Multiplex Nucleic Acid Detection Assay
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Solution Overview
Problem
Current methods for detecting multiple pathogens in clinical samples are limited by their ability to simultaneously identify and differentiate multiple agents, particularly in respiratory tract infections, where existing technologies are laborious, time-consuming, and often require expensive equipment or large setup costs, with a maximum complexity of detecting only a few pathogens at once.
Innovation Solution
A multiplex assay that allows for the simultaneous detection of up to 30 different target nucleic acid sequences in a single one-tube assay combined with real-time probe detection, using a method that involves reverse transcription, hybridization of probe sets specific for each target DNA template, forming connected probe assemblies, and real-time melting curve analysis with fluorescent donor or acceptor labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex PCR is used to detect multiple pathogens simultaneously, then the productivity and detection capability are improved, but the device complexity and optimization difficulty increase
Solution Approach 1:
The patent implements nested PCR by organizing primers into hierarchical groups (Group 1, Group 2, etc.) where outer primers define broad pathogen categories and inner primers provide specific pathogen identification. This nested structure allows multiple detection levels within a single reaction system, enabling simultaneous detection of numerous pathogens while maintaining manageable complexity through structured organization.
Solution Approach 2:
The patent employs universal primers that can amplify a wide range of pathogen DNA sequences across different pathogen types. These universal primers serve multiple functions: they initiate amplification for various pathogens, enable multiplex detection, and work in conjunction with pathogen-specific primers to achieve both broad screening and specific identification in a single assay system.
2Adaptability or versatility
If more primers are added to increase pathogen detection coverage, then the adaptability is improved, but the manufacturing precision and sensitivity maintenance become more difficult
Solution Approach 1:
The patent divides the primer set into multiple groups with distinct functions: outer primers for broad amplification and inner primers for specific pathogen identification. This segmentation allows each primer group to be optimized independently for its specific role, maintaining sensitivity for each pathogen detection while achieving comprehensive coverage through the combined action of multiple segmented primer groups.
Solution Approach 2:
The patent assigns different specificity levels to different primer groups within the same reaction system. Outer primers provide low-specificity broad coverage for multiple pathogen families, while inner primers provide high-specificity identification for individual pathogens. This local quality differentiation enables the system to simultaneously achieve broad adaptability and high precision sensitivity for each specific pathogen detection.
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
This approach enables rapid and reliable detection of multiple pathogens with improved sensitivity and specificity, reducing the risk of contamination and increasing throughput, while being cost-effective and capable of distinguishing between a large number of different amplicons in a closed system.
Implementation Method 1
a method capable of simultaneously detecting a plurality of different target DNA templates in a sample... wherein a donor or acceptor label is incorporated in the first or second tag region... providing a plurality of detection probes comprising at least one fluorescent donor label or at least one acceptor label
Implementation Method 2
bringing at least one DNA template into contact with a plurality of different probe sets, each probe set being specific for one target DNA template and allowing the at least one DNA template to hybridise with a probe set specific for the at least one DNA template
Implementation Method 3
amplifying the connected probe assembly to obtain at least one amplicon
Implementation Method 4
detecting the presence of the at least one amplicon by performing a real-time melting curve analysis... monitoring hybridisation of the labelled detection probe at at least one pre-selected temperature by measuring the fluorescence of the acceptor label
Data Source
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AI summary
The invention is in the technical field of detecting nucleic acid sequences in a sample, such as the detection of pathogenic organisms in clinical samples. More specifically, the invention relates to the field of detecting an infection caused by a pathogenic organism such as a virus or a bacterium in a clinical specimen by means of amplifying and 5 detecting specific nucleic acid sequences from said pathogenic organism. It provides a multiplex assay with the possibility to determine about 30 different target nucleic acid sequences in a single one-tube assay combined with real-time probe detection.