Multiplex Nucleic Acid Detection via Fluorescence Ratio Calculation
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Solution Overview
Problem
Current methods for detecting multiple target nucleic acids are time-consuming and costly, particularly when using independent measurements and distinguishing different labeling substances, which often require complex equipment and lengthy analysis times.
Innovation Solution
A detection method using a kit that allows for simultaneous amplification and detection of multiple genes in a single reaction vessel with a single kind of reaction solution and label, utilizing specific temperature settings and fluorescence intensity calculations to distinguish between target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If independent measurement is performed for each target nucleic acid, then measurement accuracy is improved, but analysis time and costs increase significantly
Solution Approach 1:
The patent combines multiple independent measurements into a single simultaneous measurement process. Multiple target nucleic acids are amplified and detected in one reaction vessel using a single fluorescent label, eliminating the need for separate measurement steps for each target while maintaining detection accuracy through mathematical calculation methods.
Solution Approach 2:
The invention creates a universal detection system where one reaction solution and one fluorescent label can detect multiple different target nucleic acids. The system uses universal primers and probes that can simultaneously target multiple genes, making the detection process multi-functional rather than requiring specialized reagents for each target.
2Adaptability or versatility
If different labeling substances are used to distinguish multiple target nucleic acids, then detection capability is improved, but equipment complexity and costs increase
Solution Approach 1:
Instead of changing the label itself, the invention changes detection parameters (temperature, mathematical calculations) to distinguish multiple targets. By measuring fluorescence at different temperatures and applying calculation formulas, the system can differentiate between multiple target nucleic acids using the same fluorescent label, thus avoiding the need for multiple labels and complex wavelength-setting equipment.
Solution Approach 2:
The patent introduces mathematical calculation formulas as an intermediary step between fluorescence measurement and target identification. Rather than directly identifying targets through different fluorescent colors, the system uses calculated values derived from fluorescence intensity at different temperatures to indirectly identify and distinguish multiple target nucleic acids.
3Measurement precision
If thermal melting curve analysis is performed to distinguish multiple targets, then detection accuracy is improved, but analysis time increases due to gradual temperature changes
Solution Approach 1:
The invention performs preliminary differentiation during the amplification phase itself by incorporating temperature-dependent probe binding. Instead of performing separate melting curve analysis after amplification, the system is designed to differentiate targets during the PCR process through carefully designed probe- target interactions at specific temperatures, thus eliminating the need for lengthy post-amplification temperature scanning.
Solution Approach 2:
The patent skips the gradual temperature change process required for traditional melting curve analysis. By using mathematical calculations based on fluorescence intensity at specific temperature points during amplification, the system rushes through the differentiation process without needing to slowly scan through a temperature range, thereby significantly reducing analysis time while maintaining accuracy.
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 method significantly reduces analysis time and costs by enabling efficient simultaneous detection of multiple target nucleic acids without the need for complex equipment or lengthy temperature changes, while maintaining high accuracy in distinguishing between different nucleic acid sequences.
Implementation Method 1
measuring a first fluorescence intensity value in a first target nucleic acid detection step at a first temperature; measuring a second fluorescence intensity value in a first and second target nucleic acid detection step at the first temperature
Implementation Method 2
In this method after the amplification reaction, temperature is gradually increased/decreased to make distinction and judgment with respect to the multiple target nucleic acids based on both of first temperature wherein a change rate of fluorescent signals shows a peak and second temperature wherein the target numeric acid is melt
Data Source
AI summary
The present invention provides a method for detecting multiple target nucleic acids, the method making it possible to amplify a plurality of genes using one reaction vessel containing therein one type of reaction solution and further using a single label. Computation is performed in accordance with the following formulas for every specific cycle and/or every cycle during amplification reaction using a kit for detecting the multiple target nucleic acids. (Formula 1): f1[n]=fhyb.1[n]/fden.1[n], (Formula 1′): f2[n]=fhyb.2[n]/fden.2[n], (Formula 2): Fr[n]=(a−f2 [n])/(a−f1 [n]).fhyb.1[n]: Fluorescence intensity value in elongation step of first target nucleic acid detecting step. The same hereinafter.