Single-Tube Multiplex PCR Probes for High-Plex Target Detection
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Solution Overview
Problem
Existing PCR detection methods struggle to achieve ultra-multiplex detection of multiple targets in a single reaction system without causing false positive signals from primer dimers and require complex, costly equipment.
Innovation Solution
A single-tube ultra-multiplex PCR detection system using detection probes with blocking regions and auxiliary probes that emit different signals based on hybridization, allowing for the differentiation of multiple targets and preventing primer dimer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple TaqMan hydrolysis probes labeled with fluorescence groups of different wavelengths are used for multiplex PCR detection, then the number of detectable targets increases, but the detection is limited to 4-6-plex due to the limitation of fluorescent channels in detection instruments
Solution Approach 1:
The patent transitions from detecting multiple targets through different fluorescence wavelengths (spectral dimension) to detecting multiple targets through different amplification product lengths (size dimension). By using a hybrid chip with probes of different lengths that hybridize to PCR products of corresponding lengths, the system can distinguish multiple targets without requiring multiple fluorescent channels, thereby overcoming the 4-6-plex limitation.
2Adaptability or versatility
If PCR is combined with a hybrid chip for multiplex detection, then 20 types of respiratory pathogens can be simultaneously detected, but the gene chip is complex in preparation process and expensive
Solution Approach 1:
The patent segments the detection function into modular components: universal primers for amplification, target-specific primers for length differentiation, and length-specific probes for detection. This segmentation allows the system to achieve highplex detection without requiring complex chip preparation, as the length differentiation can be achieved through simple PCR amplification conditions.
3Adaptability or versatility
If PCR is combined with capillary electrophoresis for multiplex detection, then thirty types of pathogenic bacteria can be detected, but the detection cost is greatly increased due to requiring additional equipment
Solution Approach 1:
The patent merges the amplification and detection functions into a single PCR reaction system. By designing primers that produce amplification products of different lengths for different targets and using length-specific probes for detection, the system combines what were previously separate functions (amplification by PCR and separation by electrophoresis) into one integrated process, eliminating the need for additional capillary electrophoresis equipment.
4Adaptability or versatility
If multiple specific primers are used in a single reaction system, then multiplex detection is achieved, but false positive signals from primer dimers occur
Solution Approach 1:
The patent applies local quality by making each primer pair unique in its binding specificity and amplicon length characteristics. The universal primers provide a common framework for all targets, while target-specific primers introduce local variations in amplicon length. This local differentiation ensures that each target can be specifically detected without cross-reactivity or primer dimer formation, maintaining high reliability in multiplex 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
Enables efficient, cost-effective, and rapid detection of multiple targets in a single reaction tube, reducing false positives and simplifying the detection process.
Implementation Method 1
the detection sequence binding region of the detection probe specifically binds to the detection sequence
Implementation Method 2
a detection group on the detection probe emits different signals under the condition of hybridization between the detection probe with the detection sequence or the amplification product thereof compared to the condition of non-hybridization
Implementation Method 3
when the binding site of the detection probe to the detection sequence is located downstream of the blocking region, the blocking region blocks further extension of the detection sequence
Implementation Method 4
when a DNA polymerase (taq polymerase) extends to a site at which the probe binds with the template, the TaqMan probe will be cleaved due to 5'-3' exonuclease activity of the DNA polymerase (taq polymerase)
Implementation Method 5
due to a principle of fluorescence resonance energy transfer (FRET), a fluorescence signal emitted by the fluorescence group at one end of the TaqMan probe is absorbed by the quencher group at the other end
Data Source
AI summary
The present invention provides a design method of a single-tube ultra-multiplex PCR detection primer probe, a reaction system, and a multi-target detection kit based on the method and the reaction system.