Isothermal DNA Amplification for Living Dead Cell Differentiation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for differentiating between living and dead cells in biological samples are either not sensitive, quick, or cost-effective, and often fail to accurately quantify viable cells due to issues like DNA intercalator residues, extensive sample preparation, and the inability to detect viable but non-culturable bacteria.
Innovation Solution
A method involving isothermal DNA amplification reactions that do not impair cell membrane integrity, using recombinase-polymerase-amplification (RPA) at temperatures below 50°C, allowing for direct DNA amplification from untreated samples with intact cells, and subsequent quantification using fluorescence-labeled probes or SYBR Green I, enabling the differentiation between living and dead cells without cell proliferation or expensive equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard PCR is used to detect DNA in biological samples, then the presence of bacteria can be detected, but differentiation between living and dead cells cannot be achieved since both DNA of living and dead cells is amplified
Solution Approach 1:
The patent applies local quality by making the DNA amplification reaction selective for specific locations (dead cells with compromised membranes) rather than amplifying all DNA uniformly. The isothermal amplification reaction penetrates only into dead cells where the membrane is compromised, allowing selective amplification of DNA from dead cells while leaving living cells unaffected.
Solution Approach 2:
The patent changes the temperature parameter from standard PCR (cycling through high temperatures that denature DNA and require complex thermal cycling) to isothermal conditions (constant temperature below 50°C). This parameter change enables selective DNA amplification in dead cells without impairing the membrane integrity of living cells, achieving differentiation between viable and non-viable cells.
2Measurement precision
If EMA/PMA intercalators are used to differentiate living and dead cells, then selective detection of dead cell DNA is possible, but the precision of results is diminished due to intercalator residues that inhibit PCR reactions
Solution Approach 1:
The patent removes the harmful intercalator substances (EMA/PMA) from the detection system entirely. Instead of using these chemical agents that leave inhibitory residues, the method relies on the natural physical state of cell membranes (intact vs. compromised) to enable selective DNA amplification, eliminating the need for additional extraction steps to remove intercalator residues.
Solution Approach 2:
The patent uses the cell membrane itself as an intermediary that naturally distinguishes between living and dead cells. The compromised membrane of dead cells acts as a passive gateway that allows the isothermal amplification reagents to access and amplify DNA, while the intact membrane of living cells prevents reagent penetration. This eliminates the need for chemical intercalators as mediators.
3Measurement precision
If EMA/PMA-PCR methods are used for quantitative analysis, then living and dead cells can be differentiated, but extensive and time-consuming sample preparation is required including treatment in the dark, light inactivation, and DNA isolation
Solution Approach 1:
The patent eliminates the need for preliminary treatment steps (incubation in the dark, light inactivation, DNA extraction) by using an isothermal amplification reaction that can be performed directly on untreated samples. The method is designed to work with intact cells in their native state, performing the differentiation and amplification in a single step without prior preparation.
Solution Approach 2:
The method allows the sample to serve itself by utilizing the natural properties of cell membranes (intact vs. compromised) to enable selective DNA amplification without requiring external intervention for cell lysis, DNA extraction, or intercalator removal. The isothermal amplification reagents automatically penetrate only dead cells and amplify their DNA in situ.
4Quantity of substance
If traditional PCR methods are used, then DNA amplification can be achieved, but expensive real-time thermocyclers are necessary which may be a problem for small laboratories
Solution Approach 1:
The patent replaces the complex mechanical thermal cycling system (thermocycler with heating/cooling mechanisms) with a simple isothermal reaction system. The isothermal amplification reaction proceeds at a constant temperature below 50°C, eliminating the need for repeated heating and cooling cycles, thereby replacing complex thermal mechanics with a simpler constant-temperature chemical reaction.
Solution Approach 2:
The patent changes the temperature parameter from dynamic thermal cycling (PCR requiring temperatures between 95°C and 50°C) to a static low temperature (isothermal conditions below 50°C). This parameter change simplifies the equipment requirements from expensive thermocyclers to simple incubation devices or even room temperature environments, making the method accessible to small laboratories.
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 provides a quick, sensitive, and cost-effective means to quantify living and dead cells, maintaining cell viability during amplification and allowing for accurate differentiation within 10-30 minutes without the need for expensive thermocyclers, while avoiding the limitations of traditional PCR methods.
Implementation Method 1
carrying out a DNA amplification reaction with the aliquots provided in steps (a) and (b), respectively, wherein the DNA amplification reaction does not impair the membrane integrity of living cells
Implementation Method 2
subsequent quantification using fluorescence-labeled probes or SYBR Green I
Data Source
AI summary
The present invention relates to a method for quantitatively determining living and dead cells in a biological sample. The method according to the present invention is based on the determination of the amount of DNA in the sample with the aid of a DNA amplification reaction which does not impair the membrane integrity of living cells.