Salmonella Quantification via Variable-Efficiency PCR Primers
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Solution Overview
Problem
Current methods for estimating microorganism concentrations in biological samples, such as food, are slow, laborious, and require external standard curves, making them inefficient for rapid detection and quantification of pathogens like Salmonella.
Innovation Solution
A method using sets of primers with varying specificity for target nucleic acid, combined with a specific probe, to amplify and detect Salmonella DNA in samples without the need for external standard curves, allowing for quantification of the target nucleic acid and estimation of organism concentration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If MPN methods are used to estimate microorganism concentrations, then concentration determination is achieved, but the method is slow and requires two or more days for completion
Solution Approach 1:
The patent replaces the mechanical/culture-based MPN method with a molecular biology approach (PCR-based assay) that detects microbial DNA directly. This substitution eliminates the need for lengthy cultural enrichment and colony counting, achieving concentration determination in hours rather than days while maintaining measurement precision through quantitative DNA analysis
Solution Approach 2:
The patent introduces DNA as an intermediary substance that mediates between the target microorganism and the detection system. By detecting and quantifying microbial DNA through PCR amplification and probe hybridization, the method achieves rapid concentration determination without requiring the microorganisms to be cultured, thus resolving the time-precision contradiction
2Measurement precision
If external standard curves are used for quantification, then concentration measurement is possible, but separate standard curves are needed for each sample type due to matrix effects
Solution Approach 1:
The patent employs an internal control system where the PCR amplification efficiency itself serves as the reference standard. By using primers and probes with known and consistent amplification characteristics, the method generates its own internal standard that automatically compensates for matrix effects across different sample types, eliminating the need for external standard curves and reducing complexity
Solution Approach 2:
The patent creates a universal quantification system that works across multiple sample types (food, water, clinical samples) using the same primer-probe set and analysis method. The standardized PCR-based approach with controlled amplification efficiency provides a multi-functional solution that replaces the need for sample-type-specific standard curves, thereby reducing device complexity while maintaining measurement precision
3Speed
If PCR assays are used for pathogen detection, then rapid detection is achieved, but only presence or absence results are generated without quantification capability
Solution Approach 1:
The patent utilizes fluorescent probes that emit different intensities of light (analogous to color changes) based on the amount of target DNA present. The fluorescence signal intensity is directly proportional to the microbial concentration, enabling both rapid detection and simultaneous quantification by measuring the strength of the fluorescent signal rather than merely detecting its presence
Solution Approach 2:
The patent replaces the binary presence/absence detection mechanism with a quantitative measurement system that measures fluorescence intensity. This substitution allows the same rapid PCR-based detection to simultaneously provide concentration information, eliminating the loss of information while maintaining detection speed through real-time fluorescence monitoring
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 method enables rapid and accurate detection and quantification of Salmonella in biological samples, reducing the need for lengthy enrichment periods and providing actionable results for food safety and public health.
Implementation Method 1
amplifying the target nucleic acid in each container by exposing each sample to a different set of primers in uniplex or multiplex under conditions suitable for nucleic acid amplification
Implementation Method 2
exposing the amplification product to a probe which is 100% specific for the target nucleic acid
Data Source
AI summary
Disclosed herein are methods and kits for quantifying the presence of a microorganism in a sample. Specifically, disclosed are methods for quantifying a pathogen in a sample, such as a food sample, to determine if the levels of pathogen present in the sample are within an acceptable range.