Patent ID: 8435742

Claim:
A method for detecting or quantifying a target RNA, comprising the following steps (a) to (j): (a) a step of fixing a 5′ end of a primer comprising a DNA sequence corresponding to a 5′-side target specific sequence of the target RNA on a surface of a substrate to prepare a solid-phase DNA (+) primer; (b) a step of preparing a liquid-phase cDNA (−) primer wherein an RNA-polymerase promoter sequence is added via a tag sequence to a 5′-end side of a primer comprising a cDNA sequence which is complementary to a 3′-side sequence of the target RNA, and a liquid-phase universal primer wherein an RNA-polymerase promoter sequence is added to a 5′ end of a tag sequence; (c) a step of preparing a sample RNA comprising a 3′-side sequence and 5′-side target specific sequence of the target RNA; (d) a step of allowing the liquid-phase cDNA (−) primer prepared in step (b) to contact with the sample RNA strand prepared in step (c) in a liquid phase to hybridize the liquid-phase cDNA (−) primer and the sample RNA, and then extending a DNA (−) strand by a reverse transcriptase to prepare a cDNA strand-RNA strand complex; (e) a step of allowing an RNase that specifically degrades an RNA strand in a DNA strand-RNA strand complex, to act on the cDNA strand-RNA strand complex prepared in step (d) to prepare a single-stranded DNA (−); (f) a step of allowing the single-stranded DNA (−) prepared in step (e) to contact in a liquid phase, with the solid-phase DNA (+) primer prepared in step (a) to hybridize the single-stranded DNA (−) and the solid-phase DNA (+) primer, and then extending a DNA (+) strand by an enzyme having a DNA-dependent DNA-polymerase activity to prepare a double-stranded DNA; (g) a step of allowing an RNA polymerase to act on the double-stranded DNA prepared in step (f) to amplify a single-stranded RNA (−) utilizing an RNA-polymerase promoter sequence derived from the DNA (−) strand, hybridizing an amplified single-stranded RNA (−) and a solid-phase DNA (+) primer, and then extending a DNA (+) strand by a reverse transcriptase to prepare a cDNA strand-RNA strand complex; (h) a step of allowing an RNase that specifically degrades an RNA strand in a DNA strand-RNA strand complex, to act on the cDNA strand-RNA strand complex prepared in step (g) to prepare a solid-phase single-stranded DNA (+); (i) a step of allowing the solid-phase single-stranded DNA (+) prepared in step (h) to contact with the liquid-phase universal primer prepared in step (b) in a liquid phase to hybridize the single-stranded DNA (+) and the liquid-phase universal primer, and then extending a DNA (−) strand by an enzyme having a DNA-dependent DNA-polymerase activity to prepare a double-stranded DNA; and (j) a step of quantifying the double-stranded DNAs prepared in step (f) and step (i).