Lyophilized Reagent Bead Layout for Closed AST and Amplification
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Solution Overview
Problem
Current bacterial culture methods for antimicrobial susceptibility testing (AST) are time-consuming and contribute to the rise of antimicrobial resistance (AMR), while nucleic acid amplification methods provide limited phenotypic susceptibility information, leading to inaccurate antibiotic administration.
Innovation Solution
A method using lyophilized reagent beads embedded in a reaction vessel with a phase change material, allowing spatial separation and temporal control of AST and amplification assays, enabling simultaneous phenotypic susceptibility testing and amplification in a closed system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bacterial culture methods are used for AST, then phenotypic susceptibility information can be obtained, but the test time is prolonged and contributes to antimicrobial resistance
Solution Approach 1:
The reaction vessel is divided into distinct layers: a first layer containing the amplification bead embedded in phase change material, and a second layer containing the antibiotic bead. This segmentation allows separate optimization of conditions for nucleic acid amplification and bacterial phenotypic testing, enabling rapid DNA extraction and amplification while maintaining phenotypic susceptibility assessment capabilities
Solution Approach 2:
The invention merges nucleic acid amplification and phenotypic AST into a single integrated reaction vessel and workflow. Both the amplification bead (containing polymerase, primers, dNTPs) and antibiotic bead coexist in the same vessel, allowing simultaneous or sequential execution of both assays without transferring samples between separate systems
2Loss of time
If nucleic acid amplification methods are used, then test time is reduced, but phenotypic susceptibility information is limited or inaccurate
Solution Approach 1:
The amplification bead is pre-prepared and embedded in the first layer of the reaction vessel before the actual test. This preliminary preparation includes embedding the bead containing polymerase, primers, and other amplification reagents in phase change material, so that when the sample is added, amplification can immediately proceed without preparation delays
Solution Approach 2:
The phase change material acts as an intermediary that controls the release and activation of the amplification bead. The material undergoes phase transition at a specific temperature, which triggers the release of amplification reagents from the bead into the reaction medium, thereby controlling the timing and efficiency of the amplification process
3Device complexity
If multiple reagents are placed in the same reaction vessel, then device complexity is reduced, but reagent stability and cross-contamination become problematic
Solution Approach 1:
Different regions of the reaction vessel are assigned different functions and conditions: the first layer contains amplification reagents optimized for nucleic acid amplification, while the second layer contains antibiotic reagents optimized for phenotypic testing. The phase change material creates a local environment that protects and stabilizes the amplification bead until activation is required
Solution Approach 2:
The amplification reagents are extracted from the liquid phase and incorporated into a solid amplification bead that is further embedded in phase change material. This extraction from the conventional liquid reagent format provides physical protection, reduces cross-contamination risk, and maintains reagent stability during storage and transport
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
Facilitates rapid phenotypic antimicrobial susceptibility testing and amplification in a single reaction vessel, reducing test time and minimizing user interaction, while maintaining reagent stability and accuracy.
Implementation Method 1
the first layer comprises a phase change material having a melting point temperature... increasing a temperature of the contents of the reaction vessel to be above the melting point temperature to melt the phase change material
Implementation Method 2
such that the amplification bead dissolves in the liquid layer
Implementation Method 3
adding a bacterial sample to the reaction vessel to contact the antibiotic bead in a second layer to at least partially dissolve the antibiotic bead
Data Source
AI summary
The present disclosure is directed towards using lyophilized reagent beads for a fully closed multi-step reaction to complete antibiotic susceptibility tests and amplification assays. The lyophilized reagent beads include a lyophilized amplification reagent bead and an lyophilized antibiotic bead. The lyophilized reagent bead may be embedded in solid wax layer inside the test tube to enable cell growth in antibiotic prior to release of amplification reagents. A microfluidic manifold device may be used to insert the sample into the amplification tube and enable a fully closed system.