Patent ID: 8728765

Claim:
A method of detecting the presence or absence of one or more target polynucleotides in a sample, the method comprising the steps of: amplifying in a reaction chamber of a reaction system one or more target polynucleotides from the sample using first-stage amplification reagents in a first reaction mixture to form one or more first amplicons, the first-stage amplification reagents including initial primers for each target polynucleotide, wherein the one or more first amplicons are amplified in the presence of a fluorescent indicator capable of generating an optical signal related to a quantity of an amplicon in the first-stage amplification reaction, and wherein the reaction chamber is selectively in fluid communication with a waste reservoir; monitoring the optical signal of the fluorescent indicator in the first-stage amplification reaction in the reaction chamber; automatically withholding in the reaction chamber an effective portion of the first reaction mixture and fluidly transferring remainder of the first reaction mixture to the waste reservoir when the optical signal reaches or exceeds a predetermined level; introducing second-stage amplification reagents into the effective portion of the first reaction mixture to produce a second reaction mixture in the reaction chamber; amplifying in the reaction chamber the one or more first amplicons in the second reaction mixture, the second-stage amplification reagents including at least one secondary primer for each of the one or more first amplicons, such that each secondary primer is nested in such first amplicon relative to an initial primer of such first amplicon; and detecting the one or more second amplicons to determine the presence or absence of the one or more target polynucleotides in the sample.