Primer combination and its application for detecting c-kit gene mutation in trace tissue

Abstract

The invention discloses a primer combination for detecting mutation of a C-KIT gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.9 exon mutation, a primer group for detecting No.11 exon mutation, a primer group for detecting No.13 exon mutation, a primer group for detecting No.14 exon mutation, a primer group for detecting No.17 exon mutation and a primer group for detecting No.18 exon mutation. According to a method, a trace amount of DNA is pre-amplified to obtain a high-concentration DNA template, and the mutation condition of the C-KIT gene in a sample is detected by combining a direct sequencing method; by applying the primer combination to preparation of a detection reagent for detecting mutation of the C-KIT gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.9 exon, a No.11 exon, a No.13 exon, a No.14 exon, a No.17 exon and a No.18 exon of the C-KIT gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for C-KIT gene mutation detection on a clinical tumor patient and has the very good clinical application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a C-KIT Primer combination for gene mutation detection and its application in tumor drug selection and disease diagnosis. Background technique [0002] C-KIT The gene is located on human chromosome 4q11-21 and is a proto-oncogene. Its cDNA is 5230bp in length and contains 21 exons, encoding a 145KD transmembrane tyrosine kinase receptor (RTK) protein, named CD117 . Exon 1 encodes the initiation codon and signal peptide, codons 2–9 encode the extramembrane ligand domain, exon 10 encodes the hydrophobic transmembrane domain, and exons 11–20 encode the membrane internal domain. Among them, exon 11 encodes the proximal membrane segment. C-KIT receptor belongs to the type III RTK family, distributed on the cell surface, can be detected by CD117 monoclonal antibody, and has strong homology with platelet-derived growth factor receptors (PDGFR) (Patil DT, et al, 2016)....

AI Technical Summary

Problems solved by technology

However, the application of the last base mismatch in ARMS technology cannot completely block the amplification of wild-type DNA, there is a risk of false positives, and it can only be detected for specific sites

In addition, such as high performance liquid chromatography, capillary electrophoresis, etc., require special equipment, and the operation is complicated, which is not conducive to large-scale clinical promotion

Nucleic acid sequence amplification (NASBA), self-sequencing sequence replication (3SR) and strand displacement replication (SDA) are all isothermal amplification methods, but their specificity for the amplification of target sequences is not strong

[0007] despite this, C-KIT The detection method of mutation is still based on the detection of tumor tissue samples as the gold standard, but in clinical practice, the detection is often limited due to insufficient tissue samples

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