Digital PCR Co-detection of STEC Virulence Genes
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Solution Overview
Problem
Current PCR-based detection methods are unable to effectively associate virulence genes with Shiga toxin-producing Escherichia coli (STEC) O-groups, necessitating time-consuming culture-isolation procedures for food safety testing, which takes around a week to produce results.
Innovation Solution
The development of PCR-based methods and kits for co-detection and association of multiple genes using digital PCR technology, allowing for the simultaneous identification, differentiation, and quantification of bacterial strains, including the determination of whether target nucleic acid sequences originate from a single genome or multiple genomes, without the need for sample pre-processing or extraction, enabling rapid detection of STEC O-types and virulence genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If PCR-based detection methods are used to detect virulence genes and bacterial strains, then detection speed is improved, but the ability to associate genes with specific genomes is lost
Solution Approach 1:
The sample is divided into multiple individual reaction volumes (droplets or wells), each containing a partitioned portion of the nucleic acid sample. This segmentation allows each reaction volume to potentially contain nucleic acids from a single genome, enabling association of multiple genes detected in the same reaction volume to that single genome.
Solution Approach 2:
Individual reaction volumes serve as intermediaries that physically separate and isolate nucleic acid molecules from different genomes. By performing PCR amplification in these separated reaction volumes and detecting which volumes contain positive results for multiple target genes, the method establishes gene-genome associations without requiring culture isolation.
2Measurement precision
If culture-isolation procedures are used to associate virulence genes with STEC O-groups, then gene-genome association accuracy is improved, but detection time increases to around a week
Solution Approach 1:
The method performs preliminary partitioning of the nucleic acid sample into individual reaction volumes before PCR amplification. This preliminary action establishes the spatial separation needed for gene-genome association, eliminating the need for subsequent culture isolation steps while maintaining association accuracy.
Solution Approach 2:
The invention replaces the mechanical culture-isolation system with a molecular-based digital PCR system. Instead of growing bacteria on culture media to obtain isolated colonies, the method uses partitioned PCR reactions to directly associate genes with genomes at the molecular level, dramatically reducing detection time from a week to two days or less.
3Loss of information
If digital PCR with partitioned reaction volumes is used for co-detection of multiple genes, then gene-genome association capability is improved, but device complexity increases
Solution Approach 1:
The digital PCR system is designed to perform multiple functions: it detects multiple target genes simultaneously, quantifies gene copy numbers, and associates genes with genomes all within the same partitioned reaction platform. This multi-functionality reduces the need for separate analytical steps and instruments.
Solution Approach 2:
The method changes the physical parameter of reaction volume size and number, creating many small partitioned reactions instead of a single bulk reaction. This parameter change enables the system to resolve individual genome contributions while maintaining a relatively simple overall device architecture that can be implemented on existing digital PCR platforms.
4Ease of operation
If conventional PCR methods are used for detecting multiple target genes, then ease of operation is maintained, but the ability to quantify and associate multiple genes with single genomes is lost
Solution Approach 1:
The conventional PCR approach is segmented into multiple individual reaction volumes, where each volume independently amplifies target genes from potentially single genome copies. This segmentation provides precise quantification of gene copy numbers and enables association of multiple genes to single genomes, while the overall process remains operationally simple using standard digital PCR workflows.
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 significantly reduces the detection time to two days, streamlines the sample preparation and detection processes, and provides accurate quantification of target nucleic acids, meeting current and future USDA food safety regulations by identifying STEC O-types and virulence genes with high throughput.
Implementation Method 1
A reaction mixture is formed comprising at least a portion of the sample and PCR primers and/or probes corresponding to the two or more target nucleic acid sequences. The reaction volumes are amplified (using PCR) to yield amplified reaction volumes comprising positive PCR reaction products.
Data Source
AI summary
The present invention is concerned with PCR-based detection methods and kits for the identification, differentiation, and quantification of different bacterial strains (e.g., Gram-negative bacterial strains), and also association of two or more PCR-positive genes to a single genome. The methods generally comprise carrying out PCR reactions using at least a first PCR primer set and/or probe for at least one target nucleic acid; and a second PCR primer set and/or probe for at least a second target nucleic acid. Positive PCR reaction products are then detected to determine test samples containing positive PCR reaction products for both the first and second target nucleic acids. This information can be used to calculate the gene association rate to determine whether the sample contains, for example, Shiga toxin-producing E. coli of the O-type serogroup.