Multiplex PCR Pathogen Detection with Enrichment Media
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Solution Overview
Problem
Current methods for detecting multiple foodborne pathogens, such as Escherichia coli STEC, Salmonella species, and Listeria monocytogenes, are not sufficiently sensitive or accurate and cannot be performed within a timely manner.
Innovation Solution
A method involving enrichment of samples in a rich and nonselective media, followed by multiplex PCR using specific primers and oligonucleotide probes, allows for the simultaneous detection of multiple pathogens within 28 hours, using a lysis buffer with components like TRIS, EDTA, and proteinase K to facilitate nucleic acid extraction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current molecular biology detection methods (PCR, oligonucleotide hybridizing probes) are used, then pathogen detection can be performed, but it remains difficult to simultaneously detect the presence and/or absence and screen one or more (plurality) pathogens with sufficient accuracy and precision
Solution Approach 1:
The patent divides the detection process into distinct segments: (1) enrichment of pathogen nucleic acids from food samples using selective media, (2) extraction and purification of nucleic acids, and (3) multiplex PCR amplification with pathogen-specific primers. This segmentation allows each step to be optimized independently, enabling simultaneous detection of multiple pathogens while maintaining high accuracy and precision for each pathogen type.
Solution Approach 2:
The patent develops a universal multiplex PCR system that can simultaneously detect multiple different pathogens (including Listeria, Salmonella, and other foodborne pathogens) using a single reaction mixture with multiple pathogen-specific primer pairs. This multi-functional approach allows one detection system to screen for numerous pathogens at once, improving versatility without sacrificing detection precision through the use of highly specific primers and probes for each pathogen type.
2Productivity
If current detection methods are used, then pathogen screening can be performed, but the detection process is not completed within a timely manner
Solution Approach 1:
The patent implements preliminary enrichment of pathogen nucleic acids from food samples using selective enrichment media before PCR amplification. This preliminary action concentrates the target nucleic acids and removes inhibitors, enabling faster and more reliable detection in the subsequent PCR step. The enrichment phase prepares the sample in advance, reducing the time needed for accurate pathogen detection while maintaining high reliability.
Solution Approach 2:
The patent employs continuous multiplex PCR amplification that simultaneously amplifies multiple pathogen-specific DNA sequences in a single reaction without interruption. This continuous process eliminates the need for sequential testing of different pathogens, significantly improving detection speed while maintaining reliability through the use of internal controls and validation of all primer-probe systems throughout the amplification process.
3Reliability
If enrichment is performed in selective media, then specific pathogens can be promoted, but the detection sensitivity and accuracy for multiple pathogens is reduced
Solution Approach 1:
The patent uses different enrichment media with specific selective properties tailored to different pathogen groups (e.g., selective media for Listeria, Salmonella, or other foodborne pathogens). Each media type is optimized locally for its target pathogen's growth characteristics, allowing selective promotion of specific pathogens while maintaining the ability to detect multiple pathogen types across different samples or enrichment conditions, thus preserving detection sensitivity for each pathogen group.
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 rapid and accurate detection of multiple pathogens with high sensitivity and repeatability, improving the ability to identify contaminants in food samples effectively.
Implementation Method 1
a lysis buffer with components like TRIS, EDTA, and proteinase K to facilitate nucleic acid extraction
Implementation Method 2
the amplification primers comprise one or more primer pairs, wherein a first primer of the one or more primer pairs hybridizes to a target nucleic acid sequence of the one or more pathogens
Implementation Method 3
conducting an amplification with a set of amplification primers on the lysed sample
Data Source
AI summary
Provided herein are media, methods, kits, primers and oligonucleotide probes for use in the molecular detection of pathogens. These may be used in combination for the rapid, high-throughput screening PCR-based techniques to simultaneously detect multiple pathogens. The multiplex-detection methods have improved sensitivity and specificity for the detection of multiple pathogens simultaneously. Real-time PCR assaying techniques using such primers include microarrays and multiplex arrays, the latter optionally simultaneously with oligonucleotide TaqMan probes.