Method for detecting whether fusion mutation of target gene occurs, primer combination, kit and application thereof

Abstract

The invention relates to a method for detecting whether fusion mutation of target gene occurs, a primer combination, a kit and an application thereof. A plurality of primers are sequentially designedaccording to the possible gene fusion occurrence direction by the primer design method of the invention, forming a primer combination, wherein the primers of the primer combination extend along the direction in which gene fusion occurs, can carry out directional enrichment on the target gene in which unknown fusion occurs, overcomes the disadvantage that the existing amplicon sequencing method cannot enrich the unknown fusion gene targetedly, and is conducive to the discovery and detection of the unknown fusion type of the gene; At the same time, the interval between two adjacent primers is about 0 - 130 bp, which is good for highly fragmented samples (such as cfDNA, FFPE samples). The invention also designs primer combinations for detecting fusion mutations of lung cancer genes (includingALK gene, NTRK1 gene, RET gene, ROS1 gene, FGFR3 gene and NTRK3 gene), After multiple rounds of detection and optimization of the primer sequence, the homogeneity of the sequencing data was more than85%, and the capture efficiency was more than 92%. Finally, the effective detection of unknown fusion mutation of various lung cancer genes was successfully achieved.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for detecting whether a fusion mutation occurs in a target gene, a primer combination, a kit and an application thereof. Background technique [0002] Fusion gene is a kind of gene mutation, which refers to a new gene formed by the fusion of all or part of the sequences of two genes, which may be the result of chromosomal translocation, middle deletion or chromosomal inversion. In most cases, fusion genes can lead to the production of proteins with abnormal sequences or functions, or the dysregulation of the expression of certain genes, which can cause or promote the occurrence of tumors. Since the discovery of the Philadelphia chromosome in 1973, with the development of detection technology, especially the development of sequencing technology, scientists have successively found blood cancers such as leukemia and solid tumors such as lung cancer, prostate cancer, breast can...

AI Technical Summary

Problems solved by technology

[0005] Despite many advantages, the traditional amplicon sequencing method still has many deficiencies in the detection of gene fusions. The specific performance is as follows: (1) Due to the inability to obtain the partner gene information and breakpoint position of the unknown fusion gene, the traditional amplicon sequencing method Sequencing methods are difficult to detect fusion genes with unknown breakpoints. In the prior art, there are few kits based on amplicon sequencing for fusion detection of DNA samples, especially blood samples, and it is rare in China.

(2) The primer sequences at both ends of the target capture region are fixed. For highly fragmented DNA fragments, such as FFPE, cfDNA and other samples, the capture efficiency is low, which may lead to false negative results

At present, there is still a lack of technology that can detect gene fusion mutations with breakpoints, unknown fusion directions, and highly fragmented templates.

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