Double fluorescent marker probe real-time quantitative detection method for K-ras gene 12 codon mutation and application

Abstract

The invention belongs to the technical field of biology. K-ras gene mutation plays an important role in occurrence and development of pancreatic cancer, but the current universal K-ras gene mutation detection methods comprise a limited fragment length polymorphism analysis method and an amplification blocked mutation system method, and the two methods have low specificity and cannot qualitativelyand quantitatively detect the mutation of K-ras genes at the same time. The invention aims to provide a double fluorescent marker probe real-time quantitative detection method for K-ras gene 12 codonmutation with operation convenience, high sensitivity and high specificity. The method comprises the following steps of: designing wild K-ras gene 12 codon-targeted peptide nucleic acid (PNA) and corresponding mutation detection probes, namely a K-ras-FAM Tagman MGB probe and a K-ras-VIC Tagman MGB probe; performing real-time quantitative polymerase chain reaction (PCR) detection on a plasmid standard substance of known mutation quantity by using the PNA and the probes to acquire a standard curve and a fluorescent type; and extracting and purifying sample DNA, measuring the concentration, performing real-time quantitative PCR detection, and judging K-ras gene mutation quantity and mutation type according to the standard curve and the fluorescent type.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for real-time quantitative detection of a K-ras gene 12 codon mutation with double fluorescent label probes, and an application thereof. 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 at codon 12, 13 or 61 of the K-ras gene can be detected in pancreatic cancer tissue, pancreatic juice or feces, and blood (see Takahashi, et al.Spontaneous rupture of a biliary diverticulum in the distal common bile duct, with formation of a retroperitoneal biloma, Gastrointestinal Endo 2005;61:76-9; Laghi L, et al.Common occurrence of multiple K-RAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers, Oncogene 2002;21: 4301-6; Tada M, et al. Detection of ras gene mutations in pancreatic juice and peri...

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: 1. The steps are cumbersome, and it takes 2 days to complete the identification; 2. This method can only be used for qualitative research, that is, it can only determine whether there is a K-ras gene mutation. If quantitative analysis is required, other methods are required. 3. False positives caused by incomplete digestion of the first round (see document Watanabe H, et al. Detection of K-ras point mutations at codon 12 in pure pancreatic juice for the diagnosis of pancreatic cancer by PCR-RFLP analysis, Pancreas 1996;12:18-24)

The shortcoming of this method is: 1. each kind of specific primer or probe can only be identified at a certain gene mutation type, and there can be more than ten kinds of mutation types at the 12th and 13th codons of K-ras gene, need Detect one by one; 2. There may be false positives caused by single base mismatches (see the literature Fox JC, et al. The detection of K-ras mutations in colorectal cancer using the amplification-refractory mutation system, Br J Cancer 1998; 77 :1267-74)

2. For mismatched PNA / DNA, even if there is only one base mismatch, its melting temperature will drop by about 9-10°C

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