Multi-Locus Amplicon Sequencing for Biothreat Pathogen Subtyping
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Solution Overview
Problem
Current biothreat detection systems rely on single locus PCR methods that generate false positives due to the complexity of environmental samples, where similar microorganisms confuse individual assays, and DNA sequencing is limited by incomplete knowledge of near-neighbor species.
Innovation Solution
A multi-agent multi-locus amplicon sequencing protocol targeting 79 targets to detect biothreat agents, including Bacillus anthracis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, and Yersinia pestis, with a universal amplicon indexing scheme for next-generation sequencing, enabling discrimination through multiplex amplification reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single locus PCR methods are used for biothreat detection, then the detection process is simple and fast, but false positives occur due to similar microorganisms in complex environmental samples
Solution Approach 1:
The invention segments the detection process into two distinct stages: (1) a PCR amplification stage that generates amplicons from target DNA, and (2) a next-generation sequencing stage that provides definitive identification. This segmentation allows the fast PCR method to be combined with the accurate sequencing method, resolving the contradiction between speed and accuracy by assigning each function to the most suitable technique.
Solution Approach 2:
The invention introduces amplicon sequencing as an intermediary step between PCR amplification and final pathogen identification. The amplicons serve as intermediaries that carry the amplified DNA sequences through to sequencing, enabling the transition from rapid but inaccurate PCR to precise sequencing-based identification, thereby eliminating false positives while maintaining workflow efficiency.
2Measurement precision
If DNA sequencing is used to improve pathogen identification accuracy, then near-neighbor species can be distinguished, but incomplete knowledge of near-neighbor species limits its effectiveness
Solution Approach 1:
The invention employs a universal amplicon indexing scheme that can identify multiple biothreat agents simultaneously across different species. The standardized indexing system is designed to work with a broad range of pathogens and their near-neighbors, making the system universally applicable even as new species are discovered. This universality allows the system to function effectively without requiring complete prior knowledge of all near-neighbor species.
3Adaptability or versatility
If multiple individual assays are used to detect different biothreat agents, then each agent can be specifically targeted, but the complexity of the detection system increases
Solution Approach 1:
The invention merges multiple individual assays into a single multiplexed amplicon sequencing assay. By combining the detection of multiple biothreat agents (Bacillus anthracis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, and Yersinia pestis) into one unified sequencing run, the system maintains agent-specific detection capability while dramatically reducing overall system complexity and operational burden.
Solution Approach 2:
The universal amplicon indexing scheme serves as a multi-functional platform that can detect multiple different biothreat agents simultaneously. This single system performs the functions of what would otherwise require multiple separate assays, providing versatility in detecting various agents while simplifying the overall detection architecture through standardization and integration.
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
The method achieves 100% sensitivity and 91-100% specificity in detecting biothreat agents, distinguishing between target pathogens and near neighbors, and identifying virulence factors and antibiotic resistance, with a single sequencing run across multiple samples.
Implementation Method 1
Current systems for early detection of these agents rely upon single locus Polymerase Chain Reaction (PCR) methods
Implementation Method 2
DNA sequencing offers great potential, and there is a need for primers, methods, assays, and kits with greater ability to discriminate microbial pathogens
Data Source
AI summary
The present invention provides methods of detecting a biothreat agent in a sample, comprising detecting at least one biothreat-specific amplicon in the sample. The methods also encompass confirming the absence of the biothreat agent by detecting Near Neighbor specific amplicons to avoid false positive results.