Primers, probes and kits for detecting egfr and/or k-ras gene mutations

Abstract

The invention discloses a primer and probe for detecting a human epidermal growth factor receptor (EGFR) gene and / or a K-ras gene, a kit containing the primer and the probe and a device for detecting genetic mutation on the basis of a digital PCR platform.The method for detecting the genetic mutation by means of the primer and the probe comprises the steps that the prime and the probe are provided; DNA of a sample to be detected is extracted; a fluorescent PCR reaction system capable of amplifying a mutant gene sequence is prepared; a target probe and an internal reference probe are utilized to be hybridized with amplified products respectively, and fluorescent signals of corresponding fluorescent groups are detected; existence of the genetic mutation is judged and / or the mutation rate is calculated according to the strength and proportion of the fluorescent signals of the target probe and the internal reference probe.According to the method for detecting the genetic mutation, the needed primers and probes are small in number, the optimization procedure is simple, related mutation of EGFR and / or K-ras gene can be qualitatively or quantitatively detected, and the detection sensitivity is high; a DNA sample with low initial amount can also be detected stably.

Description

technical field [0001] The present invention relates to the field of gene mutation detection, in particular to primers and probes for detecting mutations in the human epidermal growth factor receptor (EGFR) gene and / or K-ras gene, a kit comprising the primers and probes, and a A device for detecting gene mutations based on a digital PCR platform. Background technique [0002] According to the estimates of the International Cancer Research Center (IARC), the number of cancer cases will increase at an average annual rate of 3% to 5% in the future. It is estimated that there will be 20 million new cases and 12 million deaths in the world in 2020. In terms of incidence, the incidence of cancer in low- and middle-income countries is much higher than that in developed countries. Therefore, it is emphasized that countries should take necessary precautionary measures. The annual medical expenses for cancer patients in our country are about 80 billion yuan, accounting for about 20%...

AI Technical Summary

Problems solved by technology

[0017] Patent CN 103923973 A discloses a method for detecting EGFR exon 19 deletion mutations based on a digital PCR platform. The principle of probe and primer design is: use peptide nucleic acids (PNA) to lock the wild-type detection site In the region, two pairs of primer pairs and one pair of probes are used to detect the mutant type and the total DNA template that are not locked by PNA respectively, so as to calculate the mutation rate. This patent requires good specificity of PNA and high cost of reaction reagents

[0018] Patent CN 103911427 A discloses a method and kit for detecting EGFR20 (T790M) and EGFR21 (L858R) gene point mutations based on a digital PCR platform. The detection content is very limited, and a pair of probes can detect a type of point mutation, which is far from enough Meet actual testing needs

Use the amplification retardation mutation system (amplification refractory mutation system, ARMS) to perform PCR amplification on the mutated target sequence, and Taqman probes to detect the specific site of the amplified product. This method has a defect: the last base of the ARMS technology primer does not match Does not completely block the amplification of wild-type DNA, so there is a risk of false positives in this method

Clinical samples include cancer tissue, plasma, urine, effusion, etc., the sample size is extremely small, and the sample types are complex. The above methods cannot detect multiple mutation sites of the same gene in one reaction. It is an easy-to-operate and low-cost detection method for detecting multiple mutation sites of the same gene in complex samples and low-input samples

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