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Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit

A gene mutation and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., to achieve the effect of improving efficiency and reducing the cost of detection quality control

Inactive Publication Date: 2017-05-10
3D BIOMEDICINE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are no quality control products and kits for human circulating tumor DNA EGFR gene sequencing in China

Method used

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  • Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit
  • Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit
  • Quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of Quality Control Products for Human Circulating Tumor DNA EGFR Gene Variation Detection

[0040] 1.1. Six commonly used stable human tumor cell lines A549, NCI-H720, NCI-H1650, NCI-H1975, SW48 and SW1417 were purchased from ATCC. The genomic DNA of each cell line was sequenced by the Sanger method, and the heterozygous and homozygous mutation sites confirmed by the Sanger method were used as positive control sites. It has been verified that there are 12 positive mutation sites in total.

[0041] 1.2. These six human tumor cell lines were cultured in a special medium, culture conditions: 37°C constant temperature, 5% CO2, humidity 50%. Cultivate until the cell density reaches 80-90% of the area of ​​the culture dish, collect the cells in the logarithmic phase of growth, centrifuge at 1500g at 4°C for 5min, take the precipitate and discard the supernatant.

[0042] 1.3. Resuspend in pre-cooled PBS solution, centrifuge at 5000rpm at 4°C for 5min,...

Embodiment 2

[0046] Example 2: Preparation of Human Circulating Tumor DNA EGFR Gene Variation Detection Kit

[0047] 2.1. Design and synthesize multiple capture probes for different target regions on the human circulating tumor DNA EGFR gene. The collection of all capture probes can cover all coding exon regions and exon-intron junction regions of the human EGFR gene; The capture probe is labeled with biotin; the sequence of the capture probe is shown in SEQ ID NO: 1-195 below.

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] 2.2. Mix all the capture probes represented by SEQ ID NO: 1-195 in the above table in the same ratio, and dilute the mixture to a working concentration of 1.5 PM (PM = picomole / liter), and store at -20°C.

[0060] 2.3. Separately pack the capture probe mixture and the quality control obtained in Example 1.

[0061] 2.4. Prepare instructions, outer packaging, assemble and seal....

Embodiment 3

[0063] Example 3: Sequencing detection of human circulating tumor DNA EGFR gene variation

[0064] The instrument used for sequencing in this example is the gene sequence automatic analyzer CN500.

[0065] The preparation method of the quality control product is the same as that in Example 1.

[0066] 3.1. Human cfDNA was extracted from 15 positive plasma samples, and quality control products were used directly without extraction.

[0067] 3.2. Library construction

[0068] 3.2.1. End Repair

[0069] Add 30ng of extracted cfDNA sample or quality control to 0.2ml PCR reaction tube, make up to 50μL with Low EDTA TE, vortex to mix, and centrifuge briefly. Add 1 μL of end-repair enzyme mixture and 6 μL of end-repair reaction buffer, vortex and mix briefly, and then incubate at 37°C for 5 minutes, then incubate at 65°C for 30 minutes, and then at 37°C for 5 minutes for end-repair. After the reaction, take out the PCR reaction tube and transfer all the products to a new 1.5mL ce...

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Abstract

The invention discloses a quality control method for detecting human EGFR (Epidermal Growth Factor Receptor) gene variation based on high-throughput sequencing and a kit, and applications thereof. The quality control method comprises the following steps: extracting a plurality of genome DNAs of an EGFR gene variation-positive human tumor cell line; measuring variation positive sites as positive control sites through a Sanger sequencing method; fragmenting the genome DNAs and mixing according to a certain proportion to obtain a quality control product which can be applied high-throughput sequencing detection of human EGFR gene variation. The kit comprises the human EGFR gene variation detection quality control product.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a quality control method and a kit based on high-throughput detection of human circulating tumor DNA EGFR gene variation. Background technique [0002] There are free small fragments of DNA (cell-free DNA, cfDNA) in the blood, which come from dead cells. Usually dead cells will be removed, so the content of cfDNA is very low, usually 25ng cfDNA in 1ml plasma of a healthy person. The content of cfDNA in cancer patients is several times higher than normal, and part of it is ctDNA (circulating tumor DNA). The relative content of ctDNA is correlated with tumor burden and response to treatment, and can be used to identify driver genes, guide clinical treatment, monitor clinical treatment effects and cancer recurrence, reveal treatment resistance, and detect disease progression. In some respects the sensitivity of ctDNA method is even higher than traditional means. For example, trackin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
CPCC12N15/1003C12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/122
Inventor 李福根熊磊金其煌汤先念
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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