LACES Nucleic Acid Detection Without Temperature Cycling
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Solution Overview
Problem
Current nucleic acid detection methods, such as PCR, face challenges in achieving high specificity and efficiency, particularly in point-of-care settings, due to the need for temperature cycling and the generation of unwanted byproducts, which complicates the detection of viral infections like coronavirus.
Innovation Solution
The Luminescence Amplification By Continuous Elongation of DNA Strands (LACES) method uses a polymerase enzyme, ATP regenerating enzyme, and luminescence enzyme with a primer-probe to detect target nucleic acid sequences by generating luminescence through a pyrophosphate-based enzymatic loop, allowing for rapid and specific detection without temperature cycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to detect nucleic acid sequences, then the copy number of the target sequence is increased, but the process requires temperature cycling and generates unwanted byproducts that reduce specificity
Solution Approach 1:
The patent extracts and eliminates the temperature cycling step from the PCR process, using isothermal conditions instead. This removes the source of non-specific artifacts while maintaining amplification capability through strand displacement mechanisms, thereby improving detection specificity without sacrificing sensitivity.
Solution Approach 2:
The patent introduces strand displacement as an intermediary mechanism to enable amplification without traditional PCR cycling. The displacement-mediated synthesis allows for specific amplification under isothermal conditions, acting as a bridge between the need for amplification and the requirement for specificity.
2Reliability
If Tagman probes are used to improve specificity, then non-specific artifacts are reduced, but the complexity of the reaction increases due to additional probe requirements
Solution Approach 1:
The patent employs primers that perform multiple functions: they initiate amplification and simultaneously serve as displacement elements. This multi-functionality eliminates the need for separate Tagman probes while maintaining high specificity, thereby reducing reaction complexity without sacrificing reliability.
Solution Approach 2:
The patent merges the functions of primers and probes into a single primer component. The primer contains sequences that both initiate synthesis and enable specific target recognition through strand displacement, combining what were previously separate elements into one unified component.
3Loss of time
If rapid detection is achieved through simplified methods, then the detection time is reduced, but the ability to provide actionable information within the critical first hour is compromised
Solution Approach 1:
The patent performs preliminary amplification and detection steps under isothermal conditions that can be rapidly initiated and completed. The method is designed to provide preliminary actionable information within the critical first hour, enabling immediate clinical decision-making while maintaining the option for confirmatory testing.
Solution Approach 2:
The patent changes the temperature parameter from cyclic variations to a constant isothermal condition, enabling rapid reaction initiation and completion. This parameter change allows the reaction to proceed quickly at a single optimized temperature, reducing detection time while maintaining analytical performance.
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
LACES provides a rapid and specific detection of nucleic acid sequences with reduced need for copy number amplification, enabling early and effective identification of viral infections, improving point-of-care diagnostics by producing a detectable signal within minutes.
Implementation Method 1
carrying out nucleic acid elongation synthesis such that one or a plurality of nucleotide analogs are added sequentially to the template if the primer-probe hybridizes to the target nucleic acid sequence, whereby: a) a nucleotide analog associates with the polymerase, b) the nucleotide analog is incorporated on the template strand by the polymerase
Implementation Method 2
binding the ATP to a luminescence-enzyme, wherein a luminescence-substrate is catalyzed by the luminescence-enzyme to produce luminescence
Implementation Method 3
the leaving group is combined with an ATP-regenerating-enzyme-substrate by the ATP regenerating enzyme (e.g., with APS by ATP Sulfurylase; with ADP-glucose by AGPPase; with AMP+PEP by PPDK, and the like) yielding ATP
Data Source
AI summary
Provided herein are methods and systems for detecting the presence of absence of a target-nucleic acid sequence, including SARS-COV2.