PCR Data Calibration via Normalization Coefficient
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Solution Overview
Problem
Current methods for calibrating data sets in real-time PCR face limitations, including inaccurate hardware adjustments, high costs associated with reference dyes, and interference issues in multiplex reactions, which fail to effectively reduce inter- and intra-instrument variations without direct hardware adjustments or reference dyes.
Innovation Solution
A method using a normalization coefficient based on an arbitrarily determined reference value and reference cycle, applied to signal values to calibrate data sets, which also involves removing instrument blank signals to enhance accuracy and reliability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hardware adjustment method is used to calibrate analytical instruments, then inter-instrument variation is reduced, but manufacturing precision and reliability remain limited and additional calibration is needed for deterioration
Solution Approach 1:
The patent replaces hardware adjustment methods with a mathematical normalization coefficient approach. Instead of physically adjusting instrument components, the invention uses computational correction factors derived from reference dye signals to normalize data across instruments, thereby achieving higher precision without mechanical intervention.
Solution Approach 2:
The patent introduces a normalization coefficient as an intermediary element that mediates between raw instrument signals and calibrated results. This coefficient, calculated from reference dye measurements, serves as a computational bridge that corrects inter-instrument variations without requiring direct hardware modification.
2Measurement precision
If reference dye method is used for signal calibration, then signal calibration is achieved, but cost per reaction increases and interference phenomena may occur
Solution Approach 1:
The patent extracts the calibration function from the main analytical detection process. By using reference dyes with distinct emission spectra that can be separately detected and used to calculate normalization coefficients, the calibration function is separated from target analyte detection, allowing calibration without interfering with the primary measurement channels.
Solution Approach 2:
The patent changes the detection parameter by using reference dyes with different emission wavelengths than the target analyte dyes. This parameter differentiation allows simultaneous measurement of reference signals for calibration and target signals for analysis without spectral interference, resolving the contradiction between calibration accuracy and interference prevention.
3Measurement precision
If reference dye is added for calibration, then signal level is calibrated, but quantity of substance increases and interference with other dyes occurs
Solution Approach 1:
The patent utilizes the emission wavelength parameter as a distinguishing feature, allowing multiple dyes (reference and target) to coexist in the same reaction mixture without interference. By selecting reference dyes with non-overlapping emission spectra, the system achieves calibration functionality without increasing the effective concentration of interfering substances in the detection channels.
4Reliability
If intra-instrument variation is not addressed by hardware adjustment, then inter-instrument variation is reduced, but intra-instrument variation remains uncorrected
Solution Approach 1:
The patent creates a universal normalization coefficient calculation method that functions across multiple instruments and multiple reaction wells within each instrument. The same mathematical approach corrects both inter-instrument variations (between different devices) and intra-instrument variations (between different wells on the same device), providing comprehensive precision improvement through a single unified methodology.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces inter- and intra-instrument signal variations, providing more accurate and reliable calibration results without the need for hardware adjustments or reference dyes, improving the precision and competitiveness of data analysis.
Implementation Method 1
The reference dye such as ROXTM or fluorescein which constantly generates a known amount of a signal
Implementation Method 2
Analytical instrument converts the light of specific wavelength to an electric signal using photodiode or CCD
Data Source
AI summary
The present invention relates to a method for calibrating a data set of a target analyte in a sample, wherein a normalization coefficient for calibrating the data set is provided by using a reflective value, a reference cycle and the data set, and the calibrated data set is obtained by applying the normalization coefficient to the signal values of the data set. The present method is very effective in removing the inter- and intra-instrument signal values of data sets. Furthermore, since the present method can be configured in software, the instant method is capable of being applied universally to various analytical instruments (e.g., a real-time PCR instruments) regardless of manufacture. Accordingly, the method by the present invention would be very useful in diagnostic data analysis.