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A fish probe set for detecting fgfr2 fusion and its application

A probe group and probe technology, applied in recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., to achieve high probe specificity and sensitivity, accurate and reliable detection results, and high fluorescent labeling efficiency Effect

Active Publication Date: 2021-05-28
AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main variation form of FGFR2 in ICC is gene fusion. It has been found that there are more than 30 fusion partner genes of FGFR2, and the fusion methods are various. Therefore, there are certain challenges in the detection of FGFR2 fusion

Method used

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  • A fish probe set for detecting fgfr2 fusion and its application
  • A fish probe set for detecting fgfr2 fusion and its application
  • A fish probe set for detecting fgfr2 fusion and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The FGFR2 5' end probe set used two BAC clones (RP11-78A18 and RP11-436E17) as templates, and the 3' end probe set used two BAC clones (RP11-7P17 and RP11-984I17) as templates, and the above BAC clones were all Purchased from Oakland Children's Hospital Research Institute, USA. The fabrication and labeling steps of the probe set are as follows:

[0031] (1) In two 200 μL PCR reaction tubes, add the reaction materials according to the following system to prepare and label the 5′-end probe set and the 3’-end probe set:

[0032]

[0033]

[0034] The above random primers consist of 9 nucleotides, each nucleotide is A, T, C or G, and the first and / or fifth nucleotides from the 5' end are modified with fluorescence through spacer9 connection group; the formula of the above 5×Buffer is: 10mM Tris-HCl, 25mM MgCl 2 (magnesium chloride), with ddH 2 O preparation.

[0035] After mixing by pipetting, denature at 95°C for 10 minutes on a PCR instrument, immediately place ...

Embodiment 2

[0049] The steps of using a UV spectrophotometer to measure the labeling efficiency and concentration of the probes in the above-mentioned probe group are as follows:

[0050] (1) Turn on the UV spectrophotometer (NanoDrop), select the full-wavelength mode, and select the corresponding wavelength. After blank calibration, measure the A of the 5′-end probe group respectively. 260nm 、A Dye (FITC:A 494nm ) and A of the 3′ end probe set 260nm 、A Dyc (Tetramethyl-rhodamine: A 560nm ).

[0051] (2) Since the fluorescent dye also has a certain absorbance value at 260nm, in order to make the absorbance value of the probe at 260nm more accurate, use the following formula to correct A 260 Value: A Base =A 260 -(A Dye ×CF 260 ), CF 260 Refers to the correction factor, which is a constant, and each fluorescent dye has a certain CF 260 Values, such as the CF of Tetramethyl-rhodamine 260 The value is 0.06 and FITC is 0.32. (3) Calculate the probe labeling efficiency and concent...

Embodiment 3

[0056] The FGFR2 gene breakage probe system prepared in Example 1 was used to detect 50 intrahepatic cholangiocarcinoma samples with known FGFR2 gene fusion results provided by the hospital, and the detection steps were as follows:

[0057] (1) Sample dewaxing and rehydration: immerse tissue section samples in 3 tanks of xylene for 10 minutes each time for dewaxing, and then soak in 100%, 100%, 100%, 85% and 70% ethanol for 3 minutes, Finally, immerse in deionized water for 3 min.

[0058] (2) Sample pretreatment and digestion: Boil the tissue section sample in 100°C water for 20 minutes, take out the sample and digest it with pepsin for 10 minutes, transfer it to 2×SSC solution for 3 minutes, take out the sample and immerse it in 70%, 85% and 100% Ethanol dehydration, 3min each time, take out the sample to dry naturally.

[0059] (3) Hybridization of the sample with the probe: Use a pipette gun to take 10 μL of the FGFR2 gene fragmentation probe system and add it to the samp...

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Abstract

The invention discloses a FISH probe set for detecting FGFR2 fusion and its application, including a 5' end probe set and a 3' end probe set; The BAC clone of the sequence is used as a template and amplified with random primers, in which each probe is modified with the first reporter group; the 3' end probe group is based on the BAC of the nucleotide sequence on the centromere side of the FGFR2 gene Clones are used as templates and amplified with random primers in which each probe is modified with a second reporter group. The invention is quick and easy to operate, accurate and reliable in detection results, can accurately detect all fusion types of the FGFR2 gene, provides a reference for the treatment plan of patients with intrahepatic cholangiocarcinoma, is beneficial to individualized targeted therapy for patients, reduces drug side effects, and improves patient survival Quality, reduce family and social burden, suitable for large-scale promotion and application.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a FISH probe group for detecting FGFR2 fusion and its application. Background technique [0002] Intrahepatic cholangiocarcinoma (ICC) is an adenocarcinoma originating from the epithelium of the secondary bile duct and its branches. It has a relatively high degree of malignancy, accounting for about 10-15% of primary malignant tumors of the liver. The incidence rate is second only to hepatocytes. Liver cancer (Rizvi and Gores, Gastroenterology 2013). Globally, ICC is a relatively rare tumor, but in China, South Korea, Thailand and other Southeast Asian countries, ICC is a common tumor, and its incidence has been on the rise in recent years. The incidence of ICC in my country is about 7.5 / 100,000 (Cardinale et al. Hepatoma Res 2018;4:20). [0003] ICC has no obvious clinical symptoms in the early stage, and most patients are already in the advanced stage w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6841C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2525/107C12Q2537/143
Inventor 卢皇彬林煌艺朱冠山郑立谋
Owner AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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