A super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity

Abstract

The invention discloses a super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity. The invention adopts the technology of specific primers and probes, and can realize rapid detection of gene point mutations in blood samples. The advantages of the present invention include: (1) On the basis of conventional ARMS primers, LNA modification is added or additional mismatched bases are introduced at the last two to three positions of the 3', which does not affect the sensitivity of mutation detection and ensures the specificity of mutations (2) Introduce a modified blocking probe that is completely complementary to the wild type, and further reduce the background interference of the wild type on the basis of taking into account the amplification efficiency of the mutant type; (3) After introducing a highly specific On the basis of non-modified blocking probes, ARMS primers can detect different base mutations at the same site at the same time, so as to realize the merger detection of multiple sites in the same gene with only a few systems; (4) high sensitivity; (5) The detection speed is fast.

Description

technical field [0001] The invention relates to the field of clinical diagnostic molecular biology, in particular to a super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity. Background technique [0002] With the continuous deepening of people's understanding of the process of tumorigenesis, it is currently believed that the occurrence of cancer is related to gene mutations, leading to uncontrolled proliferation of tumor cells. The abnormality can serve as a targeted reminder. Chemotherapy is currently one of the most important methods for clinical treatment of malignant tumors. However, with its widespread clinical use, the problem of drug resistance has become increasingly prominent. Tumor cells often develop resistance to chemotherapeutic drugs, resulting in patients no longer responding to treatment. Sensitive, which eventually leads to chemotherapy failure or even disease recurrence, in which EGFR gene T790M mutation acco...

AI Technical Summary

Problems solved by technology

Among them, direct DNA sequencing is the gold standard for mutation detection, but this method has the following disadvantages: the detection sensitivity is not high enough, and if the content of the mutant gene accounts for less than 10% of the total genomic DNA, the mutation cannot be detected by direct sequencing The existence of samples; and the operation process is complicated, the detection time is long, and the requirements for operators are high; the non-closed tube operation involves the operation after PCR amplification, so it is easy to be contaminated, and the false negative rate is high, resulting in unsatisfactory results ; The interpretation of sequencing results is highly subjective; the number of samples tested in one experiment is limited, at most 8-24 cases

Therefore, direct sequencing is difficult to be widely used in clinical practice.

The ARMS-PCR method can achieve a sensitivity of 1%, which can meet the detection requirements for tumor tissue samples, but for blood samples that are convenient to sample, such as circulating tumor cells in plasma or blood, the tumor DNA content is often lower than 1%, ARMS-PCR The method is not enough to test the blood, which limits the judgment of clinicians on the efficacy of targeted drugs

In addition, the drug resistance mutations produced in the course of clinical drug use cannot be detected by previous methods due to insufficient sensitivity

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