3D Lattice Microarray for Multiplex Plant Pathogen Detection
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Solution Overview
Problem
Current methods for pathogen detection in plant samples are inefficient due to high costs, time-consuming processes, inaccuracies, and inability to handle multiple pathogens simultaneously, especially in plant and agricultural contexts, where conventional culture-based tests and DNA-based methods face challenges with interference from plant constituents and require extensive sample preparation.
Innovation Solution
A 3-dimensional lattice microarray system using fluorescent labeled bifunctional polymer linkers and unmodified hybridization probes for multiplex pathogen and plant DNA detection, allowing for simultaneous amplification and identification of multiple pathogens in a single assay without the need for extensive sample purification, utilizing PCR amplification followed by hybridization on a microarray for accurate and specific detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional culture-based testing is used for pathogen identification, then pathogen detection can be performed, but the process requires substantial time, effort, and cost investment
Solution Approach 1:
The patent replaces the mechanical culture-based testing system with a molecular biology-based detection system using PCR amplification and microarray hybridization. This substitution eliminates the need for prolonged cultural incubation while maintaining pathogen detection capability, thereby dramatically reducing testing time while preserving reliability
Solution Approach 2:
The patent performs preliminary DNA extraction and PCR amplification of pathogen-specific genetic markers before the actual detection step. By pre-amplifying the target DNA sequences and preparing the microarray probes in advance, the system eliminates the time-consuming culture step while ensuring sufficient analyte for detection, thus resolving the time-reliability contradiction
2Measurement precision
If DNA extraction is performed before PCR analysis, then pathogen DNA can be detected, but the process becomes time-consuming and costly
Solution Approach 1:
The patent selectively extracts only the essential pathogen DNA components needed for detection using targeted PCR amplification with pathogen-specific primers. Instead of complete DNA extraction followed by comprehensive analysis, the system extracts and amplifies only the relevant genetic markers, thereby reducing preparation time and cost while maintaining detection precision
Solution Approach 2:
The patent changes the detection parameter from analyzing total DNA or cultured organisms to detecting specific amplified DNA sequences using fluorescently-labeled probes. This parameter change allows direct detection from minimal sample preparation without requiring extensive purification steps, thus reducing time and cost while preserving measurement precision
3Ease of operation
If Colony PCR is used to eliminate pre-analysis extraction, then sample preparation is reduced, but sensitivity is lower than culture methods due to interference from specimen constituents
Solution Approach 1:
The patent introduces an intermediary purification step that selectively removes interfering specimen constituents while preserving pathogen DNA. This intermediary step bridges the gap between simple Colony PCR and sensitive detection, allowing direct PCR from minimal preparation without the interference problems that reduce sensitivity, thus resolving the contradiction between ease of operation and measurement precision
4Productivity
If qPCR is used for rapid pathogen detection, then analysis time is reduced to less than an hour, but the system is limited to analyzing only a single pathogen
Solution Approach 1:
The patent segments the detection system into multiple independent probe channels, each specific to a different pathogen. By dividing the single qPCR reaction into multiple targeted detection channels using fluorescently-labeled probes with different emission spectra, the system maintains rapid detection speed while enabling simultaneous analysis of multiple pathogens, thus resolving the productivity-versatility contradiction
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 reduces processing steps, costs, and time while maintaining accuracy and specificity, enabling the detection and quantitation of multiple pathogens in a single assay, overcoming previous limitations of interference and sample preparation requirements.
Implementation Method 1
a 3-dimensional lattice microarray system having a plurality of nucleic acid probes specific to sequence determinants in pathogen DNA
Implementation Method 2
fluorescent labeled bifunctional polymer linkers and unmodified hybridization probes for multiplex pathogen and plant DNA detection
Data Source
AI summary
Provided herein is a dual amplification method for identifying plant pathogens by analysis of pathogen DNA in an unpurified nucleic acid sample from the plant. Pathogen-specific and/or plant-specific primers are used to generate a first set of amplicons that are further amplified in a second amplification step using fluorescent tagged pathogen-specific primers. Fluorescent amplicons thus generated are hybridized with pathogen-specific nucleic acid probes that are immobilized on a solid support using bifunctional polymer linkers. The hybridized microarray is imaged to obtain fluorescent images of the amplicons and the nucleic acid probes, which are superimposed to detect the pathogen present in the plant. Also described herein is a method to identify a plant by analysis of plant DNA and a method to simultaneously detect both plant DNA and pathogen DNA in a single assay.