Microbial cfDNA Enrichment via Size-Selected ssDNA Libraries
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for diagnosing sepsis and identifying microbial pathogens are hindered by low sensitivity, long turnaround times, and the challenge of differentiating between inflammatory responses and infections, leading to the misuse of antibiotics and antimicrobial resistance.
Innovation Solution
A method involving the preparation of single-stranded DNA libraries from body fluid samples, followed by size selection to enrich for DNA fragments of 110 nucleotides or less, which enhances the ratio of non-native cell-free DNA to host DNA, thereby improving the detection of microbial and circulating tumor DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microbial cultures are used for pathogen identification, then diagnostic accuracy is improved, but turnaround time increases to three days or longer
Solution Approach 1:
The patent extracts and analyzes only the relevant component (microbial cfDNA) from the complex plasma sample by using size selection to isolate short DNA fragments. This extraction approach enables rapid pathogen identification without requiring full microbial culture processes, thus reducing turnaround time while maintaining diagnostic accuracy.
2Productivity
If broad spectrum antibiotics are administered immediately, then patient treatment is initiated, but antimicrobial resistance increases
Solution Approach 1:
The patent implements a feedback mechanism where rapid pathogen identification through size-selected cfDNA sequencing provides information that guides appropriate antibiotic selection. This feedback loop enables clinicians to narrow or adjust antibiotic therapy based on actual pathogen identity, reducing unnecessary broad-spectrum antibiotic use and thereby limiting antimicrobial resistance development.
3Measurement precision
If size selection of sequencing libraries is performed to enrich microbial cfDNA, then the microbial cfDNA signal is enhanced, but the complexity of the library preparation process increases
Solution Approach 1:
The patent changes the size parameter of DNA fragments by selecting only short fragments (≤110 nucleotides) from the total cfDNA population. This parameter-based selection enriches for microbial cfDNA which is characteristically shorter than host cfDNA, thereby improving detection sensitivity while using standard library preparation techniques to minimize added complexity.
4Measurement precision
If the fragment size threshold is reduced to 110 nucleotides or less, then the ratio of non-native cfDNA to host cfDNA is enriched, but the stringency of size selection requirements increases
Solution Approach 1:
The patent applies partial size selection by focusing on a specific size range (≤110 nucleotides) rather than attempting to separate all DNA fragments across the entire size spectrum. This partial action approach achieves sufficient enrichment of microbial cfDNA without requiring extremely precise size selection that would be difficult to implement with standard equipment.
Data Source
AI summary
Described herein is a method of enriching a body fluid sample from a host subject for a ratio of non-native cell-free DNA (e.g., microbial cell-free DNA) to host subject cell-free DNA (host cfDNA). The method can include obtaining the body fluid sample from the host subject; extracting total cell-free DNA (total cfDNA) from the body fluid sample; preparing a single-stranded DNA library from the total cfDNA, wherein the single-stranded DNA library is enriched for cfDNA existing in a single-stranded configuration; and selecting a subset of the single-stranded DNA library based on the fragment size to provide a size-selected cfDNA library, wherein the size-selected cfDNA library has a DNA fragment length of 110 nucleotides or less and is enriched for the ratio of non-native cell-free DNA to host cfDNA.