Multiplexed Fuel DNA Analysis for Rapid Microbial Detection
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Solution Overview
Problem
Existing DNA-based methods for detecting microbial contamination in fuels are complex, time-consuming, and limited in their ability to identify a broad spectrum of microbes, requiring significant manual manipulation and expertise, and are not suitable for rapid, field-deployable assays that can provide actionable results within two hours.
Innovation Solution
The development of multiplexed Rapid DNA assays that can be conducted by non-technical users, capable of simultaneously identifying multiple microbial species and strains in various fuel types, using a combination of primer pairs targeting 16S, 18S, mcrA, and dsrB genes, and employing microfluidic chips and rapid thermal cyclers for rapid analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional DNA-based methods are used for detecting microbial contamination in fuels, then detection accuracy is improved, but analysis time increases and operational complexity worsens
Solution Approach 1:
The patent segments the DNA analysis process into distinct functional modules: sample preparation, DNA extraction, PCR amplification with multiplexed primers, and detection. Each module is optimized independently, with pre-designed primer sets targeting specific microbial genes (16S rRNA, 18S rRNA, ITS regions) that can be simultaneously amplified and detected, reducing overall analysis time while maintaining accuracy
Solution Approach 2:
The patent implements preliminary action through pre-designed and validated multiplexed primer sets that target multiple microbial genetic markers simultaneously. The primer combinations are optimized in advance to amplify diverse microbial DNA in a single PCR reaction, eliminating the need for sequential testing and reducing analysis time while preserving detection accuracy
2Measurement precision
If traditional DNA-based methods are used for detecting microbial contamination in fuels, then detection accuracy is improved, but device complexity and ease of operation worsen
Solution Approach 1:
The patent creates a universal detection system with multiplexed primer sets that can simultaneously detect multiple types of microorganisms (bacteria, fungi, algae) across different fuel types (diesel, gasoline, jet fuel, biofuels). The standardized protocol and pre-optimized primer combinations enable non-technical users to perform comprehensive microbial analysis without requiring specialized expertise in molecular biology or fuel chemistry
Solution Approach 2:
The patent introduces standardized reagent kits and pre-prepared primer sets as intermediaries between the complex DNA analysis process and the end user. These intermediaries contain all necessary components (buffers, enzymes, primers) pre-optimized for specific fuel types and microbial targets, allowing non-technical users to simply follow standardized protocols without needing to understand or prepare complex reagent formulations
3Adaptability or versatility
If traditional DNA-based methods are used for detecting microbial contamination in fuels, then comprehensive microbial identification is improved, but sample processing complexity worsens
Solution Approach 1:
The patent merges multiple DNA extraction and amplification procedures into a single integrated workflow. Multiplexed PCR reactions simultaneously amplify multiple microbial genetic markers (bacterial 16S rRNA, fungal 18S rRNA, ITS regions) from a single extracted DNA sample, combining what would traditionally require multiple separate experiments into one unified process that reduces sample handling steps and complexity
Solution Approach 2:
The patent optimizes PCR reaction parameters (temperature profiles, cycle numbers, primer concentrations) specifically for multiplexed amplification in the context of fuel samples. By adjusting these parameters to account for fuel-specific inhibitors and the simultaneous amplification of multiple targets, the system achieves comprehensive microbial identification while simplifying the overall processing protocol through standardized, fuel-type-specific optimization
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 rapid, efficient detection of a wide range of microbial contaminants in fuels, allowing for quick identification of contamination sources, forensic analysis of fuel origin, and assessment of remediation efficacy, while reducing the need for extensive sample processing and technical expertise.
Implementation Method 1
The sample is subjected to a polymerase chain reaction (PCR) using a set of primer pairs
Implementation Method 2
A microfluidic chip is used to separate and detect the products of the PCR
Implementation Method 3
using a combination of primer pairs targeting 16S, 18S, mcrA, and dsrB genes, and employing microfluidic chips and rapid thermal cyclers for rapid analysis
Data Source
AI summary
Compositions, testing chambers and methods for testing a fuel sample for microbial contamination (including fuels treated with a biocide) are provided, which comprise: a quantity of hydrocarbon fuel; a microbial contamination wherein the microbial contamination further comprises nucleic acid in the form of both DNA, RNA or a combination thereof, and an analyzing solution; wherein the analyzing solution comprises at least six (6) primer pairs for amplification of at least one target locus, wherein at least one primer of each pair of primers is labeled with a fluorescent dye and wherein at least one of the primer pair binds to the nucleic acid of the microbial contamination.