Microorganism Identification via Flow Cytometry and Microarray
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Solution Overview
Problem
Current methods are inadequate for analyzing microorganisms in human or animal microbiota to correlate with pathologies and evaluating potential therapies, especially for non-surgical diseases where common drugs or techniques like mass spectrometry are ineffective.
Innovation Solution
A method involving single cell flow cytometry, microarray chips with specific media, and various analytical techniques such as RT-PCR, PCR, antigen tests, and mass spectrometry to identify and characterize bacterial and fungal cells and molecules in fecal samples, including the use of antibodies, fluorescence enzymes, and phages to determine pathogenic microorganisms and their products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods like mass spectrometry are used to analyze microorganisms, then the analysis can be performed with standard equipment, but the methods are ineffective for identifying pathogenic microorganisms in microbiota and correlating them with pathologies
Solution Approach 1:
The method segments the complex task of microorganism identification into distinct stages: sample collection, DNA extraction, PCR amplification of 16S rRNA genes, and microarray hybridization. Each stage handles a specific aspect of the analysis, making the overall process more reliable for identifying pathogenic microorganisms while managing complexity through systematic division of labor
Solution Approach 2:
The invention introduces several intermediary elements: universal primers that mediate between diverse bacterial DNA and the PCR process, microarray probes that mediate between amplified DNA sequences and detection systems, and software intermediaries that mediate between raw hybridization data and pathological correlations. These intermediaries enable reliable identification without requiring direct complex interactions between all components
2Measurement precision
If multiple analytical techniques are employed to accurately identify microorganisms and their products, then the correlation with pathologies improves, but the complexity and time required for analysis increases
Solution Approach 1:
The method performs preliminary actions by using universal primers to pre-amplify conserved 16S rRNA regions before specific identification, and by pre-designing microarray probes for multiple potential pathogens. This preliminary preparation enables faster subsequent analysis while maintaining high precision in microorganism characterization and pathology correlation
Solution Approach 2:
The invention employs universal primers that can amplify 16S rRNA genes from diverse bacterial species simultaneously, and microarray platforms that can detect multiple different pathogens in a single experiment. This multi-functionality reduces the need for separate analyses for each potential pathogen, thereby decreasing total analysis time while maintaining measurement precision
3Measurement precision
If single cell flow cytometry and microarray techniques are used to separate and identify individual bacterial and fungal cells, then the accuracy of pathogenic microorganism detection improves, but the device complexity and operational difficulty increases
Solution Approach 1:
The method employs self-service mechanisms where fluorescently labeled antibodies or stains automatically bind to and label target microorganisms based on their surface characteristics, and where the microarray hybridization process automatically distinguishes between different DNA sequences. This reduces the need for complex manual manipulation while maintaining high detection accuracy
Solution Approach 2:
The invention uses fluorescent labels that produce color changes or fluorescence signals when bound to target microorganisms or DNA sequences. This visual differentiation simplifies the detection process by providing clear, easily distinguishable signals that reduce operational complexity while maintaining high measurement precision in identifying pathogenic microorganisms
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
Enables effective correlation of microorganisms with pathologies and evaluation of therapeutic options for non-surgical diseases, particularly where conventional treatments fail, by accurately identifying and characterizing microorganisms and their products in the microbiota.
Implementation Method 1
The sample is subjected to single cell flow cytometry by means of a flow cytometer with volumetric counting using an aqueous solution of water/PES as eluent
Implementation Method 2
chips also contain antibodies and/or fluorescence enzymes attached to the bottom of the chips and which recognize the molecules found previously
Implementation Method 3
identification will be carried out with the sandwich ELISA technique, with which the antigen or molecule will be captured
Implementation Method 4
in the case of fluorescence enzymes the evidence of the presence of the molecule will be provided by the fact that these enzymes change fluorescence when they bind the molecule
Implementation Method 5
provides the use of mass spectrometry to identify the molecules in the patient's serum (e.g. toxins)
Implementation Method 6
A second possible method provides the use of RT-PCR (Real Time, Polymerase Chain Reaction) on the genetic material of individual bacteria/fungi to examine the genome and consequently the species
Data Source
AI summary
A method for searching for pathogenic microorganisms inside the human or animal microbiota, comprising the steps of collecting a human or animal biological sample wherein to search for pathogenic microorganisms, treating said biological sample by flow cytometry to individually separate the bacterial cells and the fungi present thereinside, providing a microarray with chips or wells in each of which there is in suspension a first culture medium for bacteria and a second culture medium for fungi, analysing and/or treating the material collected in said culture media.