HRM Analysis for E. coli ST131 Identification
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Solution Overview
Problem
Current methods for identifying E. coli sequence type 131 are expensive, labor-intensive, and time-consuming, requiring whole genome sequencing or pulse field gel electrophoresis, which are not cost-effective or rapid enough for clinical settings.
Innovation Solution
A High Resolution Melting (HRM) analysis method that amplifies a gene target with a fluorescent reporter dye and records fluctuations in fluorescence under an increasing temperature gradient, producing a unique melt curve to differentiate E. coli sequence type 131 without the need for sequencing PCR amplicons, using a HRM-capable thermocycler and multilocus sequence typing primers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome sequencing or MLST analysis is used to identify E. coli sequence type, then identification accuracy is improved, but cost and time consumption increase significantly
Solution Approach 1:
The patent extracts and analyzes only specific short amplicon regions (7 genes for MLST) from the entire genome, rather than sequencing the whole genome. This selective extraction of critical information regions enables rapid identification of sequence type while dramatically reducing sequencing time and cost compared to complete genome sequencing.
Solution Approach 2:
The patent uses PCR amplification to create multiple copies of target DNA regions before analysis. By amplifying the gene targets of interest exponentially, sufficient material is generated for accurate sequencing and analysis without requiring whole genome sequencing, thus reducing both time and resource requirements while maintaining identification accuracy.
2Measurement precision
If whole genome sequencing is used to identify E. coli sequence type, then identification accuracy is improved, but cost increases significantly
Solution Approach 1:
The patent extracts and sequences only specific short amplicon regions (7 genes for MLST) from the entire genome, rather than sequencing the whole genome. This selective extraction of critical information regions enables rapid identification of sequence type while dramatically reducing sequencing time and cost compared to complete genome sequencing.
Solution Approach 2:
The patent performs partial sequencing of only the essential MLST gene regions rather than complete genome sequencing. This partial action approach provides sufficient information for sequence type identification at a fraction of the cost and time of whole genome sequencing, while generating more than enough data for accurate classification.
3Measurement precision
If traditional sequencing methods are used, then sequence type identification is achieved, but labor intensity and complexity increase
Solution Approach 1:
The patent segments the complex task of bacterial identification into distinct modular components: DNA extraction, PCR amplification of specific genes, sequencing of amplicons, and bioinformatics analysis. This segmentation allows each step to be optimized independently and performed by different personnel or automated systems, reducing overall labor intensity and methodological complexity compared to whole genome sequencing.
Solution Approach 2:
The patent performs preliminary PCR amplification of target regions before sequencing, preparing the DNA in advance in a standardized format. This preliminary action simplifies the subsequent sequencing and analysis steps by ensuring adequate material is available and properly prepared, reducing labor intensity during the critical analysis phases.
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
This method provides rapid and cost-effective differentiation of E. coli sequence type 131 from other types, achieving 100% sensitivity and high specificity, reducing reagent costs and processing time, and can be performed in any lab with a HRM-capable instrument, facilitating screening in large sample populations.
Implementation Method 1
amplifying a gene target in the presence of a fluorescent reporter dye; recording fluctuations in fluorescence
Implementation Method 2
exposing the gene target and the fluorescent reporter dye to an increasing temperature gradient to denature and reduce the helicity
Data Source
AI summary
Methods for differentiating E. coli sequence type 131 are described. In one implementation, a method that employs example techniques in accordance with the present disclosure for determining E. coli sequence type 131 includes amplifying a gene target in the presence of a fluorescent reporter dye; exposing the gene target and the fluorescent reporter dye to an increasing temperature gradient to denature and reduce the helicity of a double-stranded oligo of the gene target; recording fluctuations in fluorescence using a High Resolution Melting analysis (HRM)-capable thermocycler; and producing a melt curve unique to the gene target.