Fixed Bacillus subtilis Cells for Enzymatic Lysis Control
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Solution Overview
Problem
Existing molecular assays face challenges in using internal full process controls for enzymatic cell lysis, as traditional mechanical lysis methods are not feasible or desirable, particularly in automated processes, necessitating the development of controls for chemical or enzymatic cell disruption methods.
Innovation Solution
The use of fixed bacterial cells, such as Bacillus subtilis, which are susceptible to enzymatic lysis by achromopeptidase (ACP) and other enzymes, serves as internal controls to monitor the efficacy of enzymatic cell lysis and maintain stability throughout the assay process, allowing for the verification of all steps in molecular assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mechanical lysis methods are used for cell disruption, then cell lysis efficacy is improved, but adaptability to automated processes deteriorates
Solution Approach 1:
The patent replaces mechanical lysis methods (glass bead shearing) with enzymatic lysis using achromopeptidase. This substitution enables automated processing while maintaining effective cell lysis, as the enzymatic approach can be performed in solution without requiring mechanical agitation or specialized equipment.
Solution Approach 2:
The patent changes the lysis mechanism from mechanical force to biochemical reaction. By using enzymes that specifically target cell wall components, the process transitions from physical disruption to chemical degradation, enabling automation while preserving lysis efficacy through controlled enzymatic reactions.
2Stability of the object's composition
If fixed cells are used as controls, then stability during storage is improved, but susceptibility to lysis may deteriorate
Solution Approach 1:
The patent applies fixation to control cells in advance to stabilize them for long-term storage. The fixed cells are prepared beforehand and stored stable, then undergo enzymatic lysis during the actual assay. This preliminary stabilization allows the control material to be stored without degradation while maintaining its ability to be lysed when needed for the assay.
Solution Approach 2:
The patent uses an intermediary enzymatic treatment step that reverses the fixation effect during the assay. The achromopeptidase enzyme acts as a mediator that can penetrate the fixed cell structure and degrade the cell wall, effectively overcoming the stabilization provided by fixation and enabling successful lysis of the control cells.
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 provides a reliable and stable method for monitoring enzymatic cell lysis, ensuring the integrity and consistency of molecular assays by using fixed cells that remain lyse-resistant and maintain their susceptibility to ACP-driven lysis, even after extended storage, thereby ensuring accurate detection of nucleic acids.
Implementation Method 1
The use of fixed bacterial cells, such as Bacillus subtilis, which are susceptible to enzymatic lysis by achromopeptidase (ACP) and other enzymes
Implementation Method 2
providing a control for use with a molecular assay wherein the control comprises fixed cells
Data Source
AI summary
A full process control for use with a molecular assay and a method of determine the efficacy of the molecular assay. A full process control can include a fixed cell, and specifically can include a fixed vegetative cell. A method of determining the efficacy of a molecular assay can include providing an internal control, mixing the internal control with a sample, lysing the internal control and the sample, and detecting the lysis product. The full process control and/or the internal control can be Bacillus subtilis cells.