Kit and method for detecting NTRK gene fusion

A gene fusion, ETV6-NTRK3 technology, applied in the field of biomedicine, can solve problems such as high cost, time-consuming, and inability to detect

Active Publication Date: 2020-01-21
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of NGS technology applied to NTRK gene fusion detection are: (1) high cost; (2) cumbersome operation; (3) quite time-consuming, such as based on Ion torrent TM The detection process of the NGS technology platform, the report can be obtained within 3 days at the fastest
However, the current application of this method in NTRK gene fusion d

Method used

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  • Kit and method for detecting NTRK gene fusion
  • Kit and method for detecting NTRK gene fusion
  • Kit and method for detecting NTRK gene fusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 is used for detecting the fluorescent PCR detection kit of human NTRK gene fusion

[0054] A fluorescent PCR detection kit for detecting human NTRK gene fusion, comprising PCR reaction solution I, PCR reaction solution II, PCR reaction solution III, PCR reaction solution IV, and a positive quality control product.

[0055] The preparation of the kit includes the following steps:

[0056] (1) Design and preparation of detection primers and probes: Design several pairs of specific primers and fluorescent probes for the 12 fusion types of NTRK gene, conduct pre-experiments on each primer and probe, compare sensitivity, specificity and other properties, Ultimately, primer combinations and probes for detecting 12 fusion types of human NTRK genes were selected, specifically as SEQ ID NO.1 to 36. Preferable detection primers and detection probes are stored in 100 μM mother solution, and prepared into 20 μM working solution according to detection requirements.

...

Embodiment 2

[0084] Embodiment 2: specificity analysis experiment

[0085] A fluorescent PCR detection kit for detecting human NTRK gene fusion prepared in Example 1 is used to detect the NTRK gene wild-type plasmid, NTRK fusion mutant plasmid, and internal standard β-actin plasmid with a concentration of 1250 copies / μl , load 2 μl, 2 wells. A blank control was set up, and the specificity of the kit of the present invention was analyzed according to the test results.

[0086] The NTRK gene wild-type plasmids described in this example include NTRK1-E10, NTRK1-E11, NTRK2-E12, NTRK2-E13, NTRK2-E15, NTRK2-E16, NTRK3-E14, NTRK3-E15, a total of 8 NTRK wild-type plasmids , the fusion mutant plasmids include LMNA-E2-NTRK1-E10, LMNA-E2-NTRK1-E11, LMNA-E10-NTRK1-E11, TPM3-E7-NTRK1-E10, AFAP1-E13-NTRK2-E12, NACC2- E4-NTRK2-E13, QKI-E6-NTRK2-E16, TRIM24-E12-NTRK2-E15, ETV6-E4-NTRK3-E14, ETV6-E5-NTRK3-E14, ETV6-E4-NTRK3-E15, ETV6-E5- NTRK3-E15.

[0087] The specific fusion type detection result jud...

Embodiment 3

[0100] Embodiment 3: sensitivity analysis experiment

[0101] In this example, the above-mentioned 12 kinds of NTRK fusion mutant plasmids were taken, and the concentrations were adjusted to 1×10 9 copies / μl, and then diluted to 1×10 8 , 1×10 7 , 1×10 6 , 1.25×10 5 , 1.25×10 4 , 1.25×10 3 , 1.25×10 2 , 12.5, 1.25, 1, 0.5 copies / μl.

[0102] Using a fluorescent PCR detection kit for detecting human NTRK gene fusion obtained in Example 1, the above concentrations are 0.5, 1, 1.25, 12.5, 125, 1.25 × 10 3 , 1.25×10 4 , 1.25×10 5 The 12 kinds of NTRK fusion mutant plasmids of copy / μl were used as templates (2 μl of sample loading, 3 wells) for detection respectively, and a blank control was set up, and the sensitivity of the kit of the present invention was analyzed according to the detection results.

[0103] The detection result shows that when the copy number of 12 kinds of NTRK fusion mutant plasmids is as low as 1.25 copies / μl, the detection result is still positive ...

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Abstract

The invention provides a kit and a method for detecting NTRK gene fusion. The kit includes at least one of four tubes of reagents, the 4 tubes of the reagents contain specific primers and probes designed for 12 fusion types of a NTRK gene, one kind of NTRK1 fusion, one kind of NTRK2 fusion and one kind of NTRK3 fusion are detected in each tube, interference between the primers of the same gene canbe excluded, thus the stability of a reaction system is greatly improved, the detection sensitivity and specificity are improved, and the probability of occurrence of false positives is decreased.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a kit and method for detecting NTRK gene fusion. Background technique [0002] The neurotrophic factor receptor tyrosine kinase (NTRK) family includes three proteins of the tropomyosin receptor kinase (TRK) family, TRKA, TRKB, and TRKC, which are encoded by NTRK1, NTRK2, and NTRK3 genes, and these proteins are usually expressed in nerve cells. Expressed in tissues and activated by neurotrophic factors (NTs), they play important roles in the physiology of nervous system development and function. NTs refer to the specific ligands of TRKA, nerve growth factor (NGF), the specific ligands of TRKB, brain-derived growth factor (BDGF), neurotrophic factor-4 / 5 (NT-4 / 5), and the specificity of TRKC Ligand NT-3. The combination of NTs and TRK protein can induce receptor dimerization, phosphorylation and activate TRK downstream signaling cascade pathway through PI3K, RAS / MAPK / ERK and P...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6851
CPCC12Q1/6876C12Q1/6851C12Q2600/156C12Q2563/107C12Q2531/113Y02A50/30
Inventor 吴诗扬许嘉森彭璨璨刘志明刘芳
Owner SUREXAM BIO TECH
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