Isothermal Nucleic Acid Detection System with Integrated Fluorescence
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Solution Overview
Problem
Current nucleic acid amplification technologies, such as PCR, require thermal cycling and sophisticated equipment, whereas isothermal methods like RPA offer advantages but lack efficient fluorescence detection systems for point-of-care applications.
Innovation Solution
A system comprising a lysis chamber, an amplification chamber with recombinase polymerase amplification enzymes, and a fluorescence detection device that allows for isothermal nucleic acid amplification and real-time fluorescence detection, enabling detection of DNA and RNA without separate cDNA production steps, using a combination of chambers for safe and contamination-minimized handling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR is used for nucleic acid amplification, then amplification sensitivity is improved, but device complexity and equipment requirements worsen
Solution Approach 1:
The patent replaces the thermal cycling mechanism of PCR with an isothermal amplification system that operates at constant temperature (37-42°C). This substitution eliminates the need for complex thermal cyclers while maintaining amplification capability through recombinase polymerase amplification (RPA) chemistry, directly resolving the contradiction between sensitivity and equipment complexity
Solution Approach 2:
The patent changes the temperature parameter from variable (thermal cycling between denaturation, annealing, and extension temperatures) to constant (isothermal operation at 37-42°C). This parameter change enables the use of simpler equipment while maintaining nucleic acid amplification sensitivity through optimized isothermal RPA conditions
2Device complexity
If isothermal amplification is used, then device complexity is reduced, but fluorescence detection capability worsens
Solution Approach 1:
The patent merges the isothermal amplification system with a fluorescence detection system in a single integrated device. The amplification chamber and fluorescence detection chamber are combined, allowing real-time monitoring of amplification through fluorescence signals while maintaining the simplicity of isothermal operation, thus resolving the contradiction between device simplicity and detection capability
Solution Approach 2:
The patent introduces a fluorescence probe as an intermediary that binds to the amplified nucleic acid sequences and emits fluorescence signals. This intermediary enables sensitive detection of the amplification products without requiring complex thermal cycling equipment, bridging the gap between simple isothermal amplification and effective fluorescence detection
3Adaptability or versatility
If separate cDNA production step is added, then RNA detection capability is improved, but process time and complexity worsen
Solution Approach 1:
The patent merges the reverse transcription of RNA to cDNA with the subsequent amplification step into a single simultaneous reaction. The recombinase polymerase amplification system directly amplifies RNA templates through reverse transcription, eliminating the need for separate cDNA production steps and reducing total process time while maintaining RNA detection capability
Solution Approach 2:
The patent performs reverse transcription of RNA to cDNA during the amplification process itself rather than as a preliminary separate step. The recombinase polymerase system inherently具备 the capability to perform reverse transcription, allowing RNA detection to be initiated immediately without time-consuming pre-processing steps
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
Facilitates rapid, efficient, and cost-effective detection of nucleic acids at room temperature, minimizing equipment requirements and ensuring safe handling and disposal, suitable for point-of-care or personal use.
Implementation Method 1
The amplification chamber contains a mixture that comprises a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase that causes a recombinase polymerase amplification (RPA)
Implementation Method 2
After the light source at specific wavelength illuminates on the targeted nucleic acids, the DNA-binding dyes or fluorescein- binding probes of the nucleic acids will react and enable fluorescent signals to be emitted
Implementation Method 3
The lysis chamber may contain a lysing fluid that causes lysing of the cells in a sample to thus release the nucleic acids (DNA or RNA)
Data Source
AI summary
The invention relates to a system is provided that comprises a lysis chamber, an amplification chamber and a fluorescence detection device. The fluorescence detection device (16) comprises - a detection chamber (42) configured to receive the amplification chamber (14) or the contents of the amplification chamber, - a light source (44), - an optical sensor (46), - energy supply means (48), - a wireless data interface (52) and - a controller (50).