Patent ID: 7252937
Filing Date: 2007-08-07
Classification: C12Q

Abstract:
1. A method of determining an amount of a target nucleic acid in a sample, said method comprising the steps of: a) providing said sample; b) contacting an aliquot of said sample with a solid support, wherein nucleic acid in said aliquot binds to said solid support; c) eluting said nucleic acid bound to said solid support with at least two sequential volumes of 5-25 μl to produce a concentrated nucleic acid sample; d) contacting said concentrated nucleic acid sample of step (c) with oligonucleotide primers capable of amplifying said target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and e) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles followed by a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore, wherein the kinetics of an increase in intensity of fluorescence from said acceptor fluorophore are indicative of the amount of said target nucleic acid in said sample.