Patent Publication Number: US-10767235-B2

Title: Microarray based multiplex pathogen analysis and uses thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional under 35 U.S.C. § 120 of pending application U.S. Ser. No. 16/158,181, filed Oct. 11, 2018, which is a continuation-in-part under 35 U.S.C. § 120 of pending application U.S. Ser. No. 15/916,036, filed Mar. 8, 2018, which is a continuation-In-part under 35 U.S.C. § 120 of pending non-provisional application U.S. Ser. No. 15/388,561, filed Dec. 22, 2016, which claims benefit of priority under 35 U.S.C. § 119(e) of provisional application U.S. Ser. No. 62/271,371, filed Dec. 28, 2015, all of which are hereby incorporated in their entireties. 
    
    
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present disclosure is in the technical field of DNA based pathogen and plant analysis. More particularly, the present disclosure is in the technical field of pathogen analysis for plant, agriculture, food and water material using a multiplex assay and a 3-dimensional lattice microarray technology for immobilizing nucleic acid probes. 
     Description of the Related Art 
     Current techniques used to identify microbial pathogens rely upon established clinical microbiology monitoring. Pathogen identification is conducted using standard culture and susceptibility tests. These tests require a substantial investment of time, effort, cost as well as labile products. Current techniques are not ideal for testing large numbers samples. Culture-based testing is fraught with inaccuracies which include both false positives and false negatives, as well as unreliable quantification of colony forming units (CFUs). There are issues with the presence of viable but non-culturable microorganisms which do not show up using conventional culture methods. Certain culture tests are non-specific in terms of detecting both harmful and harmless species which diminishes the utility of the test to determine if there is a threat present in the sample being tested. 
     In response to challenges including false positives and culturing of microorganisms, DNA-based diagnostic methods such as polymerase chain reaction (PCR) amplification techniques were developed. To analyze a pathogen using PCR, DNA is extracted from a material prior to analysis, which is a time-consuming and costly step. 
     In an attempt to eliminate the pre-analysis extraction step of PCR, Colony PCR was developed. Using cells directly from colonies from plates or liquid cultures, Colony PCR allows PCR of bacterial cells without sample preparation. This technique was a partial success but was not as sensitive as culture indicating a possible issue with interference of the PCR by constituents in the specimens. Although this possible interference may not be significant enough to invalidate the utility of the testing performed, such interference can be significant for highly sensitive detection of pathogens for certain types of tests. Consequently, Colony PCR did not eliminate the pre-analysis extraction step for use of PCR, especially for highly sensitive detection of pathogens. 
     It is known that 16s DNA in bacteria and the internal transcribed spacer 2 (ITS2) locus in yeast or mold DNA can be PCR amplified, and once amplified can be analyzed to provide information about the specific bacteria or specific mold or yeast contamination in or on plant material. Further, for certain samples such as blood, fecal matter and others, PCR may be performed on the DNA in such samples absent any extraction of the DNA. However, for blood it is known that the result of such direct PCR is prone to substantial sample to sample variation due to inhibition by blood analytes. Additionally, attempts to perform direct PCR analysis on plant matter have generally been unsuccessful, due to heavy inhibition of PCR by plant constituents. 
     Over time, additional methods and techniques were developed to improve on the challenges of timely and specific detection and identification of pathogens. Immuno-assay techniques provide specific analysis. However, the technique is costly in the use of chemical consumables and has a long response time. Optical sensor technologies produce fast real-time detection but such sensor lack identification specificity as they offer a generic detection capability as the pathogen is usually optically similar to its benign background. Quantitative Polymerase Chain Reaction (qPCR) technique is capable of amplification and detection of a DNA sample in less than an hour. However, qPCR is largely limited to the analysis of a single pathogen. Consequently, if many pathogens are to be analyzed concurrently, as is the case with plant, agriculture, food and water material, a relatively large number of individual tests are performed in parallel. 
     Biological microarrays have become a key mechanism in a wide range of tools used to detect and analyze DNA. Microarray-based detection combines DNA amplification with the broad screening capability of microarray technology. This results in a specific detection and improved rate of process. DNA microarrays can be fabricated with the capacity to interrogate, by hybridization, certain segments of the DNA in bacteria and eukaryotic cells such as yeast and mold. However, processing a large number of PCR reactions for downstream microarray applications is costly and requires highly skilled individuals with complex organizational support. Because of these challenges, microarray techniques have not led to the development of downstream applications. 
     It is well known that DNA may be linked to a solid support for the purposes of DNA analysis. In those instances, the surface-associated DNA is generally referred to as the “Oligonucleotide probe” (nucleic acid probe, DNA probe) and its cognate partner to which the probe is designed to bind is referred to as the Hybridization Target (DNA Target). In such a device, detection and-or quantitation of the DNA Target is obtained by observing the binding of the Target to the surface bound Probe via duplex formation, a process also called “DNA Hybridization” (Hybridization). 
     Nucleic acid probe linkage to the solid support may be achieved by non-covalent adsorption of the DNA directly to a surface as occurs when a nucleic acid probe adsorbs to a neutral surface such as cellulose or when a nucleic acid probe adsorbs to cationic surface such as amino-silane coated glass or plastic. Direct, non-covalent adsorption of nucleic acid probes to the support has several limitations. The nucleic acid probe is necessarily placed in direct physical contact with the surface thereby presenting steric constraints to its binding to a DNA Target as the desired (bound) Target-Probe complex is a double helix which can only form by wrapping of the Target DNA strand about the bound Probe DNA: an interaction which is fundamentally inhibited by direct physical adsorption of the nucleic acid probe upon the underlying surface. 
     Nucleic acid probe linkage may also occur via covalent attachment of the nucleic acid probe to a surface. This can be induced by introduction of a reactive group (such as a primary amine) into the Probe then covalent attachment of the Probe, through the amine, to an amine-reactive moiety placed upon the surface: such as an epoxy group, or an isocyanate group, to form a secondary amine or a urea linkage, respectively. Since DNA is not generally reactive with epoxides or isocyanates or other similar standard nucleophilic substitutions, the DNA Probe must be first chemically modified with “unnatural” ligands such as primary amines or thiols. While such chemistry may be readily implemented during oligonucleotide synthesis, it raises the cost of the DNA Probe by more than a factor of two, due to the cost associated with the modification chemistry. A related UV crosslinking based approach circumvents the need for unnatural base chemistry, wherein Probe DNA can be linked to the surface via direct UV crosslinking of the DNA, mediated by photochemical addition of thymine within the Probe DNA to the amine surface to form a secondary amine adduct. However, the need for high energy UV for efficient crosslinking results in substantial side reactions that can damage the nucleic acid probe beyond use. As is the case for adsorptive linkage, the covalent linkages possible between a modified nucleic acid probe and a reactive surface are short, in the order of less than 10 rotatable bonds, thereby placing the nucleic acid probe within 2 nm of the underlying surface. Given that a standard nucleic acid probe is &gt;20 bases in length (&gt;10 nm long) a Probe/linker length ratio &gt;10/1 also provides for destabilizing inhibition of the subsequent formation of the desired Target-Probe Duplex. 
     Previous Attempts at addressing these problems have not met with success. Attachment of nucleic acid probes to surfaces via their entrapment into a 3-Dimensional gel phase such as that created by polymerizing acrylamide and polysaccharides among others have been problematic due to the dense nature of the gel phases. While the pore size (about 10 nm) in these gels permit entrapment and/or attachment of the nucleic acid probes within the gel, the solution-phase DNA Target, which is typically many times longer than the nucleic acid probe, is blocked from penetrating the gel matrix thereby limiting use of these gel phase systems due to poor solution-phase access to the Target DNA. Furthermore 
     Thus, the prior art is deficient in systems and methods for detecting and identifying pathogen DNA, which uses fewer chemical and labile products, reduces processing steps and provides faster results while maintaining accuracy, specificity and reliability. The prior art is also deficient in methods to determine absolute copy numbers of pathogen-specific DNA in an unpurified nucleic acid sample comprising a multiplicity of pathogens. The present invention fulfills this long-standing need and desire in the art. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method for detecting the presence of one or more pathogens in a plant sample. The method comprises performing a first amplification of pathogen DNA on an unpurified complex nucleic acid sample followed by a second fluorescent labeling amplification step using the first amplification products as template to obtained fluorescent labeled amplicons. These are hybridized on a 3-dimensional lattice microarray system comprising fluorescent labeled bifunctional polymer linkers and unmodified hybridization probes corresponding to sequence determinants in a plurality of pathogens. A multi-color fluorescent image of the microarray is analyzed to detect and identify the pathogen present in the plant sample. The present invention is also directed to a method for identifying plant attributes and to identify the plant by repeating the amplification, hybridization and imaging steps described above for the plant DNA using fluorescent labeled hybridization probes that correspond to sequence determinants in a plurality of plants. 
     The present invention is further directed to a method for detecting pathogen DNA and plant DNA and identifying the pathogen and plant in a plant sample in a single assay. The method comprises performing a first amplification step on an unpurified complex nucleic acid sample comprising plant DNA and pathogen DNA, followed by a second fluorescent labeling amplification step using the first amplification products as template to obtained fluorescent labeled pathogen and plant-specific amplicons. These amplicons are hybridized to a 3-dimensional lattice microarray system comprising fluorescent labeled bifunctional polymer linkers and unmodified hybridization probes corresponding to sequence determinants in a plurality of pathogens and plants. A multi-color fluorescent image of the microarray is analyzed to detect and identify the pathogen and plant present in the sample. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other features, aspects, and advantages of the embodiments of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawing, wherein: 
         FIGS. 1A-1D  illustrate a covalent microarray system comprising probes and bifunctional labels printed on an activated surface.  FIG. 1A  shows the components—unmodified nucleic acid probe, amine-functionalized (NH) bifunctional polymer linker and amine-functionalized (NH) fluorescently labeled bifunctional polymer linker in a solvent comprising water and a high boiling point water-miscible liquid, and a solid support with chemically activatable groups (X).  FIG. 1B  shows the first step reaction of the bifunctional polymer linker with the chemically activated solid support where the bifunctional polymer linker becomes covalently attached by the amine groups to the chemically activated groups on the solid support.  FIG. 1C  shows the second step of concentration via evaporation of water from the solvent to increase the concentration of the reactants—nucleic acid probes and bifunctional polymer linker.  FIG. 1D  shows the third step of UV crosslinking of the nucleic acid probes via thymidine base to the bifunctional polymer linker within evaporated surface, which in some instances also serves to covalently link adjacent bifunctional polymeric linkers together via crosslinking to the nucleic acid Probe. 
         FIGS. 2A-2D  illustrate an adsorptive microarray system comprising probes and bifunctional polymeric linkers.  FIG. 2A  shows the components; unmodified nucleic acid probe and functionalized (R n ) bifunctional polymer linker and similarly functionalized fluorescent labeled bifunctional polymer linker in a solvent comprising water and a high boiling point water-miscible liquid, and a solid support, wherein the R n  group is compatible for adsorbing to the solid support surface.  FIG. 2B  shows the first step adsorption of the bifunctional polymer linker on the solid support where the bifunctional polymer linkers become non-covalently attached by the R n  groups to the solid support.  FIG. 2C  shows the second step of concentration via evaporation of water from the solvent to increase the concentration of the reactants—Nucleic acid probes and bifunctional polymer linker.  FIG. 2D  shows the third step of UV crosslinking of the nucleic acid probes via thymidine base to the bifunctional polymer linker and other nucleic acid probes within the evaporated surface which in some instances also serves to covalently link adjacent bifunctional polymeric linkers together via crosslinking to the nucleic acid Probe. 
         FIGS. 3A-3C  show experimental data using the covalent microarray system. In this example of the invention the bifunctional polymeric linker was a chemically modified 40 base long oligo deoxythymidine (OligodT) having a CY5 fluorescent dye attached at its 5′ terminus and an amino group attached at its 3′ terminus, suitable for covalent linkage with a borosilicate glass solid support which had been chemically activated on its surface with epoxysilane. The nucleic acid probes comprised unmodified DNA oligonucleotides, suitable to bind to the solution state target, each oligonucleotide terminated with about 5 to 7 thymidines, to allow for photochemical crosslinking with the thymidines in the top domain of the polymeric (oligodT) linker. 
         FIG. 3A  shows an imaged microarray after hybridization and washing, as visualized at 635 nm. The 635 nm image is derived from signals from the (red) CY5 fluor attached to the 5′ terminus of the bifunctional polymer linker (OligodT) which had been introduced during microarray fabrication as a positional marker in each microarray spot. 
         FIG. 3B  shows a microarray imaged after hybridization and washing as visualized at 532 nm. The 532 nm image is derived from signals from the (green) CY3 fluor attached to the 5′ terminus of PCR amplified DNA obtained during PCR Reaction #2 of a DNA containing sample. 
         FIG. 3C  shows an imaged microarray after hybridization and washing as visualized with both the 532 nm and 635 nm images superimposed. The superimposed images display the utility of parallel attachment of a CY5-labelled OligodT positional marker relative to the sequence specific binding of the CY3-labelled PCR product. 
         FIG. 4A  is a graphical representation of the position of PCR primers employed within the 16s locus (all bacteria) to be used to PCR amplify unpurified bacterial contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for bacterial analysis via microarray hybridization. 
         FIG. 4B  is a graphical representation of the position of PCR primers employed within the stx1 locus (pathogenic  E. coli ) to be used to PCR amplify unpurified bacterial contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for bacterial analysis via microarray hybridization. 
         FIG. 5A  is a graphical representation of the position of PCR primers employed as a two stage PCR reaction within the stx2 locus (pathogenic  E. coli ) to be used to PCR amplify unpurified bacterial contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for bacterial analysis via microarray hybridization. 
         FIG. 5B  is a graphical representation of the position of PCR primers employed within the invA locus ( Salmonella ) to be used to PCR amplify unpurified bacterial contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for bacterial analysis via microarray hybridization. 
         FIG. 6  is a graphical representation of the position of PCR primers employed within the tuf locus ( E. coli ) to be used to PCR amplify unpurified bacterial contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for bacterial analysis via microarray hybridization. 
         FIG. 7  is a graphical representation of the position of PCR primers employed within the ITS2 locus (yeast and mold) to be used to PCR amplify unpurified yeast, mold and fungal contamination obtained from  Cannabis  wash and related plant wash. These PCR primers are used to amplify and dye label DNA from such samples for yeast and mold analysis via microarray hybridization. 
         FIG. 8  is a graphical representation of the position of PCR primers employed within the ITS1 locus ( Cannabis  Plant Control) to be used to PCR amplify unpurified DNA obtained from  Cannabis  wash. These PCR primers are used to amplify and dye label DNA from such samples for DNA analysis via microarray hybridization. This PCR reaction is used to generate an internal plant host control signal, via hybridization, to be used to normalize bacterial, yeast, mold and fungal signals obtained by microarray analysis on the same microarray. 
         FIG. 9  is a flow diagram illustrating the processing of unpurified  Cannabis  wash or other surface sampling from  Cannabis  (and related plant material) so as to PCR amplify the raw  Cannabis  or related plant material, and then to perform microarray analysis on that material so as to analyze the pathogen complement of those plant samples 
         FIG. 10  is a representative image of the microarray format used to implement the nucleic acid probes. This representative format comprises 12 microarrays printed on a glass slide, each separated by a Teflon divider (left). Each microarray queries the full pathogen detection panel in quadruplicate. Also, shown is a blow-up (right) of one such microarray for the analysis of pathogens in  Cannabis  and related plant materials. The Teflon border about each microarray is fit to localize about 504 fluid sample for hybridization analysis where fluorescent labeled amplicons and placed onto the microarray for 30 min at room temperature, followed by washing at room temperature then microarray image scanning of the dye-labelled pathogen and host  Cannabis  DNA. 
         FIGS. 11A-11B  shows representative microarray hybridization data obtained from purified bacterial DNA standards ( FIG. 11A ) and purified fungal DNA standards ( FIG. 11B ). In each case, the purified bacterial DNA is PCR amplified as though it were an unpurified DNA, then hybridized on the microarray via the microarray probes described above. The data show that in this microarray format, each of the bacteria can be specifically identified via room temperature hybridization and washing. Similarly, the purified fungal DNA is PCR amplified as though it were an unpurified DNA, then hybridized on the microarray via the microarray probes described above. The data show that in this microarray format, each of the fungal DNAs can be specifically identified via room temperature hybridization and washing. 
         FIG. 12  shows representative microarray hybridization data obtained from 5 representative raw  Cannabis  wash samples. In each case, the raw pathogen complement of these 5 samples is PCR amplified, then hybridized on the microarray via the microarray probes described above. The data show that in this microarray format, specific bacterial, yeast, mold and fungal contaminants can be specifically identified via room temperature hybridization and washing. 
         FIG. 13  shows representative microarray hybridization data obtained from a representative raw  Cannabis  wash sample compared to a representative (raw) highly characterized,  candida  samples. In each case, the raw pathogen complement of each sample is PCR amplified, then hybridized on the microarray via the microarray probes described above. The data show that in this microarray format, specific fungal contaminants can be specifically identified via room temperature hybridization and washing on either raw  Cannabis  wash or cloned fungal sample. 
         FIG. 14  shows a graphical representation of the position of PCR primers employed in a variation of an embodiment for low level detection of Bacteria in the Family Enterobacteriaceae including  E. coli . These PCR primers are used to selectively amplify and dye label DNA from targeted organisms for analysis via microarray hybridization. 
         FIG. 15A  is a graphical representation of microarray hybridization data demonstrating low level detection of  E. coli  O157:H7 from certified reference material consisting of enumerated colonies of specified bacteria spiked onto  Humulus lupulus , (Hop plant). 
         FIG. 15B  is a graphical representation of microarray hybridization data demonstrating low level detection of  E. coli  O1111 from certified reference material consisting of enumerated colonies of specified bacteria spiked onto  Humulus lupulus , (Hop plant). 
         FIG. 15C  is a graphical representation of microarray hybridization data demonstrating low level detection of  Salmonella enterica  from certified reference material consisting of enumerated colonies of specified bacteria spiked onto  Humulus lupulus , (Hop plant). 
         FIG. 16  shows diagrams for sample collection and preparation from two methods. Both the tape pull and wash method are used to process samples to provide a solution for microbial detection via microarray analysis. 
         FIG. 17A  is a graphical representation of the position of PCR primers used to PCR amplify unpurified bacterial contamination in a  Cannabis  wash or related plant wash. Two sets of PCR primers are used. The first set of forward primer (FP) and reverse primer (RP) support a “Locus PCR” step wherein amplification of bacterial recombinant DNA (rDNA) is based on the 16s locus present in all bacteria. The second set of primers support a “Labeling PCR” step wherein the primers are dye labeled and specific to the bacteria of interest. The two PCR steps in this invention differs from  FIGS. 4A and 4B  in the addition of a known copy number of a synthetic DNA sequence to the sample, as an internal reference standard, prior to performing the first PCR wherein, the synthetic DNA sequence is distinguishable from the bacterial sequences in the sample and have end sequences complementary to the sequence in the FP and the RP, so that the synthetic DNA sequence is co-amplified with the bacterial sequence in the sample. 
