Using serial dilutions of reference samples to construct a reference table for sigmoidal fitting in real-time PCR copy number analysis
Inventors
Wong, Jr. Winston • Kao, Stephen Chang-Chi • Lai, Ying-Ta • Shann, Yih-Jyh • Chen, Ming-Fa • Chen, Chih-Rong
Assignees
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Publication Number
US-10510436-B2
Publication Date
2019-12-17
Expiration Date
Abstract
The present invention discloses a method of real-time quantification of a target nucleic acid in a sample by constructing a reference table of copy number vs. designated parameter from reference samples which sharing the same nucleic acid sequences with the target nucleic acid. The method includes (a) constructing a reference table of copy number vs. designated parameter from reference samples; (b) amplifying the target nucleic acid; (c) monitoring and detecting the amplification of the target nucleic acid in real-time; (d) analyzing the detected signals to get the designated parameter of the target nucleic acid; and (e) looking up and interpolating to the reference table to get the copy number of the target nucleic acid.
Core Innovation
The invention relates to a method for quantification of a target nucleic acid using real-time PCR by constructing a reference table that maps copy number versus normalized cycle number-derived parameters. Reference samples are selected to have the same nucleic acid sequence as the target nucleic acid, are amplified, and their amplifications are monitored and detected in real time. Detected signals are normalized so that the saturated range falls into 0-1, and serial dilution data are fit with a two-parameter sigmoidal PCR model to obtain t1/2 and a slope parameter τ.
Cycle number normalization is performed by the slope of the curves themselves, and repeating the curve-fitting and normalization produces t1/2 for each reference sample. A reference table is then constructed using the mapping between copy number and t1/2, based on the fitted serial dilution results. The target nucleic acid is amplified and monitored in real time, detected signals are normalized to fall into 0-1 when the amplification is saturated, and the target curve is fitted with the same sigmoidal function to obtain t1/2 after cycle number normalization by the slope of the target curve itself.
Finally, the copy number of the target nucleic acid contained in the sample is obtained by performing a look-up in the reference table using the measured t1/2 of the target. The approach provides absolute nucleic-acid quantification without requiring a per-run Ct standard curve, while using t1/2 from sigmoidal curve fitting as the basis for copy-number interpolation through the reference table. The disclosed examples indicate robustness of t1/2 against changes in primer concentration and dNTP concentration, and demonstrate target copy-number interpolation with <20% relative error.
Claims Coverage
The independent claim covers a real-time PCR absolute quantification method that builds a copy-number reference table using t1/2 derived from sigmoidal curve fitting and normalized signals, then quantifies an unknown target by measuring t1/2 and looking up copy number in the table. It also requires reference samples with the same nucleic-acid sequence as the target and includes the normalization and curve-fitting workflow for both reference samples and the target.
Reference table construction from same-sequence reference samples using normalized sigmoidal curve fitting
A reference table of copy number versus normalized cycle number is constructed from reference samples with the same nucleic acid sequence as the target nucleic acid by preparing the reference samples, amplifying them, monitoring and detecting the amplifications in real-time, normalizing detected signals to fall into 0-1 when saturated, fitting curves of serial dilution using the sigmoidal function NS=1/(1+e^{-(t-t1/2)/τ}) to obtain t1/2 and τ, normalizing cycle number by the slope of the curves themselves, and repeating to get t1/2 of each reference sample.
Real-time PCR monitoring and sigmoidal fitting to obtain target t1/2
The target nucleic acid is amplified, monitored and detected in real-time, detected signals are normalized to fall into 0-1 when saturation occurs, and the target curve is fitted using the same sigmoidal function NS=1/(1+e^{-(t-t1/2)/τ}); the target cycle number is normalized by the slope of the target curve itself and the fitting is repeated to get t1/2 of the target nucleic acid.
Copy-number look-up by target t1/2 from the reference table
The copy number of the target nucleic acid contained in the sample is obtained by performing a look-up in the reference table using the obtained t1/2, where the amplification reactions of the target nucleic acid and the reference samples are real-time PCR.
Monitoring and detecting amplifications using an optical device or a chemical sensor
Amplifications of the target nucleic acid and reference samples are monitored and detected using an optical device or a chemical sensor.
Chemical sensor defined as hydrogen ion or pyrophosphate
Monitoring and detecting of amplifications are performed using a chemical sensor selected from hydrogen ion or pyrophosphate.
Optical detection with DNA-binding/intercalating dye, probe, or molecular beacon
Monitoring and detecting of amplifications are performed using an optical device with a DNA-binding dye, an intercalating dye, a probe, or a molecular beacon.
Fluorescence signal detection from DNA-binding dye interacting with double-stranded nucleic acid
The amplification monitoring is performed with an optical device that detects a fluorescence signal emitted by a DNA-binding dye upon excitation when the dye interacts with double-stranded nucleic acid.
Across the dependent refinements, the claim set further specifies that real-time monitoring and detection can be performed either with an optical device or a chemical sensor, and narrows chemical sensing to hydrogen ion or pyrophosphate, and optical detection to fluorescence and/or named dye/probe/molecular beacon types. The core quantification mechanism remains centered on constructing a copy-number reference table using normalized sigmoidal fits to derive t1/2, then quantifying the target by measuring its t1/2 and looking up copy number.
Stated Advantages
Absolute nucleic-acid quantification without requiring a per-run Ct standard curve.
Robustness of t1/2 against primer concentration changes.
Robustness of t1/2 against dNTP concentration changes.
Target copy-number interpolation with <20% relative error.
Documented Applications
No documented applications found
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