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EGFR gene detection probe, preparation method thereof and reagent kit

A gene detection and kit technology, which is applied in the field of EGFR gene detection probes and its preparation, can solve the problems of high detection kits and lack of specificity, and achieve the goals of improving survival rate, accurate signal counting, and adjuvant treatment plan selection Effect

Inactive Publication Date: 2016-04-13
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, for EGFR gene FISH method detection, there is still a lack of highly specific detection kits

Method used

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  • EGFR gene detection probe, preparation method thereof and reagent kit
  • EGFR gene detection probe, preparation method thereof and reagent kit
  • EGFR gene detection probe, preparation method thereof and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 EGFR gene detection probe

[0034] The preparation method of the EGFR detection probe described in this implementation comprises the following steps:

[0035] Select the clones containing the target gene EGFR and the sequences at both ends, such as figure 1 shown.

[0036] GSPEGFR includes two groups, respectively including probe group 1: the first probe, the second probe, the third probe and the fourth probe; probe group 2: the first probe, the second probe and the third probe Needles, as shown in the table below, were purchased from Invitrogen RP11BAC and CTDBAC clone banks. The following two groups of detection probes were prepared respectively.

[0037] EGFRchr7:55,086,725-55,275,031,188,307bp

[0038] First group

[0039] Probe set 1

BAC

Insert segment start and end position

first probe

RP11-748O6

chr7:54,711,888-54,884,289,172,402bp

second probe

RP11-164G7

chr7:54,818,676-54,995,52...

Embodiment 2

[0053] Embodiment 2: Preparation method of EGFR gene detection kit

[0054] The EGFR gene detection kit includes two components of EGFR hybridization solution and DAPI counterstaining agent, wherein the EGFR hybridization solution contains the GSPEGFR gene probe described in Example 1 (respectively two groups of detection probes, corresponding to two kits) , CSP7 probe (identification probe for chromosome 7), buffer components for hybridization environment (promoting hybridization), COT Human DNA for blocking repetitive sequences, etc. DAPI counterstaining agent is mainly used for cell counterstaining after hybridization, in which DAPI will bind to DNA, making the nucleus show blue fluorescence, and the counterstaining agent containing p-phenylenediamine can keep the fluorescence stable.

[0055] The specific formula is as follows:

[0056] (1) Preparation of hybridization solution

[0057]

[0058] (2) DAPI counterstain preparation

[0059] Dissolve 10 mg of p-phenylene...

Embodiment 3

[0062] Embodiment 3: the detection method of EGFR gene detection kit

[0063] 1. Slide pretreatment

[0064] 1.1 Place the slides in a 65±5°C incubator and bake overnight;

[0065] 1.2 Take out the slide and put it in xylene to dewax at room temperature for 15 minutes;

[0066] 1.3 Take out the slide, and put it into another vat of xylene to continue dewaxing at room temperature for 15 minutes;

[0067] 1.4 Take out the slide, and put it in absolute ethanol at room temperature for 10 minutes to remove residual xylene;

[0068] 1.5 Take out the slide, and put it into 100%, 90%, 70% graded ethanol for rehydration at room temperature for 3 minutes each;

[0069] 1.6 Take out the slide, put it in purified water and wash it at room temperature for 3 minutes, and absorb excess water with a lint-free paper towel;

[0070] 1.7 Take out the slides, put them into purified water and boil them at 100±5°C for 25 minutes (place the slides horizontally in the container with the sample si...

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Abstract

The invention relates to an EGFR gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone is at least one of RP11-748O6, RP11-164G7, RP11-781C22 and RP11-17J15, or a selected BAC clone is at least one of CTD-2086F7, RP11-381G5 and CTD-2343B15, a plasmid is extracted from the clone to obtain DNA of the plasmid, and quantification is conducted; marking is carried out with fluorescein. The invention further discloses a reagent kit provided with the EGFR gene detection probe. By obtaining the optimal EGFR detection probe through screening, signal line counting is accurate and quick, and result repeatability is good. Defects of EGFR mutation detection in clinic are made up for, more patients benefited from targeted drug can be screened more easily, the survival rate of lung cancer patients is increased, and overall life time is prolonged.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to an EGFR gene detection probe, a preparation method and a kit thereof. Background technique [0002] Lung cancer is a current research hotspot, but the detection hotspot mainly focuses on the detection of EGFR gene mutation sites, which have potential value in small molecule TKI targeted therapy. However, for some macromolecular monoclonal antibody drugs, such as cetuximab, FISH detection of EGFR copy number changes is more valuable. Current clinical research has also found that EGFR mutation and EGFR amplification are two partially overlapping and independent targets. Evaluation of EGFR amplification can effectively predict the overall survival of patients, and it is also of positive significance in clinical medication. [0003] Fluorescence in situ hybridization (Fluorescence in situ hybridization FISH) is a non-radioactive in situ hybridization technology developed on the basis of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12N15/10C12N15/11C12Q1/68C12Q1/6886C12Q1/6811C12Q1/6841C12Q2600/106C12Q2600/166C12Q2563/107
Inventor 陈绍宇何瑰欧焕金
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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