Multiplex PCR Vessel Segmentation for Fluorochrome Overlap
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Solution Overview
Problem
Current multiplex nucleic acid amplification methods face challenges in efficiently amplifying and detecting multiple target nucleic acids simultaneously due to limitations in label usage and potential overlap of fluorochrome spectra, leading to false positives and reduced precision in real-time PCR assays.
Innovation Solution
A process involving two reaction vessels with different amplification reagents and conditions for each target nucleic acid, using a polymerase with reverse transcriptase activity, allows for simultaneous amplification and detection of multiple nucleic acids without the need for multiple labels, reducing manual intervention and contamination risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex amplification is performed in the same reaction vessel using different labels, then multiple target nucleic acids can be detected simultaneously, but false positives occur and measurement precision decreases due to fluorochrome spectrum overlap
Solution Approach 1:
The invention divides the multiplex amplification process into separate reaction vessels, with each vessel dedicated to amplifying a specific target nucleic acid. This segmentation eliminates the fluorochrome spectrum overlap problem that occurs when multiple labeled targets are amplified together in the same vessel, thereby maintaining high measurement precision while still achieving simultaneous detection of multiple targets through parallel processing.
Solution Approach 2:
The invention transitions from detecting multiple targets in a single dimension (one reaction vessel with multiple overlapping fluorochromes) to detecting multiple targets across multiple dimensions (multiple separate reaction vessels, each with a single fluorochrome). This dimensional expansion resolves the spectrum overlap issue by distributing targets across spatially separated vessels.
2Adaptability or versatility
If multiple labels and fluorochromes are used for multiplex detection, then multiple target nucleic acids can be distinguished, but device complexity and cost increase
Solution Approach 1:
By segmenting the multiplex assay into separate reaction vessels, each targeting a specific nucleic acid, the invention eliminates the need for multiple fluorochrome labels within a single vessel. Each vessel uses a simple single-fluorochrome system, dramatically reducing the overall complexity of labels and detection components while maintaining the ability to detect multiple targets in parallel.
Solution Approach 2:
The invention creates a universal reaction vessel design that can be used for any single-target amplification, with the same basic setup and single fluorochrome approach applicable to all targets. This universalization reduces complexity by avoiding the need for multiple specialized labeled probes and complex detection systems.
3Productivity
If real-time detection with modified oligonucleotides carrying reporter groups is used, then amplification and detection can be performed in the same vessel, but manual intervention increases and contamination risk rises
Solution Approach 1:
The invention segments the amplification and detection processes into separate reaction vessels, where each vessel is dedicated to a specific target. This segmentation reduces manual intervention by allowing automated parallel processing of multiple vessels and minimizes contamination risk by isolating each amplification reaction, eliminating the need for complex post-amplification handling and reducing cross-contamination opportunities.
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 reliable and efficient simultaneous amplification of multiple nucleic acids, reducing hands-on time and improving testing flexibility, leading to faster diagnosis and cost-effective clinical applications by minimizing the use of antiviral agents and hospitalizations.
Implementation Method 1
incubating in the reaction vessels the purified nucleic acids with the one or more amplification reagents for a period of time, under conditions suitable for transcription of RNA by the polymerase with reverse transcriptase activity to occur
Data Source
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.