Salmonella Enteritidis Detection via Prot6e Gene Targeting
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Solution Overview
Problem
Current methods for detecting Salmonella enterica subspecies enterica serovar Enteritidis are not sensitive enough to differentiate it from other Salmonella species, particularly Salmonella Paratyphi C, and require lengthy culture confirmation processes, which are time-consuming and prone to cross-reactivity.
Innovation Solution
Development of specific nucleic acid sequences, primers, and probes that target the Prot6e gene unique to S. Enteritidis, allowing for highly specific amplification and detection using real-time PCR assays, which reduce cross-reactivity with other Salmonella species and expedite the detection process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional culture confirmation methods are used to detect Salmonella, then detection sensitivity is improved, but detection time increases significantly and cross-reactivity with other Salmonella species occurs
Solution Approach 1:
The patent extracts and targets a specific gene sequence (invA gene) that is unique to S. Enteritidis, separating the detection target from general Salmonella detection. This allows specific amplification of S. Enteritidis DNA without cross-reacting with other Salmonella species, thereby maintaining high detection sensitivity while reducing detection time through specific PCR assays.
Solution Approach 2:
The patent replaces traditional mechanical culture confirmation methods with molecular biology techniques (PCR amplification and hybridization assays). This substitution enables rapid DNA-based detection instead of time-consuming bacterial culture, reducing detection time from days to hours while maintaining or improving detection sensitivity through specific gene targeting.
2Adaptability or versatility
If traditional detection methods are used, then detection coverage is improved, but specificity decreases due to cross-reactivity with other Salmonella species
Solution Approach 1:
The patent applies local quality by designing detection probes and primers that target specific local regions (unique gene sequences) of the S. Enteritidis genome. The invA gene target and associated probes are specifically engineered to bind only to S. Enteritidis DNA, providing high detection specificity while maintaining broad detection coverage through sensitive amplification methods.
Solution Approach 2:
The patent segments the Salmonella detection problem by identifying and targeting a specific gene segment (invA gene) that distinguishes S. Enteritidis from other Salmonella species. This segmentation allows the development of specialized assays that can detect S. Enteritidis specifically without cross-reacting with other Salmonella, thereby improving specificity while maintaining detection coverage.
3Measurement precision
If highly sensitive detection methods are developed, then detection sensitivity is improved, but method complexity increases
Solution Approach 1:
The patent applies preliminary action by performing DNA extraction and purification before the PCR amplification step. This preliminary preparation ensures high-quality template DNA is available for amplification, enabling sensitive detection. The method also includes preliminary design of specific primers and probes based on known S. Enteritidis gene sequences, which simplifies the actual detection process while maintaining high sensitivity.
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 solution enables rapid, sensitive, and specific detection of S. Enteritidis, reducing the time-to-result to under 27 hours and preventing false positives with other Salmonella species, while maintaining high specificity and sensitivity across various samples.
Implementation Method 1
contacting the extracted nucleic acid with at least one primer pair having hybridization specificity for amplifying a Prot6e gene of S. Enteritidis species, or a fragment of the Prot6e gene of S. Enteritidis species
Implementation Method 2
contacting the amplified nucleic acid with at least one probe; and detecting the hybridization of probe with the amplified nucleic acid
Data Source
AI summary
Embodiments of the disclosure relate to compositions of isolated nucleic acid sequences, methods, workflows and kits of use thereof for detection of Salmonella enterica, subspecies enterica, serovar Enteritidis (S. Enteritidis) in a sample, particularly in a food (egg, poultry) or environmental sample (including poultry-related environmental samples). Embodiments of the disclosure may also be used to differentially detect S. Enteritidis without cross-reacting with S. Paratyphi C or other closely related Salmonella species. In some embodiments, methods and kits of the disclosure may comprise a TaqMan® assay. Following sample enrichment, methods of detection may be completed in approximately 3 hours.