Novel Primers for Multiplex Pathogen Detection
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Solution Overview
Problem
Current methods lack a viable, accurate, and rapid mechanism for simultaneously detecting multiple food-borne pathogens and biothreat agents in the food supply, particularly challenging due to genome similarity and the need for optimized PCR conditions, which complicates the differentiation of closely related species and strains.
Innovation Solution
Development of novel primers for use in multiplex PCR and PCR-microplate array tests that allow for the simultaneous detection and identification of multiple high-impact food-borne pathogens, including Escherichia, Shigella, Francisella, Salmonella, Vibrio, and Yersinia species, under optimized reaction conditions, enabling high-throughput screening and accurate classification of contamination sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional PCR methods are used to detect multiple pathogens, then detection capability is limited, but the complexity of the system increases when attempting to detect multiple agents simultaneously
Solution Approach 1:
The patent combines multiple pathogen detection capabilities into a single multiplex PCR system. Multiple primer sets targeting different pathogens (Salmonella, E. coli, Listeria, etc.) are merged into one reaction mixture, allowing simultaneous detection of multiple food-borne pathogens in a single test, thereby improving detection versatility without proportionally increasing system complexity
Solution Approach 2:
The invention creates a universal detection platform that can identify multiple food-borne pathogens using a common PCR methodology. The system serves multiple functions by detecting various pathogens (bacteria, viruses, parasites) through standardized procedures, reagents, and equipment, eliminating the need for separate specialized tests for each pathogen type
2Measurement precision
If genome similarity between closely related species is considered, then detection accuracy improves, but differentiation becomes more difficult
Solution Approach 1:
The patent applies local quality by designing primers that target specific local regions (unique genetic sequences) within the genomes of different pathogens. Each primer set is customized to bind to distinctive sequences that differentiate closely related species, allowing accurate identification despite overall genome similarity. For example, primers are designed to target species-specific genes or unique regions within conserved genomic areas
Solution Approach 2:
The invention uses parameter changes by optimizing primer characteristics (melting temperature, length, GC content) and PCR reaction conditions (annealing temperature, cycle parameters) to enhance the ability to distinguish between closely related pathogens. By carefully adjusting these parameters, the system achieves specific amplification of target sequences even when the pathogens share high genomic similarity
3Speed
If rapid detection is implemented, then response time improves, but sensitivity and accuracy may be compromised
Solution Approach 1:
The patent applies preliminary action by performing DNA extraction and primer annealing optimizations beforehand to enable rapid amplification. The system uses pre-designed primer sets with optimized binding characteristics and employs a two-stage PCR approach (initial denaturation followed by rapid cycling) that reduces total detection time while maintaining sensitivity. Food samples are prepared with pre-extraction buffers that facilitate quick DNA release, and the PCR protocol is pre-optimized for fast cycling parameters
4Productivity
If multiple pathogens are detected simultaneously, then screening efficiency improves, but the complexity of primer design and PCR conditions increases
Solution Approach 1:
The patent segments the detection process by dividing it into distinct modules: sample preparation, DNA extraction, multiplex PCR amplification, and detection/analysis. Each module handles specific tasks independently, with primer sets for different pathogens organized in separate groups that can be independently validated. This segmentation allows systematic design and optimization of each component, reducing overall complexity despite detecting multiple pathogens simultaneously
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
The novel primers provide high sensitivity and specificity for detecting small amounts of target DNA, enabling rapid identification of multiple pathogens in real-time, even in complex food matrices, and can distinguish between live and dead organisms, thus enhancing food safety surveillance and bioterrorism response capabilities.
Implementation Method 1
novel primers for use in PCR and other genetic screening methodologies
Data Source
AI summary
Disclosed are novel primers for use in the molecular detection of food-threat agents and food-borne pathogens. The primers may be used in combination for the rapid, high-throughput screening PCR-based techniques to simultaneously detect multiple food safety biothreat agents. The multiplex-detection methods have improved sensitivity and specificity for the detection of multiple high-impact food-borne pathogens simultaneously. Real-time PCR assaying techniques using such primers include microarrays and multiplex single-tube arrays, the latter optionally simultaneously with TaqMan probes.