Method for detecting tumor gene mutation through combination of sequence specific blocking agent (SSHB) and specific PCR program

Abstract

The invention discloses a method for high-sensitivity detection of low-frequency tumor gene mutation through a combination of an SSHB and a specific PCR amplification program. The detection method comprises the following steps: designing of primers, probes and the SSHB; selection of Buffer and Taq enzyme; and the specific PCR amplification program. According to the invention, the SSHB is designedand is combined with the specific reaction program (wherein each amplification cycle comprises a high annealing temperature and a low annealing temperature); at a high annealing temperature, the SSHBis preferentially and fully combined with a wild type template, and ARMS primers are not combined with the wild type template; at a low annealing temperature, the SSHB and a mutant template are not combined or are slightly combined, and the ARMS primers and the mutant template are fully combined; therefore, amplification of a wild type background can be reduced, meanwhile, the detection efficiencyof the mutant type can be greatly improved (wherein a mutation frequency of 0.1% can be detected); and the method can be applied to detection of tumor gene mutation and can be used for detecting point mutation, insertion mutation, deletion mutation and the like.

Description

technical field [0001] The invention relates to a method for highly sensitive detection of tumor gene low-frequency mutations by using a sequence-specific blocker (SSHB) combined with a specific PCR program, and belongs to the technical field of gene detection. Background technique [0002] The gene mutations of tumor cells are mainly divided into germline mutations and somatic cell mutations. The latter needs to detect low-frequency gene mutations in genetic testing. The main sample types include tissue samples and blood samples. Tissue biopsy for somatic mutations is currently the gold standard for cancer diagnosis and a method for tumor genotyping. Monitoring has certain limitations. Plasma circulating tumor DNA (circulating tumor DNA, ctDNA) is single or double-stranded DNA present in plasma or serum, with a fragment size of about 167 bp. Studies have shown that ctDNA has many cancer-related molecular properties, and ctDNA-based liquid biopsy has high sensitivity and s...

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Problems solved by technology

However, amplification will also occur when the 3′ terminal base of the primer is not paired, and the amplification efficiency of different mutation base changes has a certain degree of randomness (the difference in amplification efficiency between partially and completely paired primers is relatively large, that is, in qPCR The ΔCT value is large; the difference in amplification efficiency between partial and complete paired primers is relatively small, that is, the ΔCT value in qPCR is small, or even 0), and the accuracy of some 1% mutation frequency gene loci in detection applications unable to meet requirements

[0007] Based on Block blocker and ARMS primers to detect mutation sites, there have been corresponding literature or patent reports. Block blocker is a single-stranded nucleic acid sequence that is completely complementary to wild-type template DNA, and its ends are blocked so that it cannot be amplified.

Block blockers cannot effectively distinguish mutant templates from wild-type templates, although there is a difference of one base (mutation site), at conventional primer annealing temperatures, Block blockers mainly bind to wild-type templates, and at the same time It will bind to the mutant template, thereby reducing the blocking efficiency of the wild type and the detection efficiency of the mutant

In a method and kit for detecting gene mutations based on Blocker primers and ARMS primers (patent publication number CN 107419018A), Blocker has been improved in order to improve the blocking efficiency of Blocker primers, so that the Tm value of Blocker primers is higher than the annealing temperature of mutation detection primers. 5-25°C higher, this method greatly enhances Blocker's blocking efficiency for wild type, but at the same time greatly weakens the detection rate of mutant type in low-frequency mutations

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