Normal-temperature and constant-temperature nucleic acid amplification method using fluorescence probe

Abstract

The invention relates to a normal-temperature and constant-temperature nucleic acid amplification method using a fluorescence probe. The normal-temperature and constant-temperature nucleic acid amplification method includes: under constant temperature, single-strand binding protein partially opens the double-strand parental strand into a single strand; recombinant enzyme is combined with primer to form complex, the complex is combined to the parental strand under the action of auxiliary protein, and the fluorescence probe is combined with a complementation area; DNA polymerase is combined to the 3' tail end of the primer to perform sub-strand extension; exonuclease identifies the tetrahydrofuran locus on the fluorescence probe under a double-strand state, fluorescent groups and quenching groups are separated after enzyme digestion to release fluorescence; after the fluorescence probe is cut off, the 3'-OH end originally sealed by probe 3' tail end modification is exposed, and DNA polymerase can continuously extend to form a sub-strand; qualitative and semi-quantitative detection can be performed on a to-be-detected sample by detecting the shape of an amplification curve and the strength of a fluorescence signal. The nucleic acid amplification method has the advantages that normal-temperature and constant-temperature detection can be performed, and qualitative and semi-quantitative detection can be performed on nucleic acid to-be-detected objects.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a normal temperature isothermal nucleic acid amplification method of a fluorescent probe method, which is to perform rapid, real-time and accurate detection of nucleic acid targets under normal temperature conditions, and can be applied to many fields related to biological detection , such as clinical medicine, disease prevention and control, or food safety. Background technique [0002] In medicine, especially in the field of disease prevention and control, the rapid molecular detection of pathogens (including bacteria, viruses and parasites) by traditional nucleic acid in vitro amplification technology and polymerase chain reaction (PCR) has also experienced a long period of time. And after the process of continuous technological innovation, it has been highly recognized in the field of medical molecular diagnosis. The initial post-amplification agarose gel electrophoresis detec...

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Problems solved by technology

[0005] The purpose of the present invention is to provide a fluorescent probe method at room temperature isothermal nucleic acid amplification method, to overcome the current fluorescent PCR relies heavily on precision instruments, and nucleic acid isothermal amplification technology can not achieve complete isothermal and PCR product quantification is not accurate enough

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