GelRed DNA Intercalator for Live Bacteria PCR Detection
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Solution Overview
Problem
Current DNA-based diagnostics and PCR methods fail to accurately distinguish between live and dead bacteria, leading to overestimation of live cells and potential false alarms, especially in cases of 'super-bug' antibiotic-resistant bacteria and bio-threats, as they cannot differentiate between viable and non-viable organisms.
Innovation Solution
The method involves using GelRed™, a DNA-intercalating chemical that selectively penetrates dead bacteria, inhibiting PCR amplification, allowing only live bacteria DNA to be amplified and detected through PCR, thereby differentiating between live and dead microbes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA-based diagnostics or PCR methods are used to detect bacteria, then detection speed and sensitivity are improved, but the ability to distinguish between live and dead bacteria deteriorates because DNA persists after cell death
Solution Approach 1:
The patent introduces an intermediary substance (DNA-binding chemical such as PMA, EMA, or GelRed) that selectively binds to DNA from dead bacteria but not from live bacteria. This intermediary acts as a mediator that differentiates between viable and non-viable cells by its selective penetration and binding properties, allowing PCR to subsequently detect only live bacterial DNA.
Solution Approach 2:
The patent changes the chemical state or binding properties of DNA through the introduction of DNA-binding chemicals. By altering the DNA's accessibility or chemical state through selective binding, the method enables differentiation between live and dead bacteria. The parameter change involves the transition from unbound DNA (detectable by PCR) to bound DNA (inhibitable by PCR) based on cell viability.
2Measurement precision
If traditional culture-based methods are used to determine bacterial viability, then the ability to distinguish live and dead bacteria is improved, but detection time and productivity deteriorate
Solution Approach 1:
The patent merges two previously separate methods: the viability differentiation capability of culture-based methods and the rapid detection capability of PCR. By combining DNA extraction with selective DNA-binding chemical treatment and PCR amplification, the method achieves both accurate viability distinction and rapid results within hours rather than days.
Solution Approach 2:
The patent replaces the mechanical/biological process of bacterial cultivation (growth on media plates) with a chemical and molecular biology-based system. Instead of waiting for bacteria to grow and form colonies, the method uses chemical binding and molecular amplification to rapidly detect viable bacteria, substituting a slow biological process with a faster chemical-molecular process.
3Measurement precision
If DNA-intercalating chemicals like PMA or EMA are used to detect only viable bacteria, then measurement precision is improved, but cost and safety deteriorate due to toxicity and handling requirements
Solution Approach 1:
The patent employs disposable, non-toxic alternatives to hazardous chemicals. GelRed, for example, is a safer, less toxic DNA-binding chemical that can be used without special handling precautions. This principle replaces expensive, toxic, and hazardous materials with cheaper, safer alternatives that achieve the same functional goal of selective DNA binding.
Solution Approach 2:
The patent converts a potentially harmful situation (use of toxic chemicals) into a beneficial one by selecting DNA-binding chemicals with favorable safety profiles. By choosing chemicals like GelRed that are less toxic and easier to handle, the method maintains its ability to differentiate viable bacteria while eliminating the safety hazards and cost burdens associated with highly toxic alternatives.
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 provides a rapid, accurate, and cost-effective means to detect live microbes, reducing false alarms and ensuring safety in food and environmental samples by distinguishing between viable and non-viable bacteria, with GelRed™ being safer and less toxic than existing alternatives like PMA and EMA.
Implementation Method 1
GelRedTM would have intercalated into the DNA, which inhibits a PCR amplification reaction
Implementation Method 2
GelRedTM does not penetrate the cell membrane of the live microbes but when the DNA is extracted and subjected to PCR amplification
Data Source
AI summary
The invention relates to a method of determining whether a live microbe, such as bacteria, is present in a test sample.