Cascading isothermal amplification based miRNA fluorescence detection kit and miRNA fluorescence detection method

Abstract

The invention discloses a cascading isothermal amplification based miRNA fluorescence detection kit and a miRNA fluorescence detection method. The target miRNA can specifically recognize and form a stable three-way crossing structure with a ternary probe through complementary hybridization of nucleic acids; a ternary primer in the three-way crossing structure performs strand displacement amplification (SDA) along a ternary template and generates a number of SDA template products; the SDA template product can open a difunctional hairpin probe to trigger nickase to perform signal amplification reaction finally; and a cyclophorase digestion molecular beacon can generate cascading amplified fluorescence signals. The kit can detect miRNA low to 100fM; and the detection method has superhigh specificity, can well distinguish highly homologous sequences at different mispairing positions and solves the difficult problem of false positive result caused by poor specificity in the existing detection method.

Description

technical field [0001] The invention belongs to the technical field of miRNA detection, and relates to a miRNA fluorescence detection method and a kit based on cascade constant temperature amplification. Background technique [0002] MiRNA is a class of endogenous non-protein-coding single-stranded RNA molecules with a length of about 18-25 bases and widely exists in eukaryotes. MiRNA originates from the transcription of intracellular miRNA gene, and the whole process is synchronized with the transcription of other genes, and the transcription product is pri-miRNA consisting of hundreds of nucleotides. Pri-miRNA undergoes enzyme cleavage in the nucleus, and finally forms a precursor miRNA (pre-miRNA, about 70 bases) with a hairpin structure. Afterwards, pre-miRNAs enter the cytoplasm and are processed into mature miRNAs by Dicer enzymes. The mature miRNA can interact with the non-coding region of messenger RNA (mRNA) to form a gene silencing complex (RNA-induced silencing ...

AI Technical Summary

Problems solved by technology

[0004] The problem solved by the present invention is to provide a miRNA fluorescence detection kit and method based on cascade constant temperature amplification, so as to overcome the shortcomings of the prior art such as low selectivity, complicated design, and long time consumption.

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