Fluorescence Probes for Rapid CRE Gene Detection
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Solution Overview
Problem
Current methods for detecting carbapenem-resistant Enterobacteriaceae bacteria, such as those carrying NDM1, KPC, IMP, VIM, and OXA genes, are inadequate for rapid and specific identification, leading to challenges in clinical management due to high antibiotic resistance levels.
Innovation Solution
The use of specific primers and probes designed to amplify and detect NDM1, KPC, IMP, and OXA genes in biological samples through real-time PCR, employing fluorescence-generating probes and flap primers to identify the presence of these genes, allowing for rapid and sensitive detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for carbapenem-resistant Enterobacteriaceae, then detection can be performed, but the detection speed is slow and specificity is insufficient
Solution Approach 1:
The detection method is segmented into distinct functional components: flap primers for specific amplification initiation, fluorescence-generating probes for targeted detection, and real-time monitoring capabilities. This segmentation allows each component to be optimized independently for both speed and specificity, resolving the contradiction between rapid detection and accurate identification of carbapenem-resistant bacteria
Solution Approach 2:
The patent replaces conventional mechanical or chemical detection methods with a fluorescence-based optical detection system. The fluorescence-generating probes emit light signals when bound to target DNA sequences, enabling rapid and specific detection without time-consuming mechanical separation or chemical analysis steps, thus improving both detection speed and specificity
2Reliability
If high levels of antibiotic resistance are present in bacteria, then treatment becomes difficult, but rapid detection can enable timely clinical interventions
Solution Approach 1:
The detection system is designed to be self-contained with all necessary reagents (flap primers, fluorescence probes, buffers) integrated into a single kit. The real-time PCR system automatically performs amplification and detection without requiring complex external equipment or multiple separate assays, reducing operational complexity while maintaining high reliability for detecting antibiotic-resistant bacteria to guide effective treatment
Solution Approach 2:
The detection method uses universal flap primers and fluorescence probes that can detect multiple types of carbapenem-resistant Enterobacteriaceae (including KPC, NDM, IMP, VIM, and OXA producers) through a single standardized protocol. This multi-functionality ensures reliable detection across different resistance types without requiring complex, pathogen-specific assays for each bacterium, thereby improving treatment reliability while minimizing system complexity
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 NDM1, KPC, IMP, and OXA genes, facilitating timely clinical interventions by identifying antibiotic-resistant bacteria, thereby improving treatment outcomes.
Implementation Method 1
detecting the amplified nucleic acids using a fluorescence-generating probe comprising at least the following full-length sequence: SEQ ID N° 17
Implementation Method 2
incubating the reaction mixture of step (a) under conditions sufficient to amplify the NDM1 nucleic acids, thereby generating amplified nucleic acids
Data Source
Figure 1a~1c
Figure 2
Figure 3
AI summary
Primers and probes specific to genes encoding carbapenem-resistant Enterobacteriaceae (CREs) that include KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi Metallo-beta-lactamase), VIM (Verona Integron-Mediated Metallo-β- lactamase), IMP-type carbapenemase and OXA 48 (oxacillinase), that cause resistance in Enterobacteriaceae bacteria, are described herein, with methods and kits for using these primers and probes to detect target nucleic acids. In the methods described, nucleic acids present in a clinical or test sample obtained from a biological sample or tissue suspected of containing the the NDMl, KPC, IMP, VIM and OXA genes are amplified and corresponding sequences for the NDMl, KPC, IMP, VIM and OXA genes are detected. The amplified nucleic acid can be detected by a variety of state of the art methods, including fluorescence resonance energy transfer (FRET), radiolabels, enzyme labels, and the like.