Apparatus for quantitative analysis of a nucleic acid amplification reaction

An apparatus for determining an unknown starting quantity of a target nucleic acid sequence in a test sample, the apparatus comprising:

• a) at least one detection mechanism for measuring:
• i) signals indicative of the respective quantities of the target nucleic acid sequence and of a first internal control being amplified in a first nucleic acid amplification reaction, wherein the first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence;
• ii) signals indicative of the respective quantities of a first standard and of a second internal control being amplified in a second nucleic acid amplification reaction, wherein the first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and wherein the second internal control comprises the second nucleic acid sequence;
• iii) signals indicative of the respective quantities of at least a second standard and of a third internal control being amplified in a third nucleic acid amplification reaction, wherein the second standard comprises a second known starting quantity of the calibration nucleic acid sequence, the third internal control comprises the second nucleic acid sequence, and the starting quantity of the second nucleic acid sequence is substantially equal in each of the amplification reactions;
• b) at least one controller in communication with the detection mechanism, wherein the controller is programmed to perform the steps of:
• i) determining from the measured signals respective threshold values for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample;
• ii) normalizing the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control;
• iii) normalizing the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively;
• iv) deriving a calibration curve from the known starting quantities and the normalized threshold values of the standards;
• and
• v) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target nucleic acid sequence.