Patent ID: 6541205
Filing Date: 2003-04-01
Classification: C12Q

Abstract:
A method for determining a concentration of a target RNA in a sample consisting of the steps:providing an RNA amplification system in which: (a) a double-stranded DNA is produced using, as a template, a target RNA containing a specific nucleotide sequence in a sample, said double-stranded DNA has a promoter sequence and transcribes an RNA comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence; (b) an RNA transcription product comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence is produced in the presence of an RNA polymerase; and (c) the double-stranded DNA is produced using the RNA transcription product as a template, â€ƒwherein said steps (a), (b) and (c) are carried out in the presence of a probe labeled with an intercalating fluorochrome having a sequence complementary to the RNA transcription product, measuring the fluorescence intensity in the RNA amplification system with time; calculating a time when the fluorescence intensity satisfies a prescribed criterion based on the measured change in the fluorescence intensity with time; and determining a concentration of the target RNA in the sample based on the calculated time, wherein all steps of: production of a double-stranded DNA using a target RNA; production of an RNA transcription product from the double-stranded DNA; measurement of the RNA transcription product using a probe; and production of the above double-stranded using the RNA transcription product; are carried out continuously.