Patent ID: 8628924

Claim:
A method for quantitating a target nucleic acid sequence in a test sample comprising: (a) contacting the test sample with a nucleic acid amplification reaction mixture comprising at least two primers, wherein the at least two primers hybridize to the same strand of the target nucleic acid sequence but hybridize to distinct nucleotide sequences on the strand, and each of the at least two primers is present in the reaction mixture in a different amount, and wherein the at least two primers comprise a first inner primer and a second outer primer; (b) subjecting the reaction mixture to amplification conditions under which each of the at least two primers simultaneously and independently produces one of at least two amplicons, wherein the number of copies of each amplicon produced differs by at least two orders of magnitude, wherein the at least two amplicons comprise a first amplicon produced from the first inner primer and a second amplicon produced from the second outer primer, and wherein the first amplicon is shorter than the second amplicon; (c) hybridizing the at least two amplicons with at least two probes, wherein one of the at least two probes is specific to a nucleotide sequence common to both the first and second amplicons and another of the at least two probes is specific to a nucleotide sequence contained within the second amplicon but not within the first amplicon, each probe is detectable within a detection range, wherein the sum of the detection ranges for each of the at least two probes is greater than the detection range for each probe, such that the at least two probes together are detectable across a dynamic range of from about 10 3 to 10 7 , and the at least two probes produce distinguishable detection signals; (d) detecting at least one of the at least two probes; and (e) determining the initial amount of target nucleic acid sequence in the test sample.