Nucleic Acid Detection via Probe Excision and Capture
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Solution Overview
Problem
Traditional nucleic acid detection methods, such as qPCR, rely on amplification and are prone to errors, biases, and require intensive sample preparation, limiting their speed, reliability, and multiplexing capabilities, especially in detecting viral RNA and RNA transcripts.
Innovation Solution
A method involving excising specific target probes from nucleic acids using cutting reagents and detecting them with complementary capture oligonucleotides, which avoids amplification and intensive sample preparation, enabling rapid, sensitive, and specific detection of multiple nucleic acids in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification is used for nucleic acid detection, then detection sensitivity is improved, but errors and biases are introduced
Solution Approach 1:
The invention extracts only the necessary detection function from the amplification process. Instead of amplifying the entire nucleic acid target, the method excises and detects only specific short probe sequences (10-50 nucleotides) from the target nucleic acid using cutting reagents. This extraction approach maintains detection sensitivity while eliminating amplification-related errors and biases, as the detected probes directly represent the target without enzymatic amplification artifacts.
Solution Approach 2:
The invention uses probe sequences as simplified copies or representatives of the target nucleic acid. Rather than detecting the entire target molecule through amplification, short probe sequences are excised and serve as detectable copies. These probes contain the essential identification information while being small enough for direct detection without amplification, thus avoiding amplification errors while maintaining detection capability.
2Reliability
If traditional detection methods are used, then detection capability is achieved, but intensive sample preparation and labour intensive optimization are required
Solution Approach 1:
The invention segments the detection process into distinct functional modules: (1) cutting reagents that specifically excise probes from target nucleic acid, (2) capture probes that bind to excised probes, and (3) detection of the captured probes. This segmentation allows each module to be independently optimized and simplifies the overall workflow, reducing the need for intensive sample preparation and optimization compared to traditional qPCR methods.
Solution Approach 2:
The cutting reagents and capture probes are designed to automatically perform their functions based on sequence complementarity and specific binding. The system self-assembles and self-detects without requiring extensive manual optimization or skilled user intervention. The specificity of nucleic acid hybridization and enzymatic cutting provides built-in validation, reducing the need for labor-intensive optimization steps.
3Measurement precision
If amplification-based methods are used, then detection sensitivity is improved, but multiplexing capacity is limited
Solution Approach 1:
The invention creates a universal detection platform where multiple different target nucleic acids can be detected simultaneously using the same basic methodology. Different probe sequences can be excised from different targets and detected in a single reaction mixture using multiple capture probes with different labels. This multi-functional approach enables high-capacity multiplexing without the technical limitations that constrain amplification-based methods.
Solution Approach 2:
The invention uses different fluorescent labels or detectable markers on capture probes to distinguish between different target nucleic acids in multiplexed detection. Each target can be associated with a unique label or label combination, allowing simultaneous detection of multiple targets through their distinct optical or detectable signatures. This labeling strategy enables high-capacity multiplexing beyond the limitations of amplification-based methods.
4Measurement precision
If qPCR is used for RNA detection, then gold standard detection is achieved, but the method requires intensive sample preparation and labour intensive optimization
Solution Approach 1:
The invention replaces the complex mechanical and enzymatic amplification system of qPCR with a simpler direct detection system. Instead of using polymerase enzymes and thermal cycling for amplification, the method uses cutting reagents to excise probes and capture probes to bind them for direct detection. This substitution eliminates the need for intensive sample preparation and optimization while maintaining detection accuracy through specific probe-target recognition.
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 method allows for rapid, sensitive, and specific detection of nucleic acids, including viral RNA, with reduced errors and biases, and the ability to differentiate between closely related sequences, improving detection efficiency and reducing the need for skilled users.
Implementation Method 1
contacting the sample with a cutting reagent for excising a target probe from the target nucleic acid
Implementation Method 2
an enzyme suitable for cutting the target nucleic acid at sites hybridised to cutting oligonucleotides
Implementation Method 3
contacting the excise mixture with a nucleic acid carrier comprising a capture oligonucleotide that is complementary to the target probe; and detecting binding of the target probe to the capture oligonucleotide
Data Source
AI summary
This invention relates to methods for detecting the presence or absence of target nucleic acids in samples by excising and detecting specific target probes.