Microarray based multiplex pathogen analysis and uses thereof
Inventors
Hogan, Michael Edward • May, Melissa Rose • Eggers, Frederick Henry
Assignees
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Publication Number
US-10767235-B2
Publication Date
2020-09-08
Expiration Date
Abstract
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.
Core Innovation
The invention provides methods for identifying plant DNA from a plant tissue sample and identifying a pathogen and a plant from a plant tissue sample in a single assay. Total nucleic acids comprising DNA and non-DNA nucleic acids are isolated from the plant tissue sample, and plant DNA, and in the combined method pathogen DNA, is amplified in a first amplification to generate plant-specific first amplicons and pathogen-specific first amplicons.
The amplified DNA is subjected to a second amplification to generate fluorescent labeled amplicons using fluorescent labeled primer pairs. The fluorescent labeled amplicons are hybridized with nucleic acid probes specific for signature sequence determinants in the plant DNA and pathogen DNA, where the probes are immobilized at specific known positions on a 3-dimensional lattice microarray via fluorescent labeled bifunctional polymer linkers.
A washing step and imaging step are performed on the 3-dimensional lattice microarray to detect a fluorescent signal corresponding to the fluorescent labeled amplicons and a fluorescent signal corresponding to the immobilized probes. Fluorescent signals are superimposed to obtain a superimposed signal image, and the sequence of the nucleic acid probe at one or more superimposed signal positions is compared with a database of signature sequence determinants for a plurality of plant DNA and pathogen DNA thereby identifying the plant and the at least one pathogen in the plant tissue sample.
Claims Coverage
The provided independent claims cover plant DNA identification and simultaneous pathogen and plant identification in a single assay. Across these claims, there are two core inventive feature sets: two-stage amplification with fluorescent labeled primers, probe-based hybridization on a 3-dimensional lattice microarray with signal superimposition, and database comparison to identify plant DNA, and analogous dual-target identification of pathogen DNA and plant DNA in a single assay.
Two-stage plant DNA amplification with fluorescent labeled primers and probe hybridization on a 3-dimensional lattice microarray
The method isolates total nucleic acids comprising DNA and non-DNA nucleic acids from a plant tissue sample, amplifies plant tissue DNA in a first amplification to generate plant-specific first amplicons, amplifies in a second amplification using the plant-specific first amplicons as template and fluorescent labeled second primer pairs to generate fluorescent labeled second amplicons, hybridizes the fluorescent labeled amplicons with nucleic acid probes specific for signature sequence determinants in the plant DNA immobilized at specific known positions on a 3-dimensional lattice microarray via fluorescent labeled bifunctional polymer linkers, and images to detect probe and amplicon fluorescence.
Superimposed signal imaging and database comparison of immobilized probe signature sequence determinants
After washing the 3-dimensional lattice microarray and imaging to detect fluorescent signals corresponding to the fluorescent labeled amplicons and the nucleic acid probes at known positions, the method superimposes the signals to obtain a superimposed signal image, and compares the sequence of the nucleic acid probe at one or more superimposed signal positions on the 3-dimensional lattice microarray with a database of signature sequence determinants for a plurality of plant DNA to identify the plant in the plant tissue sample.
Single-assay simultaneous pathogen and plant identification with fluorescent labeled primers and 3-dimensional lattice microarray
The method obtains a plant tissue sample comprising one or more pathogens, isolates total nucleic acids comprising DNA and non-DNA nucleic acids from the plant tissue and the pathogens, amplifies in a first amplification in a single assay the pathogen DNA and plant DNA using pathogen-specific and plant-specific primer pairs to generate pathogen-specific first amplicons and plant-specific second amplicons, amplifies in a second amplification using these amplicons as templates and fluorescent labeled third and fourth primer pairs to generate pathogen-specific fluorescent labeled third amplicons and plant-specific fluorescent labeled fourth amplicons, and hybridizes both sets of fluorescent labeled amplicons with nucleic acid probes specific for signature sequence determinants in pathogen DNA and plant DNA respectively, immobilized on a 3-dimensional lattice microarray via a fluorescent labeled bifunctional polymer linker.
Multi-signal imaging, superimposition, and database comparison for simultaneous identification
The method washes and images the 3-dimensional lattice microarray to detect signals corresponding to the pathogen-specific fluorescent labeled third amplicons, the plant-specific fluorescent labeled fourth amplicons, and the immobilized nucleic acid probes, superimposes each of the pathogen and plant signals with the probe signals, and compares the sequence of the nucleic acid probe at one or more superimposed signal positions with a database of signature sequence determinants for a plurality of pathogen DNA and a plurality of plant DNA to thereby identify the at least one pathogen and the at least one plant in the plant tissue sample.
The claims cover plant identification by two-stage amplification, fluorescent labeling, probe hybridization on a 3-dimensional lattice microarray, signal superimposition, and database comparison, and also cover simultaneous identification of a pathogen and a plant in a single assay using analogous dual-target amplification, imaging, superimposition, and database comparison.
Stated Advantages
Simultaneous identification of a pathogen and a plant in a single assay.
Documented Applications
No documented applications found
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