Microbial Cell Isolation from Blood via Selective Lysis and Adsorption
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Solution Overview
Problem
Current methods for isolating microbial cells from blood samples are inefficient, particularly in detecting low concentrations of microorganisms in the presence of high levels of eukaryotic cells, which can lead to inappropriate antimicrobial therapy and increased antibiotic resistance.
Innovation Solution
The method involves providing an aqueous solution with a blood sample, adding selective lysis reagents to lyse eukaryotic cells while preserving microbial cells, and using an insoluble solid surface with polymers and salts to displace microbial cells, followed by protease treatment to elute and isolate the microbial cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional enzymatic lysis methods are used to extract microbial DNA from blood samples, then DNA can be obtained for PCR amplification, but the high concentration of eukaryotic cells and their DNA strongly competes with the limited microbial DNA, decreasing detection sensitivity
Solution Approach 1:
The patent segments the lysis process into two distinct stages: first selective lysis of eukaryotic cells using non-ionic detergents at controlled pH and temperature, then lysis of microbial cells. This segmentation allows sequential removal of eukaryotic DNA before microbial DNA extraction, eliminating competitive inhibition and improving detection sensitivity
Solution Approach 2:
The patent performs preliminary selective lysis of eukaryotic cells before microbial cell lysis. By pre-removing eukaryotic cellular material and DNA through controlled detergent treatment and filtration, the method prepares the sample in advance to eliminate interference with subsequent microbial DNA detection
2Loss of time
If broad-spectrum antibiotics are administered empirically before pathogen identification, then immediate treatment can be provided, but inappropriate antibiotic use increases antibiotic resistance burden
Solution Approach 1:
The patent replaces the mechanical/cultural method of pathogen identification (traditional culture requiring days) with a molecular detection system using PCR and specific probes. This substitution enables rapid identification of the causative pathogen within hours, allowing targeted antibiotic selection and avoiding the need for prolonged empiric broad-spectrum therapy that drives resistance
3Quantity of substance
If the entire blood sample is processed for DNA extraction, then all microbial DNA can be recovered, but the large volume of non-microbial DNA and cellular components strongly decreases PCR reaction efficiency
Solution Approach 1:
The patent extracts and removes non-microbial cellular components (eukaryotic DNA, proteins, fats) through selective lysis and filtration steps before microbial DNA extraction. By taking out these interfering substances, the method preserves PCR reaction efficiency while recovering microbial DNA from the entire blood sample volume
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 effectively isolates microbial cells from blood samples, even at low concentrations, allowing for rapid and accurate diagnosis of sepsis and reducing the risk of inappropriate antimicrobial therapy.
Implementation Method 1
adding a first selective lysis reagent to the aqueous solution for a time period sufficient to lyse most of the higher eukaryotic erythrocytes and release hemoglobin from the erythrocytes, wherein the integrity of at least most microbial cells and higher eukaryotic leukocytes in the blood sample are preserved
Implementation Method 2
adding to the aqueous solution an insoluble solid surface, and at least one of a water soluble polymer and an inorganic salt in a quantity sufficient to cause the blood plasma, the eukaryotic lysed blood cells' components and microorganisms to displace onto the solid surface
Implementation Method 3
eluting microbial cells from the proteins coating the solid surface by incubating the sample with a basic solution comprising protease
Data Source
AI summary
This disclosure relates to methods for isolating bacterial cells, fungal cells, and single-celled parasites present in a blood sample containing higher eukaryotic cells; particularly wherein the microorganisms are present at a concentration significantly lower than the eukaryotic cells in the sample.