         FIG. 17B  is a graphical representation of the position of PCR primers used to PCR amplify unpurified bacterial contamination in a  Cannabis  wash or related plant wash. Two sets of PCR primers are used. The first set of forward primer (FP) and reverse primer (RP) support a “Locus PCR” step wherein amplification of bacterial genomic DNA (DNA) is based on the 16s locus present in all bacteria. The second set of primers support a “Labeling PCR” step wherein the primers are dye labeled and specific to the bacteria of interest. The two PCR steps in this invention differs from  FIGS. 4A and 4B  in the addition of a known copy number of a synthetic DNA sequence to the sample, as an internal reference standard, prior to performing the first PCR wherein, the synthetic DNA sequence is distinguishable from the bacterial sequences in the sample and have end sequences complementary to the sequence in the FP and the RP, so that the synthetic DNA sequence is co-amplified with the bacterial sequence in the sample. 
         FIG. 18  is a graphical representation of the position of PCR primers used to PCR amplify unpurified eukaryotic (e.g. yeast or mold) contamination in a  Cannabis  wash or related plant wash. Two sets of PCR primers are used. The first set of forward primer (FP) and reverse primer (RP) support a “Locus PCR” step wherein amplification of the eukaryotic DNA is based on the ITS2 locus present in all eukaryotes. The second set of primers support a “Labeling PCR” step wherein the primers are dye labeled and specific to the eukaryotes of interest. The two PCR steps in this invention differs from  FIGS. 4A and 4B  in the addition of a known copy number of a synthetic DNA sequence to the sample, as an internal reference standard, prior to performing the first PCR wherein, the synthetic DNA sequence is distinguishable from the eukaryotic sequences in the sample and have end sequences complementary to the sequence in the FP and the RP, so that the synthetic DNA sequence is co-amplified with the eukaryotic sequence in the sample. 
         FIG. 19A  illustrates results of a microarray analysis of  Aspergillus  lysate varied to deliver approximately 0 to 30,000 copies of ITS2 per PCR reaction performed using known amounts of Total Yeast and Mold Quantitative Control (TYM Quant Control) as the internal reference standard. The point of crossover (arrow) between the titration curves for  Aspergillus  (unknown copy number) and the TYM internal reference standard (known copy number) reveals the copy number of  Aspergillus  DNA in the sample. 
         FIG. 19B  illustrates results of a microarray analysis of  Saccharomyces  lysate varied to deliver approximately 0 to 30,000 calculated copies of ITS2 region per PCR reaction performed using known amounts of TYM Quant Control as the internal reference standard. The point of crossover (arrow) between the titration curves for  Saccharomyces  (unknown copy number) and the TYM internal reference standard (known copy number) reveals the copy number of  Saccharomyces  DNA in the sample. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In one embodiment of this invention, there is provided a 3-dimensional lattice microarray system for screening a sample for the presence of a multiplicity of DNA. The system comprises a chemically activatable solid support, a bifunctional polymer linker and a plurality of nucleic acid probes designed to identify sequence determinants in plant, animal or pathogen DNA. 
     In this embodiment, the solid support may be made of any suitable material known in the art including but not limited to borosilicate glass, a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT (PMMA-VSUVT), a cycloolefin polymers such as ZEONOR® 1060R, metals including, but not limited to gold and platinum, plastics including, but not limited to polyethylene terephthalate, polycarbonate, nylon, ceramics including, but not limited to TiO 2 , and Indium tin oxide (ITO) and engineered carbon surfaces including, but not limited to graphene. The solid support has a front surface and a back surface and may be activated on the front surface with suitable chemicals which include but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide. These are well known in the art and one of ordinary skill in this art would be able to readily functionalize any of these supports as desired. In a preferred embodiment, the solid support is epoxysilane functionalized borosilicate glass support. 
     In this embodiment, the bifunctional polymer linker has a top domain and a bottom end. On the bottom end is attached a first reactive moiety that allows covalent attachment to the chemically activatable groups in the solid support. Examples of first reactive moieties include but are not limited to an amine group, a thiol group and an aldehyde group. Preferably, the first reactive moiety is an amine group. On the top domain of the bifunctional polymer linker is provided a second reactive moiety that allows covalent attachment to the oligonucleotide probe. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. The bifunctional polymer linker may be an oligonucleotide such as OligodT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or any other polymeric compounds with dual functional groups which can be attached to the chemically activatable solid support on the bottom end, and the nucleic acid probes on the top domain. Preferably, the bifunctional polymer linker is OligodT having an amine group at the 5′ end. 
     In this embodiment, the bifunctional polymer linker may be unmodified. Alternatively, the bifunctional polymer linker has a color or fluorescent label attached covalently. Examples of fluorescent labels include, but are not limited to the fluorescent dyes CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. These may be attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. The chemistries of such reactive groups are well known in the art and one or ordinary skill can readily identify a suitable group on a selected bifunctional polymer linker for attaching the fluorescent label. Preferably, the bifunctional polymer linker is CY5-labeled OligodT having an amino group attached at its 3′ terminus for covalent attachment to an activated surface on the solid support. 
     Also in this embodiment, the present invention provides a plurality of nucleic acid probes designed with the purpose of identifying sequence determinants in plants, animals or pathogens. The nucleic acid probes are synthetic oligonucleotides and have terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling. Preferably, covalent attachment of the nucleic acid probes to the bifunctional polymer linker is by photochemical means using ultraviolet light. 
     In this embodiment, the fluorescent label (fluorescent tag) attached to the bifunctional polymer linker is beneficial since it allows the user to image and detect the position of the individual nucleic acid probes (“spot”) printed on the microarray. By using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated from the DNA being queried, the user can obtain a superimposed image that allows parallel detection of those nucleic acid probes which have been hybridized with amplicons. This is advantageous since it helps in identifying the plant or pathogen comprised in the sample using suitable computer and software, assisted by a database correlating nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants, animals or pathogens. Any emitter/acceptor fluorescent label pairs known in the art may be used. For example, the bifunctional polymer linker may be labeled with emitters such as CY5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while the amplicons may be labeled with acceptors such as CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. Preferably, the emitter is CY5 and the acceptor is CY3. 
     In another embodiment of this invention, there is provided a 3-dimensional lattice microarray system for screening a sample for the presence of a multiplicity of DNA. The system comprises a solid support, a fluorescent labeled bifunctional polymer linker and a plurality of nucleic acid probes designed to identify sequence determinants in plant, animal or pathogen DNA. In another aspect of this embodiment, there is provided a 3-dimensional lattice microarray system for quantitative screening of a sample for copy number of one or more types of DNA. The system comprises a bifunctional polymer linker, a plurality of nucleic acid probes designed to detect copy number of sequence determinants in plant, animal or pathogen DNA and, nucleic acid probes designed to detect copy number of an internal reference standard comprising a known copy number of synthetic DNA. The synthetic DNA has a central region with a nucleotide sequence distinct from signature sequence determinants in the unknown DNA being queried, and 5′ and 3′ ends sequences substantially identical to a consensus sequence in the unknown DNA. Such consensus sequences include but are not limited to the sequences shown in SEQ ID NO: 152 and 153. Such a structure for the synthetic DNA permits amplification of the synthetic DNA by the same pair of PCR primers used to amplify the hypervariable region of the unknown DNA being queried. Examples of such synthetic DNA which may be employed include but is not limited to the sequences shown in SEQ ID NOs: 154-157. 
     Further in this embodiment, the solid support may be made of any suitable material known in the art including but not limited to borosilicate glass, a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT (PMMA-VSUVT), a cycloolefin polymers such as ZEONOR® 1060R, metals including, but not limited to gold and platinum, plastics including, but not limited to polyethylene terephthalate, polycarbonate, nylon, ceramics including, but not limited to TiO 2 , and Indium tin oxide (ITO) and engineered carbon surfaces including, but not limited to graphene. The solid support has a front surface and a back surface and may be activated on the front surface with suitable chemicals which include but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide. These are well known in the art and one of ordinary skill in this art would be able to readily functionalize any of these supports as desired. In a preferred embodiment, the solid support is epoxysilane functionalized borosilicate glass support. 
     In this embodiment, the bifunctional polymer linker has a top domain and a bottom end. On the bottom end is attached a first reactive moiety that allows covalent attachment to the chemically activatable groups in the solid support. Examples of first reactive moieties include but are not limited to an amine group, a thiol group and an aldehyde group. Preferably, the first reactive moiety is an amine group. On the top domain of the bifunctional polymer linker is provided a second reactive moiety that allows covalent attachment to the oligonucleotide probe. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. The bifunctional polymer linker may be an oligonucleotide such as OligodT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or any other polymeric compounds with dual functional groups which can be attached to the chemically activatable solid support on the bottom end, and the nucleic acid probes on the top domain. Preferably, the bifunctional polymer linker is OligodT having an amine group at the 5′ end. 
     In one aspect of this embodiment, the bifunctional polymer linker is unmodified. Alternatively, the bifunctional polymer linker has a color or fluorescent label attached covalently. Examples of fluorescent labels include, but are not limited to the fluorescent dyes CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. These may be attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. The chemistries of such reactive groups are well known in the art and one or ordinary skill can readily identify a suitable group on a selected bifunctional polymer linker for attaching the fluorescent label. Preferably, the bifunctional polymer linker is CY5-labeled OligodT having an amino group attached at its 3′ terminus for covalent attachment to an activated surface on the solid support. 
     Also, in this embodiment, the present invention provides a plurality of nucleic acid probes designed with the purpose of quantitating copy number of sequence determinants in plants, animals or pathogens and, nucleic acid probes designed to detect an internal reference standard comprising a known copy number of synthetic DNA. The nucleic acid probes are synthetic oligonucleotides and have terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling. Preferably, covalent attachment of the nucleic acid probes to the bifunctional polymer linker is by photochemical means using ultraviolet light. 
     In this embodiment, the fluorescent label (fluorescent tag) attached to the bifunctional polymer linker is beneficial since it allows the user to image and detect the position of the individual nucleic acid probes (“spot”) printed on the microarray. By using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated from the DNA being queried, the user can obtain a superimposed image that allows parallel detection of those nucleic acid probes which have been hybridized with amplicons. Furthermore, by using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated, one can quantitate copy number of the DNA being queried. This feature is advantageous since it allows; identification of the plant or pathogen comprised in the sample using suitable computer and software, assisted by a database correlating nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants, animals or pathogens; and in addition, quantification of the copy number of the plant and/or pathogen DNA identified. Any emitter/acceptor fluorescent label pairs known in the art may be used for imaging and analysis. For example, the bifunctional polymer linker may be labeled with emitters such as CY5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while the amplicons may be labeled with acceptors such as CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. Preferably, the emitter is CY5 and the acceptor is CY3. 
     In another embodiment of this invention, there is provided a 3-dimensional lattice microarray system for screening a sample for the presence of a multiplicity of DNA. The system comprises a solid support, a fluorescent labeled bifunctional polymer linker and a plurality of nucleic acid probes designed to identify sequence determinants in plant, animal or pathogen DNA. 
     In this embodiment, the solid support has a front surface and a back surface. The front surface has non-covalent adsorptive properties for specific functionalized group(s) present in the fluorescent labeled bifunctional polymer linker (described below). Examples of such solid support include, but are not limited to borosilicate glass, SiO2, metals including, but not limited to gold and platinum, plastics including, but not limited to polyethylene terephthalate, polycarbonate, nylon, ceramics including, but not limited to TiO 2 , and Indium tin oxide (ITO) and engineered carbon surfaces including, but not limited to graphene. 
     In this embodiment, the fluorescent labeled bifunctional polymer linker has a top domain and a bottom end. On the bottom end is attached one or more functional groups (designated by “R n ”) that are compatible for non-covalent adsorptive attachment with the front surface of the solid support. Examples of compatible R groups include, but are not limited to, single stranded nucleic acids (example, OligodT), amine-polysaccharide (example, chitosan), extended planar hydrophobic groups (example, digoxigenin, pyrene, CY5 dye). 
     Further in this embodiment, on the top domain of the bifunctional polymer linker is provided a second reactive moiety that allows covalent attachment to the oligonucleotide probe. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. To the bottom end of the bifunctional polymer linker may be attached polymeric molecules including, but not limited to an oligonucleotide such as OligodT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or OligodT that is modified at its 5′ end with a digoxigenin, a pyrene or CY5 or any other polymeric molecules with or without chemical modification suitable for non-covalent attachment to the solid support. On the top domain of these bifunctional polymer linkers is attached, the nucleic acid probes. Preferably, the bifunctional polymer linker is OligodT. 
     In one aspect of this embodiment, the bifunctional polymer linker is unmodified. Alternatively, the bifunctional polymer linker may be a fluorescent labeled bifunctional polymer linker. The fluorescent label may be, but is not limited to the fluorescent dyes CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550 attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. The chemistries of such reactive groups are well known in the art and one or ordinary skill can readily identify a suitable group on a selected bifunctional polymer linker for attaching the fluorescent label. Preferably, the bifunctional polymer linker is CY5-labeled OligodT. 
     Also, in one aspect of this embodiment, the present invention provides a plurality of nucleic acid probes designed with the purpose of identifying sequence determinants in plants, animals or pathogens. In another aspect of this embodiment, there is additionally provided nucleic acid probes that identify sequence determinants in a synthetic DNA added as an internal reference standard to the unpurified sample being queried (see below in the embodiments comprising the claimed methods) with the purpose of quantitating a DNA copy number for the identified plants, animals or pathogens. In either embodiment, the nucleic acid probes are synthetic oligonucleotides and have terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling. Preferably, covalent attachment of the nucleic acid probes to the bifunctional polymer linker is by photochemical means using ultraviolet light. 
     Further in this embodiment, the fluorescent label (fluorescent tag) attached to the bifunctional polymer linker is beneficial since it allows the user to image and detect the position of the individual nucleic acid probes (“spot”) printed on the microarray. By using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated from the DNA being queried, the user can obtain a superimposed image that allows parallel detection of those nucleic acid probes which have been hybridized with amplicons. This is advantageous since it helps in identifying the plant or pathogen comprised in the sample using suitable computer and software, assisted by a database correlating nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants, animals or pathogens. Additionally, by using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated from the unknown DNA and the internal reference standard (synthetic DNA, which is added to the sample), one can quantitate copy number of the DNA being queried. This feature is advantageous since it allows; identification of the plant or pathogen comprised in the sample using suitable computer and software, assisted by a database of signature sequence determinants for correlating nucleic acid probe sequence and microarray location of this sequence to identify the plants, animals or pathogens; and in addition, quantification of the copy number of the plant and/or pathogen DNA identified. 
     Any emitter/acceptor fluorescent label pairs known in the art may be used. For example, the bifunctional polymer linker may be labeled with emitters such as CY5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while the amplicons may be labeled with acceptors such as CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. Preferably, the emitter is CY5 and the acceptor is CY5. 
     In yet another embodiment of this invention, there is provided a method for fabricating a 3-dimensional lattice microarray system for the purpose of screening a sample for the presence of a multiplicity of DNA in a sample. The method comprises, contacting a solid support with a formulation comprising a plurality of nucleic acid probes, a plurality of fluorescent bifunctional polymer linkers and a solvent mixture comprising water and a high boiling point, water-miscible liquid, allowing a first attachment between the fluorescent bifunctional polymer linkers and the solid support to proceed, evaporating the water in the solvent mixture thereby concentrating the nucleic acid probes and fluorescent labeled bifunctional polymer linkers, allowing a second attachment between the nucleic acid probes and the fluorescent bifunctional polymer linker, and washing the solid support with at least once to remove unattached fluorescent bifunctional polymer linkers and nucleic acid probes. 
     In this embodiment, the contacting step is achieved by standard printing methods known in the art including, but not limited to piezo-electric printing, contact printing, ink jet printing and pipetting, which allow a uniform application of the formulation on the activated support. For this, any suitable solid support known in the art including but not limited to borosilicate glass, a polycarbonate, a graphene, a gold, a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT (PMMA-VSUVT) and a cycloolefin polymer such as ZEONOR® 1060R may be employed. 
     In one aspect of this embodiment, the first attachment of the bifunctional polymer linker to the solid support is by non-covalent means such as by adsorption or electrostatic binding. In this case, the bifunctional polymer linkers with one or more functional groups (designated by “R n ”) on the bottom end, that are compatible for attachment with the front surface of the solid support will be used. Examples of compatible R groups include, but are not limited to, single stranded nucleic acids (example, OligodT), amine-polysaccharide (example, chitosan), extended planar hydrophobic groups (example, digoxigenin, pyrene, CY5 dye). In another aspect of this embodiment, the first attachment of the bifunctional polymer linker to the solid support is by covalent coupling between chemically activatable groups on the solid support and a first reactive moiety on the bottom end of the bifunctional polymer linker. Suitable chemicals including but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide may be used for activating the support. These are well known in the art and one of ordinary skill in this art would be able to readily functionalize any of these supports as desired. In a preferred embodiment, a borosilicate glass support that is epoxysilane functionalized is used. Examples of first reactive moieties amenable to covalent first attachment include, but are not limited to an amine group, a thiol group and an aldehyde group. Preferably, the first reactive moiety is an amine group. 
     In this embodiment, the bifunctional polymer linker has a second reactive moiety attached at the top domain. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. Preferably, the second reactive moiety is thymidine. In this aspect of the invention, the bifunctional polymer linker may be an oligonucleotide such as OligodT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or any other polymeric compounds with dual functional groups which can be attached to the chemically activatable solid support on the bottom end, and the nucleic acid probes on the top domain. Preferably, the bifunctional polymer linker is OligodT having an amine group at the 5′ end. 
     In this embodiment, the bifunctional polymer linkers are modified with a fluorescent label. Examples of fluorescent labels include but are not limited to the fluorescent dyes CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547 and ALEXA FLUOR® 550 attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. The chemistries of such reactive groups are well known in the art and one or ordinary skill can readily identify a suitable group on a selected bifunctional polymer linker for attaching the fluorescent label. Preferably, the bifunctional polymer linker used for fabricating the microarray is CY5-labeled OligodT. 
