Probe, primer and kit for detecting ALK (anaplastic lymphoma kinase) gene expression

Abstract

The invention discloses primer, a probe and a kit for detecting ALK (anaplastic lymphoma kinase) gene expression difference. The primer mainly comprises the following sequences: SEQ ID NO1-SEQ ID NO12. The primer and the probe disclosed by the invention can specifically detect the ALK gene expression difference, so as to determine whether ALK gene fusion exists. A real-time fluorescent PCR (polymerase chain reaction) system for detecting the ALK gene expression difference is built; qualitative evaluation of an ALK gene expression difference exon is provided by detecting the difference of 5'-end and 3'-end gene expression quantities; and the primer and the probe are suitable for a patient with a non-small cell lung cancer before entering personalized targeted therapy. The method has the advantages that (1) sensitivity is high, wherein five copies of positive plasmids can be detected; (2) operation is simple, detection is cheap, and clinical application range is wide; (3) sample detection range is wide, wherein the sample can be used as a fresh pathological tissue, a paraffin-embedded tissue (slice) or a frozen pathological slice; (4) fast detection speed is fast, wherein the detection process can be finished just by 160 minutes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting differential expression of ALK gene. Background technique [0002] Receptor-type tyrosine kinase Anaplastic lymphoma kinase (Anaplastic lymphoma kinase, ALK) was first discovered in anaplastic large cell lymphoma (Anaplastic large cell lymphoma, ALCL), which is formed by the translocation of chromosomes 2 and 5 The fusion protein contains the 3' end intracellular domain of ALK and the 5' end domain of nucleophosmin (NPM). Subsequent studies have found that normal ALK is exclusively expressed in the nervous system, such as the brain and nerve cord, especially in the brain of newborns. The ALK gene is located at chromosome 2p23. Under normal circumstances, human ALK can be transcribed to produce a 6222bp mRNA, which consists of 29 exons and encodes a 1620 amino acid sequence of 200KDa type I transmembrane protein ALK. The protein is a receptor ty...

AI Technical Summary

Problems solved by technology

The previous RT-PCR method for ALK fusion gene detection was designed and detected for the reported ALK fusion types, and the detection spectrum can only cover known ALK fusion types, therefore, it does not have unknown / unreported ALK fusions It is difficult to avoid false positives caused by amplification; the IHC method can detect the expression of tumor-specific antigens without losing the ability to distinguish normal tissues from pathological tissues However, the IHC method is aimed at the expression of ALK protein, and there may be false positives (increased expression of ALK protein may be due to the fusion of ALK gene in tumor cells, and may Due to the abnormal expression of ALK protein in tumor cells), and ALK protein expression results may appear weak and local weak positive results, there is a certain degree of subjectivity in the interpretation of the results, the weak positive results must be further verified by FISH and other techniques

FISH technology can use probes to diagnose ALK rearrangement, but this technology has high cost and long detection cycle, especially when using biopsy samples, it is not easy to interpret, and for specific fusion types, such as EML4-ALK fusion, because EML4 gene and ALK gene Close distance (12Mb) in chromosomal position, which may cause rearrangement, the segregation signal is not obvious, resulting in false negative results

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