Method for isothermal amplification of nucleic acid target sequence

Abstract

The invention provides a method for isothermal amplification of a nucleic acid target sequence. The method is suitable for the combined reaction of double-stranded DNA, single-stranded DNA and single-stranded RNA, including nickase and strand displacement enzyme, three primers and one probe are adopted during the detection of the double-stranded DNA and the single-stranded DNA, and three primers and one probe or two primers and one probe can be adopted during the detection of the single-stranded RNA. The probe is a molecular beacon, does not degrade in the amplification process, is only used for specifically binding with a target fragment, provides a fluorescence signal and guarantees the specificity of the reaction. According to the invention, a result is judged in real time by adopting the beacon probe which is not overlapped with the primer in the binding area on the target sequence, and beacon probe binding target sequence has very high specificity; meanwhile, a tube is not opened after the reaction, so that false positive is further avoided; the reaction is carried out at a constant temperature, the consumed time is short, the detection can be completed within 8 minutes, and the POCT detection requirements are met better.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and in particular relates to a method for isothermally amplifying a nucleic acid target sequence. Background technique [0002] Polymerase chain reaction (Polymerase chain reaction, PCR) is currently the most widely used nucleic acid amplification detection technology (Nucleic Acid Amplification Test, NAAT). The classic reaction of this technology includes three steps of denaturation, renaturation, and extension. It is a process that requires rapid temperature cycling. It requires a specific thermal cycler for high-precision temperature control and consumes a lot of power. At the same time, the reaction time is long, which cannot meet the requirements of point-of-care detection (POCT). Although there are products that complete the reaction in 15-30 minutes in recent years, these products rely on extremely complicated industrial designs, and the cost is too high. [0003] In order to solve ...

AI Technical Summary

Problems solved by technology

Using methods other than the fluorescence method can easily lead to false positives, and the patent labels the primers, so that the reaction cannot be carried out correctly. At the same time, the non-specific reaction of the labeled primers will bring false positive results

Due to factors such as too long reaction time and unreasonable product analysis, there is no product on the market at present

Images