Human K-ras (K-rat sarcoma) gene mutation parting type fluorescent quantitation PCR (polymerase chain reaction) detecting reagent kit and detecting method

Abstract

The invention discloses a human K-ras (K-rat sarcoma) gene mutation parting type fluorescent quantitation PCR (polymerase chain reaction) detecting reagent kit and a detecting method. The detecting reagent kit comprises PCR mixed reaction liquid, a peptide nucleic acid probe, a taqman-MGB (minor groove binder) probe, an inner control forward and reverse primer and probe, an external control forward and reverse primer and probe and a positive reference article, wherein the inner control forward and reverse primer and probe is used for K-ras gene mutation detection, and the external control forward and reverse primer and probe is used for the K-ras gene mutation detection. The PNA (peptide nucleic acid) technology and the taqman-MGB technology are combined, relevant ARMS (amplification refractory mutation system) primers are designed, PNA can be stably combined with wild K-ras genes 12 or 13 codon sequences, only mutation specimens can be amplified, and the K-ras gene mutation can be detected. The method has the advantages that the speed is high, simplicity and convenience are realized, the specificity is good, the sensitivity is high, and the method can be used for the clinical K-ras gene mutation screening.

Description

technical field [0001] The invention belongs to the technical field of clinical molecular diagnosis, in particular to a gene mutation typing fluorescent quantitative PCR detection kit and detection method, in particular to a fluorescent quantitative PCR detection kit and detection of seven common mutations of human K-ras gene method. Background technique [0002] The K-ras gene is located on human chromosome 12 and contains 4 coding exons and 1 5′ non-coding exon, which jointly encode the ras protein consisting of 189 amino acids. Because of its molecular weight of 21D, Also known as P21 protein. The most common activation method of K-ras gene is point mutation, and the mutation sites are almost all concentrated in codons 12 and 13 of exon 2. There are 7 common hot spot mutations, which are the 12th codon of K-ras gene. Six mutation types (GAT, GCT, GTT, GAC, TGT, AGT, CGT) and one mutation type at codon 13 (GAC). [0003] As a molecular switch of EGFR signaling pathway, ...

AI Technical Summary

Problems solved by technology

[0005] At present, the method for K-ras mutation detection is the traditional sanger sequencing method. However, the sequencing method has many disadvantages, including: (1) The detection cycle is long, and it takes 1-2 days from the extraction of sample DNA to the detection report time

(2) The operation process is complicated, and it needs to be operated after PCR amplification, which is easy to cause pollution

(3) The interpretation of sequencing results is complex and requires experienced people to accurately interpret, especially when the sample size is large

(4) The detection sensitivity is not high, and the proportion of false positives is high

(5) The sample size of a single test is very limited

(6) It is unsafe. There are many toxic substances in the experiment process, such as EB and acrylamide, which are harmful to the operator.

(7) The testing cost is relatively high

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