     The method of fabricating the microarray requires use of a solvent mixture comprising water and a water-miscible liquid having a boiling point above 100° C. This liquid may be any suitable water-miscible liquid with a boiling point higher than that of water, so that all the solvent is not lost during the evaporation step. This allows the molecular reactants—nucleic acid probes and bifunctional linkers to be progressively concentrated during evaporation. Such controlled evaporation is crucial to the present invention since it controls the vertical spacing between nucleic acid probes their avoiding steric hindrance during the hybridization steps thereby improving accuracy and precision of the microarray. Examples of high boiling point water-miscible solvent include but are not limited to glycerol, DMSO and propanediol. The ratio or water to high boiling point solvent is kept between 10:1 and 100:1 whereby, in the two extremes, upon equilibrium, volume of the fluid phase will reduce due to water evaporation to between 1/100th and 1/10 th  of the original volume, thus giving rise to a 100-fold to 10-fold increase in reactant concentration. In a preferred embodiment, the water-miscible solvent is propanediol and the water to propanediol ratio is 100:1. 
     Further in this embodiment, the nucleic acid probes used in the method of microarray fabrication are designed with terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling during the fabrication process. Preferably, coupling of the nucleic acid probes to the fluorescent labeled bifunctional polymer linkers is by photochemical covalent crosslinking. 
     In yet another embodiment of this invention, there is provided a customizable microarray kit. The kit comprises a solid support, a plurality of fluorescent labeled bifunctional polymer linkers, nucleic acid probes and a solvent mixture comprising water and one or more of a water-miscible liquid having a boiling point above 100° C., and instructions to use the kit. Each of the components comprising this kit may be individually customized prior to shipping based on the goals of the end user. 
     In this embodiment, the solid support has a front surface and a back surface and made of any suitable material known in the art including but not limited to borosilicate glass, a polycarbonate, a graphene, a gold, a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT (PMMA-VSUVT) and a cycloolefin polymer such as ZEONOR® 1060R. 
     In one aspect of this embodiment, the solid support is unmodified and has properties capable of non-covalent attachment to groups in the bifunctional polymer linker. Alternatively, the solid support is activated on the front surface with chemically activatable groups which include but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide. These are well known in the art and one of ordinary skill in this art would be able to readily functionalize any of these supports as desired. In a preferred embodiment, the solid support is epoxysilane functionalized borosilicate glass support. 
     In this embodiment, the bifunctional polymer linker has a top domain and a bottom end. In one aspect of this embodiment, to the bottom end of the bifunctional polymer linker are attached one or more functional groups (designated by “R n ”), which are compatible for attachment with the front surface of the solid support in a non-covalent binding. Examples of such compatible R groups include, but are not limited to, single stranded nucleic acids (example, OligodT), amine-polysaccharide (example, chitosan), extended planar hydrophobic groups (example, digoxigenin, pyrene, CY5 dye). Alternatively, to the bottom end of the bifunctional polymer linker are attached a first reactive moiety that allows covalent attachment to chemically activatable groups in the solid support. Examples of first reactive moieties include but are not limited to an amine group, a thiol group and an aldehyde group. Preferably, the first reactive moiety is an amine group. 
     Further in this embodiment, on the top domain of the bifunctional polymer linker is provided a second reactive moiety that allows covalent attachment to the oligonucleotide probe. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. The bifunctional polymer linker may be an oligonucleotide such as OligodT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or any other polymeric compounds with dual functional groups for attachment to the solid support from the bottom end, and the nucleic acid probes from the top domain. 
     In one aspect of this embodiment, the bifunctional polymer linkers are modified with a fluorescent label. Alternatively, the bifunctional polymer linker may be a fluorescent labeled bifunctional polymer linker where the fluorescent label is either of CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550 attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. The chemistries of such reactive groups are well known in the art and one or ordinary skill can readily identify a suitable group on a selected bifunctional polymer linker for attaching the fluorescent label. Preferably, the bifunctional polymer linker is CY5-labeled OligodT. 
     Also in this embodiment, the present invention provides a plurality of nucleic acid probes designed with the purpose of identifying sequence determinants in plants, animals or pathogens. The nucleic acid probes are synthetic oligonucleotides and have terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling. Preferably, covalent attachment of the nucleic acid probes to the bifunctional polymer linker is by photochemical means using ultraviolet light. In an alternative aspect of this embodiment, the present invention provides a plurality of nucleic acid probes designed with the purpose of identifying sequence determinants in plants, animals or pathogens and, a synthetic DNA internal reference standard, which is added to the sample to quantitate DNA copy number for the sequence determinants in plants, animals or pathogens. The synthetic DNA has a central region with a nucleotide sequence distinct from signature sequence determinants in the unknown DNA being queried, and 5′ and 3′ ends sequences substantially identical to a consensus sequence in the unknown DNA. Such consensus sequences include but are not limited to the sequences shown in SEQ ID NO: 152 and 153. Such a structure for the synthetic DNA permits amplification of the synthetic DNA by the same pair of PCR primers used to amplify the hypervariable region of the unknown DNA being queried. Examples of such synthetic DNA which may be employed include but is not limited to the sequences shown in SEQ ID NOs: 154-157. In either of these embodiment, the nucleic acid probes are synthetic oligonucleotides and have terminal thymidine bases at their 5′ and 3′ end. The thymidine bases permit covalent attachment of the nucleic acid probes to the bifunctional polymer linker by any standard coupling procedures including but not limited to chemical, photochemical and thermal coupling. Preferably, covalent attachment of the nucleic acid probes to the bifunctional polymer linker is by photochemical means using ultraviolet light. 
     In yet another embodiment of this invention there is provided a method for detecting the presence of one or more pathogens in a plant sample. In this embodiment, the pathogen may be a human pathogen, an animal pathogen or a plant pathogen, such as a bacterium, a fungus, a virus, a yeast, algae or a protozoan or a combination thereof. These pathogens may be present as constituents of the soil, soilless growth media, hydroponic growth media or water in which the plant sample was grown. The method comprises harvesting the pathogens from the plant sample, isolating total nucleic acids comprising pathogen DNA, performing a first amplification for generating one or more amplicons from the one or more pathogens present in the sample in a single, simultaneous step; performing a labeling amplification using as template, the one or more amplicons generated in the first amplification step to generate fluorescent labeled second amplicons; hybridizing the second amplicons with the nucleic acid probes immobilized on the fabricated self-assembled, 3-dimensional lattice microarray described above and imaging the microarray to detect the fluorescent signal, which indicates presence of the one or more pathogens in a plant sample. In this embodiment, the pathogens present on the plant surface may be harvested by washing the plant with water to recover the pathogens, followed by concentrating by filtration on a sterile 0.4 μm filter. In another aspect of this embodiment, pathogens within the plant tissue may be harvested by fluidizing the plant tissue sample and pathogens, followed by centrifuging to get a pellet of plant cells and pathogen cells. In either embodiment, the harvested sample is disrupted to release the total nucleic acids which is used in the subsequent steps without further purification. 
     Also in this embodiment, the sample comprising nucleic acids from pathogens (external pathogens) or nucleic acids from both pathogens and plant (internal pathogens) is used to perform a first amplification of pathogen DNA using pathogen-specific first primer pairs to obtain one or more pathogen-specific first amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. In one embodiment, the pathogen is a bacterium and the first primer pairs have sequences shown in SEQ ID NOS: 1-12. In another embodiment, the pathogen is a fungus and the first primer pairs have sequences shown in SEQ ID NOS: 13-16. An aliquot of first amplicons so generated is used as template for a second, labelling PCR amplification using fluorescent labeled second primer pairs. The second primer pairs are designed to amplify an internal flanking region in the one or more first amplicons to obtain one or more first fluorescent labeled second amplicons. In one embodiment, the pathogen is a bacterium and the second primer pairs have sequences shown in SEQ ID NOS: 19-30. In another embodiment, the pathogen is a fungus and the second primer pairs have sequences shown in SEQ ID NOS: 31-34. 
     Further in this embodiment, the fluorescent labeled second amplicons are hybridized on a 3-dimensional lattice microarray system having a plurality of nucleic acid probes as described in detail above. In this embodiment, the bifunctional polymer linker has a fluorescent label (that is different from the label on the second amplicon) attached whereby, imaging the microarray after hybridization and washing results in two distinct fluorescent signals—the signal from the fluorescent bifunctional polymer linker which is covalently linked to the nucleic acid probe during fabrication, which would be detected in each spot comprised in the microarray, and a second amplicon signal that would be detected only in those spots where the nucleic acid probe sequence is complementary to the second amplicon (originally derived by amplification from the pathogen DNA in the sample). Thus, superimposing the two images using a computer provides beneficial attributes to the system and method claimed in this invention since one can readily identify the plant or pathogen comprised in the sample from a database that correlates nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants or pathogens. In a preferred embodiment, the bacterial nucleic acid probes having sequences shown in SEQ ID NOS: 37-85, and fungal nucleic acid probes having sequences shown in SEQ ID NOS: 86-125 may be used for this purpose. 
     Further to this embodiment is a method for detecting plant DNA. The plant may be a terrestrial plant such as a  Humulus  or a  Cannabis , an aquatic plant, an epiphytic plant or a lithophytic plant that grows in soil, soilless media, hydroponic growth media or water. In a preferred aspect, the plant is a  Cannabis . This method comprises the steps of performing an amplification on an unpurified complex nucleic acid sample using plant-specific first primer pairs to generate plant-specific first amplicons. In one aspect of this embodiment, the first primer pair has sequences shown in SEQ ID NOS: 17-18. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. Preferably the amplification is by PCR. The first amplicons so generated are used as template for a labeling amplification step using fluorescent labeled second primer pairs that are designed to amplify an internal flanking region in the one or more of first amplicons generated in the first amplification step to generate one or more first fluorescent labeled second amplicons. In one embodiment, the second primer pair has sequences shown in SEQ ID NOS: 35-36. The second amplicons are hybridized on a 3-dimensional lattice microarray system having a plurality of plant-specific nucleic acid probes, and the microarrays imaged and analyzed as described above for identifying pathogen DNA. In one aspect of this embodiment, the hybridization nucleic acid probes have sequences shown in SEQ ID NOS: 126-128. 
     In yet another embodiment of this invention, there is provided a method for simultaneously detecting resident pathogen DNA and plant DNA in a plant sample in a single assay. In this embodiment, the pathogen may be a human pathogen, an animal pathogen or a plant pathogen, which may be a bacterium, a fungus, a virus, a yeast, algae or a protozoan or a combination thereof. These pathogens may be present as constituents of the soil, soilless growth media, hydroponic growth media or water in which the plant sample was grown. The plant may be a terrestrial plant such as a  Humulus  or a  Cannabis , an aquatic plant, an epiphytic plant or a lithophytic plant that grows in soil, soilless media, hydroponic growth media or water. Preferably, the plant is a  Cannabis.    
     In this embodiment, the method comprises harvesting a plant tissue sample potentially comprising one or more pathogens, fluidizing the plant tissue sample and the one or more pathogens and isolating total nucleic acids comprising DNA from at least the plant tissue and DNA from the one or more pathogens. In one aspect of this embodiment, the step of isolating total nucleic acids comprises centrifuging the fluidized sample to get a pellet of plant cells and pathogen cells which are disrupted to release the total nucleic acids, which are used in the subsequent steps without further purification. 
     Further in this embodiment, a first amplification is performed on the unpurified total nucleic acid sample using one or more of a first primer pair each selective for the one or more pathogen DNA and one or more of a second primer pair selective for the plant DNA to generate one or more pathogen-specific first amplicons and one or more plant-specific second amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. In one embodiment, the pathogen is a bacterium and the first primer pairs have sequences shown in SEQ ID NOS: 1-12. In another embodiment, the pathogen is a fungus and the first primer pairs have sequences shown in SEQ ID NOS: 13-16. In either of these embodiments, the plant-specific second primer pairs have sequences shown in SEQ ID NOS: 35-36. An aliquot of the first and second amplicons so generated is used as a template for a second, labeling PCR amplification step using fluorescent labeled third primer pairs having a sequence complementary to an internal flanking region in the one or more pathogen-specific first amplicons and fluorescent labeled fourth primer pairs having a sequence complementary to an internal flanking region in the one or more plant-specific second amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. In one embodiment, the pathogen is a bacterium and the third primer pairs have sequences shown in SEQ ID NOS: 19-30. In another embodiment, the pathogen is a fungus and the third primer pairs have sequences shown in SEQ ID NOS: 31-34. In either of these embodiments, the plant-specific fourth primer pairs have sequences shown in SEQ ID NOS: 35-36. The labeling PCR step results in generation of first fluorescent labeled third amplicons and second fluorescent labeled fourth amplicons corresponding to the pathogen and plant DNA respectively in the original harvested sample. These amplicons are then hybridized on a 3-dimensional lattice microarray system having a plurality of nucleic acid probes specific to sequence determinants in pathogen DNA or plant DNA. Bacterial nucleic acid probes having sequences shown in SEQ ID NOS: 37-85, fungal nucleic acid probes having sequences shown in SEQ ID NOS: 86-125 and plant nucleic acid probes having sequences shown in SEQ ID NOS: 126-128, may be used for this purpose. After hybridization, unhybridized amplicons are removed by washing and the microarray imaged. Detection of the first fluorescent labeled third amplicon signal indicates presence of pathogens in the plant sample. Detecting the second fluorescent labeled fourth amplicon indicates presence of the plant DNA. Superimposing these two signals with the third fluorescent signal from the fluorescent bifunctional polymer linker-coupled nucleic acid probes allow simultaneous identification of the pathogen and plant in the sample by correlating nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants or pathogens. These features provide beneficial attributes to the system and method claimed in this invention. 
     In yet another embodiment of this invention there is provided a method for both detecting the presence of one or more pathogens and quantitating the copy number of pathogen DNA and plant DNA in a plant sample by introducing a known copy number of a synthetic DNA sequence as an internal reference standard to the plant sample. In this embodiment, the pathogen may be a human pathogen, an animal pathogen or a plant pathogen, such as a bacterium, a fungus, a virus, a yeast, algae or a protozoan or a combination thereof. These pathogens may be present as constituents of the soil, soilless growth media, hydroponic growth media or water in which the plant sample was grown. The method comprises harvesting the pathogens from the plant sample, isolating total nucleic acids comprising pathogen DNA, performing a first amplification for generating one or more amplicons from the one or more pathogens present in the sample in a single, simultaneous step; performing a labeling amplification using as template, the one or more amplicons generated in the first amplification step to generate fluorescent labeled second amplicons; hybridizing the second amplicons with the nucleic acid probes immobilized on the fabricated self-assembled, 3-dimensional lattice microarray described above and imaging the microarray to detect the fluorescent signal, which indicates presence of the one or more pathogens in a plant sample. In this embodiment, the pathogens present on the plant surface may be harvested by washing the plant with water to recover the pathogens, followed by concentrating by filtration on a sterile 0.4 μm filter. In another aspect of this embodiment, pathogens within the plant tissue may be harvested by fluidizing the plant tissue sample and pathogens, followed by centrifuging to get a pellet of plant cells and pathogen cells. In either embodiment, the harvested sample is disrupted to release the total nucleic acids which is used in the subsequent steps without further purification. 
     In this embodiment, the synthetic DNA has a central region with a nucleotide sequence distinct from signature sequence determinants in the unknown DNA being queried, and 5′ and 3′ ends sequences substantially identical to a consensus sequence in the unknown DNA. Such consensus sequences include but are not limited to the sequences shown in SEQ ID NO: 152 and 153. Such a structure for the synthetic DNA permits amplification of the synthetic DNA by the same pair of PCR primers used to amplify the hypervariable region of the unknown DNA being queried. Examples of such synthetic DNA which may be employed include but is not limited to the sequences shown in SEQ ID NOs: 154 (fungus) and 155-157 (bacteria). 
     Also in this embodiment, a known copy number of a synthetic DNA is added to the sample comprising nucleic acids from pathogens (external pathogens) or nucleic acids from both pathogens and plant (internal pathogens) and a first amplification is performed using pathogen-specific first primer pairs to obtain one or more pathogen-specific first amplicons and synthetic DNA specific second amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. Any suitable first amplification primer pairs may be used for this purpose and one of skill in this art can easily design these primers based on the pathogen of interest. In one embodiment, the pathogen is a bacterium and the first primer pairs have sequences shown in SEQ ID NOS: 1 and 2, or SEQ ID NOS: 3 and 4, or SEQ ID NOS: 5 and 6 or SEQ ID NOS: 7 and 8, or SEQ ID NOS: 9 and 10, or SEQ ID NOS: 11 and 12, or SEQ ID NOS: 137 and 138. In another embodiment, the pathogen is a fungus and the first primer pairs have sequences shown in SEQ ID NOS: 13 and 14, or SEQ ID NOS: 15 and 16, or SEQ ID NOS: 135 and 136. An aliquot of first and second amplicons so generated is used as template for a second, labelling PCR amplification using fluorescent labeled second primer pairs. The second primer pairs are designed to amplify an internal flanking region in the one or more pathogen DNA-specific first amplicons and the synthetic DNA-specific second amplicons to obtain one or more first fluorescent labeled third amplicons and first fluorescent labeled fourth amplicons. Any suitable second amplification primer pairs may be used for this purpose and one of skill in this art can easily design these primers based on the pathogen of interest. In one embodiment, the pathogen is a bacterium and the second primer pairs have sequences shown in SEQ ID NOS: 19 and 20, or SEQ ID NOS: 21 and 22, or SEQ ID NOS: 23 and 24 or SEQ ID NOS: 25 and 26, or SEQ ID NOS: 27 and 28, or SEQ ID NOS: 29 and 30, or SEQ ID NOS: 141 and 30. In another embodiment, the pathogen is a fungus and the second primer pairs have sequences shown in SEQ ID NOS: 31 and 32, or SEQ ID NOS: 33 and 34, or SEQ ID NOS: 139 and 140. 
     Further in this embodiment, the fluorescent labeled second amplicons are hybridized on a 3-dimensional lattice microarray system having a plurality of nucleic acid probes specific to pathogen or synthetic DNA specific amplicons as described in detail above. Any suitable nucleic acid probes may be used for this purpose and one of skill in this art can easily design them based on the pathogen of interest. In one embodiment, the bacterial nucleic acid probes have sequences shown in SEQ ID NOS: 37-85 and the synthetic DNA has sequences shown in SEQ ID NO: 155, SEQ ID NO: 156 corresponding respectively to synthetic DNA specific nucleic acid probes having sequences shown in SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144. In another embodiment, the fungal nucleic acid probes having sequences shown in SEQ ID NOS: 86-125, the synthetic DNA has sequences shown in SEQ ID NO: 154 that corresponds to synthetic DNA specific nucleic acid probes having sequences shown in SEQ ID NO: 145. 
     In this embodiment, the bifunctional polymer linker has a fluorescent label (that is different from the label on the second amplicon) attached whereby, imaging the microarray after hybridization and superimposing the image results in two fluorescent signals—the signal from the fluorescent bifunctional polymer linker which is covalently linked to the nucleic acid probe during fabrication, which would be detected in each spot comprised in the microarray, and the signal from fluorescent labeled third and fourth amplicons corresponding to pathogen and synthetic DNA respectively and which would be detected only in those positions (spots) where the nucleic acid probe sequence is complementary to the pathogen specific third amplicon (originally derived by amplification from the pathogen DNA in the sample) and synthetic DNA specific fourth amplicon. Thus, superimposing the signals using a computer provides beneficial attributes to the system and method claimed in this invention since one can readily identify the plant or pathogen comprised in the sample from a database that correlates nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants or pathogens. Further to this embodiment, the relative fluorescence intensities (RFU) from the microarray image corresponding to fluorescent pathogen DNA-specific amplicons, fluorescent plant DNA-specific amplicons are analyzed and mathematically correlated with fluorescence intensity for the synthetic DNA-specific amplicons and the known copy number for the synthetic DNA added to the sample, to determine copy numbers of the pathogen DNA and plant DNA in the sample, the mathematical correlation being;
 
