Nucleic Acid Amplification Signal Normalization
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Solution Overview
Problem
Existing nucleic acid amplification techniques, such as real-time PCR, face challenges in accurately interpreting raw signals due to variations caused by reagent amounts, sample concentrations, and measurement device variations, leading to potential misinterpretations and inconsistencies across replicates and instruments.
Innovation Solution
A method and system for normalizing fluorescence intensity signals by calculating a dimensionless normalized signal through determining an intercept value, baseline, and maximum growth value, and then applying a mixed intercept growth normalization formula, which divides the signal difference by a divisor proportional to a combination of these values, allowing for precise detection and measurement of analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR signal measurement is performed, then nucleic acid amplification can be detected and quantified, but signal variations occur due to reagent amount, sample concentration, and measurement device differences
Solution Approach 1:
The patent applies parameter changes by transforming the raw RT-PCR signal through mathematical operations (baseline subtraction, normalization by maximum growth value) to convert it into a standardized parameter that eliminates variations caused by reagent amounts, sample concentrations, and measurement device differences. This transformation changes the signal from an absolute fluorescence intensity to a relative normalized value that can be consistently compared across different experimental conditions.
2Reliability
If normalization methods are applied to reduce signal variations, then measurement consistency improves, but additional processing steps and complexity are introduced
Solution Approach 1:
The patent applies preliminary action by performing baseline subtraction and normalization calculations during the real-time PCR measurement process itself, rather than as a separate post-processing step. The system automatically determines the maximum growth value and applies the normalization formula to each data point as it is collected, thereby reducing signal variations in real-time without requiring complex external processing equipment or procedures.
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 reduces signal variations, ensures clear separation of positive and negative signals, and provides accurate calculation of cycle threshold values, thereby enhancing the precision and reliability of nucleic acid amplification data interpretation.
Implementation Method 1
various fluorescent dyes, which are conjugated to the nucleic acid temporarily or permanently. This creates fluorescence signals indicating nucleic acid amplification which are measured in real time
Data Source
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AI summary
The invention relates to a method of detecting a presence and/or measuring a quantity of an analyte in a sample by a nucleic acid amplification reaction, said method comprising: acquiring a fluorescence intensity of the analyte for each amplification reaction cycle of the nucleic acid amplification reaction (810) and creating from said fluorescence intensity a growth signal being indicative of a fluorescence intensity over the cycles, determining an intercept value of the signal (820), determining a baseline of the signal (830), determining a maximum growth value of the signal (840), normalizing the signal by modifying said signal (850), wherein the modification comprises calculating a difference of the signal and the baseline and dividing the difference by a divisor, wherein the divisor is proportional to a combination of the intercept and the maximum growth value, wherein the normalized signal is dimensionless, and processing (860) the normalized signal in order to determine a cycle number at which the normalized signal exceeds the baseline (870) for detecting the presence and/or measuring the quantity of the analyte (880).