Quantitative determination method for K-ras gene mutation

Abstract

The invention discloses a quantitative detection method of K-ras gene mutation, comprising the following steps: a PNA which is hybridized with a twelfth and / or thirteenth codon in a first exon of a wild-typed K-ras gene is designed; SYBR-RT-PCR quantitative detection is carried out to the mutant-typed and wild-typed standard products with known copy numbers respectively with the PNA for obtainingstandard and melting curves; SYBR-RT-PCR quantitative detection is carried out to the sample nucleic acid to be detected with the PNA; and after the sample nucleic acid to be detected is judged to bewild type or mutant type or mutant / wild hybrid type according to the melting curve, the K-ras gene mutation quantity of the sample nucleic acid to be detected is obtained according to the standard curve. The method of the invention has simple operation, high sensitivity and high specificity, and can be applied to early auxiliary diagnosis on diseases such as pancreatic cancer, etc.

Description

technical field [0001] The invention relates to the detection of K-ras gene mutation, in particular to a quantitative detection method of K-ras gene mutation. Background technique [0002] K-ras gene (GenBank: NC_000012) mutation plays an important role in the occurrence and development of pancreatic cancer. It has been reported that point mutations in codon 12, 13 or 61 of the K-ras gene can be detected in pancreatic cancer tissue, pancreatic juice or feces, and blood (Takahashi, et al. Gastrointestinal Endo 2005.61: 76-9; Luigi L, et al. Oncogene 2002.21:4301-6; TadaM, et al. Cancer Res 1993.53:2472-4; Lu XH, et al.Natl Med J China2001.81:1050-3; N van Heek, et al.J.Clin.Pathol. 2005.58: 1315-1320). [0003] Studies have shown that detection of K-ras gene mutation in somatic DNA can be used as a method for early diagnosis of pancreatic cancer. However, in some experiments, it was found that K-ras gene mutations also exist in benign diseases such as chronic pancreatitis ...

AI Technical Summary

Problems solved by technology

[0005] (1) The steps are cumbersome and it takes 2 days to complete the identification;

[0006] (2) It can only be used for qualitative research, that is, it can only be determined whether there is a K-ras gene mutation. If quantitative research is required, other methods must be used for further testing;

[0007] (3) False positives caused by incomplete first round of enzyme digestion

[0014] (1) Each specific primer or probe can only be identified for a certain type of gene mutation, and there are more than ten types of mutations in the 12th and 13th codons of the K-ras gene, which need to be detected one by one;

[0015] (2) There may be false positives due to single base mismatches

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