Isothermal Nucleic Acid Detection with Lytic Agent Protection
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Solution Overview
Problem
Current nucleic acid amplification methods, such as PCR, require thermocycling and are time-consuming, making them unsuitable for rapid on-site testing and point-of-care diagnostics. Additionally, these methods are prone to inactivation by lytic agents and nucleases, which complicates the detection process.
Innovation Solution
The method involves contacting a sample with a lysis buffer containing lytic agents to release nucleic acids, followed by the addition of a reagent composition that includes protectants and amplification reagents. This composition allows for isothermal amplification and rapid detection of target nucleic acid sequences within 20 minutes without the need for thermocycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If thermocycling is used for nucleic acid amplification, then amplification can be achieved, but the process becomes time-consuming and requires specialized machinery
Solution Approach 1:
The patent changes the temperature parameter from cyclic variation to constant isothermal condition. The amplification reaction is performed at a single temperature (e.g., 65°C) rather than cycling through multiple temperatures, thereby eliminating the need for complex thermocycling machinery and reducing amplification time while maintaining amplification efficiency
Solution Approach 2:
The patent extracts and removes the thermocycling step from the amplification process. By using isothermal amplification methods such as LAMP or RPA, the complex temperature cycling machinery is eliminated, leaving only a simple heating device to maintain a constant temperature, thus reducing device complexity
2Productivity
If lytic agents are used to release nucleic acids, then nucleic acid extraction is achieved, but amplification reagents may be inactivated
Solution Approach 1:
The patent introduces an intermediary substance that is compatible with both lytic agents and amplification reagents. This intermediary buffer system allows the lytic agents to effectively release nucleic acids while simultaneously protecting the amplification reagents from inactivation, enabling the entire process to occur in a single tube without requiring reagent purification
Solution Approach 2:
The patent uses a composite buffer composition that combines multiple components with complementary functions. The buffer includes ingredients that stabilize amplification reagents against inactivation by lytic agents, creating a composite solution that performs multiple functions simultaneously: cell lysis, nucleic acid release, and reagent protection
3Measurement precision
If traditional PCR methods are used, then nucleic acid amplification is achieved, but the detection process takes too long for point-of-care diagnostics
Solution Approach 1:
The patent implements continuous amplification at a constant temperature without interruption for temperature cycling. The isothermal amplification process maintains continuous productive action throughout the reaction, eliminating the downtime associated with temperature transitions in PCR, thereby reducing total detection time while maintaining amplification efficiency and detection accuracy
Solution Approach 2:
The patent performs sample lysis and nucleic acid release in advance within the same reaction buffer before adding amplification reagents. This preliminary action prepares the sample in a state that is immediately ready for isothermal amplification, eliminating additional processing steps and reducing overall detection time for point-of-care applications
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 quick and efficient nucleic acid amplification and detection, preventing the inactivation of amplification reagents by lytic agents and inhibiting nuclease activity, thus facilitating rapid point-of-care diagnostics.
Implementation Method 1
the lysis buffer comprises one or more lytic agents capable of lysing biological entities to release sample nucleic acids comprised therein
Implementation Method 2
the reagent composition comprises one or more protectants and one or more amplification reagents
Implementation Method 3
amplifying a target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product
Data Source
AI summary
Disclosed herein include methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. The method can comprise the use of a lysis buffer comprising a lytic agent and/or a reducing agent for treating a sample and detecting the presence of a target nucleic acid sequence. In some embodiments, the method comprises contacting a reagent composition comprising amplification agents and one or more protectants (e.g., cyclodextrin compounds) capable of sequestering lytic agents with the treated sample to generate an amplification reaction mixture, for example under isothermal conditions, for detecting.