Nucleic Acid Detection via Nanopore Ligation
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Solution Overview
Problem
Current methods for detecting nucleic acid sequences, particularly for identifying pathogens, are time-consuming, lack specificity and sensitivity, and require PCR amplification, making them unsuitable for rapid and cost-effective on-site detection.
Innovation Solution
The method employs probe hybridization and enzymatic ligation to produce single-molecule identifiers that can be detected using solid-state nanopores, eliminating the need for PCR amplification and enabling rapid identification of nucleic acid sequences with high specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional microbiology techniques (cell culturing) are used for pathogen identification, then comprehensive pathogen detection is achieved, but the detection time is extended to days or weeks
Solution Approach 1:
The patent extracts the essential detection function from traditional multi-day culturing processes by using targeted probe hybridization to directly detect specific pathogen nucleic acid sequences, eliminating unnecessary cultivation steps while maintaining detection accuracy
Solution Approach 2:
The patent performs preliminary enrichment and concentration of target nucleic acid sequences before detection, allowing rapid specific detection within 15 minutes without requiring extended culturing periods for pathogen amplification
2Loss of time
If antibody based assays are used for rapid pathogen detection, then detection speed is improved, but sensitivity and specificity are reduced
Solution Approach 1:
The patent replaces antibody-based detection mechanisms with nucleic acid probe hybridization and enzymatic ligation systems, achieving both rapid detection and high sensitivity/specificity through sequence-specific molecular recognition rather than protein-antigen binding
Solution Approach 2:
The patent changes the detection parameter from antibody-antigen binding to nucleic acid hybridization with subsequent enzymatic amplification, enabling simultaneous achievement of rapid detection and high sensitivity through controlled thermal cycling and enzymatic reactions
3Measurement precision
If PCR amplification is used for nucleic acid detection, then sensitivity is improved, but the process complexity and cost are increased
Solution Approach 1:
The patent extracts the essential amplification function from complex PCR systems by using targeted enzymatic ligation of probes to target sequences, achieving sufficient signal amplification through simpler isothermal enzymatic reactions rather than cyclic thermal amplification
Solution Approach 2:
The patent employs disposable probe cartridges with pre-loaded nucleic acid probes and enzymatic reagents that can be discarded after single use, eliminating the need for expensive, complex PCR instrumentation while achieving comparable sensitivity through simplified chemistry
4Adaptability or versatility
If probe hybridization arrays are used for broad spectrum nucleic acid detection, then detection coverage is improved, but sensitivity is limited to 10^5 to 10^6 target molecules
Solution Approach 1:
The patent merges probe hybridization with enzymatic ligation and signal amplification steps, combining the broad coverage capability of hybridization arrays with the high sensitivity of enzyme-mediated signal amplification to achieve detection of low copy number targets across multiple pathogen types
Solution Approach 2:
The patent introduces enzymatic ligation as an intermediary step between probe hybridization and detection, where the ligase enzyme mediates signal amplification by catalyzing the formation of ligated probe products that can be detected with high sensitivity, bridging the gap between hybridization coverage and detection sensitivity
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 and accurate detection of nucleic acid sequences in under 15 minutes, with high specificity and sensitivity, and is suitable for portable, integrated systems, reducing costs and increasing multiplexing capabilities.
Implementation Method 1
contacting the sample with a capture probe and a terminal probe under conditions that provide for selective hybridization of the capture probe to a first portion of the target nucleic acid sequence, and transient hybridization of the terminal probe to a second portion of the target nucleic acid sequence
Implementation Method 2
ligating the capture probe and the terminal probe to form a target identifier
Implementation Method 3
detecting the target identifier
Data Source
AI summary
A method for the rapid identification of a target nucleic acid sequence is provided, as well as corresponding devices, products and kits. Such methods are useful for the rapid detection, identification and/or quantification of target nucleic acid sequences associated with, for example, a pathogen.