Cleavable Chimeric Probe for Real-Time Salmonella Detection
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Solution Overview
Problem
Current methods for detecting Salmonella in foods and surfaces are time-consuming, labor-intensive, and prone to false positives and false negatives, necessitating a more accurate and reliable real-time detection system.
Innovation Solution
A method combining PCR and CataCleave technologies to detect Salmonella species by targeting specific invasion genes, using amplification primers and probes with RNase H activity to form RNA:DNA heteroduplexes, allowing for real-time signal emission detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If classical microbiological assays are used to detect Salmonella, then detection can be performed with simple equipment, but the method is time-consuming and labor-intensive
Solution Approach 1:
The patent replaces classical microbiological assays with a molecular biology-based method combining PCR amplification and fluorescence detection. This substitution transforms a time-consuming culture-based mechanical process into a rapid in vitro chemical-biological reaction system that can detect Salmonella DNA within hours, dramatically improving detection speed while maintaining operational simplicity through standardized reagent kits.
Solution Approach 2:
The patent changes the detection parameter from observing bacterial growth (colony formation) to detecting specific DNA sequences through fluorescence signal amplification. By monitoring fluorescence intensity in real-time during PCR cycles, the system achieves rapid detection without requiring lengthy incubation periods, thus resolving the contradiction between simplicity and speed.
2Productivity
If PCR amplification methods are used to detect Salmonella, then detection speed and sensitivity are improved, but false positives and false negatives occur
Solution Approach 1:
The patent introduces an RNA-DNA heteroduplex as an intermediary structure in the detection system. The probe contains both RNA and DNA sequences that form a heteroduplex with the target DNA, which is then cleaved by RNase H. This intermediary mechanism adds a specific recognition step that enhances accuracy and reduces false positives while maintaining rapid detection capability.
Solution Approach 2:
The patent uses a composite probe structure combining RNA and DNA sequences (chimeric probe) with fluorescent labels. This composite design allows the probe to hybridize specifically to target DNA and be cleaved by RNase H, providing both high sensitivity and high specificity. The composite nature of the probe resolves the contradiction by enabling accurate detection through multiple molecular recognition events.
3Reliability
If real-time detection with multiple components is implemented, then accuracy and reliability are improved, but device complexity increases
Solution Approach 1:
The patent merges multiple detection components (PCR reagents, fluorescent probes, RNase H enzyme, and buffer solutions) into a single integrated real-time PCR assay system. All reactions occur in one tube with continuous fluorescence monitoring, eliminating the need for separate steps and equipment. This merging approach maintains high accuracy through multiple molecular recognition events while simplifying the overall system architecture.
Solution Approach 2:
The patent designs a universal detection platform that can detect different Salmonella species using the same basic system architecture. The chimeric probe with RNA-DNA structure and RNase H cleavage mechanism serves multiple detection functions, allowing the system to maintain high reliability across different applications without requiring separate complex systems for each detection target.
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 fast, accurate, and reliable detection of Salmonella nucleic acid sequences, suitable for high-throughput applications and user-friendly diagnostic kits, reducing the risk of false results and improving public health response to outbreaks.
Implementation Method 1
under conditions where the RNA sequences within the probe can form a RNA:DNA heteroduplex with the complimentary DNA sequences in the PCR fragment
Implementation Method 2
an RNase H activity and the probe and under conditions where the RNA sequences within the probe can form a RNA:DNA heteroduplex with the complimentary DNA sequences in the PCR fragment
Implementation Method 3
detecting a real-time increase in the emission of a signal from the label on the probe
Data Source
AI summary
A method is described for the real-time detection of Salmonella species in foods and on surfaces. Salmonella are enriched in media to increase their cell density prior to analysis. DNA is recovered by lysis in the presence of azide, proteinase K, and detergent. Real-time detection of Salmonella species is performed in a PCR reaction using gene specific primers and a cleavable chimeric fluorescent probe. The method also describes an internal control to confirm the efficiency of nucleic acid amplification and detection. The method is amenable to medium and high throughput analysis.