Isothermal Nucleic Acid Amplification with Isotachophoresis
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Solution Overview
Problem
Current nucleic acid diagnostic tests require complex sample preparation, lab-scale instrumentation, and are limited to centralized laboratories due to the need for thermocyclers and complex fluidic systems, making them unsuitable for point-of-care applications, especially in settings that lack skilled personnel and infrastructure.
Innovation Solution
Integration of isotachophoresis (ITP) with isothermal nucleic acid amplification methods like recombinase polymerase amplification (RPA) to concentrate and amplify nucleic acids within a portable device, using a porous matrix, electrodes, and electrolytes to create an ITP plug that separates and concentrates nucleic acids with RPA reagents, enabling rapid amplification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid amplification is performed using conventional PCR methods, then amplification sensitivity and specificity are improved, but device complexity and infrastructure requirements increase due to need for thermocyclers and complex fluidic systems
Solution Approach 1:
The patent extracts the thermocycling function from the amplification system by using isothermal amplification methods (RPA, LAMP, NEAR) that operate at constant temperature, eliminating the need for complex thermocyclers and reducing device complexity while maintaining amplification sensitivity
Solution Approach 2:
The patent combines sample preparation (extraction and purification) and amplification into a single integrated isothermal reaction step, eliminating the need for separate thermocycling phases and complex fluidic systems, thereby reducing device complexity while preserving diagnostic accuracy
2Measurement precision
If sample preparation is performed using conventional extraction methods, then nucleic acid purity is improved, but processing time and operational complexity increase
Solution Approach 1:
The patent merges sample extraction, purification, and amplification into a single isothermal reaction step, eliminating multiple separate processing steps and significantly reducing processing time while maintaining nucleic acid purity through the specificity of isothermal amplification
Solution Approach 2:
The isothermal amplification system performs self-purification by selectively amplifying target nucleic acids from complex samples without requiring extensive preliminary purification steps, reducing both processing time and operational complexity while maintaining diagnostic accuracy
3Measurement precision
If conventional nucleic acid amplification is performed, then amplification accuracy is improved, but ease of operation deteriorates due to requirement for skilled personnel and controlled infrastructure
Solution Approach 1:
The isothermal amplification system operates autonomously at constant temperature without requiring skilled personnel to manage complex thermocycling programs or monitor multiple parameters, maintaining amplification accuracy while dramatically improving ease of operation for use in resource-limited settings
Solution Approach 2:
The patent changes the operational parameter from variable temperature (PCR) to constant temperature (isothermal), simplifying the operational requirements and making the system easier to operate while maintaining amplification accuracy through optimized isothermal reaction conditions
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 allows for rapid, portable, and efficient extraction, purification, and detection of nucleic acids, reducing the need for complex equipment and infrastructure, enabling point-of-care diagnostics in various settings.
Implementation Method 1
In ITP, sample ions focus between leading (LE) and trailing electrolytes (TE) which have co-ions with respectively higher and lower effective electrophoretic mobilities than the sample ions. When a constant voltage or current is applied across the channel, sample ions accumulate and preconcentrate by electrophoresis into a number of contiguous zones between LE and TE zones
Implementation Method 2
sample ions accumulate and preconcentrate by electromigration into the zones previously occupied by species with another composition
Implementation Method 3
a porous matrix having a first end and a second end opposing the first end, the first end and the second end defining a first axis, the porous matrix having a first fluid pathway having a first end and extending to a second end
Data Source
AI summary
The present disclosure relates generally to the integration of isotachophoresis (ITP) and isothermal nucleic acid amplification methods such as recombinase polymerase amplification (RPA). One aspect of the disclosure relates to a method for concentrating and amplifying a nucleic acid, the method including an isotachophoresis device, the isotachophoresis device including a porous matrix, and first and second electrodes, having a leading electrolyte, a trailing electrolyte and a set of isothermal nucleic acid amplification reaction reagents disposed in the porous matrix as described herein, and applying a voltage across the first electrode and the second electrode for a time sufficient to provide a first isotachophoresis (ITP) plug comprising an amplification product of the nucleic acid, wherein the concentration of the nucleic acid is substantially higher in the first FTP plug than in the first and/or second fluids outside of the first ITP plug.