Multiplex Barcoded Nucleic Acid Analysis for Strain-Level Typing
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Solution Overview
Problem
Current methods for microbial and viral subtyping, such as whole genome sequencing (WGS) and traditional Multilocus Sequence Typing (MLST), are laborious, costly, and lack discriminatory power to differentiate closely related strains, especially in complex samples, and require skilled labor for data analysis.
Innovation Solution
A method involving multiplex barcoding amplification using target-specific primers and barcoded universal primers in a single reaction, followed by next-generation sequencing, to analyze a large panel of variable regions for high-resolution genotyping and subtyping, with a simplified workflow and automated potential.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If whole genome sequencing (WGS) is used for microbial and viral subtyping, then comprehensive genomic information is obtained, but the method is laborious, costly, and requires skilled labor for data analysis
Solution Approach 1:
The patent extracts and sequences only specific variable regions of interest from the genome rather than performing whole genome sequencing. This selective approach obtains sufficient discriminatory information for subtyping while avoiding the complexity and cost of sequencing entire genomes, directly resolving the contradiction between comprehensive information and workflow complexity
Solution Approach 2:
The patent divides the genome into specific variable regions that are targeted for sequencing. By segmenting the genomic analysis focus to only the most informative regions rather than analyzing the entire genome, the method maintains high discriminatory power while significantly reducing the labor and expertise required
2Device complexity
If traditional Multilocus Sequence Typing (MLST) is used for subtyping, then the method is simpler than WGS, but it lacks discriminatory power to differentiate closely related strains
Solution Approach 1:
The patent targets specific variable regions within the genome that have high variability and discriminatory power. By focusing sequencing efforts on these locally optimized regions rather than using traditional MLST loci, the method achieves superior strain differentiation while maintaining relative simplicity
Solution Approach 2:
The patent changes the parameters of what regions are sequenced - specifically targeting variable regions with higher discriminatory power compared to traditional MLST. This parameter change in region selection enables differentiation of closely related strains while keeping the overall approach simpler than WGS
3Measurement precision
If a large panel of variable regions is analyzed for high-resolution genotyping, then accurate strain and sub-strain differentiation is achieved, but the workflow becomes more complex and resource-intensive
Solution Approach 1:
The patent performs preliminary selection and design of target variable regions before the actual sequencing process. By pre-defining the specific regions to be amplified and sequenced, the method achieves high-resolution genotyping without adding complexity during the execution phase, as the targets are predetermined and optimized
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 efficient, cost-effective, and high-resolution detection and typing of microbial and viral strains, reducing the need for specialized resources and expertise, and providing accurate strain and sub-strain differentiation.
Implementation Method 1
hybridizing a plurality of target-specific primers with nucleic acid from the sample
Implementation Method 2
The multiplex PCR (polymerase chain reaction) method allows for the simultaneous amplification of multiple target DNA sequences in a single reaction. It involves the use of multiple primer pairs, each specific to a different target sequence, along with a DNA polymerase enzyme and nucleotides.
Data Source
AI summary
The present disclosure relates to compositions and methods for detection, identification, sequence analysis and quantification of biological organisms in a single amplification reaction. The disclosed method utilizes next-generation sequencing (NGS) to sequence amplified products. The present disclosure is also directed to kits containing primers specific to microbial and viral organisms, cancer, genetic disorders and forensics.