Metagenomic Pathogen Detection With Host Nucleic Acid Depletion
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Solution Overview
Problem
Current metagenomic methods for pathogen detection in clinical samples are time-consuming and inefficient due to the need to distinguish and sequence both human and microbial nucleic acids, often requiring centrifugation and chemical lysis, which can contaminate and complicate the sample.
Innovation Solution
A method involving mechanical disruption to lyse mammalian cells while preserving microbial nucleic acids, followed by nuclease treatment to digest host nucleic acids, allowing simultaneous detection of DNA and RNA from various pathogens without centrifugation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If chemical lysis is used to release host nucleic acid, then host DNA is released for depletion, but microbial nucleic acid is also damaged and contamination increases
Solution Approach 1:
The patent segments the lysis process into two distinct stages: first, mechanical disruption (bead beating) selectively lyses host cells while preserving microbial cells; second, enzymatic digestion releases and depletes host nucleic acid without affecting microbial nucleic acid. This segmentation resolves the contradiction by separating the harmful effects of chemical lysis from the desired host DNA release.
Solution Approach 2:
The patent introduces mechanical disruption (bead beating) as an intermediary step between sample collection and chemical/enzymatic processing. This intermediary mechanical lysis selectively breaks host cells while leaving microbial cells intact, enabling subsequent specific depletion of host nucleic acid without contaminating or damaging microbial nucleic acid.
2Ease of manufacture
If centrifugation is used to separate microorganisms, then bacteria can be sedimented, but viruses are lost in supernatant and sample manipulation increases
Solution Approach 1:
Instead of using centrifugation to separate and concentrate microorganisms (which loses viruses in supernatant), the patent inverts the approach: it processes the entire sample homogenate through mechanical disruption and enzymatic depletion, then sequences all nucleic acid without separation. This inversion eliminates the need for centrifugation while capturing both bacterial and viral pathogens.
Solution Approach 2:
The patent employs a universal processing method (mechanical disruption followed by nuclease depletion) that works for all microorganism types in the sample without requiring separate handling for bacteria, viruses, or other pathogens. This multi-functional approach maintains total microorganism integrity while enabling comprehensive detection.
3Adaptability or versatility
If metagenomic sequencing is performed on total nucleic acid, then all pathogens can be detected, but host DNA overwhelms microbial signal and time required increases
Solution Approach 1:
The patent extracts and selectively depletes host nucleic acid using nuclease treatment after mechanical disruption. This extraction/removal of the overwhelming host DNA signal allows metagenomic sequencing to efficiently detect microbial pathogens without being swamped by host genetic material, reducing both time and computational burden.
Solution Approach 2:
The patent performs preliminary mechanical disruption (bead beating) and enzymatic depletion of host nucleic acid before metagenomic sequencing. This preliminary action removes the dominant host DNA signal in advance, enabling faster and more efficient pathogen detection during sequencing without requiring extended analysis time.
4Object-affected harmful factors
If mechanical disruption is used to lyse host cells, then host nucleic acid is released without chemical contamination, but energy input is required
Solution Approach 1:
The patent replaces chemical lysis methods with mechanical disruption (bead beating) to release host nucleic acid. This substitution eliminates chemical contaminants while the mechanical energy input is controlled and localized, providing a cleaner sample for downstream processing despite the energy requirement.
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 detection of multiple pathogens in 7 hours with high genome coverage, reducing contamination risks and sample manipulation, and supporting broad-range pathogen identification in diverse samples.
Implementation Method 1
subjecting the biological sample to mechanical disruption such that the mammalian host cells are lysed to thereby release host's nucleic acid
Implementation Method 2
contacting the mechanically disrupted sample with a nuclease, to thereby digest the host's released nucleic acid
Data Source
AI summary
The present invention relates to metagenomics methods. The invention also relates to their use in the detection and/or diagnosis of a wide range of infectious diseases, for example, caused by a pathogenic virus, bacterium, fungus or protozoan. The invention also extends to methods for sample preparation and microorganism detection and/or identification, and methods for host cell nucleic acid depletion. The invention further relates to kits and apparatus used in these methods. The invention is especially useful in clinical diagnostic and veterinary medicine.