Displacement Probe Multiplex Detection for Isothermal Amplification
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Solution Overview
Problem
Current isothermal nucleic acid amplification techniques, such as LAMP, face challenges in detecting multiple targets in a single sample due to limited sensitivity and specificity, especially with existing real-time detection methods that suffer from non-specific quenching and require extensive probe design and optimization, making them less effective for field diagnostics.
Innovation Solution
A composition and method using a combination of oligonucleotides with quencher or fluorescent labels, where the ratio of labeled to unlabeled oligonucleotides is optimized, allowing for stable duplex formation and efficient amplification, enabling target-specific detection of multiple polynucleotides in a multiplex reaction without the need for additional primer optimization or probe design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If real-time detection methods using non-specific quenching are used, then detection capability is provided, but sensitivity is reduced and fluorophore selection is limited
Solution Approach 1:
The patent introduces a displacement probe as an intermediary element that mediates between the amplification product and the fluorophore. The probe contains a fluorophore and a quencher, and its fluorescence is restored when displaced by the amplification product, providing sensitive and specific detection without the limitations of non-specific quenching methods
Solution Approach 2:
The patent changes the detection mechanism from non-specific quenching to specific displacement-based fluorescence restoration. By modifying the detection parameter from general quenching to sequence-specific displacement, the method achieves both high sensitivity and broad fluorophore selection
2Adaptability or versatility
If qPCR probes are used for multiplex detection, then multiple targets can be detected, but extensive design and optimization is required
Solution Approach 1:
The patent creates a universal detection system using displacement probes that can be applied to various amplification methods including LAMP and PCR. The probe design follows consistent principles across different applications, enabling multiplex detection without requiring extensive re-optimization for each specific case
Solution Approach 2:
The detection system is segmented into distinct functional components: the amplification primers, the displacement probe with fluorophore-quencher pair, and the amplification product. This segmentation allows independent optimization of each component and simplifies the overall design process for multiplex applications
3Measurement precision
If LAMP reaction vessels are not opened to avoid contamination, then sensitivity is maintained, but additional processing and instrumentation is required for multiplex detection
Solution Approach 1:
The displacement probe system enables the LAMP reaction to self-detect its own amplification products through fluorescence restoration. The probe automatically binds to the amplification product and restores fluorescence without requiring external intervention, opening the reaction vessel, or additional processing steps, thus maintaining sensitivity while enabling simple multiplex detection
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 enhances the sensitivity and speed of nucleic acid amplification reactions, allowing for robust, target-specific detection of multiple targets in a single reaction, maintaining sensitivity across a wide range of amplification techniques and reducing the complexity of probe design, thereby improving diagnostic capabilities.
Implementation Method 1
A first oligonucleotide comprises a primer sequence which is also a target sequence for priming an amplification reaction, the first oligonucleotide having a quencher or fluorescent label; a second oligonucleotide having a sequence suitable for hybridizing to a portion of the first oligonucleotide under stringent conditions to form a stable duplex, the second oligonucleotide having a fluorescent label if the first oligonucleotide has a quencher label, or having a quencher label if the first oligonucleotide has a fluorescent label
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.