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EWSR1-TFEB fusion gene as well as detection primer and application thereof

A technology of fusing genes and primer pairs, applied in the field of medical testing, can solve the problems of scarcity of testing platforms, long testing cycles, and high requirements for sample quality, and achieve the goal of expanding the testing range, improving the detection rate, accuracy, and reliable detection rate. Effect

Active Publication Date: 2019-12-13
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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AI Technical Summary

Problems solved by technology

[0005] At present, high-throughput sequencing is the only detection method that can clarify unknown translocation sites. However, high-throughput sequencing is expensive, the detection cycle is long, the detection platform is scarce, and the requirements for sample quality are high, which is not conducive to popularization and promotion. For most patients It is not the preferred detection method

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  • EWSR1-TFEB fusion gene as well as detection primer and application thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 is verified for the case of definite diagnosis:

[0024] For cases in which new fusion sites of EWSR1exon6-TFEB exon5 were detected by high-throughput sequencing RNA-seq, the primers we designed were used for verification.

[0025] 1. Extraction of RNA:

[0026]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, cent...

Embodiment 2

[0030] Embodiment 2 detects for control group case

[0031] We analyzed 30 well-diagnosed control cases (including 10 cases of clear cell RCC, 5 cases of papillary RCC, 5 cases of chromophobe RCC, 5 cases of TFE3 translocation-associated RCC and 5 cases of MALAT1-TFEB translocation-associated renal carcinoma, theoretically no EWSR1-TFEB gene fusion) were detected using the primer combination of the present invention, and the RNA extraction, reverse transcription PCR and sequencing methods were the same as above.

[0032] Results: No EWSR1-TFEB fusion gene was detected by using the primer combination designed in the present invention, which proves that the primers designed in this project have high specificity.

[0033] Evaluation: The primer combination of the present invention is a supplement to the original TFEB translocation renal cell carcinoma fusion gene primers, expands the types of TFEB translocation renal cell carcinoma fusion genes, and increases the detection rate o...

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Abstract

The invention discloses a EWSR1-TFEB fusion gene as well as a detection primer and an application thereof. The fusion gene comprises a nucleotide sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4. Theinvention discloses a primer composition for diagnosing EWSR1-TFEB translocation renal cell carcinoma, which is a PCR primer for detecting EWSR1-TFEB gene translocation, and is preferably shown as SEQ ID NO.1 and SEQ ID NO.2. The invention also discloses the application of the primer of the EWSR1-TFEB fusion gene in preparation of a EWSR1-TFEB translocation renal cell carcinoma diagnostic reagent. According to the invention, a new translocation partner of TFEB is cloned, and is reported for the first time in TFEB translocation renal cell carcinoma. A specific PCR primer is designed for new fusion sites found through high-throughput sequencing, a detection range of the original primer composition is expanded, and after the primer is applied to clinic, and the detection rate and accuracy ofdiagnosis of the tumors can be improved.

Description

technical field [0001] The invention belongs to the field of medical testing, and relates to an EWSR1-TFEB fusion gene, detection primers and applications thereof. Background technique [0002] In 2016, WHO revised the histopathological classification of renal cell carcinoma in 2004 and added TFEB gene translocation-associated renal carcinoma (TFEB translocation renal carcinoma). The TFEB gene is located at 6p21.1, which is the only driver gene of TFEB translocation renal cancer, and its pathogenic mechanism is clear: all these tumors involve the translocation of the TFEB gene located at 6p21.1 with other chromosomes and the resulting fusion genes , TFEB fusion protein is highly expressed through promoter transformation, and TFEB, as a transcription factor, combines with specific DNA structures to transcriptionally regulate the expression of various genes in the body and eventually cause disease. Only one single translocation form, MALAT1-TFEB, is currently included in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11C12N15/62
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2531/113
Inventor 饶秋夏秋媛叶胜兵李锐王璇魏雪
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A