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Probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma and application of probe assembly

A probe and fusion gene technology, applied in the field of fluorescent in situ hybridization probe application, can solve the problems of no reports, etc., and achieve the effect of improving accuracy and reliable success rate

Active Publication Date: 2019-09-20
中国人民解放军东部战区总医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports at home and abroad on the method of detecting EWSR1-TFEB fusion gene by fluorescence in situ hybridization (FISH)

Method used

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  • Probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma and application of probe assembly
  • Probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma and application of probe assembly
  • Probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma and application of probe assembly

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 is verified for the case of definite diagnosis:

[0035] For cases in which new fusion sites of EWSR1 exon6-TFEB exon5 were detected by high-throughput sequencing RNA-seq, the primers we designed were used for verification.

[0036] 1. Extraction of RNA:

[0037] Strictly follow the RNeasy FFPE Kit operating instructions for extraction. ① Dewaxing: dewax the collected slides with xylene, rinse with absolute ethanol, air-dry and scrape off with a scalpel blade and put them into a 1.5ml EP tube; Proteinase K, mix well, enzymatically digest at 56°C for 15min, then at 80°C for 15min, cool on ice; ③add 16μl DNase buffer, then add 10μl DNase I, mix well, let stand at room temperature for 15min, centrifuge at 12000rpm for 15min, and take the supernatant;④ Add 320μl of binding solution and 720μl of absolute ethanol, mix well, transfer to the adsorption column twice, centrifuge at 8000rpm for 1min, discard the waste liquid; ⑤Wash: add 500μl of washing solution, ce...

Embodiment 2

[0042] Embodiment 2: the preparation of DNA probe combination:

[0043] Select two BAC clone fragments that can be connected at the telomeric side of the TFEB gene on chromosome 6 and the centromere side of the EWSR1 gene on chromosome 22, and control the farthest distance between the probes at both ends within 1500kb, and keep the distance between the BAC clone fragments A certain distance does not overlap, and the fragments are similar in size. The cloned fragments were obtained from the Human BAC Cloning Center of EmpireGenomics ( http: / / www.empiregenomics.com / helixhq / clonecentral / search / human ). The BAC clone probe on the centromere side of EWSR1 is RP11-480L23 (fragment length 155.0kb), and the BAC clone probe on the TFEB telomeric side is RP11-81C24 (fragment length 176kb). These fragments are bacterial artificial chromosomes (Bacterial artificial chromosome, BAC) clone, its location on human chromosomes has been published, respectively, RP11-480L23 is located on chro...

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Abstract

The invention discloses a probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma and application of the probe assembly. A EWSR1-TFEB fusion gene comprises a nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO.4. The probe assembly for diagnosis of EWSR1-TFEB translocation renal cell carcinoma comprises a BAC clone probe RP11-480L23 and a BAC clone probe RP11-81C24. By adoption of the probe assembly for detection of EWSR1-TFEB translocation renal cell carcinoma, convenience, quickness, reliability and high success rate are realized. The probe assembly can be used for preparing a EWSR1-TFEB translocation renal cell carcinoma diagnosis kit to provide a new tool for quick and accurate diagnosis of EWSR1-TFEB translocation renal cell carcinoma.

Description

technical field [0001] The invention belongs to the application field of fluorescent in situ hybridization probes, and relates to a fluorescent in situ hybridization (FISH) fusion gene probe combination for diagnosing EWSR1-TFEB translocation renal cancer and its use in preparing EWSR1-TFEB translocation renal cancer Application in cancer diagnostic reagents. Background technique [0002] In 2016, a new type of renal cell carcinoma was added to the new WHO classification of kidney tumors by histopathology: MiT family translocation renal cell carcinoma. The MiT family is the abbreviation of the microphthalmia-associated transcription factor family, which includes MITF, TFE3, TFEB and TFEC genes. [0003] MiT family translocation renal cell carcinomas that have been discovered so far include Xp11.2 translocation / TFE3 gene fusion-related renal cell carcinoma and t(6;11)(p21;q12) translocation / TFEB gene fusion-related renal cell carcinoma. [0004] The pathogenic mechanism of ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12Q1/6886C12Q1/6841C12N15/11
CPCC12Q1/6886C12Q1/6841C12Q2563/107C12Q2543/10
Inventor 夏秋媛王小桐方茹叶胜兵潘瑞饶秋
Owner 中国人民解放军东部战区总医院