Multiplex PCR Primer Set for Beta-Lactamase Gene Detection
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Solution Overview
Problem
Current methods for detecting β-lactamase genes in bacteria are limited by their inability to identify all clinically-important genes, leading to delayed detection of antibiotic resistance and potential therapeutic challenges.
Innovation Solution
Development of a large-scale PCR primer pair set, known as LARGE-SCALEblaFinder, which includes 54 optimized primer pairs capable of detecting all clinically-important β-lactamase genes with high specificity and sensitivity, allowing for rapid identification of resistance genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classical culture-based phenotypic tests are used to determine susceptibility or resistance, then the method is generally applicable in clinical microbiological laboratories, but the procedure is time consuming and cannot easily detect ESBL and carbapenemase production
Solution Approach 1:
The patent replaces the mechanical/culture-based phenotypic testing system with a molecular-based diagnostic system using PCR amplification and detection methods. This substitution enables direct detection of resistance genes (bla genes) at the molecular level, achieving both rapid results (within hours rather than days) and high accuracy in detecting ESBL and carbapenemase production.
Solution Approach 2:
The patent introduces molecular biomarkers (specific bla genes) as intermediaries to indicate resistance phenotypes. Instead of directly observing phenotypic resistance through culture tests, the method detects the presence of specific resistance genes as molecular intermediaries that reliably predict phenotypic outcomes, enabling faster and more accurate detection.
2Productivity
If previous molecular-based diagnostic methods are implemented, then the speed and accuracy of detecting resistance genes is increased, but these methods are restricted to the identification of several bla genes and cannot detect all clinically-important bla genes
Solution Approach 1:
The patent develops a universal molecular diagnostic platform that can detect multiple clinically-important bla genes (including but not limited to ESBLs and carbapenemases) within a single testing framework. The method uses a panel of primers and probes targeting various bla gene families, enabling one system to perform multiple detection functions simultaneously, thus achieving both high productivity and broad adaptability.
Solution Approach 2:
The patent segments the detection of different bla gene types into specific molecular targets, with dedicated primers and probes for each gene family (e.g., TEM, SHV, CTX-M for ESBLs; KPC, NDM, VIM for carbapenemases). This segmentation allows the system to maintain high detection speed for each specific target while collectively achieving comprehensive coverage of all clinically-important bla genes.
3Loss of time
If previous molecular-based diagnostic methods are used, then detection speed is improved, but they cannot replace the susceptibility test because they detect only partial types of bla genes
Solution Approach 1:
The patent changes the detection parameters by expanding the scope of targeted bla genes from partial types to comprehensive clinically-important types. The method incorporates detection of multiple gene families with varying prevalence and clinical significance, adjusting the detection parameters (primer sets, probe targets) to ensure complete coverage while maintaining rapid detection capabilities.
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 effectively detects 24 additional unreported β-lactamase genes, providing comprehensive resistance profiling and aiding in prompt therapeutic decisions by overcoming the limitations of previous detection methods.
Implementation Method 1
a pair of nucleic acid molecules that are complementary to each other
Implementation Method 2
multiplex polymerase chain reaction (PCR)
Data Source
AI summary
The present invention provides kits and primers for colony multiplex PCR for the detection of class A, B, C, and D β-lactamase genes. The rapid detection of bla genes by using the kits and primers according to the present invention allows appropriate prescribing of antibiotics, which can reduce patient mortality and minimize antibiotic resistance. The present invention provides kits and primers for a rapid and accurate molecular method to overcome (a) to detect all clinically-important bla genes and (b) to explain phenotypic tests' results well by using 54 primer pairs, which are designed through novel and elaborate optimization processes. With perfect specificity and sensitivity in 172 control strains and 403 clinical strains, the present invention provides prompt and clinical application to the identification of all bla genes in bacterial pathogens.