Spectrally Encoded Microbeads for Rapid Multiplex Pathogen Detection
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Solution Overview
Problem
Current methods for identifying pathogens in blood infections, such as those causing sepsis, are slow and require sample compartmentalization, leading to reduced sensitivity and potential false positives, while next-generation sequencing is too time-consuming.
Innovation Solution
A method involving amplifying polynucleotides from a sample, combining them with microbeads having unique lanthanide spectral signatures and capture polynucleotides, and detecting signal-generating moieties to identify microbial strains rapidly and accurately.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If PCR-based nucleic acid tests are used to identify pathogens, then detection speed is improved (hours vs days), but multiplexing capability is limited (1-4 pathogens simultaneously)
Solution Approach 1:
The invention divides the detection system into multiple distinct reaction compartments, each containing a different capture oligonucleotide sequence. This segmentation allows simultaneous detection of multiple pathogens (up to 96) in parallel while maintaining the speed advantages of PCR-based methods. Each compartment acts as an independent detection unit with its own microbead population and capture sequence.
Solution Approach 2:
The invention adds a spatial dimension to multiplexed detection by using microbeads with different spectral signatures (fluorescent, phosphorescent, or magnetic properties) to encode multiple targets within a single reaction. This dimensional encoding allows distinction between multiple pathogens through their unique spectral signatures rather than requiring separate reactions for each pathogen.
2Adaptability or versatility
If sample splitting and compartmentalization are used to test for multiple pathogens, then multiplexing is enabled, but sensitivity is reduced due to statistical sampling of blood volumes
Solution Approach 1:
The invention merges multiple capture oligonucleotides and their corresponding microbead populations into a single reaction compartment. This consolidation allows the entire blood sample volume to be utilized for detecting all targets simultaneously, maintaining high sensitivity while enabling multiplexed detection of multiple pathogens without the need for sample splitting.
3Measurement precision
If pre-amplification steps are used to compensate for reduced sensitivity, then detection sensitivity is improved, but false-positive detections increase and detection complexity is compounded
Solution Approach 1:
The invention performs preliminary capture of target nucleic acids onto microbeads before detection, concentrating the targets from the entire sample volume onto a small fraction of the total volume. This pre-concentration step achieves the necessary sensitivity enhancement without requiring additional amplification cycles, thereby reducing the risk of false positives and maintaining assay reliability.
4Adaptability or versatility
If next-generation sequencing is used for unbiased pathogen discovery, then detection comprehensiveness is improved, but detection time increases to many hours or days
Solution Approach 1:
The invention extracts and detects specific pathogen signatures using targeted capture oligonucleotides that hybridize to conserved regions of pathogen genomes. This targeted approach extracts only the relevant pathogen information from the sample, enabling rapid detection within hours rather than requiring the comprehensive but time-consuming whole-genome sequencing approach.
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 rapid, sensitive, and specific detection of multiple pathogens in a single reaction, reducing false positives and increasing throughput, with the ability to detect up to 96 samples in 2 hours or less.
Implementation Method 1
each of the microbeads has a lanthanide spectral signature
Implementation Method 2
detecting signal from immobilized signal-generating moieties of individual microbeads
Implementation Method 3
wherein the combining is conducted under conditions in which at least some amplicons are captured onto the beads by the capture polynucleotides
Implementation Method 4
the amplicons are labeled with one or more signal-generating moieties
Implementation Method 5
detecting signal from immobilized signal-generating moieties
Data Source
AI summary
Methods for characterizing a population of microbes in a sample are described. The methods include: amplifying microbial polynucleotides obtained from the sample to form a plurality of amplicons and combining the amplicons with a plurality of microbeads, wherein each of the microbeads has a lanthanide spectral signature paired with the sequence of capture polynucleotides immobilized on the microbead. At least some of the capture polynucleotides comprise a sequence substantially complementary to a microbe-identifying sequence in one or more amplicons, such that at least some amplicons are captured onto beads by the capture polynucleotides and the microbe can be identified based on the lanthanide spectral signature of with which the capture polynucleotide is paired. Further described are methods for the identification of pathogens present in a sample by evaluating patterns of hybridization of a capture oligonucleotide to amplicons. Microbead compositions and methods for the preparation thereof are also described.