Reagent for nucleic acid real-time isothermal amplification, amplification system and amplification method and application of amplification system

Abstract

The invention relates to a reagent for nucleic acid isothermal amplification. The reagent comprises RNA polymerase, DNA polymerase with reverse transcription activity and RNase H, wherein the DNA polymerase with reverse transcription activity has 5'-3' DNA polymerization activity and reverse transcription activity and 5'-3' exonuclease activity. The reagent further comprises primers and a fluorescence probe, wherein the primers comprise the primer with an RNA promoter recognition sequence and the second cDNA synthesis primer, and the fluorescent probe is selected from a TaqMan probe and a Molecular Beacon probe. The invention further relates to a nucleic acid isothermal amplification system comprising the reagent and an amplification method thereof, and a kit comprising the nucleic acid isothermal amplification system and application thereof. By using the RNA polymerase, the DNA polymerase with reverse transcription activity and the RNase H, controllable RNA and DNA isothermal amplification is realized; the isothermal amplification is combined with the fluorescent probe technology, which can realize real-time fluorescence detection in the target RNA amplification process; besides,the whole process is completed in the same reaction system, and quantitative detection of nucleic acid is realized. The reagent, the amplification system and the kit are suitable for clinical promotion and application.

Description

technical field [0001] The present invention relates to the field of biochemistry and molecular biology, in particular to specific nucleic acid amplification detection technology, in particular to a reagent, amplification system, amplification method and application for real-time constant temperature amplification of nucleic acid. Background technique [0002] Since the birth of Polymerase Chain Reaction (PCR) technology in 1985, it has greatly promoted the development of nucleic acid detection technology. With the maturity of the enzyme process, the fluorescent quantitative PCR technology has gradually become dominant in the field of nucleic acid detection with the emergence of fluorescent dyes. When fluorescent PCR technology detects nucleic acid, it needs to go through repeated (about 40 times) temperature change process "95 degrees" to "55-60 degrees" to achieve exponential amplification, and the amplification amount is 2n times. In addition to fluorescent PCR technolog...

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Problems solved by technology

However, there is no literature report on the real-time fluorescent constant temperature amplification technology using DNA polymerase with reverse transcription function instead of reverse transcriptase AMV or MMLV

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