Microarray Pathogen Detection Using Synthetic DNA Standards
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Solution Overview
Problem
Current methods for pathogen detection in plant, agriculture, food, and water samples are hindered by inaccuracies, high costs, and inefficiencies, including false positives and negatives, and the inability to analyze unpurified samples effectively, particularly due to interference from plant constituents and the need for multiple tests for concurrent pathogen analysis.
Innovation Solution
A method utilizing a 3-dimensional lattice microarray system with synthetic DNA as an internal reference standard for absolute copy number determination, involving tandem PCR amplification and fluorescent labeling, followed by hybridization with probes immobilized on the microarray, allowing for simultaneous detection and quantitation of pathogens in unpurified samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA extraction is performed before PCR analysis, then pathogen detection accuracy is improved, but processing time and cost increase
Solution Approach 1:
The invention extracts only the necessary DNA amplification step from the traditional extraction-PCR workflow by using Colony PCR that amplifies DNA directly from colonies without requiring prior DNA extraction and purification, thereby eliminating time-consuming extraction steps while maintaining detection accuracy
Solution Approach 2:
The method performs colony formation and preliminary DNA amplification in a single step by adding PCR reagents directly to colonial growth, allowing the system to prepare and amplify pathogen DNA simultaneously before microarray analysis, thus reducing overall processing time
2Loss of time
If Colony PCR is used to eliminate extraction step, then processing time is reduced, but detection sensitivity decreases due to interference from specimen constituents
Solution Approach 1:
The microarray probe acts as an intermediary that specifically binds to amplified pathogen DNA sequences, enabling specific detection even in the presence of interfering specimen constituents, thus maintaining sensitivity without requiring DNA extraction
Solution Approach 2:
The invention changes the detection parameter from direct DNA visualization to fluorescent signal detection via microarray hybridization, which provides higher sensitivity and specificity by detecting fluorescent labels bound to pathogen-specific sequences rather than relying on raw PCR product analysis
3Measurement precision
If multiple individual qPCR tests are performed for concurrent pathogen analysis, then pathogen identification specificity is improved, but device complexity and cost increase
Solution Approach 1:
The invention merges multiple individual qPCR tests into a single microarray-based multiplex assay that can simultaneously detect and identify multiple pathogens in one experiment, reducing the number of separate tests from many individual qPCRs to a single integrated platform
Solution Approach 2:
The microarray platform serves multiple functions simultaneously: it performs PCR amplification, hybridization, and detection of multiple pathogens in a single system, replacing the need for multiple separate qPCR machines and protocols with one universal diagnostic platform
4Reliability
If standard culture methods are used for pathogen detection, then reliability of pathogen identification is improved, but time consumption and false positives increase
Solution Approach 1:
The invention replaces the mechanical culture-based identification system with a molecular biology-based microarray system that uses DNA amplification and hybridization to identify pathogens, providing faster results while maintaining or improving reliability through sequence-specific detection
Solution Approach 2:
The method uses fluorescent color changes and signal intensities on the microarray to indicate pathogen presence and identity, replacing the visual colony morphology assessment with quantitative fluorescent detection that provides more reliable and faster identification
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 approach reduces processing steps, minimizes chemical use, and provides accurate, specific, and reliable detection of pathogens, enabling faster results while determining absolute copy numbers without the need for sample purification.
Implementation Method 1
hybridization with probes immobilized on the microarray
Implementation Method 2
tandem PCR amplification
Implementation Method 3
fluorescent labeling
Data Source
AI summary
Provided herein is an internal standard method for determining copy number of a pathogen DNA in an unpurified nucleic acid sample by using a known copy number of synthetic DNA that shares a consensus region sequence with the pathogen. The sample is subject to two amplification steps using locus-specific primers and fluorescent primers respectively to obtain fluorescent amplicons for the pathogen and synthetic DNA. These are hybridized with immobilized pathogen-specific and synthetic DNA-specific nucleic acid probes and imaged to obtain fluorescent signals for pathogen-specific and synthetic DNA-specific amplicons. Signal intensities are correlated with the known copy number of synthetic DNA to determine copy number of pathogen DNA in the plant. Also described herein is a method to simultaneously quantitate using the above method, copy numbers of both pathogen and plant DNA in a sample.