A digital PCR detection kit for a T790M mutation site of the EGFR gene

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 low T790M mutation content and high sensitivity requirements

Inactive Publication Date: 2019-01-11
XIAMEN SPACEGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the T790M 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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  • A digital PCR detection kit for a T790M mutation site of the EGFR gene
  • A digital PCR detection kit for a T790M mutation site of the EGFR gene
  • A digital PCR detection kit for a T790M mutation site of the EGFR gene

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Experimental program
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Embodiment 1

[0021] The mutation standard was prepared in the following way: the genomic DNA of the known mutated EGFR gene T790M mutant cell line and the wild-type cell line genomic DNA were mixed in different proportions, then added to the Covaris ultrasonic tube and subjected to ultrasonic treatment to obtain Fragmented DNA of about 180 bp, which is close to the length of the ctDNA fragment. The wild-type standard is obtained by adding the genomic DNA of the EGFR gene T790M mutant cell line known to be mutated 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 T790M mutation is established. This method mainly includes the following steps:

[0023] (1) According to the T790M mutation of the EGFR gene and the wild-type DN...

Embodiment 2

[0037] A digital PCR detection kit for the EGFR gene T790M 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 a T790M mutation site of the EGFR gene. As the mutation of the EGFR gene T790M is taken as a detection object. By optimizing the combination ofspecific primers and fluorescent probe, so that accuracy is achieve, A T790M mutation of that EGFR gene is detect simultaneously simply and quickly, Moreover, the mutation detection rate was as high as 0.1%, which could detect T790M mutation in free DNA of patients effectively. The short DNA fragments obtained from formaldehyde-immobilized paraffin-embedded samples still had the same amplificationand detection ability as that of fresh tissue samples.

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 T790M mutation site of 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] Early studies have found that 50% of tumors that progress after EGFR-TKI treatment have a mutation at the 790th position of exon 20 of the EGFR gene, that is, the T790M gene mutation. Therefore, clinically, ...

Claims

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Application Information

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