Real-time amplification and monitoring system incorporating vector-based algorithm for analyzing polynucleotide-containing samples
View Patent ↗System for analyzing a polynucleotide-containing sample using real-time amplification and monitoring. The system includes a computer or processing device with software for performing a vector analysis of growth curves. The vector analysis advantageously simplifies polynucleotide quantitation by circumventing the need to establish thresholds used for calculating initiation of the growth phase, or to calculate derivatives.
1. A system for analyzing a polynucleotide-containing sample by time-dependent monitoring of an in vitro nucleic acid amplification reaction, said system comprising:
an apparatus comprising a temperature-controlled incubator and a fluorescence monitoring device, said apparatus generating a data set comprising values proportional to the amount of analyte amplicon present in the in vitro nucleic acid amplification reaction as a function of time, said data set defining a growth curve;
a processing device that analyzes the data set defining the growth curve, said processing device comprising software instructions to perform a vector analysis of at least a portion of said growth curve to identify a time-dependent feature indicating a transition from the baseline phase into the growth phase of said growth curve, said vector analysis comprising the steps of,
(a) establishing a plurality of pairs of first and second vectors at different points on a time dimension of the growth curve,
wherein each vector of a single pair of vectors among said plurality has the same origin, and
wherein the head of the first vector and the head of the second vector of said single pair of vectors among said plurality are each positioned at different points on the growth curve,
(b) calculating for each pair of vectors the value of an angle therebetween, thereby resulting in a plurality of calculated angles, with each point on the time dimension of the growth curve being associated with a different one of said plurality of calculated angles,
(c) comparing the plurality of calculated angles with each other to identify a characteristic angle, and
(d) identifying said time-dependent feature of said growth curve as the point on the time dimension of the growth curve that is associated with the characteristic angle, said characteristic angle being either the maximum angle among the plurality of calculated angles or the minimum angle among the plurality of calculated angles; and
an output device in communication with the processing device.
2. The system of claim 1 , wherein the fluorescence monitoring device comprises a multiplexed fluorimeter.
3. The system of claim 1 , wherein the temperature-controlled incubator is a thermal cycling incubator.
4. The system of claim 1 , wherein said temperature-controlled incubator maintains constant temperature during the in vitro nucleic acid amplification reaction, said in vitro nucleic acid amplification reaction being an isothermal in vitro nucleic acid amplification reaction.
5. The system of claim 1 , wherein the temperature-controlled incubator is configured to hold a multiwell plate.
6. The system of claim 1 , wherein said processing device is an integral component of said apparatus.
7. The system of claim 1 , wherein said processing device is selected from the group consisting of a computer linked to said apparatus, and a computer that is an integral part of said apparatus.
8. The system of claim 7 , wherein the temperature-controlled incubator of said apparatus is a thermal cycling incubator.
9. The system of claim 1 , wherein said output device comprises a video monitor.
10. The system of claim 1 , wherein said processing device further comprises software instructions that direct said output device to display a quantitative value selected from the group consisting of a TArc value, an O-TArc value, a concentration of analyte polynucleotide, and an amount of analyte polynucleotide.
11. The system of claim 1 , wherein said processing device further comprises software instructions to perform the step of,
creating a calibration curve that correlates the time-dependent feature identified in step (d) and a starting amount of analyte polynucleotide present in the polynucleotide-containing sample.
12. The system of claim 1 , wherein said processing device further comprises software instructions to perform the steps of:
comparing the time-dependent feature identified in step (d) with a standard calibration curve; and
determining from said time-dependent feature of said growth curve and said standard calibration curve the amount of analyte polynucleotide present in the polynucleotide-containing sample.