 C   n   /C   o   =P ( S   n   /S   o ) x  where,  Equation #1
 
     C n =the number of microbial DNA copies of each type (n) present in the original sample mixture added to the first of two tandem PCR reactions used to prepare amplicons for microarray analysis. 
     C o =the number of known synthetic DNA copies (internal reference standard) added to the first of two PCR reactions used to prepare amplicons for microarray analysis. C o  may be set at any value including but not limited to 100, 500, 3,000 and 5,000 depending on the range of unknown microbial copies which might be encountered. In a preferred embodiment C o =3000. 
     S n =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the nth microbial species, followed by image analysis. 
     S o =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the synthetic DNA species, followed by image analysis. 
     X=a complex exponential factor which defines the functional relationship between the Experimental Microarray Data Ratio (S n /S o ) to the underlying ratio of microbial DNA copies vs synthetic DNA standard copies present in the original sample (C n /C o ). X may be a linear function or exponential or related functional form or a constant which is itself a function of amplification parameters and conditions of microarray analysis and imaging. In one aspect, X is an exponential factor ranging from about 1 to about 3. 
     P=A constant which relates the Experimental Microarray Data ratio (S n /S o ) to the concentration of amplified PCR product which binds to the microarray. In one aspect, P may range from about 0.1 to about 10. 
     In yet another embodiment of this invention, there is provided a method for simultaneously detecting and quantitating resident pathogen DNA and plant DNA in a plant sample in a single assay by introducing a known copy number of a synthetic DNA sequence as an internal reference standard to the plant sample. In this embodiment, the pathogen may be a human pathogen, an animal pathogen or a plant pathogen, which may be a bacterium, a fungus, a virus, a yeast, algae or a protozoan or a combination thereof. These pathogens may be present as constituents of the soil, soilless growth media, hydroponic growth media or water in which the plant sample was grown. The plant may be a terrestrial plant such as a  Humulus  or a  Cannabis , an aquatic plant, an epiphytic plant or a lithophytic plant that grows in soil, soilless media, hydroponic growth media or water. Preferably, the plant is a  Cannabis.    
     In this embodiment, the method comprises harvesting a plant tissue sample potentially comprising one or more pathogens, fluidizing the plant tissue sample and the one or more pathogens and isolating total nucleic acids comprising DNA from at least the plant tissue and DNA from the one or more pathogens. In one aspect of this embodiment, the step of isolating total nucleic acids comprises centrifuging the fluidized sample to get a pellet of plant cells and pathogen cells which are disrupted to release the total nucleic acids, which are used in the subsequent steps without further purification. To this unpurified total nucleic acid sample is added a known copy number of synthetic DNA. The synthetic DNA has a central region with a nucleotide sequence distinct from signature sequence determinants in the unknown DNA being queried, and 5′ and 3′ ends sequences substantially identical to a consensus sequence in the unknown DNA. Such consensus sequences include but are not limited to the sequences shown in SEQ ID NO: 152 and 153. Such a structure for the synthetic DNA permits amplification of the synthetic DNA by the same pair of PCR primers used to amplify the hypervariable region of the unknown DNA being queried. Examples of such synthetic DNA which may be employed include but is not limited to the sequences shown in SEQ ID NOs: 154 (fungus) and 155-157 (bacteria). 
     Further in this embodiment, a first amplification is performed on the unpurified total nucleic acid sample using one or more of a first primer pair selective for the pathogen DNA and the synthetic DNA and one or more of a second primer pair selective for the plant DNA to generate one or more pathogen-specific first amplicons and one or more plant-specific second amplicons and synthetic DNA-specific third amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. Any suitable first amplification primer pairs may be used for this purpose and one of skill in this art can easily design these primers based on the pathogen and plant of interest. In one embodiment, the pathogen is a bacterium and the first primer pairs have sequences shown in SEQ ID NOS: 1 and 2, or SEQ ID NOS: 3 and 4, or SEQ ID NOS: 5 and 6 or SEQ ID NOS: 7 and 8, or SEQ ID NOS: 9 and 10, or SEQ ID NOS: 11 and 12, or SEQ ID NOS: 137 and 138. In another embodiment, the pathogen is a fungus and the first primer pairs have sequences shown in SEQ ID NOS: 13 and 14, or SEQ ID NOS: 15 and 16, or SEQ ID NOS: 135 and 136. In either of these embodiments, the plant-specific second primer pairs have sequences shown in SEQ ID NOS: 17 and 18. An aliquot of the first, second, and third amplicons so generated is used as a template for a second, labeling PCR amplification step using a first fluorescent labeled third primer pairs having a sequence complementary to an internal flanking region in the first amplicons and third amplicons and second fluorescent labeled fourth primer pairs having a sequence complementary to an internal flanking region in the one or more plant-specific second amplicons to obtain pathogen DNA-specific first fluorescent labeled fourth amplicons, plant DNA-specific second fluorescent labeled fifth amplicons and synthetic DNA-specific first fluorescent labeled sixth amplicons. Any DNA amplification methodology, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) that can selectively amplify the DNA complement in the sample may be employed. In a preferred embodiment, the amplification is by PCR. Any suitable second amplification primer pairs may be used for this purpose and one of skill in this art can easily design these primers based on the pathogen and plant of interest. In one embodiment, the pathogen is a bacterium and the second primer pairs have sequences shown in SEQ ID NOS: 19 and 20, or SEQ ID NOS: 21 and 22, or SEQ ID NOS: 23 and 24 or SEQ ID NOS: 25 and 26, or SEQ ID NOS: 27 and 28, or SEQ ID NOS: 29 and 30, or SEQ ID NOS: 141 and 30. In another embodiment, the pathogen is a fungus and the second primer pairs have sequences shown in SEQ ID NOS: 31 and 32, or SEQ ID NOS: 33 and 34, or SEQ ID NOS: 139 and 140. In either of these embodiments, the plant-specific fourth primer pairs have sequences shown in SEQ ID NOS: 35-36. 
     Further in this embodiment, the fourth, fifth and sixth amplicons are then hybridized on a 3-dimensional lattice microarray system having a plurality of nucleic acid probes specific to sequence determinants in pathogen DNA, plant DNA or synthetic DNA. Any suitable nucleic acid probes may be used for this purpose and one of skill in this art can easily design them based on the pathogen of interest. In one embodiment, the bacterial nucleic acid probes have sequences shown in SEQ ID NOS: 37-85 and the synthetic DNA has sequences shown in SEQ ID NO: 155, SEQ ID NO: 156 corresponding respectively to synthetic DNA specific nucleic acid probes having sequences shown in SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144. In another embodiment, the fungal nucleic acid probes having sequences shown in SEQ ID NOS: 86-125, the synthetic DNA has sequences shown in SEQ ID NO: 154 that corresponds to synthetic DNA specific nucleic acid probes having sequences shown in SEQ ID NO: 145. In either embodiment, plant nucleic acid probes having sequences shown in SEQ ID NOS: 126-128. In this embodiment, the nucleic acid probes are attached to the microarray via a third fluorescent label bifunctional polymer linker has a (third fluorescent label is different from the first and second fluorescent label on the amplicons). Thereby, imaging of the hybridized amplicons on the microarray gives fluorescent signals—the third fluorescent signal from the nucleic acid probes that are attached to the bifunctional polymer linker, first fluorescent signal from the hybridized pathogen-specific fourth, and synthetic DNA-specific sixth amplicons and second fluorescent signal from the hybridized plant-specific fifth amplicons. Superimposing each of the first and second fluorescent third signals with the third fluorescent signal from the nucleic acid probe using a computer provides beneficial attributes to the system and method claimed in this invention since one can readily identify the plant or pathogen comprised in the sample from a database that correlates nucleic acid probe sequence and microarray location of this sequence with a known DNA signature in plants or pathogens. Further to this embodiment, the relative fluorescence intensities (RFU) from the microarray image corresponding to fluorescent pathogen DNA-specific amplicons, fluorescent plant DNA-specific amplicons are analyzed and mathematically correlated with fluorescence intensity for the synthetic DNA-specific amplicons and the known copy number for the synthetic DNA added to the sample, to determine copy numbers of the pathogen DNA and plant DNA in the sample, the mathematical correlation being;
 
