Real-Time PCR Fluorescence Background Subtraction
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Solution Overview
Problem
Existing real-time PCR systems face challenges in accurately measuring fluorescence due to apparatus errors such as optical system errors, concentration errors, and contamination, which lead to unbalanced amplification curves and require the use of a second fluorescence signal for correction, increasing complexity and cost.
Innovation Solution
An apparatus that detects fluorescence strength by subtracting background fluorescence measurements from raw measurements in adjacent regions, allowing for precise calculation of the original fluorescence strength without the need for a second fluorescence signal, and normalizes fluorescence data using the maximum value to correct for apparatus errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a second fluorescence signal is used for correction of apparatus errors, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The invention extracts and removes the background fluorescence signal from the total fluorescence measurement by detecting fluorescence in adjacent non-reaction regions and subtracting it from the reaction region measurements. This eliminates the need for a second fluorescence signal while achieving accurate correction of apparatus errors.
Solution Approach 2:
The invention uses adjacent non-reaction regions as copies of the reaction regions to measure background fluorescence. These adjacent regions serve as reference copies that contain the same optical path and apparatus characteristics but without the reaction signal, allowing for background subtraction.
2Measurement precision
If a second fluorescence signal is used for correction, then measurement precision is improved, but cost increases
Solution Approach 1:
The invention extracts and removes the background fluorescence signal from the total fluorescence measurement by detecting fluorescence in adjacent non-reaction regions and subtracting it from the reaction region measurements. This eliminates the need for a second fluorescence signal while achieving accurate correction of apparatus errors.
3Productivity
If background fluorescence is not corrected, then data acquisition time is reduced, but measurement precision deteriorates
Solution Approach 1:
The invention merges the background measurement process with the reaction fluorescence measurement process by simultaneously detecting fluorescence in both reaction and adjacent non-reaction regions. This allows background correction to be performed without additional measurement time, maintaining high productivity while improving precision.
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 enables accurate and precise measurement of fluorescence strength, reducing data acquisition time and costs, while ensuring correct amplification curve quantitation and reliable calculation of threshold cycle numbers, even in the presence of apparatus errors.
Implementation Method 1
excite fluorescent material by irradiating a PCR product with excitation light
Data Source
AI summary
There is provided an apparatus for detecting a nucleic acid amplification product in real time, which is capable of effectively excluding or reducing apparatus error factors without using a second fluorescence signal used for correction. A plurality of wells 7A are given with temperature cycles and fluorescence strength from a nucleic acid amplification product is detected in real time in each well 7A. A fluorescence measurement value [DNA]raw obtained from the well 7A and a fluorescence measurement value [DNA]bg obtained from a connection wall near the well 7A are detected, and the fluorescence measurement value [DNA]bg is subtracted from the fluorescence measurement value [DNA]raw to determine fluorescence strength [DNA]real of the well 7A.