T-Structure Invasive Cleavage Assays for Broad Dynamic Range Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection systems face limitations in dynamic range, struggling to detect low concentrations of targets effectively while avoiding saturation at high concentrations, requiring multiple systems and increasing costs and labor in biological specimen analysis.
Innovation Solution
The method involves using multiple probes with different binding affinities and concentrations to hybridize with viral nucleic acids in a single reaction vessel, generating signals through invasive cleavage assays and amplification methods like PCR, allowing for the detection of low copy numbers of viral sequences and expanding the dynamic range of detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single detection system is used, then device complexity is reduced, but measurement precision deteriorates at extreme concentrations
Solution Approach 1:
The detection system is segmented into multiple detection channels, each optimized for specific concentration ranges. The method divides the detection task across multiple probes with different affinities, allowing each segment to excel at its designated range while collectively covering the full dynamic range from 10 to 10^9 copies/mL.
Solution Approach 2:
The invention creates a universal detection system that can handle multiple concentration ranges within a single assay. By incorporating probes with varying affinities and using competitive hybridization mechanisms, the system achieves multi-functionality, serving both low-copy-number detection and high-concentration quantification without requiring separate specialized systems.
2Measurement precision
If multiple detection systems are used to cover different concentration ranges, then measurement precision is improved, but device complexity increases
Solution Approach 1:
Multiple detection capabilities are merged into a single integrated system. The invention combines probes with different affinities, multiple reporter sequences, and competitive hybridization mechanisms into one cohesive assay that simultaneously provides precision across the full dynamic range, eliminating the need for separate detection systems.
Solution Approach 2:
The system employs dynamic probe selection through competitive hybridization. Depending on the target concentration, different probes naturally dominate the hybridization equilibrium, allowing the system to dynamically adapt its detection mechanism. At low concentrations, high-affinity probes prevail; at high concentrations, lower-affinity probes become dominant, providing precision across ranges without mechanical system changes.
3Measurement precision
If multiple detection systems are used sequentially, then measurement precision is improved, but productivity decreases
Solution Approach 1:
The detection process maintains continuous useful action by performing all detection steps in a single parallel reaction mixture. Instead of sequentially running multiple assays, the invention enables simultaneous detection across all concentration ranges in one continuous process, maintaining precision while eliminating the time losses associated with sequential testing and sample handling between assays.
4Measurement precision
If multiple detection systems are used, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The system performs preliminary differentiation of target concentrations through competitive hybridization kinetics. By designing probes with predetermined affinity differences and using a competitive assay format, the system pre-establishes detection pathways that automatically resolve the appropriate concentration range during the initial hybridization phase, eliminating the need for sequential testing and reducing total detection time while maintaining precision.
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 the detection of viral nucleic acids across a broader range, reducing the need for multiple systems, decreasing costs, and improving the efficiency of nucleic acid detection in biological specimens by effectively handling low and high concentrations within a single assay.
Implementation Method 1
the first probes hybridize to first regions of the target nucleic and are cleaved by the cleavage agent
Implementation Method 2
the first probes hybridize to first regions of the target nucleic and are cleaved by the cleavage agent thereby generating first 5′ cleaved portions
Implementation Method 3
the first and second regions of the target nucleic acid are amplified prior to step a)
Data Source
AI summary
The present invention relates to systems, methods and kits for low-level detection of nucleic acids, detecting at least two different viral sequences in a single reaction vessel, and increasing the dynamic range of detection of a viral target nucleic acid in a sample. The present invention also relates to T-structure invasive cleavage assays, as well as T-structure related target dependent non-target amplification methods and compositions. The present invention further relates to methods, compositions, devices and systems for consistent nucleic acid dispensing onto surfaces.