Multiplex Real-Time PCR Assay for β-Lactamase Resistance Detection
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Solution Overview
Problem
Existing methods for detecting bacterial resistance to antibiotics, particularly β-lactamases, are time-consuming, limited in the number of gene targets identified, and lack sensitivity and specificity, especially in multiplex polymerase chain reaction (PCR) assays, making it difficult to accurately identify and characterize resistance mechanisms.
Innovation Solution
A multiplex real-time PCR assay using specific primers and probes for multiple β-lactamase gene families, including MOX-like, FOX-like, ACC-like, and others, with optimized reagent concentrations and cycling protocols, enabling comprehensive detection of β-lactamase gene targets in a single test.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional polymerase chain reaction with agarose gel detection is used, then the method is simple to perform, but the resolution and sensitivity for detecting β-lactamase gene targets are poor
Solution Approach 1:
The patent replaces the mechanical agarose gel electrophoresis system with a real-time fluorescent detection system. Instead of visualizing DNA fragments after electrophoresis through staining and imaging, the assay uses fluorogenic probes that emit fluorescence signals during the PCR amplification process itself, enabling real-time monitoring without post-PCR processing
Solution Approach 2:
The patent introduces fluorogenic probes as intermediary molecules that mediate between the DNA target and the detection system. These probes hybridize to specific β-lactamase gene sequences and generate fluorescent signals upon binding, serving as intermediaries that translate molecular recognition into detectable optical signals with high sensitivity and specificity
2Adaptability or versatility
If multiplex PCR is used to detect multiple β-lactamase gene families, then the number of gene targets identified increases, but the assay complexity and difficulty in interpreting data increase
Solution Approach 1:
The patent designs a universal real-time PCR platform that can simultaneously detect multiple β-lactamase gene families (AmpC, ESBLs, carbapenemases, metallo-β-lactamases) using a single assay system. Different fluorogenic probes with distinct fluorescent labels target different gene families, allowing one multiplex reaction to perform the function of multiple separate assays
Solution Approach 2:
The patent employs fluorogenic probes labeled with different fluorescent dyes that emit distinct colors or wavelengths when excited. Each probe type generates a characteristic fluorescence signal, enabling simultaneous detection and differentiation of multiple β-lactamase gene families through spectral discrimination, simplifying the interpretation of multiplex results
3Productivity
If real-time polymerase chain reaction is used, then the sensitivity and speed of detection improve, but the cost and complexity of the assay increase
Solution Approach 1:
The patent implements real-time monitoring of PCR amplification throughout the entire thermal cycling process. Fluorescent signals are continuously measured during each amplification cycle, allowing detection to occur continuously rather than requiring interruption for sample processing and analysis, thereby significantly reducing total detection time
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 assay provides rapid, sensitive, and specific detection of various β-lactamase gene families, facilitating accurate identification of antibiotic-resistant bacteria, supporting effective treatment strategies and infection control.
Implementation Method 1
polymerase chain reaction to identify nucleic acid characteristics of family specific β-lactamase enzymes in samples
Implementation Method 2
Real-time polymerase chain reaction allows for monitoring of reaction products as they are formed
Data Source
AI summary
Assays and methods for detecting resistance to beta-lactam antibiotics including detection of multiple β-lactamase family specific gene targets by polymerase chain reaction or microarray. One or more kits including primers and/or probes for identification of β-lactamase genes selected from the group consisting of one or more of the following: MOX-like, FOX-like, ACC-like, ACT/MIR-like, CMY-2-like, DHA-like, CTX-M-14-like, CTX-M-15-like, VIM-like, NDM-like, IMP-like, KPC-like, and OXA-48-like, OXA-51-like, OXA-143-like, OXA-58-like, OXA-23-like, OXA-24/40-like, TEM-like, and SHV-like. A kit may also include one or more primers and/or probes for the identification a non-beta lactamase gene family which confers antibiotic resistance, such as the MCR-1 gene.