 C   n   /C   o   =P ( S   n   /S   o ) x  where,  Equation #1
 
     C n =the number of microbial DNA copies of each type (n) present in the original sample mixture added to the first of two tandem PCR reactions used to prepare amplicons for microarray analysis. 
     C o =the number of known synthetic DNA copies (internal reference standard) added to the first of two PCR reactions used to prepare amplicons for microarray analysis. C o  may be set at any value including but not limited to 100, 500, 3,000 and 5,000 depending on the range of unknown microbial copies which might be encountered. In a preferred embodiment C o =3000. 
     S n =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the nth microbial species, followed by image analysis. 
     S o =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the synthetic DNA species, followed by image analysis. 
     X=a complex exponential factor which defines the functional relationship between the Experimental Microarray Data Ratio (S n /S o ) to the underlying ratio of microbial DNA copies vs synthetic DNA standard copies present in the original sample (C n /C o ). X may be a linear function or exponential or related functional form or a constant which is itself a function of amplification parameters and conditions of microarray analysis and imaging. In one aspect, X is an exponential factor ranging from about 1 to about 3. 
     P=A constant which relates the Experimental Microarray Data ratio (S n /S o ) to the concentration of amplified PCR product which binds to the microarray. In one aspect, P may range from about 0.1 to about 10. 
     In yet another embodiment of the present disclosure there is provided a method for DNA based pathogen analysis. The embodiments of the present disclosure use DNA amplification methodologies, including loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) tests that can selectively amplify the DNA complement of that plant material using unpurified plant and pathogen material. The embodiments are also based on the use of aforementioned PCR-amplified DNA as the substrate for microarray-based hybridization analysis, wherein the hybridization is made simple because the nucleic acid probes used to interrogate the DNA of such pathogens are optimized to function at room temperature. This enables the use of the above-mentioned microarray test at ambient temperature, thus bypassing the prior art requirement that testing be supported by an exogenous temperature-regulating device. 
     The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. 
     Example 1 
     Fabrication of 3-Dimensional Lattice Microarray Systems 
     The present invention teaches a way to link a nucleic acid probe to a solid support surface via the use of a bifunctional polymeric linker. The nucleic acid probe can be a PCR amplicon, synthetic oligonucleotides, isothermal amplification products, plasmids or genomic DNA fragment in a single stranded or double stranded form. The invention can be sub-divided into two classes, based on the nature of the underlying surface to which the nucleic acid probe would be linked. 
     Covalent Microarray System with Activated Solid Support 
     The covalent attachment of any one of these nucleic acid probes does not occur to the underlying surface directly, but is instead mediated through a relatively long, bi-functional polymeric linker that is capable of both chemical reaction with the surface and also capable of efficient UV-initiated crosslinking with the nucleic acid probe. The mechanics of this process is spontaneous 3D self assembly and is illustrated in  FIG. 1A - FIG. 1D . As seen in  FIG. 1A , the components required to fabricate this microarray system are: 
     (a) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCR or isothermal amplicon, plasmid or genomic DNA; 
     (b) a chemically activatable surface 1 with chemically activatable groups (designated “X”) compatible for reacting with a primary amine such as, epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde. 
     (c) bifunctional polymer linkers 2 such as a natural or modified OligodT, amino polysaccharide, amino polypeptide suitable for coupling to chemically activatable groups on the support surface, each attached with a fluorescent label 4; and 
     (d) a solvent comprising water and a high boiling point, water-miscible liquid such as glycerol, DMSO or propanediol (water to solvent ratio between 10:1 and 100:1). 
     Table 1 shows examples of chemically activatable groups and matched reactive groups on the bifunctional polymer linker for mere illustration purposes only and does not in any way preclude use of other combinations of matched reactive pairs. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Covalent Attachment of Bifunctional Polymeric Linker to an Activated Surfaces 
               
            
           
           
               
               
               
            
               
                 Activated Surface 
                 Matched Reactive Group 
                 Specific Implementation as Bifunctional 
               
               
                 Moiety 
                 on Bifunctional Linker 
                 polymeric linker 
               
               
                   
               
               
                 Epoxysilane 
                 Primary Amine 
                 (1) Amine-modified OligodT (20-60 bases) 
               
               
                   
                   
                 (2) Chitosan (20-60 subunits) 
               
               
                   
                   
                 (3) Lysine containing polypeptide (20-60aa) 
               
               
                 EDC Activated 
                 Primary Amine 
                 (4) Amine-modified OligodT (20-60 bases) 
               
               
                 Carboxylic Acid 
                   
                 (5) Chitosan (20-60 subunits) 
               
               
                   
                   
                 (6) Lysine containing polypeptide (20-60aa) 
               
               
                 N-hydroxysuccinimide 
                 Primary Amine 
                 (7) Amine-modified OligodT (20-60 bases) 
               
               
                 (NHS) 
                   
                 (8) Chitosan (20-60 subunits) 
               
               
                   
                   
                 (9) Lysine containing polypeptide (20-60aa) 
               
               
                   
               
            
           
         
       
     
     When used in the present invention, the chemically activatable surface, bifunctional polymer linkers and unmodified nucleic acid probes are included as a solution to be applied to a chemically activated surface 4 by ordinary methods of fabrication used to generate DNA Hybridization tests such as contact printing, piezo electric printing, ink jet printing, or pipetting. 
     Microarray fabrication begins with application of a mixture of the chemically activatable surface, bifunctional polymer linkers and unmodified nucleic acid probes to the surface. The first step is reaction and covalent attachment of the bifunctional linker to the activated surface ( FIG. 1B ). In general, the chemical concentration of the bi-functional linker is set to be such that less than 100% of the reactive sites on the surface form a covalent linkage to the bi-functional linker. At such low density, the average distance between bi-functional linker molecules defines a spacing denoted lattice width (“LW” in  FIG. 1B ). 
     In the second step, the water in the solvent is evaporated to concentrate the DNA and bifunctional linker via evaporation of water from the solvent ( FIG. 1C ). Generally, use of pure water as the solvent during matrix fabrication is disadvantageous because water is quickly removed by evaporation due to a high surface area/volume ratio. To overcome this, in the present invention, a mixture of water with a high boiling point water-miscible solvent such as glycerin, DMSO or propanediol was used as solvent. In this case, upon evaporation, the water component will evaporate but not the high boiling point solvent. As a result, molecular reactants—DNA and bifunctional linker are progressively concentrated as the water is lost to evaporation. In the present invention, the ratio or water to high boiling point solvent is kept between 10:1 and 100:1. Thus, in the two extreme cases, upon equilibrium, volume of the fluid phase will reduce due to water evaporation to between 1/100th and 1/10 th  the original volume, thus giving rise to a 100-fold to 10-fold increase in reactant concentration. Such controlled evaporation is crucial to the present invention since it controls the vertical spacing (Vertical Separation, “VG” in  FIG. 1C ) between nucleic acid probes, which is inversely related to the extent of evaporative concentration. 
     In the third step, the terminal Thymidine bases in the nucleic acid probes are UV crosslinked to the bifunctional linker within the evaporated surface ( FIG. 1D ). This process is mediated by the well-known photochemical reactivity of the Thymidine base that leads to the formation of covalent linkages to other thymidine bases in DNA or photochemical reaction with proteins and carbohydrates. If the bifunctional crosslinker is OligodT, then the crosslinking reaction will be bi-directional, that is, the photochemistry can be initiated in either the nucleic acid probe or the bifunctional OligodT linker. On the other hand, if the bifunctional linker is an amino polysaccharide such as chitosan or a polyamino acid, with a lysine or histidine in it, then the photochemistry will initiate in the nucleic acid probe, with the bifunctional linker being the target of the photochemistry. 
     Microarray System with Unmodified Solid Support for Non-Covalent Attachment 
     In this microarray system, attachment of the nucleic acid probes does not occur to the underlying surface directly, but is instead mediated through a relatively long, bi-functional polymeric linker that binds non-covalently with the solid support, but covalently with the nucleic acid probes via UV-initiated crosslinking. The mechanics of this process is spontaneous 3D self assembly and is illustrated in  FIGS. 2A-2D . As seen in  FIG. 2A , the components required to fabricate this microarray system are:
         (1) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCR or isothermal amplicon, plasmid or genomic DNA;   (2) an unmodified solid support 1   (3) bifunctional polymer linkers 2 such as OligodT or a amino polysaccharide, amino polypeptide, that inherently have or are modified to have functional groups (designated “R”) compatible for adsorptive binding to the solid support, each having a fluorescent label 4; and   (4) a solvent comprising water and a high boiling point, water-miscible liquid such as glycerol, DMSO or propanediol (water to solvent ratio between 10:1 and 100:1);       

     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Non-Covalent Attachment of Bi-Functional 
               
               
                 Polymeric Linker to an Inert Surface 
               
            
           
           
               
               
               
            
               
                 Representative 
                 Matched Adsorptive Group 
                 Specific Bifunctional 
               
               
                 support surface 
                 on Bifunctional Linker (R n ) 
                 polymeric linker 
               
               
                   
               
               
                 glass 
                 Single Stranded Nucleic 
                 OligodT (30-60 bases) 
               
               
                   
                 Acid &gt; 10 bases 
               
               
                 glass 
                 Amine-Polysaccharide 
                 Chitosan (30-60 
               
               
                   
                   
                 subunits) 
               
               
                 glass 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. Digoxigenin 
                 5′-Digoxigenin 
               
               
                 polycarbonate 
                 Single Stranded Nucleic 
                 Oligo-dT (30-60 bases) 
               
               
                   
                 Acid &gt; 10 bases 
               
               
                 polycarbonate 
                 Amine-Polysaccharide 
                 Chitosan (30-60 
               
               
                   
                   
                 subunits) 
               
               
                 polycarbonate 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. Digoxigenin 
                 5′-Digoxigenin 
               
               
                 graphene 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. pyrene 
                 5′pyrene 
               
               
                 graphene 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. CY-5 dye 
                 5′-CY-5 dye 
               
               
                 graphene 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. Digoxigenin 
                 5′-Digoxigenin 
               
               
                 gold 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. pyrene 
                 5′pyrene 
               
               
                 gold 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. CY-5 dye 
                 5′ CY-5 dye 
               
               
                 gold 
                 Extended Planar Hydrophobic 
                 OligodT (30-60 bases)- 
               
               
                   
                 Groups e.g. Digoxigenin 
                 5′ Digoxigenin 
               
               
                   
               
            
           
         
       
     
     Table 2 shows examples of unmodified support surfaces and matched absorptive groups on the bifunctional polymer linker for mere illustration purposes only and does not in any way precludes the use of other combinations of these. 
     When used in the present invention, components 1-3 are included as a solution to be applied to the solid support surface by ordinary methods of fabrication used to generate DNA Hybridization tests such as contact printing, piezo electric printing, ink jet printing, or pipetting. 
     Microarray fabrication begins with application of a mixture of the components (1)-(3) to the surface. The first step is adsorption of the bifunctional linker to the support surface ( FIG. 2B ). The concentration of the bi-functional linker is set so the average distance between bi-functional linker molecules defines a spacing denoted as lattice width (“LW” in  FIG. 2B ). 
     In the second step, the water in the solvent is evaporated to concentrate the DNA and bifunctional linker via evaporation of water from the solvent ( FIG. 2C ). Generally, use of pure water as the solvent during matrix fabrication is disadvantageous because water is quickly removed by evaporation due to a high surface area/volume ratio. To overcome this, in the present invention, a mixture of water with a high boiling point water-miscible solvent such as glycerin, DMSO or propanediol was used as solvent. In this case, upon evaporation, the water component will evaporate but not the high boiling point solvent. As a result, molecular reactants—DNA and bifunctional linker are progressively concentrated as the water is lost to evaporation. In the present invention, the ratio or water to high boiling point solvent is kept between 10:1 and 100:1. Thus, in the two extreme cases, upon equilibrium, volume of the fluid phase will reduce due to water evaporation to between 1/100th and 1/10 th  the original volume, thus giving rise to a 100-fold to 10-fold increase in reactant concentration. 
     In the third step, the terminal Thymidine bases in the nucleic acid probes are UV crosslinked to the bifunctional linker within the evaporated surface ( FIG. 2D ). This process is mediated by the well-known photochemical reactivity of the Thymidine base that leads to the formation of covalent linkages to other thymidine bases in DNA or photochemical reaction with proteins and carbohydrates. If the bifunctional crosslinker is OligodT, then the crosslinking reaction will be bi-directional, that is, the photochemistry can be initiated in either the nucleic acid probe or the bifunctional OligodT linker. On the other hand, if the bifunctional linker is an amino polysaccharide such as chitosan or a polyamino acid, with a lysine or histidine in it, then the photochemistry will initiate in the nucleic acid probe, with the bifunctional linker being the target of the photochemistry. 
     Although such non-covalent adsorption described in the first step is generally weak and reversible, when occurring in isolation, in the present invention it is taught that if many such weak adsorptive events between the bifunctional polymeric linker and the underlying surface occur in close proximity, and if the closely packed polymeric linkers are subsequently linked to each other via Thymidine-mediated photochemical crosslinking, the newly created extended, multi-molecular (crosslinked) complex will be additionally stabilized on the surface, thus creating a stable complex with the surface in the absence of direct covalent bonding to that surface. 
     The present invention works efficiently for the linkage of synthetic oligonucleotides as nucleic acid probes to form a microarray-based hybridization device for the analysis of microbial DNA targets. However, it is clear that the same invention may be used to link PCR amplicons, synthetic oligonucleotides, isothermal amplification products, plasmid DNA or genomic DNA fragment as nucleic acid probes. It is also clear that the same technology could be used to manufacture hybridization devices that are not microarrays. 
     DNA nucleic acid probes were formulated as described in Table 3, to be deployed as described above and illustrated in  FIG. 1  or  FIG. 2 . A set of 48 such probes (Table 4) were designed to be specific for various sequence determinants of microbial DNA and each was fabricated so as to present a string of 5-7 T bases at each end, to facilitate their UV-crosslinking to form a covalently linked microarray element, as described above and illustrated in  FIG. 1 . Each of the 48 different probes was printed in triplicate to form a 144 element (12×12) microarray having sequences shown in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Representative Conditions of use of the Present Invention 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 5′ labelled 
               
               
                   
                   
                 Unique sequence 
                 OligodT 
               
               
                   
                   
                 Oligonucleotide 
                 Fluorescent 
               
               
                 Nucleic acid probe 
                   
                 30-38 bases Long 
                 marker 30 bases 
               
               
                 Type 
                   
                 7 T&#39;s at each end 
                 Long(marker) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Nucleic acid probe 
                 50 
                 mM 
                 0.15 mM 
               
               
                 Concentration 
               
            
           
           
               
               
               
            
               
                 Bifunctional Linker 
                 OligodT 30 bases 
                   
               
               
                   
                 long Primary 
               
               
                   
                 amine at 3′ terminus 
               
            
           
           
               
               
               
               
            
               
                 Bifunctional Linker 
                 1 
                 mM 
                   
               
               
                 Concentration 
               
            
           
           
               
               
               
            
               
                 High Boiling point 
                 Water:Propanediol, 
                   
               
               
                 Solvent 
                 100:1 
               
               
                 Surface 
                 Epoxysilane on 
               
               
                   
                 borosilicate glass 
               
            
           
           
               
               
               
               
            
               
                 UV Crosslinking Dose 
                 300 
                 millijoule 
                   
               
               
                 (mjoule) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Nucleic acid probes Linked to the Microarray Surface via the Present Invention 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 132 
                 Negative control 
                 TTTTTTCTACTACCTATGCTGATTCACTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 129 
                 Imager Calibration 
                 TTTTCTATGTATCGATGTTGAGAAATTTTTTT 
               
               
                   
                 (High) 
                   
               
               
                   
               
               
                 SEQ ID NO: 130 
                 Imager Calibration 
                 TTTTCTAGATACTTGTGTAAGTGAATTTTTTT 
               
               
                   
                 (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 131 
                 Imager Calibration 
                 TTTTCTAAGTCATGTTGTTGAAGAATTTTTTT 
               
               
                   
                 (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 126 
                   Cannabis  ITS1 DNA 
                 TTTTTTAATCTGCGCCAAGGAACAATATTTTTTT 
               
               
                   
                 Control 1 
                   
               
               
                   
               
               
                 SEQ ID NO: 127 
                   Cannabis  ITS1 DNA 
                 TTTTTGCAATCTGCGCCAAGGAACAATATTTTTT 
               
               
                   
                 Control 2 
                   
               
               
                   
               
               
                 SEQ ID NO: 128 
                   Cannabis  ITS1 DNA 
                 TTTATTTCTTGCGCCAAGGAACAATATTTTATTT 
               
               
                   
                 Control 3 
                   
               
               
                   
               
               
                 SEQ ID NO: 86 
                 Total Yeast and Mold 
                 TTTTTTTTGAATCATCGARTCTTTGAACGCATTTT 
               
               
                   
                 (High sensitivity) 
                 TTT 
               
               
                   
               
               
                 SEQ ID NO: 87 
                 Total Yeast and Mold 
                 TTTTTTTTGAATCATCGARTCTCCTTTTTTT 
               
               
                   
                 (Low sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 88 
                 Total Yeast and Mold 
                 TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT 
               
               
                   
                 (Medium sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 132 
                 Negative control 
                 TTTTTTCTACTACCTATGCTGATTCACTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 92 
                   Aspergillus fumigatus  1 
                 TTTCTTTTCGACACCCAACTTTATTTCCTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 90 
                   Aspergillus flavus  1 
                 TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 95 
                   Aspergillus niger  1 
                 TTTTTTCGACGTTTTCCAACCATTTCTTTT 
               
               
                   
               
               
                 SEQ ID NO: 100 
                   Botrytis  spp. 
                 TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTT 
               
               
                   
                   
                 TTTTT 
               
               
                   
               
               
                 SEQ ID NO: 108 
                   Fusarium  spp. 
                 TTTTTTTTAACACCTCGCRACTGGAGATTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 89 
                   Alternaria  spp 
                 TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 123 
                   Rhodoturula  spp. 
                 TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 117 
                 
                   Penicillium paxilli 
                 
                 TTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 116 
                 
                   Penicillium oxalicum 
                 
                 TTTTTTACACCATCAATCTTAACCAGGCCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 118 
                   Penicillium  spp. 
                 TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 102 
                   Candida  spp. Group 1 
                 TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT 
               
               
                   
               
               
                 SEQ ID NO: 103 
                   Candida  spp. Group 2 
                 TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT 
               
