Novel translocation companion of TFEB as well as detection primer and application thereof

A TFEB-E2-R, a companion technology, 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, to expand the detection range, reliable detection rate, improve detection rate and accuracy. rate effect

Active Publication Date: 2018-12-18
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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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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  • Novel translocation companion of TFEB as well as detection primer and application thereof

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Embodiment 1

[0024] Example 1 Verification of cases with definite diagnosis:

[0025] For cases in which new fusion sites of ACTB exon3-TFEB exon2 were detected by high-throughput sequencing RNA-seq, the primers we designed were used for verification.

[0026] 1. Extraction of RNA:

[0027] 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, centrif...

Embodiment 2

[0031] Example 2 Detection for the control group cases

[0032] 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 ACTB-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.

[0033] Results: No ACTB-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.

[0034] 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...

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Abstract

The invention discloses a novel translocation companion of TFEB as well as a detection primer and application thereof. The novel translocation companion of the TFEB is an ACTB-TFEB gene subjected to translocation, gene fusion happens between an exon 3 of ACTB and an exon 2 of a TFEB gene, and the novel translocation companion of the TFEB comprises a nucleotide sequence as shown in SEQ ID NO.3. ThePCR primers for detecting the novel translocation companion of the TFEB are composed of ACTB-E3-F as shown in SEQ ID NO.1 and TFEB-E2-R as shown in SEQ ID NO.2. The invention further discloses application of an agent for detecting the translocation companion of the TFEB in preparing an ACTB-TFEB translocation renal cell carcinoma diagnosis agent. A novel tool is provided for fast and accurately diagnosing an ACTB-TFEB translocation tumor.

Description

technical field [0001] The invention belongs to the field of medical testing, and relates to a new translocation partner of TFEB, 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 includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/686C12Q1/6886
CPCC12Q1/686C12Q1/6886C12Q2531/113
Inventor 夏秋媛饶秋李锐鲍炜王小桐李芳秋周晓军
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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