Digital PCR detection kit for EGFR gene L858R mutation site

A technology for detecting kits and mutation sites, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems of extremely high sensitivity requirements and low L858R mutation content

Inactive Publication Date: 2019-02-01
XIAMEN SPACEGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the L858R mutation content in the cell-free DNA of patients is often extremely low, and the sensitivity of the detection methodology is extremely high, and conventional detection methods cannot meet this demand well.

Method used

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  • Digital PCR detection kit for EGFR gene L858R mutation site
  • Digital PCR detection kit for EGFR gene L858R mutation site
  • Digital PCR detection kit for EGFR gene L858R mutation site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The mutation standard was prepared in the following way: the genomic DNA of the known mutated EGFR gene L858R mutant cell line and the wild-type cell line genomic DNA were mixed in different proportions, then added to a Covariis ultrasonic tube and subjected to ultrasonic treatment to obtain a Fragmented DNA of about 180 bp, which is close to the length of the ctDNA fragment. The wild-type standard product is obtained by adding the genomic DNA of the known mutated EGFR gene L858R mutant cell line into a Covaris ultrasonic tube and performing ultrasonic treatment to obtain fragments of about 180 bp that are close to the length of the ctDNA fragment DNA. Standards with mutation ratios of 10%, 5% and 0.1% were obtained according to the above method.

[0022] A reaction system for digital PCR detection of EGFR gene L858R mutation is established. This method mainly includes the following steps:

[0023] (1) According to the EGFR gene L858R mutation and the wild-type DNA ge...

Embodiment 2

[0037] A digital PCR detection kit for the EGFR gene L858R mutation site, comprising a detection primer pair and a fluorescent probe set, the detection primer pair includes a forward primer and a reverse primer shown in SEQ ID NO 01 and 02, and the fluorescent probe The needle set includes a first fluorescent probe and a second fluorescent probe as shown in SEQ ID NO 03 and 04 respectively, and the 5' ends of the first fluorescent probe and the second fluorescent probe are respectively labeled with first fluorescent lights different from each other group and a second fluorescent group, the 3' end is labeled with a third fluorescent group that is mutually quenched with the above-mentioned first and second fluorescent groups, and the first fluorescent probe and the second fluorescent probe are different from each other bases have LNA labels,

[0038] Its reaction system is as follows:

[0039]

[0040] The first fluorescent group is FAM. The second fluorescent group is HEX....

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Abstract

The invention discloses a digital PCR detection kit for an EGFR gene L858R mutation site. According to the invention, EGFR gene L858R mutation is adopted as the detection object, through optimized combination of specific primers and fluorescent probe, accurate, simple and rapid simultaneous detection of EGFR gene L858R mutation can be achieved, and the ability of mutation detection is as high as 0.1%. The kit can effectively detect L858R mutation in patients' free DNA, and still has the same amplification and detection ability on a short fragment DNA obtained from formaldehyde-fixed and paraffin-embedded sample as that of a fresh tissue sample DNA.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a digital PCR detection kit for the L858R mutation site of the EGFR gene. Background technique [0002] In recent years, a milestone change in the treatment of non-small cell lung cancer (NSCLC) is the use of EGFR-tyrosine kinase inhibitors (TKIs) for advanced patients with positive epidermal growth factor receptor (EGFR) gene mutations. Patients who benefited from treatment showed prolonged disease progression-free period (PFS), increased objective response rate (ORR), and greatly improved quality of life. Despite a good response to initial EGFR-TKI therapy, most patients develop resistance to these drugs after an average of 10 months of treatment. [0003] Exon 21 mutations account for 40.3% of the total number of epithelial growth factor receptor gene mutations in non-small cell lung cancer in my country, and 94.4% of the mutations are base substitutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2563/159C12Q2563/107
Inventor 甘煌灿康灿昆陈琰
Owner XIAMEN SPACEGEN BIOTECH CO LTD
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