               
                   
               
               
                 SEQ ID NO: 124 
                   Stachybotrys  spp 
                 TTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 125 
                   Trichoderma  spp. 
                 TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT 
               
               
                   
               
               
                 SEQ ID NO: 105 
                   Cladosporium  spp. 
                 TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 121 
                   Podosphaera  spp. 
                 TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 132 
                 Negative control 
                 TTTTTTCTACTACCTATGCTGATTCACTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 37 
                 Total Aerobic bacteria 
                 TTTTTTTTTCCTACGGGAGGCAGTTTTTTT 
               
               
                   
                 (High) 
                   
               
               
                   
               
               
                 SEQ ID NO: 38 
                 Total Aerobic bacteria 
                 TTTTTTTTCCCTACGGGAGGCATTTTTTTT 
               
               
                   
                 (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 39 
                 Total Aerobic bacteria 
                 TTTATTTTCCCTACGGGAGGCTTTTATTTT 
               
               
                   
                 (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 47 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTT 
               
               
                   
                 negative (High) 
                 T 
               
               
                   
               
               
                 SEQ ID NO: 48 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT 
               
               
                   
                 negative (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTT 
               
               
                   
                 negative (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 53 
                 Coliform/ 
                 TTTTTTCTATTGACGTTACCCGCTTTTTTT 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 81 
                 stx1 gene 
                 TTTTTTCTTTCCAGGTACAACAGCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 82 
                 stx2 gene 
                 TTTTTTGCACTGTCTGAAACTGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 59 
                 etuf gene 
                 TTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 132 
                 Negative control 
                 TTTTTTCTACTACCTATGCTGATTCACTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 65 
                   Listeria  spp. 
                 TTTTCTAAGTACTGTTGTTAGAGAATTTTT 
               
               
                   
               
               
                 SEQ ID NO: 56 
                   Aeromonas  spp. 
                 TTATTTTCTGTGACGTTACTCGCTTTTATT 
               
               
                   
               
               
                 SEQ ID NO: 78 
                 
                   Staphylococcus aureus 
                 
                 TTTATTTTCATATGTGTAAGTAACTGTTTTATTT 
               
               
                   
                 1 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                   Campylobacter  spp. 
                 TTTTTTATGACACTTTTCGGAGCTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 72 
                   Pseudomonas  spp. 3 
                 TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 53 
                   Clostridium  spp. 
                 TTTTCTGGAMGATAATGACGGTACAGTTTT 
               
               
                   
               
               
                 SEQ ID NO: 42 
                   Escherichia coli / 
                 TTTTCTAATACCTTTGCTCATTGACTCTTT 
               
               
                   
                   Shigella  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 74 
                   Salmonella enterica / 
                 TTTTTTTGTTGTGGTTAATAACCGATTTTT 
               
               
                   
                   Enterobacter  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 61 
                 invA gene 
                 TTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT 
               
               
                   
               
            
           
         
       
     
     The set of 48 different probes of Table 4 were formulated as described in Table 3, then printed onto epoxysilane coated borosilicate glass, using an Gentics Q-Array mini contact printer with Arrayit SMP pins, which deposit about 1 nL of formulation per spot. As described in  FIG. 1 , the arrays thus printed were then allowed to react with the epoxisilane surface at room temperature, and then evaporate to remove free water, also at room temperature. Upon completion of the evaporation step (typically overnight) the air-dried microarrays were then UV treated in a STATOLINKER® UV irradiation system: 300 mjoules of irradiation at 254 nm to initiate thymidine-mediated crosslinking. The microarrays are then ready for use, with no additional need for washing or capping. 
     Example 2 
     Using the 3-Dimensional Lattice Microarray System for DNA Analysis 
     Sample Processing 
     Harvesting Pathogens from plant surface comprises the following steps: 
     1. Wash the plant sample or tape pull in 1× phosphate buffered saline (PBS); 
     2. Remove plant material/tape; 
     3. Centrifuge to pellet cells and discard supernatant; 
     4. Resuspend in PathogenDx® Sample Prep Buffer pre-mixed with Sample Digestion Buffer; 
     5. Heat at 55° C. for 45 minutes; 
     6. Vortex to dissipate the pellet; 
     7. Heat at 95° C. for 15 minutes; and 
     8. Vortex and centrifuge briefly before use in PCR. 
     Amplification by PCR 
     The sample used for amplification and hybridization analysis was a  Cannabis  flower wash from a licensed  Cannabis  lab. The washed flower material was then pelleted by centrifugation. The pellet was then digested with proteinase K, then spiked with a known amount of  Salmonella  DNA before PCR amplification. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 PCR Primers and PCR conditions used in amplification 
               
               
                   
               
             
            
               
                 PCR primers (P1) for PCR Reaction #1 
               
               
                   Cannabis  ITS1 1° FP*-TTTGCAACAGCAGAACGACCCGTGA 
               
               
                 (SEQ 1D NO: 17) 
               
               
                   
               
               
                   Cannabis  ITS1 1° RP*-TTTCGATAAACACGCATCTCGATTG (SEQ 1D NO: 18) 
               
               
                   
               
               
                   Enterobacteriaceae  16S 1° FP-TTACCTTCGGGCCTCTTGCCATCRGATGTG 
               
               
                 (SEQ 1D NO: 11) 
               
               
                   
               
               
                   Enterobacteriaceae  16S 1° RP-TTGGAATTCTACCCCCCTCTACRAGACTCAAGC 
               
               
                 (SEQ 1D NO: 12) 
               
               
                   
               
               
                 PCR primers (P2) for PCR Reaction #2 
               
               
                   Cannabis  ITS1 2° FP-TTTCGTGAACACGTTTTAAACAGCTTG 
               
               
                 (SEQ 1D NO: 5) 
               
               
                   
               
               
                   Cannabis  ITS1 2° RP-(CY3)TTTCCACCGCACGAGCCACGCGAT 
               
               
                 (SEQ 1D NO: 36) 
               
               
                   
               
               
                   Enterobacteriaceae  16S 2° FP-TTATATTGCACAATGGGCGCAAGCCTGATG 
               
               
                 (SEQ 1D NO: 29) 
               
               
                   
               
               
                   Enterobacteriaceae  16S 2° RP-(CY3)TTTTGTATTACCGCGGCTGCTGGCA 
               
               
                 (SEQ 1D NO: 30) 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 Secondary 
               
               
                   
                 Primary PCR 
                 PCR 
               
               
                 PCR Reagent 
                 Concentration 
                 Concentration 
               
               
                   
               
               
                 PCR Buffer 
                 1X 
                 1X 
               
               
                   
               
               
                 MgCl 2   
                   2.5 mM 
                   2.5 mM 
               
               
                   
               
               
                 BSA 
                   0.16 mg/mL 
                   0.16 mg/mL 
               
               
                   
               
               
                 dNTP&#39;s 
                 200 mM 
                 200 mM 
               
               
                   
               
               
                 Primer mix 
                 200 nM each 
                  50 nM- 
               
               
                   
                   
                 FP/200 nM RP 
               
               
                   
               
               
                 Taq 
                   1.5 Units 
                   1.5 Units 
               
               
                 Polymerase 
                   
                   
               
               
                   
               
            
           
           
               
            
               
                 Program for PCR Reaction #1 
               
            
           
           
               
               
               
               
               
            
               
                 95° C., 4 min 
                 98° C., 30s 
                 61° C., 30s 
                 72° C., 60s 
                 72° C., 7 min 
               
            
           
           
               
               
               
            
               
                   
                 25X 
                   
               
               
                   
               
            
           
           
               
            
               
                 Program for PCR Reaction #2 
               
            
           
           
               
               
               
               
               
            
               
                 95° C., 4 min 
                 98° C., 20s 
                 61° C., 20s 
                 72° C., 30s 
                 72° C., 7 min 
               
            
           
           
               
               
               
            
               
                   
                 25X 
                   
               
               
                   
               
               
                 *FP, Forward Primer; *RP, Reverse Primer 
               
            
           
         
       
     
     The  Salmonella  DNA spiked sample was then amplified with PCR primers (P1-Table 5) specific for the 16s region of Enterobacteriaceae in a tandem PCR reaction to first isolate the targeted region (PCR Reaction #1) and also PCR primers (P1-Table 5) which amplify a segment of  Cannabis  DNA (ITS) used as a positive control. 
     The product of PCR Reaction #1 (14) was then subjected to a second PCR reaction (PCR Reaction #2) which additionally amplified and labelled the two targeted regions (16s, ITS) with green CY3 fluorophore labeled primers (P2-Table 5). The product of the PCR Reaction #2 (504) was then diluted 1-1 with hybridization buffer (4×SSC+5×Denhardt&#39;s solution) and then applied directly to the microarray for hybridization. 
     Hybridization 
     Because the prior art method of microarray manufacture allows DNA to be analyzed via hybridization without the need for pre-treatment of the microarray surface, the use of the microarray is simple, and involves 6 manual or automated pipetting steps. 
     1) Pipette the amplified DNA+binding buffer onto the microarray 
     2) Incubate for 30 minutes to allow DNA binding to the microarray (typically at room temperature, RT) 
     3) Remove the DNA+binding buffer by pipetting 
     4) Pipette 50 uL of wash buffer onto the microarray (0.4×SSC+0.5×Denhardt&#39;s) and incubate 5 min at RT. 
     5) Remove the wash buffer by pipetting 
     6) Repeat steps 4 and 5 
     7) Perform image analysis at 532 nm and 635 nm to detect the probe spot location (532 nm) and PCR product hybridization (635 nm). 
     Image Analysis 
     Image Analysis was performed at two wavelengths (532 nm and 635 nm) on a raster-based confocal scanner: GenePix 4000B Microarray Scanner, with the following imaging conditions: 33% Laser power, 400PMT setting at 532 nm/33% Laser Power, 700PMT setting at 635 nm.  FIG. 3  shows an example of the structure and hybridization performance of the microarray. 
       FIG. 3A  reveals imaging of the representative microarray, described above, after hybridization and washing, as visualized at 635 nm. The 635 nm image is derived from signals from the (red) CY5 fluor attached to the 5′ terminus of the bifunctional polymer linker OligodT which had been introduced during microarray fabrication as a positional marker in each microarray spot (see  FIG. 1  and Table 3). The data in  FIG. 3A  confirm that the CY5-labelled OligodT has been permanently linked to the microarray surface, via the combined activity of the bi-functional linker and subsequent UV-crosslinking, as described in  FIG. 1 . 
       FIG. 3B  reveals imaging of the representative microarray described above after hybridization and washing as visualized at 532 nm. The 532 nm image is derived from signals from the (green) CY3 fluor attached to the 5′ terminus of PCR amplified DNA obtained during PCR Reaction #2. It is clear from  FIG. 3B  that only a small subset of the 48 discrete probes bind to the CY3-labelled PCR product, thus confirming that the present method of linking nucleic acid probes to form a microarray ( FIG. 1 ) yields a microarray product capable of sequence specific binding to a (cognate) solution state target. The data in  FIG. 3B  reveal the underlying 3-fold repeat of the data (i.e., the array is the same set of 48 probes printed three times as 3 distinct sub-arrays to form the final 48×3=144 element microarray. The observation that the same set of 48 probes can be printed 3-times, as three repeated sub-domains show that the present invention generates microarray product that is reproducible. 
       FIG. 3C  reveals imaging of the representative microarray, described above, after hybridization and washing, as visualized with both the 532 nm and 635 nm images superimposed. The superimposed images display the utility of parallel attachment of a CY5-labelled OligodT positional marker relative to the sequence specific binding of the CY3-labelled PCR product. 
     Example 3 
     First PCR Amplification Step 
       FIG. 4A  shows an exemplar of the first PCR step. As is standard, such PCR reactions are initiated by the administration of PCR Primers. Primers define the start and stopping point of the PCR based DNA amplification reaction. In this embodiment, a pair of PCR reactions is utilized to support the needed DNA amplification. In general, such PCR amplification is performed in series: a first pair of PCRs, with the suffix “P1” in  FIG. 4A  are used to amplify about 1 μL of any unpurified DNA sample, such as a raw  Cannabis  leaf wash for example. About 1 μL of the product of that first PCR reaction is used as the substrate for a second PCR reaction that is used to affix a fluorescent dye label to the DNA, so that the label may be used to detect the PCR product when it binds by hybridization to the microarray. The primer sequences for the first and second PCRs are shown in Table 6. The role of this two-step reaction is to avert the need to purify the pathogen DNA to be analyzed. The first PCR reaction, with primers “P1” is optimized to accommodate the raw starting material, while the second PCR primer pairs “P2” are optimized to obtain maximal DNA yield, plus dye labeling from the product of the first reaction. Taken in the aggregate, the sum of the two reactions obviates the need to either purify or characterize the pathogen DNA of interest. 
       FIG. 4A  reveals at low resolution the 16s rDNA region which is amplified in an embodiment, to isolate and amplify a region which may be subsequently interrogated by hybridization. The DNA sequence of this 16s rDNA region is known to vary greatly among different bacterial species. Consequently, having amplified this region by two step PCR, that sequence variation may be interrogated by the subsequent microarray hybridization step. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 First and Second PCR Primers 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO. 
                 Primer target 
                 Primer sequence 
               
               
                   
               
            
           
           
               
            
               
                 First PCR Primers (P1) for the first amplification step 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 1 
                 16s rDNA HV3 
                 TTTCACAYTGGRACTGAGACACG 
               
               
                   
                 Locus (Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 2 
                 16s rDNA HV3 
                 TTTGACTACCAGGGTATCTAATCCTGT 
               
               
                   
                 Locus (Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 3 
                 Stx1 Locus 
                 TTTATAATCTACGGCTTATTGTTGAACG 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 4 
                 Stx1 Locus 
                 TTTGGTATAGCTACTGTCACCAGACAATG 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 5 
                 Stx2 Locus 
                 TTTGATGCATCCAGAGCAGTTCTGCG 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 6 
                 Stx2 Locus 
                 TTTGTGAGGTCCACGTCTCCCGGCGTC 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 7 
                 InvA Locus 
                 TTTATTATCGCCACGTTCGGGCAATTCG 
               
               
                   
                 ( Salmonella ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 8 
                 InvA Locus 
                 TTTCTTCATCGCACCGTCAAAGGAACCG 
               
               
                   
                 ( Salmonella ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 9 
                 tuf Locus (All  E . 
                 TTTCAGAGTGGGAAGCGAAAATCCTG 
               
               
                   
                   coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 10 
                 tuf Locus (All  E . 
                 TTTACGCCAGTACAGGTAGACTTCTG 
               
               
                   
                   coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 11 
                 16s rDNA 
                 TTACCTTCGGGCCTCTTGCCATCRGATGTG 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
                 HV3 Locus 
                   
               
               
                   
               
               
                 SEQ ID NO: 12 
                 16s rDNA 
                 TTGGAATTCTACCCCCCTCTACRAGACTCAAGC 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
                 HV3 Locus 
                   
               
               
                   
               
               
                 SEQ ID NO: 13 
                 ITS2 Locus (All 
                 TTTACTTTYAACAAYGGATCTCTTGG 
               
               
                   
                 Yeast, 
                   
               
               
                   
                 Mold/Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 14 
                 ITS2 Locus (All 
                 TTTCTTTTCCTCCGCTTATTGATATG 
               
               
                   
                 Yeast, 
                   
               
               
                   
                 Mold/Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 15 
                 ITS2 Locus 
                 TTTAAAGGCAGCGGCGGCACCGCGTCCG 
               
               
                   
                 ( Aspergillus   
                   
               
               
                   
                 species) 
                   
               
               
                   
               
               
                 SEQ ID NO: 16 
                 ITS2 Locus 
                 TTTTCTTTTCCTCCGCTTATTGATATG 
               
               
                   
                 ( Aspergillus   
                   
               
               
                   
                 species) 
                   
               
               
                   
               
               
                 SEQ ID NO: 17 
                 ITS1 Locus 
                 TTTGCAACAGCAGAACGACCCGTGA 
               
               
                   
                 ( Cannabis /Plant) 
                   
               
               
                   
               
               
                 SEQ ID NO: 18 
                 ITS1 Locus 
                 TTTCGATAAACACGCATCTCGATTG 
               
               
                   
                 ( Cannabis /Plant) 
                   
               
               
                   
               
            
           
           
               
            
               
                 Second PCR Primers (P2) for the second labeling amplification step 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 19 
                 16s rDNA HV3 
                 TTTACTGAGACACGGYCCARACTC 
               
               
                   
                 Locus (All Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 20 
                 16s rDNA HV3 
                 TTTGTATTACCGCGGCTGCTGGCA 
               
               
                   
