Patent ID: 7831417

Claim:
A method of preparing a parametric calibration curve for quantifying an analyte polynucleotide contained in a test sample, comprising the steps of: forming a plurality of standard samples, each containing a constant quantity of a nucleic acid calibrator and a known starting quantity of an analyte polynucleotide standard; coamplifying the nucleic acid calibrator and the analyte polynucleotide standard in an in vitro nucleic acid amplification reaction for each of the plurality of standard samples; determining indicia of amplification for the nucleic acid calibrator and the analyte polynucleotide standard that coamplified in each in vitro nucleic acid amplification reaction of the coamplifying step, whereby there is obtained a collection of determined indicia of amplification for the nucleic acid calibrator as a function of the known starting quantity of the analyte polynucleotide standard, and a collection of determined indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard; optimizing a first parametric equation to fit a first curve to the collection of determined indicia of amplification for the nucleic acid calibrator as a function of the known starting quantity of the analyte polynucleotide standard, thereby resulting in a first fitted equation; optimizing a second parametric equation to fit a second curve to the collection of determined indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard, thereby resulting in a second fitted equation; solving the first and second fitted equations at incremental values of the known starting quantity of the analyte polynucleotide standard to result in fitted indicia of amplification for the nucleic acid calibrator as a function of the known starting quantity of the analyte polynucleotide standard, and fitted indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard; and preparing a three-dimensional parametric calibration curve that comprises (a) fitted indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard in a first dimension, (b) fitted indicia of amplification for the nucleic acid calibrator as a function of the known starting quantity of the analyte polynucleotide standard in a second dimension, and (c) a function of the known starting quantity of the analyte polynucleotide standard in a third dimension.