Single-Cell Digital HRM for Accurate Microbial Cell Counting
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Solution Overview
Problem
Existing methods for microbial detection, such as digital High Resolution Melting (dHRM) and digital PCR, struggle with accurate quantification and linkage of antibiotic resistances due to DNA extraction biases, species-specific differences, and contamination by non-microbial DNA, leading to inaccurate cell counts and loss of linkage information.
Innovation Solution
A method involving selective enrichment of microbial cells, partitioning them into fixed reaction chambers for in situ lysis and amplification, followed by digital High Resolution Melting (dHRM) analysis, which allows for simultaneous identification and quantification of microbial cells and their genetic markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If DNA extraction is performed in bulk, then amplification can be carried out, but linkage information between genetic markers is lost due to fragmentation and separation
Solution Approach 1:
The patent divides the sample into individual reaction chambers (partitioning) before lysis, so that each chamber contains material from a single cell or organism. This segmentation prevents the mixing and fragmentation that occurs in bulk extraction, thereby preserving linkage information between genetic markers within each partition.
Solution Approach 2:
The patent performs partitioning of the sample into individual reaction chambers before carrying out lysis and DNA extraction. This preliminary action ensures that linkage information is preserved from the outset, as the physical separation occurs prior to any fragmentation that would occur during subsequent processing steps.
2Measurement precision
If conventional PCR-based methods are used, then amplification of target DNA can be achieved, but accurate cell counts cannot be obtained due to genetic ploidy differences and DNA extraction biases
Solution Approach 1:
By partitioning the sample into many individual reaction chambers, the patent enables digital counting of amplification events. Each positive reaction chamber represents a single starting template (cell), allowing for accurate cell count measurement that is independent of genetic ploidy differences and DNA extraction efficiencies.
Solution Approach 2:
The patent replaces the mechanical/biochemical process of quantifying DNA amount (which is affected by ploidy and extraction bias) with a digital counting approach based on partitioned amplification events. This substitution of measurement methodology eliminates the sources of bias inherent in conventional PCR-based quantification.
3Measurement precision
If non-microbial DNA is present in the sample, then DNA extraction can be performed, but detection accuracy decreases due to inhibition and background noise
Solution Approach 1:
The patent selectively removes or enriches for microbial cells before partitioning and lysis. By taking out the non-microbial DNA through selective enrichment steps prior to the partitioning process, the background noise and inhibition are reduced, thereby improving detection accuracy of the target microbial sequences.
4Measurement precision
If digital PCR with TaqMan probes is used, then specific detection can be achieved, but quantitative information about mixed samples is lost
Solution Approach 1:
The patent uses a universal amplification approach that can detect and quantify multiple different targets in the same reaction chamber. By combining partitioning with universal amplification and melt curve analysis, the system achieves both specific detection of individual targets and quantitative information about mixed samples, eliminating the trade-off present in TaqMan probe-based digital PCR.
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 cell counts and linkage of genetic markers, reducing biases from DNA extraction and enabling precise treatment strategies for multi-resistant organisms by maintaining linkage information within the same reaction chamber.
Implementation Method 1
The HRM process in its simplest form entails 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 microscopic organism to release nucleic acid
Data Source
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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.