Abstract: Provided is a method for a two-step multiplex DNA amplification reaction which allows bacterial or fungal DNA analysis without first extracting DNA from the sample, nor without need to enrich microbes by laboratory culture prior to analysis. Without additional DNA purification or analysis, the PCR amplified DNA is administered directly to a microarray designed to interrogate a large panel of meaningful bacteria or fungi as a single multiplex test. Microarray analysis is then performed at ambient temperature, thus enabling substantial simplification of the testing process. It is contemplated that analysis may be conducted on unprocessed and processed leaf wash and similar surface sampling of plant material, cannabis, vegetables, fruit, nuts, spices, grains, other agriculture samples, food samples, or water samples, so as to detect bacterial, yeast, mold or viral, plant or human pathogen contamination.

Abstract: Provided herein is a method for manufacturing a microarray system, for example, 3-dimensional lattice microarray system, for DNA sequence detection and analysis. A solid support, such as a plastic substrate, is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer linkers, such as oligothymidine linkers, and a solvent mixture of water and a water-miscible liquid. The bifunctional polymer linkers are attached to the solid support and the water is evaporated. Then the nucleic acid probes are attached to the bifunctional polymer linker.

Abstract: Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5? and 3? ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a method for fabricating the microarray system by first attaching the bifunctional polymer linkers to the solid support, followed by photochemical coupling of the nucleic probes to the microarray. A customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.

Abstract: Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5? and 3? ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.

Abstract: Provided herein is a method for manufacturing a microarray system, for example, 3-dimensional lattice microarray system, for DNA sequence detection and analysis. A solid support, such as a plastic substrate, is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer linkers, such as oligothymidine linkers, and a solvent mixture of water and a water-miscible liquid. The bifunctional polymer linkers are attached to the solid support and the water is evaporated. Then the nucleic acid probes are attached to the bifunctional polymer linker.

Abstract: Provided herein is a method for manufacturing a 3-dimensional lattice microarray system for DNA sequence detection and analysis. A solid support is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer liners and a solvent mixture of water and a water-miscible liquid. In a first attachment reaction the bifunctional polymer linkers are attached to the solid support and the water is evaporated. In a second attachment reaction the nucleic acid probes are attached to the bifunctional polymer linker.

Abstract: Provided is a method for a two-step multiplex DNA amplification reaction which allows bacterial or fungal DNA analysis without first extracting DNA from the sample, nor without need to enrich microbes by laboratory culture prior to analysis. Without additional DNA purification or analysis, the PCR amplified DNA is administered directly to a microarray designed to interrogatea large panel of meaningful bacteria or fungi as a single multiplex test. Microarray analysis is then performed at ambient temperature, thus enabling substantial simplification of the testing process. It is contemplated that analysis may be conducted on unprocessed and processed leaf wash and similar surface sampling of plant material, cannabis, vegetables, fruit, nuts, spices, grains, other agriculture samples, food samples, or water samples, so as to detect bacterial, yeast, mold or viral, plant or human pathogen contamination.

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.

Abstract: 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.

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.

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.

Abstract: 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.

Abstract: 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.

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.

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.

Abstract: 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.

Abstract: Provided herein is a method for manufacturing a 3-dimensional lattice microarray system for DNA sequence detection and analysis. A solid support is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer liners and a solvent mixture of water and a water-miscible liquid. In a first attachment reaction the bifunctional polymer linkers are attached to the solid support and the water is evaporated. In a second attachment reaction the nucleic acid probes are attached to the bifunctional polymer linker.

Abstract: Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5? and 3? ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.

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.

Abstract: Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5? and 3? ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a method for fabricating the microarray system by first attaching the bifunctional polymer linkers to the solid support, followed by photochemical coupling of the nucleic probes to the microarray. A customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.