Single-Cell Digital HRM Profiling for Polymicrobial Samples
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Solution Overview
Problem
Existing PCR-based technologies struggle to accurately identify and quantify microbial genomes in polymicrobial samples due to genetic ploidy variations, species-specific biases, DNA extraction issues, and the presence of inhibitors, which hinders the linkage of antibiotic resistance markers with specific species, leading to inaccurate treatment recommendations.
Innovation Solution
A method involving the selective enrichment of microbial cells, partitioning them into fixed reaction chambers for in situ lysis and digital high-resolution melt (dHRM) analysis, allowing for simultaneous amplification and melt curve analysis in each chamber, followed by computer algorithm comparison with known sequences to identify and quantify microbial cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA extraction is performed in bulk to enable PCR-based detection, then amplification can proceed, but linkage information between antibiotic resistance markers and species markers is lost due to fragmentation and separation
Solution Approach 1:
The patent divides the sample into multiple individual reaction chambers (e.g., 96 wells), with each chamber containing a single microbial cell. This segmentation prevents bulk DNA extraction and maintains the physical linkage between species markers and antibiotic resistance markers within each cell, allowing accurate association of resistance phenotypes with specific species.
Solution Approach 2:
The patent performs single-cell sorting and partitioning into individual reaction chambers before any lysis or amplification steps. This preliminary action ensures that each reaction chamber contains material from a single cell, preserving the integrity of linkage information between different genetic markers that would otherwise be separated during bulk DNA extraction.
2Measurement precision
If bulk DNA extraction is used to detect microbial genomes, then amplification can occur, but accurate cell counts cannot be generated due to species-specific biases and genetic ploidy variations
Solution Approach 1:
By partitioning the sample into individual reaction chambers containing single cells, the patent eliminates the need to account for species-specific biases and genetic ploidy variations that plague bulk extraction methods. Each chamber's amplification signal directly reflects the presence of one cell, enabling accurate cell counting without complex correction factors.
Solution Approach 2:
The patent changes the fundamental parameter of sample organization from bulk homogeneous mixture to individual segregated units. This parameter change transforms the detection approach from measuring average signals across many cells to counting discrete individual events, thereby achieving accurate cell counts despite variations in genetic ploidy and species-specific amplification efficiencies.
3Measurement precision
If TaqMan probes are used for digital PCR, then specific detection can occur, but quantitative information about mixed samples is limited by probe requirements
Solution Approach 1:
The patent removes the requirement for TaqMan probes by using alternative detection methods such as SYBR Green dye-based fluorescence or melt curve analysis. This extraction of the probe requirement simplifies the assay design for mixed samples, allowing simultaneous quantification of multiple species without the need for multiple specific probes, thereby improving quantification accuracy while reducing assay complexity.
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 provides accurate, unbiased cell counts and linkage information between genotypic markers, enabling targeted treatment of antibiotic-resistant organisms by profiling multiple species and variants within a heterogeneous sample, while minimizing contamination and reaction-to-reaction variation.
Implementation Method 1
a melting step during which precise control of a heating element allows for the sequence-dependent denaturation of double-stranded DNA (dsDNA) to single stranded DNA (ssDNA)
Implementation Method 2
lysis of the microbial cells in the reaction mixture in each of the reaction chambers to create a lysed sample comprising a nucleic acid
Data Source
AI summary
Provided are devices, systems, and methods for the identification, quantification, and profiling of microscopic organisms. The methods for the identification, quantification, and profiling of microscopic organisms include, for example, the selective enrichment of microscopic organisms from a heterogeneous sample; subsequent loading of the microscopic organisms into microfluidic channels or reaction chambers; direct amplification of nucleic acids from single, isolated microscopic organisms; and examination of amplification products using digital High Resolution Melting (HRM) analysis.