Abstract: Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells and cell free nucleic acids. The intact cells and cell free nucleic acids are captured and concentrated within the pores of a silicate matrix in a microporous silicate filter. Within the silicate matrix the cell free nucleic acids are degraded with a nuclease, the intact cells are lysed with the released DNA binding to the silicate, the nuclease treated cell free nucleic acids and contaminants from the lysed are washed from the silicate matrix and the DNA bound to the silicate is eluted therefrom to form an isolated DNA product.

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 method for detecting the presence of a COVID-19 virus RNA or other pathogenic respiratory viruses, such as an influenza virus, or other RNA of interest in a sample. Nucleic acids are obtained from the sample and are used as a template in a combined isothermal reverse transcription, RNAse H and isothermal amplification reaction to generate single stranded RNA amplicons containing sequences complementary to fluorescent labeled detector probes. The single-stranded RNA amplicons hybridize to the detector probe and to hybridization probes with sequences complementary to a sequence determinant in the COVID-19 or other virus RNAs. The microarray is imaged to detect fluorescent signals thereby identifying the virus.

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 detecting the presence of a COVID-19 virus in a human sample or an environmental sample having one or more viruses and bacterial pathogens. Samples are processed to obtain total nucleic acids. A combined reverse transcription and asymmetric PCR amplification reaction is performed to obtain fluorescent labeled COVID-19 virus specific amplicons. The amplicons are detected by microarray hybridization near the lowest limit of detection. Also provided is a method for detecting concurrently with COVID-19 virus, the presence of respiratory disease-causing pathogens including viruses, bacteria and fungus in a single assay using the above method.

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 are methods for isolating, detecting and analyzing a virus in a sample. Generally, the virus is isolated via centrifuging, incubating, aggregating, and lysing the sample to obtain a crude neutralized lysate. The crude neutralized lysate is analyzed and the virus(es) are detected by performing one of a first polymerase chain reaction (PCR) amplification followed by a second PCR amplification, by an isothermal amplification or by the reverse transcription reaction and the first polymerase chain reaction amplification together utilizing fluorescently labeled primer pairs and hybridizing the resultant fluorescent labeled amplicons to complementary nucleic acid probes.

Abstract: Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells and cell free nucleic acids. The intact cells and cell free nucleic acids are captured and concentrated within the pores of a silicate matrix in a microporous silicate filter. Within the silicate matrix the cell free nucleic acids are degraded with a nuclease, the intact cells are lysed with the released DNA binding to the silicate, the nuclease treated cell free nucleic acids and contaminants from the lysed are washed from the silicate matrix and the DNA bound to the silicate is eluted therefrom to form an isolated DNA product.

Abstract: Provided herein is a method for detecting the presence of clade variants in the COVID-19 virus in a human sample and/or an environmental sample. Samples are processed to obtain total RNA. The RNA is used as a template in a combined reverse transcription and amplification reaction to obtain fluorescent COVID-19 virus amplicons. These amplicons are hybridized on a microarray with nucleic acid probes having sequences that discriminate among the various clade variants. The microarray is imaged to detect the clade variant and each clade variant is distinguished from others by generating an intensity distribution profile from the image, which is unique to each of the clade variants.

Abstract: Provided herein is a method for detecting the presence of clade variants in the COVID-19 virus in a human sample and/or an environmental sample. Samples are processed to obtain total RNA. The RNA is used as a template in a combined reverse transcription and amplification reaction to obtain fluorescent COVID-19 virus amplicons. These amplicons are hybridized on a microarray with nucleic acid probes having sequences that discriminate among the various clade variants. The microarray is imaged to detect the clade variant and each clade variant is distinguished from others by generating an intensity distribution profile from the image, which is unique to each of the clade variants. Also provided are methods for detecting and genotyping SARS-CoV-2.

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 method for detecting the presence of clade variants in the COVID-19 virus in a human sample and/or an environmental sample. Samples are processed to obtain total RNA. The RNA is used as a template in a combined reverse transcription and amplification reaction to obtain fluorescent COVID-19 virus amplicons. These amplicons are hybridized on a microarray with nucleic acid probes having sequences that discriminate among the various clade variants. The microarray is imaged to detect the clade variant. Also provided is a method of distinguishing each clade variant from others by generating an intensity distribution profile from the image, which is unique to each of the clade variants.

Abstract: Provided herein is a method for detecting the presence of a COVID-19 virus in a human sample or an environmental sample having one or more viral and bacterial pathogens. Samples processed to obtain total nucleic acids. The nucleic acids are used as a template in a reverse transcription-amplification reaction to obtain cDNA, which is used in a PCR amplification reaction to obtain fluorescent COVID-19 virus specific amplicons. These amplicons are detected by microarray hybridization near the lowest limit of detection. Also provided is a method for detecting in addition to the COVID-19 virus, the presence of respiratory disease-causing pathogens including viruses, bacteria and fungus in a single assay using the above method.

Abstract: Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells, contamination dead cells, cell debris, and biofilm using two separation steps, either by centrifugation or filtration, performed in sequentially. Also provided is a method for isolating nucleic acids from intact cells using a first separation step followed by treatment with a nuclease and then a second separating step. Provided herein is a related method for isolating DNA from intact cells using a nuclease that produces DNA cuts on double stranded DNA, followed by a second separating step.

Abstract: Provided herein is a method of quantitating a fungus in a plant, plant product or agricultural product. Total nucleic acids are isolated from a sample of the plant or plant product, and an asymmetric PCR amplification reaction is performed using fluorescent labeled primer pairs to obtain fluorescent labeled fungal amplicons. These amplicons are hybridized to fungus specific nucleic acid probes that are attached on a microarray support. The microarray is imaged to detect fluorescent signals from the fluorescent labeled fungal amplicons. The fluorescent signal intensity is correlated to the quantity of fungus.

Abstract: Provided herein is a method for identifying a mastitis-causing microbe in a subject. A milk sample is centrifuged to form a microbial pellet, total nucleic acids are extracted from the pellet and a microarray analysis of extracted DNA from which the mastitis-causing microbe is identified from DNA hybridization to mastitis-causing microbe species-specific gene probes. Also provided is a method for diagnosing a bovine mastitis infection in a dairy cow after identifying the bovine mastitis-causing microbe in a raw milk sample from the dairy cow.

Abstract: Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells, contamination dead cells, cell debris, and biofilm using two separation steps, either by centrifugation or filtration, performed in sequentially. Also provided is a method for isolating nucleic acids from intact cells using a first separation step followed by treatment with a nuclease and then a second separating step. Provided herein is a related method for isolating DNA from intact cells using a nuclease that produces DNA cuts on double stranded DNA, followed by a second separating step.

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.