Universal method to determine real-time pcr cycle threshold values
AssigneeRoche Molecular Systems, Inc.
InventorRonald T. KURNIK
Life of this patent
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Abstract
A single technique for determining Ct is provided that can be used for standard sigmoidal growth curves and for problematic growth curves, such as parabolic curves. The Ct value can be determined as the intersection of a line tangent to the growth curve at the maximum of the second derivative with a baseline of the growth curve. Such a Ct value is usable for sigmoidal curves and parabolic curves, and can provide linear calibration curves to achieve accuracy in determining initial concentrations of a sample.
Claims20
A method of detecting a target analyte in a biological sample, the method comprising:
- detecting signal strengths of signals generated in a plurality of growth cycles of a polymerase chain reaction (PCR) growth process for the biological sample in a PCR system;
- generating a dataset representing a growth curve for the PCR growth process based on the detected signal strengths, the dataset including a plurality of data points, each data point having a pair of coordinate values that includes a cycle number and a signal strength of the PCR growth process at the cycle number, the growth curve including a linear baseline and a nonlinear region after the baseline;
- creating a function that approximates the dataset by adjusting parameters of the function to fit the dataset;
- determining the baseline of the growth curve;
- identifying a first point of the function where a maximum in a second derivative of the function occurs;
- determining a tangent line that is tangent to the function at the first point;
- identifying an intersection point of the tangent line and the baseline, a cycle number of the intersection point representing a cycle threshold value Ct of the PCR growth process for the biological sample;
- and
- determining a quantity of the target analyte in the biological sample based on the cycle threshold value Ct.
A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that, when executed, control a computer system to detecting a target analyte in a biological sample, the instructions comprising:
- obtaining a dataset representing a growth curve measured from a polymerase chain reaction (PCR) growth process for the biological sample in a PCR system, the dataset including a plurality of data points, each data point having a pair of coordinate values that includes a cycle number and a signal strength of the PCR growth process at the cycle number, the growth curve including a linear baseline and a nonlinear region after the baseline;
- creating a function that approximates the dataset by adjusting parameters of the function to fit the dataset;
- determining the baseline of the growth curve;
- identifying a first point of the function where a maximum in a second derivative of the function occurs;
- determining a tangent line that is tangent to the function at the first point;
- identifying an intersection point of the tangent line and the baseline, a cycle number of the intersection point representing a cycle threshold value Ct of the PCR growth process for the biological sample;
- and
- determining a quantity of the target analyte in the biological sample based on the cycle threshold value Ct.
A Polymerase Chain Reaction (PCR) system, comprising:
- a PCR data acquiring device configured to obtain a dataset representing a growth curve of PCR amplification for a biological sample, the dataset including a plurality of data points, each data point having a pair of coordinate values that includes a cycle number and a signal strength of the PCR amplification at the cycle number, wherein the growth curve includes a linear baseline and a nonlinear region after the baseline;
- and
- a computer system configured to process the dataset to detect a target analyte in the biological sample by:
- receiving the dataset representing the growth curve;
- creating a function that approximates the dataset by adjusting parameters of the function to fit the dataset;
- determining the baseline of the growth curve;
- identifying a first point of the function where a maximum in a second derivative of the function occurs;
- determining a tangent line that is tangent to the function at the first point;
- identifying an intersection point of the tangent line and the baseline, a cycle number of the intersection point representing a cycle threshold value Ct of the growth curve of the PCR amplification for the biological sample;
- and
- determining a quantity of the target analyte in the biological sample based on the cycle threshold value Ct.
Dependencies are inferred from the claim text.