Selective Lysis of Eukaryotic Cells in Blood Samples
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Solution Overview
Problem
Current methods for detecting pathogens in blood samples are hindered by the high concentration of eukaryotic cells, which interfere with DNA-based detection methods, increase sample viscosity, and form complexes that prevent filtration, making it challenging to detect low concentrations of microorganisms without extensive sample manipulation or purification.
Innovation Solution
A method involving the selective lysis of eukaryotic cells using a non-ionic detergent and alkaline buffer at a pH of 9.5 or higher, followed by filtration through a 0.7 μm or smaller pore size filter, allowing for the retention of microorganisms while minimizing sample dilution and enzymatic or heat-related processes, and subsequent neutralization to facilitate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If large volumes of blood are used for pathogen detection, then the detection sensitivity is improved, but the sample viscosity increases and filtration becomes difficult
Solution Approach 1:
The invention extracts and removes eukaryotic cells from the blood sample through selective lysis, separating the interfering cellular material from the target pathogens. This extraction step reduces the background DNA and viscosity while maintaining the integrity of bacterial cells for subsequent detection, directly resolving the contradiction between detection sensitivity and filtration ease.
2Measurement precision
If eukaryotic cells are lysed to remove background DNA, then detection sensitivity is improved, but proteins and membranes form complexes that prevent filtration
Solution Approach 1:
The invention changes the pH parameter to alkaline conditions (pH 9.5-11.5) to achieve selective lysis of eukaryotic cells while preserving bacterial cell integrity. This parameter change enables differential treatment of cell types, allowing background DNA removal without forming filtration-blocking complexes, thus resolving the contradiction between detection sensitivity and filtration ease.
3Measurement precision
If traditional lysis methods are used, then bacterial DNA is released for detection, but human DNA interference increases and PCR activity decreases
Solution Approach 1:
The invention applies local quality by treating different cell types differently through selective lysis. Bacterial cells are preserved with intact DNA for detection, while eukaryotic cells are selectively lysed to remove their DNA. This spatial and chemical differentiation resolves the contradiction between bacterial DNA detection and human DNA interference.
4Measurement precision
If extensive DNA purification is performed to remove mammalian DNA, then detection accuracy is improved, but the manipulation time and complexity increase
Solution Approach 1:
The invention performs preliminary action by removing eukaryotic cells and their DNA through selective lysis before the PCR detection step. This pre-cleanup approach eliminates the need for extensive post-sample purification steps, reducing manipulation time while maintaining detection accuracy. The selective lysis creates a pre-conditioned sample that is ready for direct PCR amplification.
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 enables the efficient detection of microorganisms in large volumes of blood with minimal sample manipulation, reducing background DNA interference and maintaining bacterial integrity, thus allowing for the processing of larger sample volumes and automated procedures.
Implementation Method 1
adding a non-ionic detergent and a buffer to the sample to obtain a solution with a pH of about 9.5 or more, and incubating the solution for a time period sufficiently long to lyse the eukaryotic cells
Implementation Method 2
a method involving the selective lysis of eukaryotic cells using a non-ionic detergent and alkaline buffer at a pH of 9.5 or higher
Implementation Method 3
followed by filtration through a 0.7 μm or smaller pore size filter, allowing for the retention of microorganisms
Data Source
AI summary
Methods and devices for detection of micro-organisms present or suspected to be present within a mammalian blood sample are provided. A selective lysis is obtained by incubating the sample in a non-ionic detergent under alkaline conditions.