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Method for detecting sensitive mutation of EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) based on extracellular vesicle DNA (Deoxyribonucleic Acid)

A technology of EGFR-TKI and cells, which is applied in the field of biomedicine to achieve high detection sensitivity and specificity and improve accuracy

Inactive Publication Date: 2018-12-21
上海浦美医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the formation mechanism of EVs is still not particularly clear, studies have found that EVs carry various proteins, nucleic acids, and even whole-genome DNA.

Method used

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  • Method for detecting sensitive mutation of EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) based on extracellular vesicle DNA (Deoxyribonucleic Acid)
  • Method for detecting sensitive mutation of EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) based on extracellular vesicle DNA (Deoxyribonucleic Acid)
  • Method for detecting sensitive mutation of EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) based on extracellular vesicle DNA (Deoxyribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Arms PCR was used to detect the EGFR-TKI sensitive mutation detection of blood EVs-DNA and cfDNA in patients with non-small cell lung cancer, and compared them. The patient information is shown in Table 1.

[0020] 1. Take 5ml of blood from the patient's vein, centrifuge at 2600g for 15min within 30min, and take 1ml of the upper plasma.

[0021] 2. Add an equal volume of 15% PEG (w / v) NaCl (1M) solution to 1 ml of plasma. After mixing evenly, let stand at 4°C for 1h.

[0022] 3. After standing still, centrifuge at 3000g for 15min at 4°C, and discard the supernatant.

[0023] 4. Add 200 μl of 1% SDS lysate to the pellet, and incubate at 56°C for 30 minutes.

[0024] 5. Add an equal volume of saturated phenol solution to the incubation solution and shake gently for 5 minutes. Centrifuge at 5000 g for 15 min at 4°C. Take the supernatant.

[0025] 6. Add an equal volume of chloroform to the supernatant and shake vigorously for 5 minutes. Centrifuge at 5000 g for 5 min...

Embodiment 2

[0046] Detection and comparison of EGFR-TKI sensitive mutations in blood EVs-DNA and cfDNA of patients with non-small cell lung cancer detected by next-generation sequencing.

[0047] 1. Take 5ml of blood from the patient's vein, centrifuge at 2600g for 15min within 30min, and take 1ml of the upper plasma.

[0048] 2. Add an equal volume of 15% PEG (w / v) NaCl (1M) solution to 1 ml of plasma. After mixing evenly, let stand at 4°C for 1h.

[0049] 3. After standing still, centrifuge at 3000g for 15min at 4°C, and discard the supernatant.

[0050] 4. Add 200 μl of 1% SDS lysate to the pellet, and incubate at 56°C for 30 minutes.

[0051] 5. Add an equal volume of saturated phenol solution to the incubation solution and shake gently for 5 minutes. Centrifuge at 5000 g for 15 min at 4°C. Take the supernatant.

[0052]6. Add an equal volume of chloroform to the supernatant and shake vigorously for 5 minutes. Centrifuge at 5000 g for 5 min at 4°C. Take the supernatant.

[005...

Embodiment 3

[0065] Detection of EGFR-TKI sensitive mutations in blood EVs-DNA and cfDNA of patients with non-small cell lung cancer detected by next-generation sequencing, and compared.

[0066] 1. Take 10ml of blood from the patient's vein, centrifuge at 2600g for 15min within 30min, and take 5ml of the upper plasma.

[0067] 2. Add an equal volume of 15% PEG (w / v) NaCl (1M) solution to 5 ml of plasma. After mixing evenly, let stand at 4°C for 1h.

[0068] 3. After standing still, centrifuge at 3000g for 15min at 4°C, and discard the supernatant.

[0069] 4. Add 200 μl of 1% SDS lysate to the pellet, and incubate at 56°C for 30 minutes.

[0070] 5. Add an equal volume of saturated phenol solution to the incubation solution and shake gently for 5 minutes. Centrifuge at 5000 g for 15 min at 4°C. Take the supernatant.

[0071] 6. Add an equal volume of chloroform to the supernatant and shake vigorously for 5 minutes. Centrifuge at 5000 g for 5 min at 4°C. Take the supernatant.

[00...

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PUM

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Abstract

The invention discloses a method for detecting sensitive mutation of an EGFR-TKI (Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor) based on extracellular vesicle DNA (Deoxyribonucleic Acid). The method comprises the following specific steps: (1) extracting extracellular vesicles EVs in body fluid of a tumor patient through a conventional extracellular vesicles EVs extraction method, soas to obtain the extracellular vesicles EVs; (2) damaging structures of the extracellular vesicles EVs under the action of a surfactant and releasing DNA; obtaining target DNA through a conventional DNA extraction method; (3) detecting through a common EGFR-TKI sensitive mutation method. The method disclosed by the invention is used for detecting EGFR mutation and has an accurate result and high sensitivity and specificity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting EGFR-TKI sensitive mutations based on extracellular vesicle DNA. Background technique [0002] Epidermal growth factor receptor EGFR (epidermal growth factor receptor) plays an important regulatory role in cell physiological processes. EGFR is anchored on the cell membrane and transmits signals downward through tyrosine kinase (TK). EGFR plays an important role in the occurrence of tumors. The targeted inhibitor of EGFR-TK (EGFR-TK inhibitor, EGFR-TKI) blocks the signal transduction of tumor cells, thereby inhibiting the proliferation, metastasis and angiogenesis of tumor cells etc., promote the apoptosis of tumor cells. However, not all tumor cells respond to EGFR-TKI. Only when EGFR-TKI-sensitive mutations occur in exon 18-21 of the EGFR gene can EGFR-TKI exert a better tumor suppressive effect. -45% of the entire East Asian population....

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806
Inventor 雷豪志万源张群祝琳
Owner 上海浦美医学科技有限公司
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