Multiplex PCR Segmentation for Signal Isolation
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Solution Overview
Problem
Current multiplex nucleic acid amplification methods in clinical diagnostics face challenges such as limited capacity for multiple targets due to label overlap issues in real-time PCR, requiring careful selection of fluorophores and increasing the risk of false positives, and manual intervention which can lead to contamination.
Innovation Solution
The method involves simultaneous amplification of multiple nucleic acids in separate reaction vessels using polymerase with reverse transcriptase activity at varying temperatures, allowing for independent signal detection and reducing the need for multiple labels, thereby increasing the number of targets that can be amplified simultaneously while minimizing manual handling and contamination risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiplex amplification is performed in the same reaction vessel using real-time detection, then the number of targets that can be detected is limited, but the device complexity and manual intervention are reduced
Solution Approach 1:
The patent divides the amplification process into separate reaction vessels for different target types (RNA targets in one vessel, DNA targets in another vessel). This segmentation eliminates signal overlap between different fluorophores and allows independent optimization of amplification conditions for each target type, thereby increasing the number of reliably detectable targets while reducing false positives.
2Adaptability or versatility
If multiple fluorophores are used for multiplex detection, then the number of distinguishable targets is limited, but the amplification process is simplified
Solution Approach 1:
By segregating RNA and DNA target amplifications into separate reaction vessels, the patent reduces the number of fluorophores needed per vessel. This allows use of fewer, more distinct fluorophores with minimal spectral overlap, simplifying the fluorophore selection process while maintaining the ability to detect multiple targets through the combination of vessels.
Solution Approach 2:
The patent extracts the DNA amplification process from the RNA amplification vessel, creating separate reaction systems. This extraction allows each vessel to use a simplified set of fluorophores optimized for its specific target type, reducing the overall complexity of fluorophore management while enabling detection of multiple targets across the separated systems.
3Ease of operation
If manual intervention is used in multiplex amplification, then flexibility in protocol adjustment is improved, but contamination risk increases
Solution Approach 1:
The patent combines multiple amplification reactions (RNA and DNA targets) into a coordinated automated workflow that processes multiple reaction vessels simultaneously. This merging of processes into an automated system maintains protocol flexibility through programmable control while eliminating manual transfer steps that cause contamination, as the system automatically manages all reaction vessels without human intervention.
4Adaptability or versatility
If separate reaction vessels are used for different target amplification, then the number of simultaneously amplifiable targets increases, but the hands-on time and processing complexity increase
Solution Approach 1:
The patent merges the processing of multiple reaction vessels into a coordinated automated workflow. The system simultaneously manages RNA amplification in one vessel and DNA amplification in another vessel using automated liquid handling and thermal cycling, thereby increasing the number of simultaneously amplifiable targets while minimizing hands-on time through automation.
Solution Approach 2:
The patent employs a universal automated system that can handle multiple reaction vessel types and configurations. This multi-functional platform performs amplification, detection, and data analysis across different target types without requiring separate manual protocols, thereby increasing target capacity while reducing the time investment needed for each additional target.
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 efficient, reliable, and cost-effective simultaneous amplification of multiple nucleic acids, reducing hands-on time, improving testing flexibility, and decreasing the need for repeated tests, leading to faster diagnosis and reduced antiviral agent use and hospital stays.
Implementation Method 1
contacting nucleic acids from said sample with one or more amplification reagents comprising a polymerase with reverse transcriptase activity in at least two reaction vessels for amplification of the at least first and second target nucleic acids in the at least two reaction vessels; e. incubating in said reaction vessels said nucleic acids with said one or more amplification reagents for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur, wherein the incubation of the polymerase with reverse transcriptase activity is carried out at 55°C, 60°C, and 65°C
Implementation Method 2
f. incubating in said reaction vessels said nucleic acids with said one or more amplification reagents for a period of time and under conditions sufficient for an amplification reaction indicative of the presence or absence of said first and second target nucleic acid to occur while amplifying the first, but not the second target nucleic acid in the first reaction vessel and the second, but not the first target nucleic acid in the second reaction vessel
Data Source
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AI summary
The present invention provides a method for the amplification of at least a first and a second target nucleic acid that may be present in a fluid sample. The invention further provides a kit and an analytical system for carrying out said amplification.