Parametric system for quantifying analyte polynucleotides
View Patent ↗System for quantifying analyte polynucleotides employs computer-implemented analysis of real-time amplification data using a calibration curve defined by parametric equations.
1. A system for quantifying an analyte nucleic acid that may be present in a sample undergoing testing, comprising:
an apparatus that amplifies nucleic acids and performs time-dependent monitoring of amplicon production, said apparatus comprising a temperature-controlled incubator and an optical detector;
a computer operably linked to said apparatus, said computer being programmed to execute the steps of
(a) processing results from a test amplification reaction carried out with a test reaction mixture that comprises an internal calibrator and said sample undergoing testing,
whereby there is generated a test sample data point comprising first and second coordinates respectively determined by time-dependent indicia of amplification for said analyte nucleic acid and said internal calibrator that coamplified in said test amplification reaction;
(b) comparing said test sample data point with a calibration curve to locate a point thereon that comprises first and second coordinates, and that is minimally distant from said test sample data point in a measurement plane that comprises said calibration curve and said test sample data point,
wherein said calibration curve comprises
(i) first dimension coordinates comprising solutions to a first optimized parametric equation that specifies time-dependent indicia of amplification for an analyte polynucleotide standard as a function of the parameter representing the known starting quantity of said analyte polynucleotide standard in each of a plurality of calibration reactions that comprise said internal calibrator and known starting quantities of said analyte polynucleotide standard, and
(ii) second dimension coordinates comprising solutions to a second optimized parametric equation that specifies time-dependent indicia of amplification for said internal calibrator as a function of the parameter representing the known starting quantity of said analyte polynucleotide standard in each of said plurality of calibration reactions, and
(c) determining a value for the parameter representing the known starting quantity of said analyte polynucleotide standard that results in said point located on said calibration curve, whereby said value estimates the quantity of said analyte nucleic acid in said sample undergoing testing.
2. The system of claim 1 , wherein said computer controls operation of said apparatus that amplifies nucleic acids and performs time-dependent monitoring of amplicon production.
3. The system of claim 1 , wherein said computer is selected from the group consisting of a freestanding computer, and a computer that is an integral component of said apparatus.
4. The system of claim 1 , wherein said temperature-controlled incubator is configured to maintain a constant temperature during performance of nucleic acid amplification reactions.
5. The system of claim 1 , wherein said temperature-controlled incubator is configured for temperature cycling during performance of nucleic acid amplification reactions.
6. The system of claim 1 , wherein said optical detector detects fluorescent signals from a plurality of fluorescent labels.
7. The system of claim 1 , wherein said temperature-controlled incubator is configured to hold a plurality of reaction tubes.
8. The system of claim 1 , wherein said temperature-controlled incubator is configured to hold a multiwell plate.
9. The system of claim 1 , wherein said computer receives an output of said optical detector for data storage and manipulation.
10. The system of claim 1 , wherein each of said first and second optimized parametric equations respectively comprises a plurality of optimized coefficients.
11. The system of claim 1 , further comprising an output device controlled by said computer.
12. The system of claim 11 , wherein said computer controls operation of said apparatus that amplifies nucleic acids and performs time-dependent monitoring of amplicon production.
13. The system of claim 12 , wherein said computer is selected from the group consisting of a freestanding computer, and a computer that is an integral component of said apparatus.
14. The system of claim 12 , wherein said temperature-controlled incubator is configured to maintain a constant temperature during performance of nucleic acid amplification reactions.
15. The system of claim 12 , wherein said temperature-controlled incubator is configured for temperature cycling during performance of nucleic acid amplification reactions.
16. The system of claim 12 , wherein said temperature-controlled incubator is configured to hold a plurality of reaction tubes.
17. The system of claim 12 , wherein said temperature-controlled incubator is configured to hold a multiwell plate.
18. The system of claim 11 , wherein said output device comprises a video monitor.
19. The system of claim 11 , wherein said output device displays a result generated by said computer.
20. The system of claim 19 , wherein said result comprises either an amount of said analyte nucleic acid or a concentration of said analyte nucleic acid.
21. The system of claim 11 , wherein said computer is selected from the group consisting of a freestanding computer, and a computer that is an integral component of said apparatus.