Patent ID: 6911327

Claim:
A method for determining an unknown starting quantity of a target nucleic acid sequence in a test sample, the method comprising the steps of: a) amplifying the unknown starting quantity of the target nucleic acid sequence in the test sample; b) amplifying a plurality of known starting quantities of a calibration nucleic acid sequence in respective calibration samples; c) determining a respective threshold value for each of the known starting quantities of the calibration nucleic acid sequence in the calibration samples and for the target nucleic acid sequence in the test sample, wherein the threshold value is determined for each nucleic acid sequence in a respective sample by; i) measuring, at a plurality of different times during amplification, at least one signal whose intensity is related to the quantity of the nucleic acid sequence being amplified in the sample; ii) storing signal values defining a growth curve for the nucleic acid sequence; iii) calculating a derivative of the growth curve; iv) identifying a positive peak of the derivative; v) determining if the positive peak exceeds a noise-based threshold level; and vi) if the positive peak exceeds the threshold level, then calculating the threshold value corresponding to the positive peak of the derivative; d) deriving a calibration curve from the threshold values determined for the known starting quantities of the calibration nucleic acid sequence in the calibration samples; and e) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the target sequence.