Real-Time PCR Detection of Clostridium difficile
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Solution Overview
Problem
Current methods for detecting Clostridium difficile infections are not sufficiently sensitive and specific, particularly in identifying the 'epidemic' BI/NAP1/027 strain, which is a hyperproducer of toxins A and B, leading to increased virulence and severity of the infection.
Innovation Solution
The use of real-time polymerase chain reaction (PCR) with specific primers and probes, including tcdC primers and probes, to amplify and detect C. difficile nucleic acid in biological samples, employing fluorescence resonance energy transfer (FRET) for rapid and accurate identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for C. difficile, then the detection process is simpler, but the sensitivity and specificity are insufficient
Solution Approach 1:
The patent replaces conventional mechanical detection methods with a molecular biological detection system based on real-time PCR. This substitution enables highly specific detection of C. difficile nucleic acids through sequence-specific primer and probe hybridization, achieving superior sensitivity and specificity while maintaining operational simplicity through automated real-time monitoring
Solution Approach 2:
The patent introduces fluorescently labeled oligonucleotide probes as intermediaries that specifically bind to C. difficile nucleic acid sequences. These probes act as mediators between the target nucleic acid and the detection system, enabling highly specific detection through fluorescence resonance energy transfer (FRET) while the real-time PCR system automates the detection process
2Productivity
If conventional detection methods are used, then the equipment and procedure are simpler, but the detection speed is slower
Solution Approach 1:
The patent implements real-time continuous monitoring of PCR amplification through fluorescent signal detection during each cycling step. This continuous action eliminates the need for post-PCR analysis, enabling rapid detection within a single real-time PCR run while the automated system manages the complexity of continuous monitoring
Solution Approach 2:
The patent replaces slow conventional detection procedures with automated real-time fluorescent detection. The system substitutes manual analysis steps with automated optical detection and real-time data processing, significantly accelerating detection speed while the integrated system manages operational complexity
3Measurement precision
If specific primers and probes are used for epidemic strain detection, then the diagnostic accuracy is improved, but the risk of contamination is increased
Solution Approach 1:
The patent performs real-time continuous monitoring of PCR amplification, allowing detection to be completed in a single closed-system run. This continuous action minimizes the number of times the system opens to external environments, thereby reducing contamination risk while maintaining high diagnostic accuracy through real-time fluorescent signal monitoring
Solution Approach 2:
The patent uses fluorescently labeled probes as intermediaries that remain contained within the closed PCR system throughout the reaction. These probes enable specific detection of epidemic strains through sequence-specific hybridization while the closed-system architecture prevents external contamination, reconciling high diagnostic accuracy with low contamination risk
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 provides a more sensitive and specific detection method for C. difficile, capable of identifying the 'epidemic' strain and differentiating between wild-type and mutant strains, reducing the risk of contamination and improving diagnostic accuracy and speed.
Implementation Method 1
wherein the members of said pair of tcdC probes hybridize within no more than five nucleotides of each other
Implementation Method 2
amplifying step comprises contacting said sample with a pair of tcdC primers to produce a tcdC amplification product
Implementation Method 3
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first tcdC probe and said acceptor fluorescent moiety of said second tcdC probe
Data Source
Figure 1
AI summary
The invention provides methods to detect C. difficile in biological samples using real-time PCR. Primers and probes for the detection of C. difficile are provided by the invention. Articles of manufacture containing such primers and probes for detecting C. difficile are further provided by the invention.