                 Locus (All Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 21 
                 Stx1 Locus 
                 TTTATGTGACAGGATTTGTTAACAGGAC 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 22 
                 Stx1 Locus 
                 TTTCTGTCACCAGACAATGTAACCGCTG 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 23 
                 Stx2 Locus 
                 TTTTGTCACTGTCACAGCAGAAG 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 24 
                 Stx2 Locus 
                 TTTGCGTCATCGTATACACAGGAGC 
               
               
                   
                 (Pathogenic  E .  coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 25 
                 InvA Locus (All 
                 TTTTATCGTTATTACCAAAGGTTCAG 
               
               
                   
                   Salmonella ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 26 
                 InvA Locus (All 
                 TTTCCTTTCCAGTACGCTTCGCCGTTCG 
               
               
                   
                   Salmonella ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 27 
                 tuf Locus (All  E . 
                 TTTGTTGTTACCGGTCGTGTAGAAC 
               
               
                   
                   coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 28 
                 tuf Locus (All  E . 
                 TTTCTTCTGAGTCTCTTTGATACCAACG 
               
               
                   
                   coli ) 
                   
               
               
                   
               
               
                 SEQ ID NO: 29 
                 16s rDNA 
                 TTATATTGCACAATGGGCGCAAGCCTGATG 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
                 HV3 Locus 
                   
               
               
                   
               
               
                 SEQ ID NO: 30 
                 16s rDNA 
                 TTTTGTATTACCGCGGCTGCTGGCA 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
                 HV3 Locus 
                   
               
               
                   
               
               
                 SEQ ID NO: 31 
                 ITS2 Locus (All 
                 TTTGCATCGATGAAGARCGYAGC 
               
               
                   
                 Yeast, 
                   
               
               
                   
                 Mold/Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 32 
                 ITS2 Locus (All 
                 TTTCCTCCGCTTATTGATATGC 
               
               
                   
                 Yeast, 
                   
               
               
                   
                 Mold/Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 33 
                 ITS2 Locus 
                 TTTCCTCGAGCGTATGGGGCTTTGTC 
               
               
                   
                 ( Aspergillus   
                   
               
               
                   
                 species) 
                   
               
               
                   
               
               
                 SEQ ID NO: 34 
                 ITS2 Locus 
                 TTTTTCCTCCGCTTATTGATATGC 
               
               
                   
                 ( Aspergillus   
                   
               
               
                   
                 species) 
                   
               
               
                   
               
               
                 SEQ ID NO: 35 
                 ITS1 Locus 
                 TTTCGTGAACACGTTTTAAACAGCTTG 
               
               
                   
                 ( Cannabis /Plant) 
                   
               
               
                   
               
               
                 SEQ ID NO: 36 
                 ITS1 Locus 
                 TTTCCACCGCACGAGCCACGCGAT 
               
               
                   
                 ( Cannabis /Plant) 
               
               
                   
               
            
           
         
       
     
       FIG. 4B  displays the stx1 gene locus which is present in the most important pathogenic strains of  E. coli  and which encodes Shigatoxin 1. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed bacterial samples to present the stx1 locus for analysis by microarray-based DNA hybridization. 
       FIG. 5A  displays the stx2 gene locus which is also present in the most important pathogenic strains of  E coli  and which encodes Shigatoxin 2. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed bacterial samples so as to present the stx2 locus for analysis by microarray-based DNA hybridization. 
       FIG. 5B  displays the invA gene locus which is present in all strains of  Salmonella  and which encodes the InvAsion A gene product. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed bacterial samples so as to present the invA locus for analysis by microarray-based DNA hybridization. 
       FIG. 6  displays the tuf gene locus which is present in all strains of  E. coli  and which encodes the ribosomal elongation factor Tu. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed bacterial samples so as to present the tuf locus for analysis by microarray-based DNA hybridization. 
       FIG. 7  displays the ITS2 locus which is present in all eukaryotes, including all strains of yeast and mold and which encodes the intergenic region between ribosomal genes 5.8S and 28S. ITS2 is highly variable in sequence and that sequence variation can be used to resolve strain differences in yeast, and mold. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed yeast and mold samples so as to present the ITS2 locus for analysis by microarray-based DNA hybridization. 
       FIG. 8  displays the ITS1 gene locus which is present in all eukaryotes, including all plants and animals, which encodes the intergenic region between ribosomal genes 18S and 5.8S. ITS1 is highly variable in sequence among higher plants and that sequence variation can be used to identify plant species. Employing the same two-step PCR approach, a set of two PCR primer pairs were designed which, in tandem, can be used to amplify and label unprocessed  Cannabis  samples so as to present the ITS1 locus for analysis by microarray-based DNA hybridization. The identification and quantitation of the  Cannabis  sequence variant of ITS1 is used as an internal normalization standard in the analysis of pathogens recovered from the same  Cannabis  samples. 
     Table 7 displays representative oligonucleotide sequences which are used as microarray probes in an embodiment for DNA microarray-based analysis of bacterial 16s locus as described in  FIG. 4 . The sequence of those probes has been varied to accommodate the cognate sequence variation which occurs as a function of species difference among bacteria. In all cases, the probe sequences are terminated with a string of T&#39;s at each end, to enhance the efficiency of probe attachment to the microarray surface, at time of microarray manufacture. Table 8 shows sequences of the Calibration and Negative controls used in the microarray. 
     Table 9 displays representative oligonucleotide sequences which are used as microarray probes in an embodiment for DNA microarray-based analysis of eukaryotic pathogens (fungi, yeast and mold) based on their ITS2 locus as described in  FIG. 7 . Sequences shown in Table 8 are used as controls. The sequence of those probes has been varied to accommodate the cognate sequence variation which occurs as a function of species difference among fungi, yeast and mold. In all cases, the probe sequences are terminated with a string of T&#39;s at each end, to enhance the efficiency of probe attachment to the microarray surface, at time of microarray manufacture. 
     Table 10 displays representative oligonucleotide sequences which are used as microarray probes in an embodiment for DNA microarray-based analysis of  Cannabis  at the ITS1 locus ( Cannabis  spp.). 
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Oligonucleotide probe sequence for the 16S Locus 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 37 
                 Total Aerobic bacteria 
                 TTTTTTTTTCCTACGGGAGGCAGTTTTTTT 
               
               
                   
                 (High) 
                   
               
               
                   
               
               
                 SEQ ID NO: 38 
                 Total Aerobic bacteria 
                 TTTTTTTTCCCTACGGGAGGCATTTTTTTT 
               
               
                   
                 (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 39 
                 Total Aerobic bacteria 
                 TTTATTTTCCCTACGGGAGGCTTTTATTTT 
               
               
                   
                 (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 40 
                 
                   Enterobacteriaceae 
                 
                 TTTATTCTATTGACGTTACCCATTTATTTT 
               
               
                   
                 (Low sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 41 
                 
                   Enterobacteriaceae 
                 
                 TTTTTTCTATTGACGTTACCCGTTTTTTTT 
               
               
                   
                 (Medium sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 42 
                   Escherichia   coli / 
                 TTTTCTAATACCTTTGCTCATTGACTCTTT 
               
               
                   
                   Shigella  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 43 
                   Escherichia   coli / 
                 TTTTTTAAGGGAGTAAAGTTAATATTTTTT 
               
               
                   
                   Shigella  2 
                   
               
               
                   
               
               
                 SEQ ID NO: 44 
                   Escherichia   coli / 
                 TTTTCTCCTTTGCTCATTGACGTTATTTTT 
               
               
                   
                   Shigella  3 
                   
               
               
                   
               
               
                 SEQ ID NO: 45 
                   Bacillus  spp. Group1 
                 TTTTTCAGTTGAATAAGCTGGCACTCTTTT 
               
               
                   
               
               
                 SEQ ID NO: 46 
                   Bacillus  spp. Group2 
                 TTTTTTCAAGTACCGTTCGAATAGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 47 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTT 
               
               
                   
                 negative (High) 
                 TTT 
               
               
                   
               
               
                 SEQ ID NO: 48 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT 
               
               
                   
                 negative (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                 Bile-tolerant Gram- 
                 TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTT 
               
               
                   
                 negative (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 50 
                   Campylobacter  spp. 
                 TTTTTTATGACACTTTTCGGAGCTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 51 
                   Chromobacterium  spp. 
                 TTTTATTTTCCCGCTGGTTAATACCCTTTATTTT 
               
               
                   
               
               
                 SEQ ID NO: 52 
                   Citrobacter  spp. 
                 TTTTTTCCTTAGCCATTGACGTTATTTTTT 
               
               
                   
                 Group1 
                   
               
               
                   
               
               
                 SEQ ID NO: 53 
                   Clostridium  spp. 
                 TTTTCTGGAMGATAATGACGGTACAGTTTT 
               
               
                   
               
               
                 SEQ ID NO: 54 
                 Coliform/ 
                 TTTTTTCTATTGACGTTACCCGCTTTTTTT 
               
               
                   
                 
                   Enterobacteriaceae 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 55 
                 
                   Aeromonas 
                 
                 TTTTTGCCTAATACGTRTCAACTGCTTTTT 
               
               
                   
                   salmonicida / hydrophilia   
                   
               
               
                   
               
               
                 SEQ ID NO: 56 
                   Aeromonas  spp. 
                 TTATTTTCTGTGACGTTACTCGCTTTTATT 
               
               
                   
               
               
                 SEQ ID NO: 57 
                   Alkanindiges  spp. 
                 TTTTTAGGCTACTGRTACTAATATCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 58 
                 
                   Bacillus pumilus 
                 
                 TTTATTTAAGTGCRAGAGTAACTGCTATTTTATT 
               
               
                   
               
               
                 SEQ ID NO: 59 
                 etuf gene 
                 TTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 60 
                   Hafnia  spp. 
                 TTTTTTCTAACCGCAGTGATTGATCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 61 
                 invA gene 
                 TTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 62 
                 
                   Klebsiella oxytoca 
                 
                 TTTTTTCTAACCTTATTCATTGATCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 63 
                 
                   Klebsiella pneumoniae 
                 
                 TTTTTTCTAACCTTGGCGATTGATCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 64 
                   Legionella  spp. 
                 TTTATTCTGATAGGTTAAGAGCTGATCTTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 65 
                   Listeria  spp. 
                 TTTTCTAAGTACTGTTGTTAGAGAATTTTT 
               
               
                   
               
               
                 SEQ ID NO: 66 
                 
                   Panteoa agglomerans 
                 
                 TTTTTTAACCCTGTCGATTGACGCCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 67 
                 
                   Panteoa stewartii 
                 
                 TTTTTTAACCTCATCAATTGACGCCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 68 
                 
                   Pseudomonas 
                 
                 TTTTTGCAGTAAGTTAATACCTTGTCTTTT 
               
               
                   
                 
                   aeruginosa 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 69 
                 
                   Pseudomonas 
                 
                 TTTTTTTACGTATCTGTTTTGACTCTTTTT 
               
               
                   
                 
                   cannabina 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 70 
                   Pseudomonas  spp. 1 
                 TTTTTTGTTACCRACAGAATAAGCATTTTT 
               
               
                   
               
               
                 SEQ ID NO: 71 
                   Pseudomonas  spp. 2 
                 TTTTTTAAGCACTTTAAGTTGGGATTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 72 
                   Pseudomonas  spp. 3 
                 TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 73 
                 
                   Salmonella bongori 
                 
                 TTTTTTTAATAACCTTGTTGATTGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 74 
                 
                   Salmonella 
                 
                 TTTTTTTGTTGTGGTTAATAACCGATTTTT 
               
               
                   
                   enterica / Enterobacter  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 75 
                 
                   Salmonella 
                 
                 TTTTTTTAACCGCAGCAATTGACTCTTTTT 
               
               
                   
                   enterica / Enterobacter  2 
                   
               
               
                   
               
               
                 SEQ ID NO: 76 
                 
                   Salmonella 
                 
                 TTTTTTCTGTTAATAACCGCAGCTTTTTTT 
               
               
                   
                   enterica / Enterobacter  3 
                   
               
               
                   
               
               
                 SEQ ID NO: 77 
                   Serratia  spp. 
                 TTTATTCTGTGAACTTAATACGTTCATTTTTATT 
               
               
                   
               
               
                 SEQ ID NO: 78 
                 
                   Staphylococcus aureus 
                 
                 TTTATTTTCATATGTGTAAGTAACTGTTTTATTT 
               
               
                   
                 1 
                   
               
               
                   
               
               
                 SEQ ID NO. 79 
                 
                   Staphylococcus aureus 
                 
                 TTTTTTCATATGTGTAAGTAACTGTTTTTT 
               
               
                   
                 2 
                   
               
               
                   
               
               
                 SEQ ID NO: 80 
                   Streptomyces  spp. 
                 TTTTATTTTAAGAAGCGAGAGTGACTTTTATTTT 
               
               
                   
               
               
                 SEQ ID NO: 81 
                 stx1 gene 
                 TTTTTTCTTTCCAGGTACAACAGCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 82 
                 stx2 gene 
                 TTTTTTGCACTGTCTGAAACTGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 83 
                   Vibrio  spp. 
                 TTTTTTGAAGGTGGTTAAGCTAATTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 84 
                   Xanthamonas  spp. 
                 TTTTTTGTTAATACCCGATTGTTCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 85 
                 
                   Yersinia pestis 
                 
                 TTTTTTTGAGTTTAATACGCTCAACTTTTT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Calibration and Negative Controls 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 129 
                 Imager 
                 TTTTCTATGTATCGATGTTGAGAAATTTTTTT 
               
               
                   
                 Calibration (High) 
                   
               
               
                   
               
               
                 SEQ ID NO: 130 
                 Imager 
                 TTTTCTAGATACTTGTGTAAGTGAATTTTTTT 
               
               
                   
                 Calibration (Low) 
                   
               
               
                   
               
               
                 SEQ ID NO: 131 
                 Imager 
                   
               
               
                   
                 Calibration 
                 TTTTCTAAGTCATGTTGTTGAAGAATTTTTTT 
               
               
                   
                 (Medium) 
                   
               
               
                   
               
               
                 SEQ ID NO: 132 
                 Negative control 
                 TTTTTTCTACTACCTATGCTGATTCACTCTTTTT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Oligonucleotide probe sequence for the ITS2 Locus 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 86 
                 Total Yeast 
                 TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT 
               
               
                   
                 and Mold 
                   
               
               
                   
                 (High 
                   
               
               
                   
                 sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 87 
                 Total Yeast 
                 TTTTTTTTGAATCATCGARTCTCCTTTTTTT 
               
               
                   
                 and Mold (Low 
                   
               
               
                   
                 sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 88 
                 Total Yeast 
                 TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT 
               
               
                   
                 and Mold 
                   
               
               
                   
                 (Medium 
                   
               
               
                   
                 sensitivity) 
                   
               
               
                   
               
               
                 SEQ ID NO: 89 
                   Alternaria  spp. 
                 TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 90 
                 
                   Aspergillus 
                 
                 TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT 
               
               
                   
                   flavus  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 91 
                 
                   Aspergillus 
                 
                 TTTTTTTCTTGCCGAACGCAAATCAATCTTTTTTTTTT 
               
               
                   
                   flavus  2 
                 TT 
               
               
                   
               
               
                 SEQ ID NO: 92 
                 
                   Aspergillus 
                 
                 TTTCTTTTCGACACCCAACTTTATTTCCTTATTT 
               
               
                   
                   fumigatus  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 93 
                 
                   Aspergillus 
                 
                 TTTTTTTGCCAGCCGACACCCATTCTTTTT 
               
               
                   
                   fumigatus  2 
                   
               
               
                   
               
               
                 SEQ ID NO: 94 
                 
                   Aspergillus 
                 
                 TTTTTTGGCGTCTCCAACCTTACCCTTTTT 
               
               
                   
                 
                   nidulans 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 95 
                 
                   Aspergillus 
                 
                 TTTTTTCGACGTTTTCCAACCATTTCTTTT 
               
               
                   
                   niger  1 
                   
               
               
                   
               
               
                 SEQ ID NO: 96 
                 
                   Aspergillus 
                 
                 TTTTTTTTCGACGTTTTCCAACCATTTCTTTTTT 
               
               
                   
                   niger  2 
                   
               
               
                   
               
               
                 SEQ ID NO: 97 
                 
                   Aspergillus 
                 
                 TTTTTTTCGCCGACGTTTTCCAATTTTTTT 
               
               
                   
                   niger  3 
                   
               
               
                   
               
               
                 SEQ ID NO: 98 
                 
                   Aspergillus 
                 
                 TTTTTCGACGCATTTATTTGCAACCCTTTT 
               
               
                   
                 
                   terreus 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 99 
                 
                   Blumeria 
                 
                 TTTATTTGCCAAAAMTCCTTAATTGCTCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 100 
                   Botrytis  spp 
                 TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTT 
               
               
                   
                   
                 TT 
               
               
                   
               
               
