Real-Time PCR Multi-Target Detection via Fluorescence Signal Ratios
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Solution Overview
Problem
Current methods for detecting and quantifying multiple targets in a sample using PCR are limited to qualitative analysis and require multiple steps, making it challenging to analyze a large number of targets efficiently and accurately.
Innovation Solution
A method involving a reaction solution with multiple primer pairs and fluorescent probes, each tagged with different fluorophores, allows for simultaneous detection and quantification of multiple targets in a single real-time PCR step by calculating fluorescence signal ratios and using calibration curves to determine target presence and concentration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple targets are detected using traditional PCR methods, then detection capability is improved, but the number of steps and complexity increases
Solution Approach 1:
The patent combines multiple detection targets into a single PCR reaction system by using multiple primer pairs and fluorescent probes with different fluorophores that can be detected simultaneously. This merging of multiple detection steps into one reaction directly resolves the contradiction by improving detection capability while reducing the number of steps.
Solution Approach 2:
The PCR system is designed with multi-functionality to detect multiple targets simultaneously using a single reaction setup. The universal primer pairs and fluorescent probes with different fluorophores enable the system to perform multiple detection functions in one experiment, thereby improving adaptability while reducing complexity.
2Adaptability or versatility
If multiple targets are detected using traditional PCR methods, then detection capability is improved, but analysis time increases
Solution Approach 1:
By merging multiple detection targets into a single PCR reaction, the patent eliminates the need for separate detection steps for each target. This consolidation directly reduces analysis time while maintaining the ability to detect multiple targets simultaneously.
Solution Approach 2:
The patent enables continuous detection of multiple targets throughout the single PCR reaction cycle, rather than requiring sequential detection steps. This continuous action improves detection capability while minimizing time loss by keeping the reaction system actively detecting all targets simultaneously.
3Adaptability or versatility
If multiple targets are detected using traditional PCR methods, then detection capability is improved, but measurement precision decreases
Solution Approach 1:
The patent assigns different fluorophores with distinct emission wavelengths to different targets, allowing each target to be detected with specific local quality markers. This differentiation enables precise measurement of each target's concentration or presence independently, thereby improving measurement precision while maintaining multi-target detection capability.
Solution Approach 2:
The use of fluorescent probes with different fluorophores that emit at different wavelengths creates distinct color signals for different targets. This color differentiation enables precise measurement and identification of multiple targets simultaneously, improving both detection capability and measurement 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
Enables efficient detection and quantification of multiple targets in a single PCR step, reducing analysis time and increasing accuracy, with the potential to detect up to 15 targets using four fluorophores and apply to various fields like infectious diseases and cancer screening.
Implementation Method 1
The fluorophore bound to a specific probe is used to generate fluorescence during the PCR process, and then a fluorescence detection system is used to detect the amount of fluorescence released by the fluorophore in each cycle.
Data Source
AI summary
A method for detecting multiple targets in the same sample includes the following steps. A sample is provided, and the sample includes a nucleic acid of at least one target. A reaction solution is provided, which includes target primer pairs and target fluorescent probes. The target fluorescent probes include at least two fluorophores. After mixing the sample and the reaction solution, a real-time polymerase chain reaction is performed. A fluorescence signal value ratio of the at least two fluorophores is calculated to determine whether the at least one target is present in the sample.