Multiplex Pathogen Detection via Integrated Lysis and PCR
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Solution Overview
Problem
Existing methods for detecting multiple pathogens in samples, such as food products, are inefficient due to the need for high temperature lysis steps that require waiting times and complex purification processes, which can lead to resource-intensive and time-consuming procedures.
Innovation Solution
A method involving a pre-enrichment step followed by a combination of thermal, chemical, enzymatic, and mechanical lysis to break down pathogen cell walls, with the lysis terminated using protease inhibitors and dilution, allowing for a multiplex PCR detection without the need for DNA purification, and a kit containing pre-enrichment medium, lysis reagents, protease inhibitors, and PCR reagents for simultaneous detection of pathogens like Salmonella, Listeria, and E. coli.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If high temperature lysis steps are used to disrupt pathogen cell walls, then cell wall disruption is achieved, but waiting times are required and the process becomes time-consuming
Solution Approach 1:
The patent combines multiple lysis methods (thermal, chemical, enzymatic, and mechanical) into a single integrated lysis step. By using a lysis buffer containing chaotropic salts, detergents, and enzymes like proteinase K and lysozyme, along with mechanical stress from bead beating, the method achieves complete cell wall disruption without requiring sequential high-temperature steps, thereby eliminating waiting times while maintaining reliable lysis.
Solution Approach 2:
The lysis buffer is formulated as a composite solution containing multiple active components: chaotropic salts (e.g., guanidine hydrochloride) for chemical disruption, detergents (e.g., SDS) for membrane solubilization, enzymes (proteinase K, lysozyme) for protein and cell wall degradation, and reducing agents. This composite approach enables complete lysis at lower temperatures without time delays.
2Measurement precision
If complex purification processes are implemented for DNA extraction, then detection accuracy is improved, but the procedure becomes resource-intensive and time-consuming
Solution Approach 1:
The patent extracts only the essential function of DNA purification by incorporating specific inhibitors directly into the lysis buffer. These inhibitors (e.g., polyvinylpolypyrrolidone, activated charcoal, or silica-based agents) selectively bind to substances that interfere with PCR (such as polysaccharides, polyphenols, and humic acids) while leaving pathogen DNA intact and accessible for amplification. This eliminates the need for separate purification steps while maintaining detection accuracy.
Solution Approach 2:
The lysis buffer acts as an intermediary medium that simultaneously performs multiple functions: it disrupts cell walls, protects DNA from degradation, and removes PCR-inhibiting substances. By embedding purification functionality within the lysis buffer itself, the method bridges the gap between simple lysis and complex purification, achieving both speed and accuracy.
3Reliability
If multiple lysis steps are performed sequentially, then complete cell disruption is achieved, but the process becomes resource-intensive
Solution Approach 1:
The patent merges thermal energy input with chemical and mechanical lysis mechanisms into a single step. Instead of performing sequential thermal lysis, enzymatic lysis, and mechanical lysis as separate energy-consuming steps, the method uses mild heating (50-60°C) combined with a lysis buffer containing chaotropic salts, detergents, and enzymes, along with mechanical bead beating. This integrated approach achieves complete cell disruption while minimizing total energy consumption and resource usage.
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 efficient and time-saving detection of multiple pathogens by stopping lysis at a constant or decreasing temperature, eliminating the need for purification and reducing waiting times, thus enhancing the speed and simplicity of the process while ensuring accurate results.
Implementation Method 1
Lysis can be achieved in various ways. For example, in thermal lysis, the cell walls can be disrupted by an elevated temperature.
Implementation Method 2
In chemical lysis, cell disruption occurs, for example, through the addition of chaotropic salts, detergents, or other chemical agents.
Implementation Method 3
Enzymatic lysis uses an enzyme, such as proteinase K and/or lysozyme, to disrupt the cells.
Implementation Method 4
the lysis step is terminated before the detection step... It is known in the art to terminate enzymatic lysis by increasing the temperature
Data Source
Figure 1
AI summary
A method for detecting multiple different pathogens in a sample is proposed, wherein in a pre-enrichment step (1) the sample is placed in a pre-enrichment medium, in a lysis step (2) the cell walls of the pathogens in the pre-enriched sample are disrupted, thus producing a lysate, and in a detection step (3) the majority of the pathogens are detected by means of a multiplex PCR assay. The pre-enrichment step (1), the lysis step (2), and the detection step (3) are performed in this order, and the lysis is terminated without an increase in temperature before the detection step (3). Furthermore, a kit (100) for the detection of multiple different pathogens is proposed.