Primer combination and its application for detecting kras gene mutation in trace tissue

Abstract

The invention discloses a primer combination for detecting mutation of a KRAS gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.2 exon mutation and a primer group for detecting No.3 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 KRAS 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 KRAS gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.2 exon and a No.3 exon of the KRAS gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for KRAS 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 KRAS Primer combination for gene mutation detection and its application in tumor drug selection and disease diagnosis. Background technique [0002] Ras is a proto-oncogene tumor, which was originally cloned from rat sarcoma virus. There are generally three RAS oncogene family members in mammalian genomes: H-RAS, K-RAS, and N-RAS. The proteins encoded by these three genes have about 90% amino acid homologous sequences, and the molecular weight is 21kDa, so they are called For RASp21 protein. KRAS The gene is one of the three oncogenes in the RAS gene family. It is located on chromosome 12 and contains 4 coding exons and 1 5' non-coding exon, which jointly encode the RAS protein with 189 amino acids. KRAS is the downstream molecule of the epidermal growth factor receptor functional signal, which belongs to the membrane-bound GTP / GDP binding protein, and is regula...

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

[0006] Nevertheless, the detection method of KRAS 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 the insufficient amount of tissue samples obtained.

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