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FISH probe set for detecting NTRK fusion and application thereof

A probe group and probe technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of continuous activation of kinase region, abnormal expression of TRK, low expression level, etc. , improve the quality of life, the effect of high probe specificity and sensitivity

Active Publication Date: 2019-07-09
AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression level of TRK in normal tissues is low, but when fusion occurs (the partner gene is fused with the kinase region at the 3' end of NTRK as the 5' end), it can lead to abnormal expression of TRK, resulting in continuous activation of the kinase region and triggering tumors

Method used

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  • FISH probe set for detecting NTRK fusion and application thereof
  • FISH probe set for detecting NTRK fusion and application thereof
  • FISH probe set for detecting NTRK fusion and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Such as figure 1 As shown, the NTRK1 5'-end probe set (NTRK1 gene centromere side) was based on two BAC clones (RP11-1054F3 and RP11-66D17) as templates, and the NTRK1 3'-end probe set (NTRK1 gene telomeric side ) with 2 BAC clones (RP11-1038N13 and RP11-110J1) as templates; NTRK2 5' end probe (NTRK2 gene centromere side) with 2 BAC clones (RP11-324L15 and CTD-2509D1) as templates, The NTRK2 3' end probe (NTRK2 gene telomere side) uses 2 BAC clones (RP11-152D13 and CTD-2339E11) as templates; NTRK3 5' end probe (NTRK3 gene telomere side) uses 2 BAC clones (RP11-110O23 and RP11-97O12) were used as templates, and the NTRK3 3' end probe (centromere side of NTRK3 gene) used two BAC clones (RP11-113C11 and CTD-2526N1) as templates. The 5′-end probes of the three genes are all labeled with FITC (the first fluorescent reporter group) as green signals, and the 3’-end probes are all labeled with Tetramethyl-rhodamine (the second fluorescent reporter group) as red signals. The ab...

Embodiment 2

[0062] 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:

[0063] (1) Turn on the UV spectrophotometer (NanoDrop), select the full-wavelength mode, and select the corresponding wavelength. After the blank calibration, measure the A2 of the 5′-end probe group respectively. 60nm 、A Dye (FITC:A 494nm ) and A of the 3′ end probe set 260nm 、A Dye (Tetramethyl-rhodamine: A 560nm ).

[0064] (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 con...

Embodiment 3

[0069] Using the NTRK1 gene break probe system and the NTRK3 gene break probe system prepared in Example 1 to detect 50 cases of papillary thyroid cancer samples provided by the hospital, the NTRK3 gene break probe system to detect 10 cases of salivary gland mammary secretory carcinoma samples, NTRK2 The gene breakage probe system was used to detect 20 samples of pontine glioma, and the NTRK probe of Empire Genomics was used to detect these samples at the same time. The detection steps are as follows:

[0070] (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.

[0071] (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 ...

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Abstract

The invention discloses an FISH probe set for detecting NTRK fusion and application thereof. The set comprises a NTRK1 5' end probe set, a NTRK1 3' end probe set, a NTRK2 5' end probe set, a NTRK 2 3'end probe set, a NTRK3 5' end probe set and a NTRK3 3' end probe set. The set has the advantages of rapid and simple operation, accurate and reliable detection results and accurate detection of all fusion types of NTRK1, NTRK2 and NTRK3 genes, provides a reference for the treatment of tumor patients, is beneficial to individualized targeted therapy, reduces drug side effects, improves quality oflife of patients, reduces the burden on families and society, and is 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 set for detecting NTRK fusion and its application. Background technique [0002] NTRK1, NTRK2, and NTRK3 belong to the neurotrophic receptor tyrosine kinase (NTRK) family, located on chromosomes 1, 9, and 15, and are responsible for encoding the tropomyosin receptor kinase (TRK) family proteins TrkA, TrkB, and TrkC, respectively. When the extracellular ligand neurotrophic factor binds to the Trk protein receptor, it can induce the receptor to form a dimer and undergo phosphorylation, activate RAS / MAPK / ERK, P13K, PLCγ and other downstream signaling pathways, and participate in cell growth, proliferation, etc. process. The expression level of TRK in normal tissues is low, but when fusion occurs (the partner gene is fused with the kinase region at the 3' end of NTRK as the 5' end), it can lead to abnormal expression of TRK, resulting in continuo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6841C12N15/11
CPCC12Q1/6886C12Q1/6841
Inventor 卢皇彬林煌艺朱思琪郑立谋
Owner AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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