal control TaqMan probe

0.25

0.125μM

2*SuperReal PreMix(Probe)

10

1*

h 2 o

5.9

[0067] 1 quality control tube: ...

Embodiment 2

[0106] Extract DNA from the peripheral blood, frozen sections, and fresh tissues of 41 patients in Example 1 respectively, and use it to detect template DNA. The template DNA extracted from fresh tissues and paraffin sections can be detected, and the results are the same as those in Example 1, with little difference.

Embodiment 3

[0108]19 deletion mutants and wild-type plasmid standard items in the EGFR gene exon 19 prepared in Example 1 were mixed with the wild-type plasmid according to the copy number percentage of 1% and 10% of the mutant plasmid (total plasmid amount) For 2.4*10-5ng) prepare sample, adopt the detection method of embodiment 1 to detect, specific result is as follows:

[0109]

[0110]

[0111] Table 1

[0112]

[0113] Table 2

[0114]

[0115]

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