Neisseria Gonorrhoeae Detection Using Nested PCR and FRET Probes
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Solution Overview
Problem
Current methods for detecting Neisseria gonorrhoeae (NG) are inadequate, particularly in identifying the presence or absence of the bacterium in biological samples, and there is a need for a more efficient and specific detection method that can differentiate NG from other Neisseria species.
Innovation Solution
A method involving real-time polymerase chain reaction (PCR) with specific primers and probes targeting the Pilin Inverting protein (PivNg) gene, including amplification, hybridization, and fluorescence resonance energy transfer (FRET) for rapid and accurate detection of NG, using primers and probes designed to discriminate against other Neisseria species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for Neisseria gonorrhoeae, then the detection process is simpler, but the sensitivity and specificity are insufficient to differentiate NG from other Neisseria species
Solution Approach 1:
The invention segments the detection process into multiple specific steps: initial amplification using outer primers, nested amplification using inner primers, and detection using species-specific probes. This segmentation allows each step to contribute to overall specificity, with the nested approach enabling differentiation between NG and other Neisseria species through increasingly specific amplification and detection stages.
Solution Approach 2:
The invention introduces nested primers and species-specific fluorescent probes as intermediaries between the sample and the detection system. These intermediaries enable specific identification of NG by hybridizing to unique sequences, with the probes serving as mediators that translate molecular presence into detectable fluorescent signals while distinguishing NG from other Neisseria species.
2Productivity
If rapid detection is implemented, then the detection time is reduced, but the accuracy and reliability of NG identification may be compromised
Solution Approach 1:
The invention performs preliminary amplification using outer primers that target conserved regions before the specific detection step. This preliminary action enriches the target DNA and ensures sufficient material is available for the subsequent specific nested amplification and probe hybridization, enabling rapid yet accurate detection by preparing the sample in advance for specific identification.
Solution Approach 2:
The nested PCR approach maintains continuous useful action by seamlessly transitioning from outer primer amplification to inner primer amplification and finally to probe-based detection. This continuous process eliminates intermediate steps that would require separate instrumentation or processing, allowing rapid progression through all detection stages while maintaining reliability through each successive level of specificity.
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 method provides rapid and accurate detection of NG, enhancing sensitivity and specificity, allowing for effective identification of NG in complex samples through repeated amplification and hybridization cycles, and utilizing FRET for real-time detection.
Implementation Method 1
performing an amplifying step including contacting the sample with a set of primers designed to target a specific NG gene to produce an amplification product
Implementation Method 2
performing a hybridizing step including contacting the amplification product with one or more detectable probes to the target NG gene
Implementation Method 3
utilizing FRET for real-time detection
Data Source
AI summary
Methods for the rapid detection of the presence or absence of NG Pilin Inverting protein (PivNg) gene in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the NG PivNg genes, along with kits are provided that are designed for the detection of Neisseria gonorrhoeae (NG).