METHOD FOR DETECTING NUCLEIC ACID USING ASYMMETRIC ISOTHERMAL AMPLIFICATION OF NUCLEIC ACID AND SIGNAL PROBE

摘要

The present invention relates to a method for accurately detecting a target nucleic acid by asymmetrically and isothermally amplifying the target nucleic acid using an external primer set, an internal primer set having different percentages of forward and reverse DNA-RAN-DNA hybrid primers, and a DNA-RNA-DNA hybrid signal probe and amplifying a signal of the probe at the same time. According to the present invention, the signal of the probe can be efficiently amplified compared with the method of the prior art, a symmetric iTPA method, which is an isothermal primer and probe amplification method using the same percentage of primers. Therefore, the present invention is applied to the accurate detection and confirmation of pathogens, detection of gene modification inducing identified phenotypes, diagnosis of susceptibility to genetic diseases, evaluation of gene expression, and various genome projects, and thus is useful in the molecular biological researches and disease diagnosis.

主权项

1. A method for isothermal detection of target DNA, comprising:
(i) preparing target DNA; (ii) preparing an external primer set having a base sequence complementary to the target DNA; (iii) preparing a DNA-RNA-DNA hybrid primer set having a base sequence complementary to the target DNA at the 3′-terminal end and non-complementary to the target DNA at the 5′-terminal end; (iv) preparing a DNA-RNA-DNA hybrid primer set having an excess amount either of the forward or reverse DNA-RNA-DNA hybrid primers uses to induce asymmetric amplification; and (v) adding an enzymatic reaction mixture solution containing RNase, DNA polymerase capable of performing strand displacement and a DNA-RNA-DNA hybrid signal probe having a base sequence complementary to the amplification product produced by said external primer set and said hybrid primer set, and then simultaneously amplifying said target DNA and said signal probe and detecting said target DNA at isothermal temperature.