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

A liquid phase chip and probe technology, applied in the field of molecular biology, can solve the problems that hinder the detection of mutant genes, and achieve the effects of avoiding interference, good specificity of detection results, and convenient sampling

Active Publication Date: 2013-06-05
广州益善医学检验所有限公司
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  • Summary
  • 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-phase chip for detection of EGFR gene mutation

[0078] 1. Probe sequence design and microsphere coating

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

[0080]

[0081]

[0082] The specific steps of each microsphere coating are as follows:

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

[0084] (2) with ddH 2Dissolve O 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) Aliquot into 10 μl and 2 μl and store at -20...

Embodiment 2

[0133] Detection of non-small cell lung cancer serum samples using the liquid chip for EGFR gene mutation detection in Example 1

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

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

[0136] (1) Take about 2.5ml of anticoagulated venous blood or pleural effusion from the patient, 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 500 μl of AP1 buffer solution to the centrifuge tube, vortex and mix well;

[0138] (3) Add 100 μl of AP2 buffer, vortex and mix well;

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

[0140] (5) Carefully draw the supernatant into the adsorption column AxyPrep placed on a 2ml collection tube, cover the lid, and centrifuge at 6,000rpm for 1...

Embodiment 3

[0237] Detection of lung cancer tissue samples using the liquid chip for EGFR gene mutation detection in Example 1

[0238] Get the lung cancer tissue samples of No. 1-10 patients used in the above-mentioned embodiment 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 samples after lung cancer surgery or biopsy, grind them, and wash twice with PBS solution with pH 7.4; the washed tissue samples are resuspended in 1ml of digestive solution (50mmol / L Tris, 1mmo / LNa 2 EDTA, 0.5% Tween-20, 200ug / ml protease K200, pH 8.5), digest in 55°C water bath for 1 hour, inactivate proteinase K in 99°C water bath for 15 min; Chloroform-isoamyl alcohol extraction and ethanol precipitation were used to obtain DNA samples for PCR reactions. DNA can also be extracted by micro spin column method;

[0241] 2. PCR amplification and enzyme digestion enrichment...

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Abstract

Probes, liquidchip-based products, and methods for detection of EGFR gene mutation are disclosed. The liquid chip for detection of EGFR gene mutation mainly includes: microspheres coated with probes; primers for amplifying target sequence in exon 19 and / or exon 21.The liquid chip and methods can detect mutations with relatively higher frequency in EGFR gene, and detect mutations in exon 19 and 21 simultaneously or separately. The assay has high specificity and sensitivity with uniform reaction condition. Detection has over 90 percent accuracy and takes less time. The method can be applied not only to detection of EGFR gene mutations in tumor tissue samples but also to those in humour samples from cancer patients. Thus it can monitor dynamicly.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a detection probe for EGFR gene mutation sites, a liquid phase chip and a detection method thereof. technical background [0002] Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membranes of various human tissues. It has tyrosine kinase activity and is one of the four members of the ErbB family, so it is also known as HER1 or ErbB1. After being activated by ligands, EGFR initiates intracellular signal transduction, and through the cascade reaction of adapter proteins and enzymes in the cytoplasm, it regulates the transcription of transcription factor-activated genes and guides cell migration, adhesion, proliferation, differentiation and apoptosis. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors, which provides a theoretical basis and experimen...

Claims

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Application Information

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Patent Type & Authority Patents(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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