Detection probe, liquid phase chip and detection method thereof for EGFR gene mutation sites

A liquid phase chip and detection method technology, applied in the field of molecular biology, can solve problems such as hindering the detection of mutant genes, and achieve the effects of avoiding interference, improving detection accuracy, and convenient sampling

Active Publication Date: 2009-06-03
广州益善医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the free nucleic acid may only contain a very small amount of mutant genes, but contains a large number of wild-type genes, and a large number of mixed wild-type genes will hinder the detection of mutant genes, so a highly sensitive and specific mutant detection method is required to evaluate for EGFR gene mutations

Method used

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  • Detection probe, liquid phase chip and detection method thereof for EGFR gene mutation sites
  • Detection probe, liquid phase chip and detection method thereof for EGFR gene mutation sites
  • Detection probe, liquid phase chip and detection method thereof for EGFR gene mutation sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Preparation of liquid chip for EGFR gene mutation detection

[0078] 1. Probe sequence design and microsphere coating

[0079] For the wild-type and mutant sequences of exons 19 and 21 of the EGFR gene, specific oligonucleotide probes were designed. The 5'end of the probe is an amino group, followed by a 10 T spacer. The probe was synthesized by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. The probes are respectively coupled with different color-coded microspheres (purchased from Luminex) through covalent bonding (coating process). The probe sequence is shown in the following table:

[0080]

[0081]

[0082] The specific steps for coating each kind of microspheres are as follows:

[0083] (1) Centrifuge the dry probe powder at 10,000 rpm for 1 min;

[0084] (2) Use ddH 2 O dissolved to 0.1mM (0.1nmol / μl, about 70μl);

[0085] (3) Centrifuge for a short time to gather the solution at the bottom of the tube;

[0086] (4) Divide into 10μl and 2μl, ...

Embodiment 2

[0133] Using the liquid chip for EGFR gene mutation detection in Example 1 to detect non-small cell lung cancer serum samples

[0134] 1. Preparation of the sample to be tested (extraction of free nucleic acid from plasma, serum and pleural fluid):

[0135] Refer to the instructions of AxyPrep Whole Blood Genome Small Amount Extraction Kit, the detailed steps are as follows:

[0136] (1) Take about 2.5ml of the patient's anticoagulated venous blood or pleural effusion, centrifuge at 3000rpm for 15 minutes, take 300μl of supernatant and add it to a 1.5ml clean and sterile centrifuge tube;

[0137] (2) Add 500μl AP1 buffer to the centrifuge tube, vortex and shake to mix well;

[0138] (3) Add 100μl AP2 buffer and vortex to mix thoroughly;

[0139] (4) Centrifuge at 12,000 rpm for 10 minutes at room temperature;

[0140] (5) Carefully aspirate the supernatant and add it to the adsorption column AxyPrep on a 2ml collection tube, cover it, and centrifuge at 6,000rpm for 1 minute;

[01...

Embodiment 3

[0237] Using the liquid-phase chip for EGFR gene mutation detection in Example 1 to detect lung cancer tissue samples

[0238] Take the lung cancer tissue samples of patients 1-10 used in Example 2 for detection, and the specific process is as follows:

[0239] 1. Preparation of samples to be tested

[0240] Extraction of DNA from lung cancer tissue samples: Take 5-50 mg of tissue specimens after lung cancer surgery or biopsy, and after grinding, wash them with pH 7.4 PBS solution twice; the washed tissue specimens are resuspended in 1 ml of digestive juice (50 mmol / L Tris, 1mmo / LNa 2 EDTA, 0.5% Tween-20, 200ug / ml proteinase K 200, pH 8.5), digested in a water bath at 55°C for 1 hour, and inactivated proteinase K in a water bath at 99°C for 15 minutes; centrifuged at 12000 rpm for 10 minutes; take the supernatant and pass Phenol-chloroform-isoamyl alcohol extraction method, ethanol precipitation method to obtain DNA samples for PCR reaction. DNA can also be extracted by micro spi...

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Abstract

The invention discloses a detection probe, a liquid phase chip and a detection method thereof for EGFR gene mutation sites. The EGFR gene mutation detection liquid phase chip mainly comprises a microsphere for wrapping the probe; and a primer used for being amplified with a target sequence with 19 exons and/or 21 exons. By adopting the EGFR gene mutation detection liquid phase chip and the detection method, the gene mutation sites with higher relative frequency can be detected simultaneously, the 19 exons and the 21 exons can be detected separately, and also can be detected simultaneously, the reaction conditions during the detection are uniform, the specificity of the detected result is good, the sensitivity is high, the detection accuracy reaches above 90 percent, and the detection timeis short. The detection method not only can detect the EGFR gene mutation in the tumor tissue sample, but also can detect the EGFR gene mutation in the body fluid of the patient with tumor and also can perform dynamic detection.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to detection probes, liquid-phase chips and detection methods of EGFR gene mutation sites. technical background [0002] Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of various tissues in the human body. It has tyrosine kinase activity. It is one of the four members of the ErbB family, so it is also known as HER1 or ErbB1. EGFR is activated by ligands to initiate intracellular signal transduction, and through the cascade of adaptor proteins and enzymes in the cytoplasm, it regulates the transcription of transcription factors to activate gene transcription, and guides cell migration, adhesion, proliferation, differentiation and apoptosis. Studies have shown that there are high or abnormal expressions of EGFR in many solid tumors, which provides a theoretical basis and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/156C12Q1/6886
Inventor 许嘉森杨惠夷林一群徐军
Owner 广州益善医学检验所有限公司
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