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US 7,680,604 B2Granted
PCR elbow determination by rotational transform after zero slope alignment
AssigneeRoche Molecular Systems, Inc.
InventorRonald T. Kurnik
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AssigneeRoche Molecular Systems, Inc.
InventorRonald T. Kurnik
Estimating the term…
Systems and methods for determining the elbow or Ct value in a real-time, or kinetic, PCR amplification curve data set. A PCR data set may be visualized in a two-dimensional plot of fluorescence intensity vs. cycle number. The data set may be adjusted to have a zero slope. In one aspect, a data set is fit to a double sigmoid curve function with the function parameters determined using a Levenberg-Marquardt regression process. The determined parameters are used to subtract off the linear growth portion from the data set to provide a modified data set. For multiple data sets, all the data curves can be aligned in this manner to have a common baseline slope, e.g., a slope of zero. A rotation transform is applied to a modified data set to rotate the data about a defined coordinate such as the origin so that the data point representing the Ct value becomes a minimum or a maximum along the intensity axis. The data point representing the elbow or Ct value of the curve is identified, and this data point is then rotated back and the cycle number of the data point is returned or displayed.
A computer-implemented method of determining the elbow or cycle threshold (Ct) value in a region of a Polymerase Chain Reaction (PCR) data curve, the method comprising the steps all of which are, implemented in a computer system having a processor, of:
A computer-implemented method of determining the cycle threshold (Ct) values for a plurality of the Polymerase Chain Reaction (PCR) curves, the method comprising the steps all of which are, implemented in a computer system having a processor, of:
A tangible computer-readable medium that stores code for controlling a processor to determine a cycle threshold (Ct) value in a kinetic Polymerase Chain Reaction (PCR) amplification curve, the code including instructions to:
A kinetic Polymerase Chain Reaction (PCR) system, comprising:
A Polymerase Chain Reaction (PCR) system, comprising:
Dependencies are inferred from the claim text.
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ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression: polymerase chain reaction [PCR]; Primer or probe…
Digital computing or data processing equipment or methods, specially adapted for specific functions: complex mathematical operations
Complex mathematical operations: function evaluation by approximation methods, e.g. inter- or extrapolation, smoothing, least mean square method
Ict specially adapted for hybridisation
About 10 months of term left · ends 29 Aug 2027 (estimated)
The present invention relates generally to systems and methods for processing data representing sigmoid or growth curves, and more particularly to systems and methods for determining characteristic cycle threshold (Ct) or elbow values in PCR amplification curves.
The Polymerase Chain Reaction (PCR) is an in vitro method for enzymatically synthesizing or amplifying defined nucleic acid sequences. The reaction typically uses two oligonucleotide primers that hybridize to opposite strands and flank a template or target DNA sequence that is to be amplified. Elongation of the primers is catalyzed by a heat-stable DNA polymerase. A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in an exponential accumulation of a specific DNA fragment. Fluorescent probes or markers are typically used in the process to facilitate detection and quantification of the amplification process.
A typical real-time PCR curve is shown in FIG. 1, where fluorescence intensity values are plotted vs. cycle number for a typical PCR process. In this case, the formation of PCR products is monitored in each cycle of the PCR process. The amplification is usually measured in thermocyclers which include components and devices for measuring fluorescence signals during the amplification reaction. An example of such a thermocycler is the Roche Diagnostics LightCycler (Cat. No. 20110468). The amplification products are, for example, detected by means of fluorescent labelled hybridization probes which only emit fluorescence signals when they are bound to the target nucleic acid or in certain cases also by means of fluorescent dyes that bind to double-stranded DNA.
FIG. 1 illustrates an example of a typical PCR growth curve, plotted as fluorescence intensity vs. cycle number.
FIG. 2 shows the PCR growth curve data of FIG. 1 rotated by 25 degrees clockwise.
FIG. 3 shows a superposition of FIGS. 1 and 2.
One example of an amplification curve 10 in the context of a PCR process is shown in FIG. 1. As shown, the curve 10 includes a lag phase region 15, and an exponential phase region 25. Lag phase region 15 is commonly referred to as the baseline or baseline region. Such a curve 10 includes a transitionary region of interest 20 linking the lag phase and the exponential phase regions. Region 20 is commonly referred to as the elbow or elbow region. The elbow region typically defines an end to the baseline and a transition in the growth or amplification rate of the underlying process. Identifying a specific transition point in region 20 can be useful for analyzing the behavior of the underlying process. In a typical PCR curve, identifying a transition point referred to as the elbow value or cycle threshold (Ct) value is extremely useful for understanding efficiency characteristics of the PCR process. Other processes that may provide similar sigmoid or growth curves include bacterial processes, enzymatic processes and binding processes. In bacterial growth curves, for example, the transition point of interest has been referred to as the time in lag phase, θ. Thus, although the remainder of this document will discuss the invention in terms of its applicability to PCR curves, it should be appreciated that the present invention may be applied to data curves related to these other processes.
As shown in FIG. 1, data for a typical PCR growth curve can be represented in a two-dimensional coordinate system, for example, with PCR cycle number defining the x-axis and an indicator of accumulated polynucleotide growth defining the y-axis. Typically, as shown in FIG. 1, the indicator of accumulated growth is a fluorescence intensity value as the use of fluorescent markers is perhaps the most widely used labeling scheme. However, it should be understood that other indicators may be used depending on the particular labeling and/or detection scheme used. Examples of other useful indicators of accumulated signal growth include luminescence intensity, chemiluminescence intensity, bioluminescence intensity, phosphorescence intensity, charge transfer, voltage, current, power, energy, temperature, viscosity, light scatter, radioactive intensity, reflectivity, transmittance and absorbance. The definition of cycle can also include time, process cycles, unit operation cycles and reproductive cycles.
According to the present invention, one embodiment of a process for determining a transitionary value in a single sigmoid curve, such as the elbow value or Ct value of a kinetic PCR amplification curve, can be described briefly as follows. In a first operation, an experimental data set representing the amplification curve (e.g., fluorescence curve in FIG. 1) is acquired. The data set is rotated by an angle theta (θ) so that the rotated data set forms a minimum in the region of the elbow. As an example, FIG. 2 shows the resulting data from FIG. 1 after rotation by 25 degrees clockwise relative to the origin coordinate. FIG. 3 shows a superposition of FIGS. 1 and 2. It is apparent from FIGS. 2 and 3 that by rotating the amplification curve in this manner, the search for the elbow becomes a much simpler task of identifying the location of the minimum in the rotated curve. Any data noise in the region prior to the elbow would have little influence, as the relevant parameter is the minimum in the curve.