Isothermal Amplification with Universal Detection Probes
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Solution Overview
Problem
Foldback primer amplification technologies, such as LAMP, SMAP, and GEAR, are complex and challenging for multiplex detection due to the requirement of multiple primers and strand displacing polymerases, which complicates the use of hydrolysis probes and differentiation of multiple amplification targets.
Innovation Solution
The introduction of specific detection probes and universal detection probes that interact to monitor isothermal amplification in real-time, allowing for the detection of multiple targets without additional primers or complex probes, and the use of extruding sequences on foldback primers to enhance detection and amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If foldback primer amplification technologies (LAMP, SMAP, GEAR) are used, then isothermal amplification can be achieved without expensive thermocyclers, but the requirement of multiple primers and strand displacing polymerases complicates multiplex detection and probe design
Solution Approach 1:
The invention divides the detection system into two independent parts: (1) amplification primers that remain simple foldback primers without extruding sequences, and (2) separate detection probes (specific and universal) that handle the complexity of multiplex detection. This segmentation allows each component to be optimized independently, reducing overall system complexity.
Solution Approach 2:
The invention introduces specific detection probes with specific probe sequences as intermediaries that bridge the amplification reaction and detection system. These probes hybridize to amplification products and interact with universal detection probes, enabling multiplex detection without complicating the amplification primers themselves.
2Reliability
If multiple primers with six or eight regions of homology are used for each amplicon, then foldback amplification can be achieved, but this complicates multiplexing due to the requirement for multiple primers for each reaction
Solution Approach 1:
The invention segments the multiplexing function from the amplification primers to separate detection probes. Each amplicon uses simple foldback primers for reliable amplification, while multiplexing is achieved through multiple specific detection probes that each target specific probe sequences associated with different amplicons.
Solution Approach 2:
The invention introduces universal detection probes that can interact with multiple specific detection probes through a common interaction mechanism (specific probe sequence hybridization). This universal probe design enables a single detection system to handle multiple targets, providing multi-functionality without requiring complex primer designs for each target.
3Temperature
If strand displacing polymerases are used for foldback amplification, then isothermal amplification can be achieved, but this prohibits the use of hydrolysis probes that rely upon 5′ to 3′ exonuclease activity
Solution Approach 1:
The invention uses specific detection probes as intermediaries that hybridize to amplification products through annealing rather than requiring exonuclease activity. These probes interact with the amplification system through hybridization and strand displacement, mechanisms compatible with strand displacing polymerases, thereby enabling probe-based detection without 5′ to 3′ exonuclease activity.
Solution Approach 2:
The invention changes the detection mechanism from exonuclease-based (requiring 5′ to 3′ activity) to hybridization-based (using specific probe sequences that anneal to amplification products). This parameter change in the detection approach makes the system compatible with strand displacing polymerases while maintaining probe design flexibility.
4Productivity
If conventional detection methods are used for foldback amplification, then amplification can proceed, but real-time monitoring and differentiation of multiple amplification targets is difficult
Solution Approach 1:
The invention implements real-time feedback monitoring through specific detection probes that continuously hybridize to amplification products during the reaction. The accumulation of probe-bound products provides real-time feedback on amplification progress, enabling kinetic monitoring without interfering with amplification speed.
Solution Approach 2:
The invention uses fluorescently labeled specific detection probes that produce detectable color/fluorescence changes when they hybridize to amplification products. This optical signal change enables real-time detection and differentiation of multiple targets based on their specific probe sequence interactions.
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 real-time monitoring of isothermal amplification and improves the speed and sensitivity of foldback primer amplification, facilitating multiplex detection and reducing the complexity of probe design and amplification processes.
Implementation Method 1
a specific detection probe that, under the suitable amplification conditions, hybridizes to the template nucleic acid, its complement, the amplicon nucleic acid or its complement
Implementation Method 2
monitoring during or after step (ii) interaction between (c) a specific detection probe and (d) a universal detection probe
Data Source
AI summary
The present disclosure provides compositions, methods and kits for Omega amplification technologies. In addition, the present disclosure provides compositions, methods and kits for universal FQ probe and for G-quadruplex detection methods for use in isothermal amplification technologies.