Patent ID: 9139870
Filing Date: 2015-09-22
Classification: C12P,C12Q

Abstract:
1. A method of quantifying a target nucleic acid sequence in a sample, comprising the steps of: (a) contacting the sample with a first amplification oligonucleotide, specific for a first portion of the target nucleic acid sequence, under conditions allowing hybridization of the first amplification oligonucleotide to the first portion of the target nucleic acid sequence, thereby generating a pre-amplification hybrid that comprises the first amplification oligonucleotide and the target nucleic acid sequence; (b) isolating the pre-amplification hybrid by target capture onto a solid support followed by washing to remove any of the first amplification oligonucleotide that did not hybridize to the first portion of the target nucleic acid sequence in step (a); (c) amplifying, in a first phase amplification reaction mixture, at least a portion of the target nucleic acid sequence of the pre-amplification hybrid isolated in step (b) in a first phase, substantially isothermal, transcription-associated amplification reaction under conditions that support linear amplification thereof, but do not support exponential amplification thereof, thereby resulting in a reaction mixture comprising a first amplification product, (d) combining the reaction mixture comprising the first amplification product with at least one component that participates in exponential amplification of the first amplification product, but that is lacking from the reaction mixture comprising the first amplification product, to produce a second phase amplification reaction mixture, (e) performing, in a second phase, substantially isothermal, transcription-associated amplification reaction in the second phase amplification reaction mixture, an exponential amplification of the first amplification product, thereby synthesizing a second amplification product; (f) detecting, with the sequence-specific hybridization probe at regular time intervals, synthesis of the second amplification product in the second phase amplification reaction mixture; and (g) quantifying the target nucleic acid sequence in the sample using results from step (f).