Nucleic acid reactions and related methods and compositions

A method of detecting the presence or absence of each of a plurality of target nucleic acid analytes in a sample, the method comprising:

• a) adding a primer set, the primer set comprising at least two primer pairs, to the sample, at least one of the primer pairs comprising a limiting primer, and each primer pair defining a region of one of the target nucleic acid analytes to be amplified;
• b) performing an amplification reaction on the sample;
• c) measuring a signal generated during the amplification reaction;
• and
• d) making a determination as to whether each of the plurality of target nucleic acid analytes is present based on the measured signal.

A method of detecting the presence or absence of two or more of a plurality of target nucleic acid analytes in a sample, the method comprising:

• a) adding a primer set comprising first and second primer pairs to the sample, the first primer pair having a first annealing temperature and the second primer pair having a second annealing temperature which is lower than the first annealing temperature, and the first primer pair defining a region of one of the target nucleic acid analytes, the second primer pair defining a region of another of the nucleic acid analytes;
• b) performing a thermocycling amplification reaction on the sample, the reaction comprising a set of temperature cycles comprising at least one temperature cycle having a minimum temperature which is both (i) not greater than the first annealing temperature, and (ii) greater than the second annealing temperature, the reaction further comprising at least one other temperature cycle having a minimum temperature which is not greater than the second annealing temperature;
• c) measuring a signal generated during the amplification reaction;
• and
• d) making a determination as to the presence or absence of the two or more of the nucleic acid analytes based on the generated signal.

A method of detecting a nucleic acid analyte, comprising:

• a. performing a nested asymmetric amplification reaction on a sample, wherein said nested asymmetric amplification reaction comprises:
• at least three nested primers, wherein at least one pair of primers of said at least three nested primer comprises a limiting primer and an excess primer;
• b. measuring a signal generated during said nested asymmetric amplification reaction;
• and
• c. determining whether said analyte is present based on said signal.