Stabilized FISH Control Preparation for Microbial Detection
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Solution Overview
Problem
Current methods for detecting biofilms in microbiological pathology face challenges such as the inability to accurately depict complex ecological conditions in the human body, lack of standardized control preparations for fluorescence in situ hybridization (FISH) probes, and the instability of RNA, leading to reproducibility issues and difficulties in maintaining the cold chain for shipping.
Innovation Solution
A control preparation comprising section bodies joined by a polymer, with each section body containing a 100% identical and a nearly identical nucleic acid sequence for positive and negative controls, respectively, allowing for specific binding of FISH probes and eliminating nonspecific binding, and being stable at room temperature for extended storage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If control preparations are stored at -20°C in fixation solution, then RNA stability is improved, but the complexity of storage and shipping increases due to cold chain requirements
Solution Approach 1:
The patent changes the storage temperature parameter from -20°C to room temperature by incorporating stabilizing agents (such as borax, sodium azide, and specific buffer compositions) into the control preparation formulation. This allows the RNA-containing control preparations to maintain stability without requiring cold chain storage, thereby resolving the contradiction between RNA stability and storage complexity
Solution Approach 2:
The patent introduces stabilizing agents as intermediary substances that mediate between the RNA and the storage environment. These agents (including borax, sodium azide, and specific buffer components) protect the RNA from degradation while allowing storage at room temperature, thus eliminating the need for complex cold chain storage
2Reliability
If control strains are kept in culture by users, then fresh control material is available, but the difficulty of maintaining culture increases particularly for hard-to-culture oral flora
Solution Approach 1:
The patent performs preliminary actions by preparing and stabilizing control preparations in advance under optimized conditions, then distributing them in a stable form that does not require further culturing by the end user. The control strains are fixed, embedded, and stabilized before distribution, eliminating the need for users to maintain complex cultures of hard-to-culture organisms
Solution Approach 2:
The patent creates stable copies or representations of the control strains in a fixed and embedded state that retain the necessary nucleic acid sequences for FISH probe validation. These copied control preparations can be stored and transported without active culture, yet still serve their intended purpose of validating FISH probe specificity and sensitivity
3Adaptability or versatility
If ad-hoc control preparations are prepared, then specific experimental needs are met, but the shelf life decreases due to RNA instability
Solution Approach 1:
The patent changes the chemical parameters of the control preparation formulation by incorporating specific stabilizing agents (borax, sodium azide, optimized buffer compositions) that extend the shelf life from days to years while maintaining RNA integrity. This allows control preparations to be prepared in advance and stored stably without requiring frequent re-preparation
Solution Approach 2:
The patent performs preliminary stabilization and preservation actions during the control preparation manufacturing process, embedding the control strains in stabilizing matrices and adding protective agents before distribution. This preliminary action ensures long-term stability without requiring the user to prepare fresh controls frequently
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 routine FISH analysis by providing stable, standardized control preparations that can be stored at room temperature, reducing the need for refrigeration and enabling efficient detection and visualization of biofilms and microorganisms, thereby improving diagnostic accuracy and reproducibility.
Implementation Method 1
The principle of FISH is based on oligonucleotide probes coupled to a fluorescent dye. These probes bind, in dependency of the sequence match, to bacterial sequences
Implementation Method 2
control preparation comprising target sequences for fluorescence in situ hybridization (FISH) probes encased in a polymer body
Data Source
AI summary
The invention relates to a control preparation (e.g. comprising microorganism control cells embedded in a cold polymerising resin or plastic) for use in a method for detection of a target microorganism in microbiological pathology, comprising a plurality of section bodies joint by a joining polymer, wherein each section body comprises a matrix polymer (e.g. a cold polymerising resin or plastic). A first section body comprises a nucleic acid sequence specific for the target microorganism (e.g. microorganism cells used as positive control). A second section body comprises a second nucleic acid sequence, which in comparison to said first nucleic acid sequence, contains a deletion, an additional nucleoside or a different nucleoside in one, two, three or four positions of said second nucleic acid sequence and which does not hybridize to said first nucleic acid sequence under stringent conditions (e.g. cells from a related microorganism having 1-4 mismatches as compared to the target region of a FISH probe for the target microorganism). The invention further relates to methods for detection and discrimination of two microorganism species of the same genus in tissue sections, and to kits for use in this method.