Salmonella Detection Oligonucleotide Set Multiplex PCR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting Salmonella bacteria, such as those outlined in European Standard ISO EN 6579:2002 and molecular biology techniques, are lengthy, complex, and lack sensitivity, particularly in real-time multiplex amplification, which is essential for rapid and accurate detection of various Salmonella serovars.
Innovation Solution
Development of a set of oligonucleotides that enable real-time multiplex PCR, allowing for the detection of seven Salmonella groups, including all group I serovars, with increased sensitivity to dilutions of 10−7 or 10−8 without compromising specificity, using specific primer and probe sets that can operate in a single tube.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional microbiology methods (ISO EN 6579:2002) are used for Salmonella detection, then specificity and reliability are maintained, but detection time and procedure complexity increase significantly
Solution Approach 1:
The patent replaces traditional microbiological culture methods with molecular biology techniques (PCR amplification). This substitution eliminates the need for lengthy culture steps while maintaining detection reliability through specific primer binding to Salmonella DNA sequences.
Solution Approach 2:
The invention changes the detection parameter from cultural growth (requiring incubation time) to nucleic acid amplification (detectable within hours). By detecting genetic material directly and amplifying it through PCR, the method achieves rapid detection without sacrificing reliability.
2Reliability
If traditional microbiology methods are used, then detection reliability is maintained, but the number of procedure steps and complexity increase
Solution Approach 1:
The patent extracts and amplifies only the essential genetic information (nucleic acids) from Salmonella cells, eliminating the need for complex culture media, selective incubation conditions, and multiple staining procedures. This extraction approach simplifies the overall procedure while maintaining detection reliability.
Solution Approach 2:
Molecular biology techniques replace complex microbiological procedures, reducing the number of steps from multiple culture and staining operations to a streamlined PCR-based detection system that maintains reliability through specific primer design.
3Productivity
If molecular biology methods are used for Salmonella detection, then detection speed improves, but sensitivity and specificity may be compromised
Solution Approach 1:
The patent employs specific primer sequences (lag3-2, lag6-2, lag3-2C, lag6-2C1) designed to bind to conserved genomic regions of Salmonella. This localized targeting ensures high sensitivity and specificity by focusing amplification on unique bacterial DNA sequences, preventing false positives while detecting low concentrations of Salmonella.
Solution Approach 2:
The invention incorporates probe molecules that provide feedback on amplification success and specificity. The probes bind to amplified DNA products, allowing real-time monitoring and confirmation of specific Salmonella sequence amplification, thereby ensuring both sensitivity and accuracy.
4Adaptability or versatility
If multiplex PCR is used to detect multiple Salmonella groups, then detection versatility improves, but primer and probe design complexity increases
Solution Approach 1:
The patent designs primer pairs that can detect multiple Salmonella groups (I-VI) and serovars through conserved genomic regions. The primers lag3-2/lag6-2 and their variants (lag3-2C, lag6-2C1) serve universal detection functions across different Salmonella types, eliminating the need for separate primer sets for each group.
Solution Approach 2:
The invention uses conservative primer variants (lag3-2C, lag6-2C1) that may detect additional Salmonella groups beyond the primary target, providing excess detection capability. This ensures comprehensive coverage of all Salmonella groups while using the same primer set, avoiding the complexity of designing separate primers for each 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
The oligonucleotide set provides a wide detection range and high sensitivity, enabling accurate identification of Salmonella strains even at low concentrations, as demonstrated by improved detection ranges and sensitivity compared to existing methods, facilitating rapid and reliable diagnosis.
Implementation Method 1
The invention provides oligonucleotides that enable the detection of Salmonella by nucleic acid hybridization
Implementation Method 2
notably by nucleic acid amplification, more particularly, by PCR
Implementation Method 3
by multiplex amplification (e.g., multiplex PCR), very advantageously, by real-time multiplex amplification (e.g., real-time multiplex PCR)
Data Source
AI summary
The invention relates to the detection of Salmonella by nucleic acid amplification. The invention provides primer and probe oligonucleotides that can be used in multiplex to detect Salmonella in real-time amplification. The oligonucleotides of the invention detect all group I serovars, and have an increased Salmonella detection range: they enable to cover the seven Salmonella groups. They also have an increased sensitivity, without loss in specificity.