Liquid-Phase FISH Microorganism Detection via Flow Cytometry
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Solution Overview
Problem
Current methods for detecting microorganisms, such as fluorescence in situ hybridization (FISH), require extensive rinsing steps, well-trained personnel, and are not suitable for high-throughput analysis, limiting the number of samples that can be processed quickly and accurately.
Innovation Solution
A method involving fluorescence in situ hybridization in the liquid phase using a flow-through cytometer, where fluorescence-marked and quencher-marked nucleic acid probes are used in a single reaction vessel, eliminating the need for rinsing steps and allowing direct quantification of microorganisms, with the quencher probe quenching non-specific fluorescence signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If classic FISH technology is used with microscope detection, then specific detection of microorganisms is achieved, but the analysis requires well-trained personnel and considerable time, severely limiting the number of samples that can be analyzed per day
Solution Approach 1:
The patent replaces the mechanical microscope-based detection system with an automated flow cytometer system. The flow cytometer uses optical detection (light scattering and fluorescence) to automatically measure and analyze cells in liquid suspension, eliminating the need for manual microscope observation and significantly increasing throughput while maintaining detection accuracy
Solution Approach 2:
The patent transfers the FISH hybridization protocol from solid substrate (slide) to liquid phase, creating a liquid-phase copy of the classic FISH method. This allows the same specific detection chemistry to be performed in a format compatible with automated flow cytometry, enabling high-throughput analysis without losing specificity
2Productivity
If liquid phase FISH with flow-through cytometer is used, then productivity is improved and automated evaluation is enabled, but rinsing steps are required which increase work effort and require well-instructed laboratory personnel
Solution Approach 1:
The patent extracts and removes the rinsing step from the liquid-phase FISH protocol. Instead of requiring manual rinsing to remove unbound probes, the method uses a combination of optimized hybridization conditions and automated flow cytometry with appropriate gating strategies to distinguish specific from non-specific binding, eliminating this labor-intensive step
Solution Approach 2:
The patent enables the system to automatically differentiate between specific and non-specific fluorescence signals through flow cytometer gating and data analysis algorithms. The automated evaluation system performs the discrimination function that would otherwise require trained personnel visual inspection, making the process self-sufficient and easier to operate
3Measurement precision
If centrifugation is used to remove hybridization and rinsing solutions, then detection specificity is improved, but a further processing parameter is introduced that requires standardization and affects result assessment
Solution Approach 1:
The patent maintains cells in continuous liquid suspension throughout the entire FISH and detection process, eliminating the discontinuous centrifugation step. This continuous liquid-phase approach allows direct loading onto the flow cytometer without interruption, maintaining signal integrity while simplifying the protocol and reducing sources of variability
4Extent of automation
If fluorescence in situ hybridization is performed in liquid phase with flow-through cytometer, then automated evaluation and high throughput are achieved, but non-specific autofluorescence occurs that is problematic for evaluation
Solution Approach 1:
The patent uses cell-specific parameters measured by the flow cytometer (such as forward scatter for cell size and side scatter for granularity) to locally distinguish specific fluorescent signals from non-specific autofluorescence. By analyzing multiple parameters simultaneously and applying appropriate gating strategies, the system can identify and analyze only those events representing truly positive hybridization events, filtering out autofluorescent background
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 method enables rapid, specific, and objective detection of microorganisms with reduced technological complexity, suitable for high-throughput analysis, and provides precise quantification without the need for extensive rinsing or trained personnel.
Implementation Method 1
The execution of the method comprises the following steps: (d) bringing the fixed cells in contact with a solution of a fluorescence marked nucleic acid probe that is specific for the microorganism that is to be detected
Implementation Method 2
wherein the quencher-marked nucleic acid probe comprises a quencher that at least partially quenches the fluorescence of the fluorescence-marked nucleic acid probe
Implementation Method 3
placing the second reaction mixture in a flow-through cytometer after step (g), and detecting the fluorescence emitted from the cells of the microorganism that is to be detected containing the fluorescence-marked nucleic acid probe
Data Source
AI summary
The invention relates to a method for the specific detection of a microorganism or a group of microorganisms via in situ hybridisation by means of flow cytometry.