Single-Cell Digital HRM for Microbial Identification and Counts
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Solution Overview
Problem
Existing PCR-based technologies struggle to provide accurate, unbiased cell counts and linkage information for microbial species due to genetic ploidy variations, species-specific biases, and the presence of inhibitors in samples, leading to inaccurate quantification and identification of antibiotic resistances.
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, and using computer algorithms to identify and quantify microbial cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If DNA extraction is performed in bulk before PCR analysis, then amplification can be performed, but accurate cell counts and linkage information are lost due to fragmentation and separation of markers
Solution Approach 1:
The patent divides the sample into individual reaction chambers (e.g., 96 or more chambers) where each chamber contains a single or few microbial cells. This segmentation allows analysis of individual cells without bulk extraction, preserving cell count accuracy and marker linkage information that would be lost in bulk processing.
Solution Approach 2:
The patent performs cell lysis and marker analysis within the reaction chambers before traditional bulk DNA extraction. By lysing cells in situ in each chamber and analyzing markers (such as fluorescent tags or molecular probes) directly in the chamber, the method avoids the fragmentation and separation issues of bulk extraction while maintaining the ability to perform amplification and detection.
2Measurement precision
If TaqMan probes are used in digital PCR, then detection sensitivity is improved, but the method is limited by species-specific biases and cannot provide unbiased quantification
Solution Approach 1:
The patent employs universal detection methods that work across multiple microbial species without requiring species-specific TaqMan probes. The reaction chambers can detect various microbial markers using common fluorescent probes or molecular techniques that are not biased toward specific species, enabling unbiased quantification while maintaining high detection sensitivity through digital partitioning.
3Adaptability or versatility
If inhibitors are present in samples such as blood or soil, then the sample represents real-world conditions, but amplification accuracy is compromised
Solution Approach 1:
By dividing the sample into many small reaction chambers, the patent dilutes and distributes inhibitors across numerous chambers. This segmentation reduces the impact of inhibitors on individual amplification reactions, allowing accurate detection even in complex samples like blood or soil that would otherwise contain inhibitory substances.
4Ease of manufacture
If bulk DNA extraction is performed, then DNA can be analyzed, but linkage information between antibiotic resistance markers and chromosomal species markers is lost
Solution Approach 1:
The patent maintains linkage information by analyzing individual cells or few cells per reaction chamber before and after lysis. Markers such as fluorescent tags bound to cells or membrane proteins are detected in the same chamber where the cell resides, preserving the spatial relationship and linkage between species markers and antibiotic resistance markers that would be lost during bulk extraction and DNA fragmentation.
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 accurate identification and quantification of microbial cells, preserves linkage information between genotypic markers, and allows for phenotypic profiling, overcoming biases from DNA extraction and providing more treatment options for antibiotic-resistant infections.
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)
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.