Multiplexed Analyte Detection With Universal Probe Binding Sites
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing nucleic acid detection methods face challenges in efficiently and accurately detecting multiple analytes with high sensitivity and flexibility, particularly in multiplexed assays, often requiring target-specific probes and optimized designs for each target.
Innovation Solution
The use of oligonucleotides with distinct probe binding sites and universal probes allows for multiplexed detection by generating specific signals through hybridization and probe binding, enabling flexible and sensitive detection of multiple analytes using tailed primers and molecular inversion oligonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If target-specific probes are used for each analyte, then detection specificity is improved, but device complexity and assay design difficulty increase
Solution Approach 1:
The patent employs universal probes that can bind to multiple different analytes through a common binding site architecture. Each probe contains a standardized probe binding site that can hybridize to complementary sequences from different analytes, allowing a single probe design to detect multiple targets. This universal probe approach maintains detection specificity while significantly reducing the number of probe designs needed compared to traditional target-specific probe methods.
Solution Approach 2:
The detection system is segmented into distinct functional components: analyte-specific primer regions and universal probe binding sites. The primers contain target-specific sequences that bind to different analytes, while the probe binding sites provide a standardized interface for universal probe recognition. This segmentation allows the system to maintain specificity for multiple analytes using a standardized probe platform, reducing overall assay complexity.
2Adaptability or versatility
If multiple probes are used to detect multiple analytes, then detection versatility is improved, but signal detection complexity increases
Solution Approach 1:
Universal probes with standardized binding sites enable a single probe type to detect multiple analytes. The probes contain conserved sequences that hybridize to complementary regions in different analyte amplicons, allowing multiplexed detection without requiring unique probes for each target. This approach maintains versatility while simplifying the detection system compared to using entirely distinct probes for each analyte.
Solution Approach 2:
The system employs fluorophore-labeled probes that generate detectable signals through fluorescence emission. Different fluorophores with distinct emission wavelengths allow simultaneous detection of multiple analytes in a single reaction. The universal probe design combined with multi-color fluorophores enables versatile multiplexed detection while maintaining relatively simple signal detection through wavelength-specific measurement.
3Productivity
If conventional PCR amplification is used, then nucleic acid amplification efficiency is improved, but detection sensitivity for low-abundance analytes deteriorates
Solution Approach 1:
The system incorporates pre-amplification steps using target-specific primers that enrich analyte sequences before the main detection amplification. The primers are designed with high specificity for low-abundance targets, and the reaction conditions are optimized to maximize amplification of rare sequences. This preliminary enrichment step improves detection sensitivity for low-abundance analytes while maintaining overall amplification efficiency.
Solution Approach 2:
The amplification reaction parameters are optimized to enhance sensitivity for low-abundance targets. This includes adjusting annealing temperatures, extension times, and polymerase concentrations to favor amplification of rare sequences. The probe hybridization conditions are also tuned to detect low levels of amplified product with high sensitivity, allowing conventional PCR to achieve improved detection limits through parameter optimization.
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 achieves high sensitivity and flexibility in detecting multiple analytes, with sensitivities up to 99% and the ability to differentiate between analytes at various concentrations, facilitating efficient multiplexed nucleic acid detection.
Implementation Method 1
a primer oligonucleotide comprising a first region, wherein the first region hybridizes to said analyte
Implementation Method 2
annealing (i) a second primer oligonucleotide and (ii) one or more probes to said probe binding nucleic acid
Data Source
AI summary
The present disclosure provides methods, systems, and compositions for the multiplexed detection and quantification of multiple analytes from a sample. Analytes may be nucleic acid analytes. Detection of analytes may comprise contacting one or more samples with primers and/or hybridization probes to generate cumulative signal measurements. The methods may comprise digital PCR or may comprise partitioning a sample into multiple partitions.