Multiplex Respiratory Pathogen Detection Primers and Probes
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Solution Overview
Problem
Current multiplex nucleic acid detection methods for respiratory pathogens are limited by long detection times and the ability to detect only a few targets simultaneously, making them unsuitable for rapid and high-throughput clinical diagnostics.
Innovation Solution
The development of specific primers and probes for RPA technology, designed to amplify target sequences quickly in one tube, using probes labeled with different fluorescent groups and optimized melting curves to differentiate between targets in the same fluorescence channel, allowing for the simultaneous detection of multiple respiratory pathogens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional multiplex PCR methods are used, then detection specificity is maintained, but detection time is prolonged and the number of detectable targets is limited
Solution Approach 1:
The patent combines multiple target detections into a single reaction tube using isothermal amplification technology. Multiple primer-probe sets specific to different respiratory pathogens are mixed in one reaction system, enabling simultaneous detection of multiple targets without requiring separate PCR reactions for each pathogen, thus reducing detection time while increasing target capacity
Solution Approach 2:
The patent changes the fundamental parameter of amplification methodology from traditional thermal cycling PCR to isothermal amplification. This parameter change allows the reaction to proceed at a constant temperature (37-42℃) rather than requiring repeated heating and cooling cycles, dramatically reducing detection time from hours to minutes while maintaining the ability to detect multiple targets through optimized probe design with distinct Tm values
2Speed
If isothermal amplification with multiple probes is used, then detection speed is improved, but probe design complexity increases
Solution Approach 1:
The patent applies local quality by designing probes with specific local characteristics - each probe has a defined loop region (15-33 bp) and stem region with specific Tm values. The loop region contains the target-specific sequence while the stem region provides structural stability. This localized functional differentiation allows multiple probes to coexist in the same reaction without cross-interference, managing complexity through structured design rules
Solution Approach 2:
The patent segments each probe into distinct functional regions: a loop region that binds to the target sequence and a stem region that forms a stable structure. This segmentation allows independent optimization of each region - the loop for specificity and the stem for stability - simplifying the overall design process by breaking down the complex probe structure into manageable, functionally-defined segments
3Measurement precision
If probes with different Tm values are used in the same channel, then target differentiation is improved, but probe sequence optimization becomes more difficult
Solution Approach 1:
The patent utilizes Tm value as a key differentiating parameter for probes detecting the same target. By optimizing probe sequences to have distinct Tm values (e.g., 60-65℃, 65-70℃, 70-75℃), the system can differentiate between multiple targets within the same fluorescence channel. This parameter-based differentiation simplifies the overall detection strategy compared to requiring entirely distinct fluorescent channels for each target
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 detection of up to 12 target genes within 25 minutes with high sensitivity and specificity, overcoming the limitations of traditional PCR methods by reducing detection time and increasing the number of targets that can be detected simultaneously.
Implementation Method 1
the probes are complementary to target sequences to ensure that the probes are preferentially hybridized to the target sequences
Implementation Method 2
different target nucleic acids detected in the same fluorescence channel are amplified by different probes labeled with the same fluorescent group
Data Source
AI summary
Disclosed in the present application are primers, probes and kits for multiplex detection of respiratory pathogen target nucleic acids, and methods of use thereof. The primers, probes and kits for multiplex detection of respiratory pathogen target nucleic acids and methods of use thereof, include two or more of the following: specific primers and probes for genes of influenza A virus, influenza B virus, human metapneumovirus, human coronavirus HKU1, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus type A, respiratory syncytial virus type B, adenovirus and enterovirus EV. The primers, probes, kits and methods provided by the present disclosure realize the simultaneous detection of a plurality of target nucleic acids, greatly improve the detection throughput, significantly reduce the detection time and cost, and improve both specificity and sensitivity of the detection.