Microarray based multiplex pathogen analysis and uses thereof
First Claim
1. A method for identifying pathogens in a plant sample comprising the steps of:
- a) harvesting pathogens from a plant tissue sample;
b) isolating total nucleic acids comprising DNA and non-DNA nucleic acids from the pathogens harvested from the plant tissue sample;
c) amplifying, in a first amplification in a single assay, the pathogen DNA in the total nucleic acids using at least one first primer pair selective for at least one pathogen-specific DNA isolated from the harvested pathogens to generate one or more pathogen-specific first amplicons;
d) amplifying, in a second amplification using the one or more pathogen-specific first amplicons as a template and at least one first fluorescent labeled second primer pair to generate first fluorescent labeled second amplicons;
e) hybridizing the first fluorescent labeled second amplicons with nucleic acid probes specific for signature sequence determinants in the pathogen DNA, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via second fluorescent labeled bifunctional polymer linkers;
f) washing the 3-dimensional lattice microarray at least once;
g) imaging the 3-dimensional lattice microarray to detect a first fluorescent signal corresponding to the first fluorescent labeled second amplicons and a second fluorescent signal corresponding to the nucleic acid probes immobilized at the specific known positions on the 3-dimensional lattice microarray via the second fluorescent labeled bifunctional polymer linkers;
h) superimposing the first fluorescent signal with the second fluorescent signal to obtain a superimposed signal image; and
i) comparing 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 pathogen DNA, thereby identifying the pathogens in the sample.
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Abstract
Provided herein is a dual amplification method for detecting plant pathogens by analysis of pathogen DNA in an unpurified nucleic acid sample from the plant. Pathogen-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 simultaneously detect both plant DNA and pathogen DNA in a single assay.
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Citations
10 Claims
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1. A method for identifying pathogens in a plant sample comprising the steps of:
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a) harvesting pathogens from a plant tissue sample; b) isolating total nucleic acids comprising DNA and non-DNA nucleic acids from the pathogens harvested from the plant tissue sample; c) amplifying, in a first amplification in a single assay, the pathogen DNA in the total nucleic acids using at least one first primer pair selective for at least one pathogen-specific DNA isolated from the harvested pathogens to generate one or more pathogen-specific first amplicons; d) amplifying, in a second amplification using the one or more pathogen-specific first amplicons as a template and at least one first fluorescent labeled second primer pair to generate first fluorescent labeled second amplicons; e) hybridizing the first fluorescent labeled second amplicons with nucleic acid probes specific for signature sequence determinants in the pathogen DNA, said nucleic acid probes immobilized at specific known positions on a 3-dimensional lattice microarray via second fluorescent labeled bifunctional polymer linkers; f) washing the 3-dimensional lattice microarray at least once; g) imaging the 3-dimensional lattice microarray to detect a first fluorescent signal corresponding to the first fluorescent labeled second amplicons and a second fluorescent signal corresponding to the nucleic acid probes immobilized at the specific known positions on the 3-dimensional lattice microarray via the second fluorescent labeled bifunctional polymer linkers; h) superimposing the first fluorescent signal with the second fluorescent signal to obtain a superimposed signal image; and i) comparing 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 pathogen DNA, thereby identifying the pathogens in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification