Calibration method, apparatus and computer program product
Method and system for quantifying target nucleic acids using real-time amplification and internal calibration adjustment. The approach employs a single fixed data point in combination with a single adjustment calibrator amplified on the instrument that is to be calibrated for approximating a complete calibration curve.
1. A method of calibrating a local instrument that quantifies an analyte polynucleotide using results from a real time nucleic acid amplification reaction, the method comprising the steps of:
(a) obtaining an adjustment calibrator comprising a known amount of the analyte polynucleotide and a fixed amount of an internal calibrator;
(b) coamplifying the analyte polynucleotide and the internal calibrator of the adjustment calibrator with the local instrument to determine a normalized indicia of amplification; and
(c) establishing a linear calibration curve that comprises first and second data points, each of the data points being defined by a pair of coordinates specifying an amount of the analyte polynucleotide and a normalized indicia of amplification,
wherein the pair of coordinates of the first data point comprises the known amount of the analyte polynucleotide of the adjustment calibrator and the normalized indicia of amplification from step (b), and
wherein the second data point is a fixed-point determined using an instrument other than the local instrument, thereby calibrating the local instrument, and
wherein the linear calibration curve established in step (c) is used to quantify the analyte polynucleotide present in a test sample that comprises an unknown amount of the analyte polynucleotide.
2. The method of claim 1 , wherein the local instrument comprises a memory, and amplifies nucleic acid and monitors amplicon synthesis as amplification is occurring, and wherein the pair of coordinates of the fixed-point is stored in the memory of the local instrument.
3. The method of claim 2 , wherein step (a) comprises obtaining a kit that comprises the adjustment calibrator and a tangible embodiment of the pair of coordinates of the fixed-point.
4. The method of claim 3 , further comprising the steps of:
obtaining a test sample comprising an unknown quantity of the analyte polynucleotide;
combining the test sample with the fixed amount of the internal calibrator, thereby creating a test reaction mixture;
coamplifying the analyte polynucleotide and the internal calibrator of the test reaction mixture to determine a normalized indicia of amplification; and
comparing the normalized indicia of amplification determined for the test reaction mixture with the linear calibration curve established in step (c) to quantify the analyte polynucleotide in the test sample.
5. The method of claim 2 , wherein the normalized indicia of amplification determined in step (b) results from dividing a threshold-based indicia of amplification determined for the analyte polynucleotide by a threshold-based indicia of amplification determined for the internal calibrator.
6. The method of claim 5 , wherein the linear calibration curve established in step (c) is used to quantify the analyte polynucleotide present in a test sample that comprises an unknown amount of the analyte polynucleotide.
7. The method of claim 5 , further comprising the steps of:
obtaining a test sample comprising an unknown quantity of the analyte polynucleotide;
combining the test sample with the fixed amount of the internal calibrator, thereby creating a test reaction mixture;
coamplifying the analyte polynucleotide and the internal calibrator of the test reaction mixture to determine a normalized indicia of amplification; and
comparing the normalized indicia of amplification determined for the test reaction mixture with the linear calibration curve established in step (c) to quantify the analyte polynucleotide in the test sample.
8. The method of claim 1 , wherein step (a) comprises obtaining a kit that comprises the adjustment calibrator and a tangible embodiment of the pair of coordinates of the fixed-point.
9. The method of claim 8 , wherein the tangible embodiment comprises a machine-readable barcode.
10. The method of claim 1 , further comprising the steps of:
obtaining a test sample comprising an unknown quantity of the analyte polynucleotide;
combining the test sample with the fixed amount of the internal calibrator, thereby creating a test reaction mixture;
coamplifying the analyte polynucleotide and the internal calibrator of the test reaction mixture to determine a normalized indicia of amplification; and
comparing the normalized indicia of amplification determined for the test reaction mixture with the linear calibration curve established in step (c) to quantify the analyte polynucleotide in the test sample.
11. The method of claim 1 , wherein the normalized indicia of amplification determined in step (b) results from dividing a threshold-based indicia of amplification determined for the analyte polynucleotide by a threshold-based indicia of amplification determined for the internal calibrator.
12. The method of claim 1 , wherein step (b) comprises coamplifying in an isothermal nucleic acid amplification reaction.
13. The method of claim 12 , wherein the isothermal nucleic acid amplification reaction is a transcription-associated amplification reaction.
14. The method of claim 1 , wherein the normalized indicia of amplification in step (b) is calculated by dividing a Ttime value determined for the analyte polynucleotide by a Ttime value determined for the internal calibrator.
15. The method of claim 1 , wherein the local instrument comprises a temperature-controlled incubator that maintains a constant temperature.
16. The method of claim 1 , wherein the local instrument comprises a temperature-controlled incubator that undergoes temperature cycling.
17. The method of claim 1 , wherein the local instrument amplifies nucleic acids and monitors amplicon production as a function of either cycle number or time.
18. The method of claim 17 , wherein the local instrument monitors amplicon production by optical monitoring of fluorescence.
19. The method of claim 1 , wherein step (b) comprises coamplifying the analyte polynucleotide and the internal calibrator using independent primer sets that do not comprise any of the same primers.