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Rapid fluorescence detection method for 19# exon deleted mutation of gene EGFR and application

A technology of exon deletion and exon, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of low-cost and convenient detection, slow hydrolysis rate of double-stranded DNA, It can only achieve about 0.1% and other problems, and achieve the effect of increasing the number of determinations and data processing time, low reagent cost and fast speed

Active Publication Date: 2019-11-29
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The sensitivity of NGS has been greatly improved, but its high instrument cost and long detection cycle make it difficult to achieve low-cost and convenient detection in the laboratory
The cost of ARMS is low, but its reliable relative detection limit is still only about 0.1%; the digital PCR detection result is accurate, but its sample consumption is large, the cost is high, and the reliable relative detection limit can only reach about 0.1%
[0006] Lambda exonuclease has 5' to 3' DNA exonuclease activity, which can rapidly hydrolyze the DNA single strand with 5' phosphorylated end in double-stranded DNA (dsDNA), but for single-stranded DNA (ssDNA) itself or 5' The hydrolysis rate of double-stranded DNA at the -OH end is very slow

Method used

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  • Rapid fluorescence detection method for 19# exon deleted mutation of gene EGFR and application
  • Rapid fluorescence detection method for 19# exon deleted mutation of gene EGFR and application
  • Rapid fluorescence detection method for 19# exon deleted mutation of gene EGFR and application

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1

[0052] In order to achieve high-sensitivity detection of low-abundance mutant chains, the most critical issue is to improve the ability of the probe to distinguish between wild and mutant chains. In the design of the present invention, the main factors affecting the discrimination of the probes are: the relative position of the 5' end of the probe and the mutation region, the opening degree of the two bases at the 5' end, and the total length of the probe. The length of the probe sequence is generally 12-15 nt, too long or too short will lead to a decrease in the discrimination between the mutant strand and the wild strand. For EGFR 19del, the length of the probe was designed to be 15 nt due to the low GC content (about 40%) of the sequence near the mutation region. In order to reduce the fluorescence response of the probe to the wild strand as much as possible, the mutation position was initially designed to be 5-6 bases away from the 5' end of the probe....

Embodiment 2

[0057] Example 2

[0058]In order to use the above probes directly for the detection of EGFR 19del in genomic samples and obtain quantitative and reliable results, we first optimized the PCR conditions for EGFR 19del. For common point mutations, the molecular weight difference between the mutant chain and the wild chain is usually only tens of g / mol. For the EGFR 19del mutation, a deletion of 15 nucleotides was involved, resulting in a molecular weight difference of 4674.1 g / mol between the mutant chain and the wild chain. In order to reduce the DNA quantification error that may be caused by this difference, we designed a longer EGFR 19del amplicon sequence to reduce the relative difference in molecular weight between the mutant and wild-type chains, so that the average molecular weight of the mutant and wild-type chains can be used Calculate the amount concentration of the substance of the amplified sequence. Considering that if the protruding sequence of the 3' end of the...

Embodiment 3

[0064] Example 3

[0065] The above probes are used to detect the abundance of circulating free DNA (cfDNA) EGFR 19del mutation in actual samples, and the operation steps are as follows:

[0066] (1) The cfDNA in the sample was extracted using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, Hilden, Germany), and the extracted DNA was quantified using a NanoDrop 2000 micro-ultraviolet photometer.

[0067] (2) In a 200 μL PCR tube, add 10 μL 10×Q5 Reaction Buffer, 5 μL 19del forward primer (final concentration 500 nM), 5 μL 19del reverse primer (final concentration 500 nM), 1 μL dNTPs (10 mM each dNTP), 1 μL 10 ×LCGreen, 1 μL of 19del standard ssDNA with different abundances of mutant strands (total final concentration of mutant strands and wild strands is 10 pM, change the abundance of mutant strands) or actual samples (final DNA concentration of 0.5 to 1.0 μg / mL), Q5 DNA polymerase (20U / mL), water (up to 50μL). The PCR process (93°C for 8s, 60°C for 20s, 72°C for 20s, 35 c...

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Abstract

The invention discloses a rapid fluorescence detection method for 19# exon deleted mutation of a gene EGFR and an application. The method disclosed by the invention has the properties that by using Lambda exonuclease, an outstanding structure, of which 5' terminals are two bases, and a single strand labeled with fluorophore in DNA double strands can be rapidly hydrolyzed, and a DNA single strand with a 5' phosphorylated terminal in double-stranded DNA can be continuously hydrolyzed; and mutated base identification, PCR product single-stranding and fluorescence signal amplification processes are completed in a combined manner, so that entire detection flows are greatly simplified, the detection time is shortened, the relative detection limit is lowered to 0.01%, the sensitivity is high, andthus, the method has a very good application prospect in the aspects of clinical diagnosis, targeted medication instruction, postoperative accompanied detection and the like.

Description

technical field [0001] The invention relates to a rapid and high-sensitivity detection method for detecting EGFR gene exon 19 deletion mutation (EGFR 19del), which belongs to the field of gene mutation detection and can be used for early diagnosis of lung cancer, guidance of targeted drug administration, and postoperative care. Accompanied by detection and other fields. Background technique [0002] Lung cancer is one of the most common cancers in the world today, of which non-small cell lung cancer (NSCLC) accounts for 75-80% [1] , is an important type of lung cancer. Human epidermal growth factor receptor (EGFR) mutations occur in up to 35% of Asian non-small cell lung cancer patients [2] , is an important marker of lung cancer. EGFR is a glycoprotein receptor bound by human epidermal growth factor (EGF) in the process of promoting cell proliferation. After EGF binds to EGFR, it will dimerize. Several tyrosines in the intracellular tyrosine kinase domain of EGFR Phosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2521/319C12Q2563/107C12Q2545/113
Inventor 赵美萍杨子煜陈维吴曈勃
Owner PEKING UNIV
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