Displacement Probe Multiplex Detection LAMP
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Solution Overview
Problem
Current isothermal nucleic acid amplification techniques, such as LAMP, face challenges in detecting multiple targets simultaneously due to sensitivity to carryover contamination and limitations in real-time detection methods, which are often non-specific and less sensitive, especially when using fluorescence-based approaches.
Innovation Solution
A composition and method involving oligonucleotides with quencher or fluorescent labels, where the molar ratio of specific oligonucleotides is optimized to enable stable duplex formation, allowing for sensitive and specific detection of amplification products in multiplex reactions using strand displacement polymerases, including archeal polymerases, to facilitate real-time monitoring of DNA amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence-based detection methods are used for real-time monitoring of DNA amplification, then detection sensitivity is improved, but specificity and reliability deteriorate due to non-specific quenching and limited fluorophore selection
Solution Approach 1:
The patent introduces a displacement probe as an intermediary molecule that mediates between the amplification product and the fluorophore. The probe contains a fluorophore and a quencher, and its fluorescence is activated only when displaced by the specific amplification product through hybridization. This intermediary mechanism ensures that fluorescence signal is generated only from specific targets, eliminating non-specific quenching issues while maintaining high detection sensitivity.
2Productivity
If multiple targets are detected simultaneously in a single LAMP reaction, then productivity is improved, but measurement precision deteriorates due to interference between multiple amplification reactions
Solution Approach 1:
The patent applies local quality by assigning different displacement probes with distinct fluorophores to different target sequences within the same reaction mixture. Each probe is specifically designed to hybridize only with its complementary target, creating localized specific detection zones. This allows multiple targets to be detected simultaneously in a single LAMP reaction without cross-interference, maintaining high measurement precision while improving productivity through multiplexing.
3Measurement precision
If qPCR probes are used for multiplex detection, then measurement precision is improved, but device complexity and ease of manufacture worsen due to extensive design and optimization requirements
Solution Approach 1:
The patent creates a universal displacement probe system that can be applied to various LAMP and PCR amplification reactions without requiring extensive re-optimization for each application. The probe design follows standardized principles: a fluorophore, a quencher, and a hybridization region complementary to the target. This universal approach allows the same basic probe structure to function across different amplification methods and target sequences, significantly reducing design complexity while maintaining multiplex detection accuracy.
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 robust, sensitive, and specific detection of multiple targets in a single reaction, maintaining high sensitivity and reducing the need for additional primer optimization or probe design, with the ability to perform size detection and internal quantification, suitable for various amplification techniques like LAMP and PCR.
Implementation Method 1
A first oligonucleotide (Oligo 1) is labeled with a fluorophore and a quencher... A second oligonucleotide (Oligo 2) is labeled with a quencher or fluorophore... allowing for sensitive and specific detection of amplification products
Data Source
AI summary
Compositions and methods are provided for quantitative detection of amplification products, the methods being suitable for multiplexing. A first oligonucleotide that includes a primer sequence for priming an amplification reaction and also is labeled with a fluorescent label or quencher is mixed with a second oligonucleotide which has a sequence suitable for hybridizing to a portion of the first oligonucleotide and has a fluorescent label if the first oligonucleotide has a quencher or a quencher if the first oligonucleotide has a fluorescent label; and a third nucleotide which includes some or all the primer sequence contained in the first oligonucleotide but is not labeled, the first and third oligonucleotide being combined in a molar ratio of 2.8 to 8.2.