Universal Probe Multiplex Digital PCR Background Signal Reduction
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Solution Overview
Problem
Current nucleic acid detection methods for pathogenic microorganisms and resistant genes, such as real-time quantitative PCR, face challenges in specificity and multiplex detection due to high background signals and limited target detection capabilities, especially when using traditional TaqMan probes.
Innovation Solution
A digital PCR platform utilizing a 7-color fluorescence channel and multiplex digital PCR technology, combined with a universal probe structure and specific primer-probe sets, allows for the simultaneous detection of multiple bacterial strains and drug resistance sites by using universal probes and single-modified fluorescence-free probes to generate fluorescent signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional TaqMan probes are used for multiplex detection, then specificity is improved, but background signal increases and number of detectable targets is limited
Solution Approach 1:
The probe is divided into two separate components: a universal probe containing the fluorophore and a target-specific probe containing the quenching group. This segmentation allows the universal probe to provide consistent fluorescence while the target-specific probe provides specificity through hybridization, reducing background signal from non-specific binding.
Solution Approach 2:
The universal probe serves multiple functions: it provides the fluorophore for signal detection, acts as a common binding element for multiple target-specific probes, and maintains consistency across different detection targets. This multi-functionality reduces the total number of probes needed and simplifies the detection system.
2Adaptability or versatility
If multiple probes labeled with the same fluorescence are used in one channel, then multiplex detection capability is improved, but signal contrast decreases
Solution Approach 1:
By separating the universal probe (with fluorophore) from the target-specific probe (with quenching group), the system maintains distinct fluorescent signals for different targets even when detected in the same channel. The target-specific probes prevent cross-talk between targets by providing unique hybridization specificities.
Solution Approach 2:
The universal probe acts as an intermediary element that is common to all target-specific probes. It provides the fluorescent signal while the target-specific probes mediate the specific binding to different targets, allowing multiple targets to be detected with maintained signal contrast through the intermediary's consistent fluorescent output.
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 accurate and specific detection of 12 pathogenic microorganisms and 9 resistant genes, improving the accuracy and feasibility of multiplex digital PCR by reducing background signals and expanding the number of detectable targets.
Implementation Method 1
the TaqMan method detection involves an addition of a fluorescent probe, which works through fluorescence resonance energy transfer (FRET)
Data Source
AI summary
The present application relates to a field of a biological detection technology, in particular, relates to a universal probe, a primer-probe set and a kit. The present application provides a universal probe, in which, from 5′ end to 3′ end, the universal probe sequentially comprises: quenching group-fragment A -fluorophore-fragment B-C3; the fragment A has a nucleotide sequence selected from a group consisting of SEQ ID NO:1-7; and the fragment B has a nucleotide sequence selected from a group consisting of SEQ ID NO:8-14. The present application further provide a primer-probe set, an application and the kit.