Enterococci Detection via 23S rRNA Oligomer Amplification
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Solution Overview
Problem
Conventional methods for detecting Enterococcus species, such as E. faecalis and E. faecium, in environmental and biological samples are slow and inefficient due to the need for bacterial cultivation and the ability of enterococci to enter a viable but nonculturable state, making it difficult to rapidly assess fecal contamination and antibiotic resistance.
Innovation Solution
The use of specific nucleic acid oligomers as primers and probes for amplifying and detecting 23S rRNA sequences of indicator enterococci, allowing for rapid, sensitive, and specific detection and quantification of E. faecalis, E. faecium, and other relevant species without detecting non-indicator enterococci, using methods like transcription-mediated amplification and real-time detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If conventional cultivation methods are used to detect Enterococcus species, then the detection process is simple and does not require complex equipment, but the detection time is prolonged to 1-2 days and sensitivity is reduced due to viable but nonculturable state
Solution Approach 1:
The patent replaces mechanical cultivation methods with molecular biological methods (nucleic acid amplification and detection). Specifically, it uses transcription-mediated amplification (TMA) of 23S rRNA sequences followed by detection with fluorescently labeled oligonucleotide probes, eliminating the need for bacterial cultivation while achieving rapid detection within hours rather than days.
Solution Approach 2:
The patent creates multiple copies of the target 23S rRNA sequences through transcription-mediated amplification. The TMA process generates numerous RNA copies from the original bacterial nucleic acid, enabling detection of even small amounts of enterococci in the sample without requiring bacterial cultivation.
2Measurement precision
If conventional cultivation methods are used, then all Enterococcus species can be detected, but non-indicator species cause false positives and reduce measurement precision
Solution Approach 1:
The patent applies local quality by designing species-specific detection probes that target unique sequences in indicator enterococci (E. faecalis, E. faecium, E. durans, E. gallinarum, E. mundtii). The fluorescently labeled oligonucleotide probes are complementary to specific regions of the 23S rRNA gene that differ between indicator and non-indicator species, enabling precise identification of only the relevant pathogenic strains.
Solution Approach 2:
The patent segments the detection process into two distinct steps: first, amplification of the 23S rRNA target sequence, and second, specific detection using fluorescent probes. This segmentation allows the use of different oligonucleotide sequences for amplification versus detection, with the detection probes being specifically designed to bind only to indicator species, thereby improving specificity.
3Reliability
If cultivation methods are used, then the detection process is straightforward, but injured and stressed organisms cannot be detected due to poor culturability
Solution Approach 1:
The patent replaces mechanical cultivation with molecular detection of nucleic acids. Since nucleic acid extraction and amplification can detect bacterial genetic material regardless of whether the bacteria are alive, injured, or in a viable but nonculturable state, this method reliably detects all enterococci present in the sample, including those that cannot grow on artificial media.
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
Enables rapid and specific detection of indicator enterococci, improving the assessment of fecal contamination and antibiotic resistance, while avoiding false positives from non-indicator species, thereby enhancing the quality control of recreational waters and infection prevention.
Implementation Method 1
using methods like transcription-mediated amplification and real-time detection
Implementation Method 2
The use of specific nucleic acid oligomers as primers and probes for amplifying and detecting 23S rRNA sequences
Data Source
AI summary
The disclosed invention includes nucleic acid oligomers that may be used as amplification oligomers, including primers, capture probes for sample preparation, and detection probes for detection of indicator enterococci 23S rRNA sequences in samples by using methods of specific nucleic acid amplification and detection.