                 SEQ ID NO: 101 
                 
                   Candida 
                 
                 TTTTTTTTTGAAAGACGGTAGTGGTAAGTTTTTT 
               
               
                   
                 
                   albicans 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 102 
                   Candida  spp. 
                 TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT 
               
               
                   
                 Group 1 
                   
               
               
                   
               
               
                 SEQ ID NO: 103 
                   Candida  spp. 
                 TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT 
               
               
                   
                 Group 2 
                   
               
               
                   
               
               
                 SEQ ID NO: 104 
                 
                   Chaetomium 
                 
                 TTTCTTTTGGTTCCGGCCGTTAAACCATTTTTTT 
               
               
                   
                 spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 105 
                 
                   Cladosporium 
                 
                 TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT 
               
               
                   
                 spp 
                   
               
               
                   
               
               
                 SEQ ID NO: 106 
                   Erysiphe  spp. 
                 TTTCTTTTTACGATTCTCGCGACAGAGTTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 107 
                 
                   Fusarium 
                 
                 TTTTTTTCTCGTTACTGGTAATCGTCGTTTTTTT 
               
               
                   
                 
                   oxysporum 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 108 
                   Fusarium  spp 
                 TTTTTTTTAACACCTCGCRACTGGAGATTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 109 
                 
                   Golovinomyces 
                 
                 TTTTTTCCGCTTGCCAATCAATCCATCTCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 110 
                 
                   Histoplasma 
                 
                 TTTATTTTTGTCGAGTTCCGGTGCCCTTTTATTT 
               
               
                   
                 
                   capsulatum 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 111 
                   Isaria  spp. 
                 TTTATTTTTCCGCGGCGACCTCTGCTCTTTATTT 
               
               
                   
               
               
                 SEQ ID NO: 112 
                 
                   Monocillium 
                 
                 TTTCTTTTGAGCGACGACGGGCCCAATTTTCTTT 
               
               
                   
                 spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 113 
                   Mucor  spp. 
                 TTTTCTCCAVVTGAGYACGCCTGTTTCTTTT 
               
               
                   
               
               
                 SEQ ID NO: 114 
                 
                   Myrothecium 
                 
                 TTTATTTTCGGTGGCCATGCCGTTAAATTTTATT 
               
               
                   
                 spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 115 
                 
                   Oidiodendron 
                 
                 TTTTTTTGCGTAGTACATCTCTCGCTCATTTTTT 
               
               
                   
                 spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 116 
                 
                   Penicillium 
                 
                 TTTTTTACACCATCAATCTTAACCAGGCCTTTTT 
               
               
                   
                 
                   oxalicum 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 117 
                 
                   Penicillium 
                 
                 TTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT 
               
               
                   
                 
                   paxilli 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 118 
                   Penicillium  spp 
                 TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 119 
                   Phoma / 
                 TTTTTTTGCAGTACATCTCGCGCTTTGATTTTTT 
               
               
                   
                   Epicoccum  spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 120 
                 
                   Podosphaera 
                 
                 TTTTTTGACCTGCCAAAACCCACATACCATTTTT 
               
               
                   
                 spp 
                   
               
               
                   
               
               
                 SEQ ID NO: 121 
                 
                   Podosphaera 
                 
                 TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT 
               
               
                   
                 spp. 
                   
               
               
                   
               
               
                 SEQ ID NO: 122 
                 
                   Pythium 
                 
                 TTTTATTTAAAGGAGACAACACCAATTTTTATTT 
               
               
                   
                 
                   oligandrum 
                 
                   
               
               
                   
               
               
                 SEQ ID NO: 123 
                 
                   Rhodoturula 
                 
                 TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT 
               
               
                   
                 spp 
                   
               
               
                   
               
               
                 SEQ ID NO: 124 
                 
                   Stachybotrys 
                 
                 TTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT 
               
               
                   
                 spp 
                   
               
               
                   
               
               
                 SEQ ID NO: 125 
                 
                   Trichoderma 
                 
                 TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT 
               
               
                   
                 spp 
               
               
                   
               
            
           
         
       
     
     Table 11 displays representative oligonucleotide sequences which are used as microarray probes in an embodiment for DNA microarray-based analysis of bacterial pathogens (stx1, stx2, invA, tuf) and for DNA analysis of the presence host  Cannabis  at the ITS1 locus ( Cannabis  spp.). It should be noted that this same approach, with modifications to the ITS1 sequence, could be used to analyze the presence of other plant hosts in such extracts. 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Oligonucleotide probe sequence for the  Cannabis  ITS1 Locus 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 126 
                   Cannabis  ITS1 
                 TTTTTTAATCTGCGCCAAGGAACAATATTTTTTT 
               
               
                   
                 DNA Control 1 
                   
               
               
                   
               
               
                 SEQ ID NO: 127 
                   Cannabis  ITS1 
                 TTTTTGCAATCTGCGCCAAGGAACAATATTTTTT 
               
               
                   
                 DNA Control 2 
                   
               
               
                   
               
               
                 SEQ ID NO: 128 
                   Cannabis  ITS1 
                 TTTATTTCTTGCGCCAAGGAACAATATTTTATTT 
               
               
                   
                 DNA Control 3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Representative Microarray Probe Design for the Present Invention: 
               
               
                 Bacterial Toxins, ITS1 ( Cannabis ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 81 
                 stx1 gene 
                 TTTTTTCTTTCCAGGTACAACAGCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 82 
                 stx2 gene 
                 TTTTTTGCACTGTCTGAAACTGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 59 
                 etuf gene 
                 TTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 61 
                 invA gene 
                 TTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT 
               
               
                   
               
               
                 SEQ ID NO: 126 
                   Cannabis  ITS1 
                 TTTTTTAATCTGCGCCAAGGAACAATATTTTTTT 
               
               
                   
                 DNA Control 1 
               
               
                   
               
            
           
         
       
     
       FIG. 9  shows a flow diagram to describe how an embodiment is used to analysis the bacterial pathogen or yeast and mold complement of a  Cannabis  or related plant sample. Pathogen samples can be harvested from  Cannabis  plant material by tape pulling of surface bound pathogen or by simple washing of the leaves or buds or stems, followed by a single multiplex “Loci Enhancement” Multiplex PCR reaction, which is then followed by a single multiplex “Labelling PCR”. A different pair of two step PCR reactions is used to analyze bacteria, than the pair of two step PCR reactions used to analyze fungi, yeast and mold. In all cases, the DNA of the target bacteria or fungi, yeast and mold are PCR amplified without extraction or characterization of the DNA prior to two step PCR. Subsequent to the Loci Enhancement and Labelling PCR steps, the resulting PCR product is simply diluted into binding buffer and then applied to the microarray test. The subsequent microarray steps required for analysis (hybridization and washing) are performed at lab ambient temperature. 
       FIG. 10  provide images of a representative implementation of microarrays used in an embodiment. In this implementation, all nucleic acid probes required for bacterial analysis, along with  Cannabis  DNA controls (Tables 7 and 10) are fabricated into a single 144 element (12×12) microarray, along with additional bacterial and  Cannabis  probes such as those in Table 10. In this implementation, all nucleic acid probes required for fungi, yeast and mold analysis along with  Cannabis  DNA controls were fabricated into a single 144 element (12×12) microarray, along with additional fungal probes shown in Table 9. The arrays are manufactured on PTFE coated glass slides as two columns of 6 identical microarrays. Each of the 12 identical microarrays is capable of performing, depending on the nucleic acid probes employed, a complete microarray-based analysis bacterial analysis or a complete microarray-based analysis of fungi, yeast and mold. Nucleic acid probes were linked to the glass support via microfluidic printing, either piezoelectric or contact based or an equivalent. The individual microarrays are fluidically isolated from the other 11 in this case, by the hydrophobic PTFE coating, but other methods of physical isolation can be employed. 
       FIGS. 11A-11B  display representative DNA microarray analysis of an embodiment. In this case, purified bacterial DNA or purified fungal DNA has been used, to test for affinity and specificity subsequent to the two-step PCR reaction and microarray-based hybridization analysis. As can be seen, the nucleic acid probes designed to detect each of the bacterial DNA (top) or fungal DNA (bottom) have bound to the target DNA correctly via hybridization and thus have correctly detected the bacterium or yeast (Table 12 and 13).  FIG. 12  displays representative DNA microarray analysis of an embodiment. In this case, 5 different unpurified raw  Cannabis  leaf wash samples were used to test for affinity and specificity subsequent to the two-step PCR reaction and microarray-based hybridization analysis. Both bacterial and fungal analysis has been performed on all 5 leaf wash samples, by dividing each sample into halves and subsequently processing them each for analysis of bacteria or for analysis of fungi, yeast and mold. The data of  FIG. 12  were obtained by combining the outcome of both assays.  FIG. 12  shows that the combination of two step PCR and microarray hybridization analysis, as described in  FIG. 9 , can be used to analyze the pathogen complement of a routine  Cannabis  leaf wash. It is expected, but not shown that such washing of any plant material could be performed similarly. 
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Representative microarray hybridization data 
               
               
                 obtained from purified bacterial DNA standards 
               
               
                 PURIFIED DNA BACTERIA PANEL 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 
                   Aeromonas 
                 
                 
                   Bacillus 
                 
                 
                   Campylobactor 
                 
               
               
                   
                 
                   hydrophila 
                 
                 
                   subtilus 
                 
                 ssp. 
               
               
                   
               
               
                 Low Pan Bacteria 
                 4434 
                 15943 
                 38700 
               
               
                 Control 
               
               
                 Medium Pan Bacteria 
                 7893 
                 33069 
                 28705 
               
               
                 Control 
               
               
                 High Pan Bacteria 
                 14934 
                 23469 
                 32936 
               
               
                 Control 
               
               
                 Low Bile tolerant gram 
                 5364 
                 947 
                 867 
               
               
                 negative 
               
               
                 High Bile tolerant gram 
                 55228 
                 339 
                 422 
               
               
                 negative 
               
               
                 Total Coliform 
                 106 
                 101 
                 145 
               
               
                 
                   E. coli 
                 
                 104 
                 121 
                 127 
               
               
                   E. Coli  specific gene 
                 318 
                 255 
                 422 
               
               
                   E. Coli  Stx1 
                 106 
                 116 
                 158 
               
               
                   E. Coli  Stx2 
                 100 
                 100 
                 126 
               
               
                 Enterobacteriacea 
                 885 
                 125 
                 211 
               
               
                 
                   Salmonella/Enterobacter 
                 
                 115 
                 99 
                 124 
               
               
                   Salmonella  specific 
                 189 
                 175 
                 217 
               
               
                 gene 
               
               
                 
                   Aeromonas 
                 
                 10335 
                 120 
                 123 
               
               
                 
                   Pseudomonas 
                 
                 106 
                 107 
                 120 
               
               
                 
                   Pseudomonas 
                 
                 169 
                 228 
                 173 
               
               
                 
                   aeriginosa 
                 
               
               
                 
                   Xanthomonas 
                 
                 98 
                 188 
                 122 
               
               
                 
                   Listeria 
                 
                 117 
                 263 
                 144 
               
               
                 
                   Campylobacter 
                 
                 148 
                 120 
                 65535 
               
               
                   Bacillus  Group 2 
                 143 
                 34517 
                 121 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 
                   E. coli 
                 
                   E. coli  0157:H7 
                   Listeria  ssp. 
               
               
                   
               
               
                 Low Pan Bacteria 
                 4215 
                 1745 
                 14140 
               
               
                 Control 
               
               
                 Medium Pan Bacteria 
                 8349 
                 3638 
                 35237 
               
               
                 Control 
               
               
                 High Pan Bacteria 
                 9827 
                 4327 
                 16726 
               
               
                 Control 
               
               
                 Low Bile tolerant gram 
                 2803 
                 1801 
                 817 
               
               
                 negative 
               
               
                 High Bile tolerant gram 
                 24172 
                 14746 
                 1482 
               
               
                 negative 
               
               
                 Total Coliform 
                 8276 
                 9175 
                 139 
               
               
                 
                   E. coli 
                 
                 55419 
                 47805 
                 151 
               
               
                   E. Coli  specific gene 
                 57638 
                 57112 
                 521 
               
               
                   E. Coli  Stx1 
                 134 
                 65535 
                 151 
               
               
                   E. Coli  Stx2 
                 169 
                 52041 
                 135 
               
               
                 Enterobacteriacea 
                 58323 
                 36641 
                 179 
               
               
                 
                   Salmonella/Enterobacter 
                 
                 190 
                 160 
                 144 
               
               
                   Salmonella  specific 
                 208 
                 392 
                 212 
               
               
                 gene 
               
               
                 
                   Aeromonas 
                 
                 127 
                 139 
                 163 
               
               
                 
                   Pseudomonas 
                 
                 130 
                 126 
                 133 
               
               
                 
                   Pseudomonas 
                 
                 318 
                 1217 
                 208 
               
               
                 
                   aeriginosa 
                 
               
               
                 
                   Xanthomonas 
                 
                 133 
                 143 
                 143 
               
               
                 
                   Listeria 
                 
                 136 
                 128 
                 24783 
               
               
                 
                   Campylobacter 
                 
                 139 
                 153 
                 224 
               
               
                   Bacillus  Group 2 
                 128 
                 150 
                 137 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 
                   Pseudomonas 
                 
                 
                   Salmonella 
                 
                 
                   Xanthomonas 
                 
               
               
                   
                 
                   aeruginosa 
                 
                 
                   enterica 
                 
                 ssp. 
               
               
                   
               
               
                 Low Pan Bacteria 
                 26431 
                 11167 
                 22152 
               
               
                 Control 
               
               
                 Medium Pan Bacteria 
                 39002 
                 17682 
                 24141 
               
               
                 Control 
               
               
                 High Pan Bacteria 
                 38682 
                 28596 
                 22072 
               
               
                 Control 
               
               
                 Low Bile tolerant gram 
                 4852 
                 4453 
                 461 
               
               
                 negative 
               
               
                 High Bile tolerant gram 
                 36337 
                 32579 
                 356 
               
               
                 negative 
               
               
                 Total Coliform 
                 145 
                 204 
                 196 
               
               
                 
                   E. coli 
                 
                 144 
                 83 
                 147 
               
               
                   E. Coli  specific gene 
                 695 
                 641 
                 461 
               
               
                   E. Coli  Stx1 
                 142 
                 196 
                 145 
               
               
                   E. Coli  Stx2 
                 147 
                 117 
                 132 
               
               
                 Enterobacteriacea 
                 375 
                 23847 
                 204 
               
               
                 
                   Salmonella/Enterobacter 
                 
                 138 
                 37520 
                 144 
               
               
                   Salmonella  specific 
                 211 
                 8124 
                 231 
               
               
                 gene 
               
               
                 
                   Aeromonas 
                 
                 142 
                 99 
                 146 
               
               
                 
                   Pseudomonas 
                 
                 25866 
                 77 
                 153 
               
               
                 
                   Pseudomonas 
                 
                 64437 
                 135 
                 424 
               
               
                 
                   aeriginosa 
                 
               
               
                 
                   Xanthomonas 
                 
                 221 
                 80 
                 41903 
               
               
                 
                   Listeria 
                 
                 144 
                 79 
                 131 
               
               
                 
                   Campylobacter 
                 
                 144 
                 88 
                 160 
               
               
                   Bacillus  Group 2 
                 139 
                 81 
                 134 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Representative microarray hybridization data 
               
               
                 obtained from purified bacterial DNA standards 
               
               
                 PURIFIED DNA FUNGAL PANEL 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 
                   Fusarium 
                 
                 
                   Penicillium 
                 
                   
               
               
                   
                 
                   A. fumigatus 
                 
                 
                   A. flavus 
                 
                 
                   A. niger 
                 
                 spp. 
                 spp. 
                 
                   Mucor 
                 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Low Pan 
                 4269 
                 6097 
                 5252 
                 13907 
                 3929 
                 3073 
               
               
                 Fungal 
                   
                   
                   
                   
                   
                   
               
               
                 Control 
                   
                   
                   
                   
                   
                   
               
               
                 Medium 
                 27006 
                 30445 
                 19746 
                 30972 
                 30947 
                 49986 
               
               
                 Pan Fungal 
                   
                   
                   
                   
                   
                   
               
               
                 Control 
                   
                   
                   
                   
                   
                   
               
               
                 High Pan 
                 64940 
                 64679 
                 54483 
                 47268 
                 65535 
                 63932 
               
               
                 Fungal 
                   
                   
                   
                   
                   
                   
               
               
                 Control 
                   
                   
                   
                   
                   
                   
               
               
                 Negative 
                 119 
                 127 
                 151 
                 107 
                 117 
                 118 
               
               
                 control 
                   
                   
                   
                   
                   
                   
               
               
                 
                   A. fumigatus 
                 
                 62018 
                 232 
                 114 
                 604 
                 126 
                 228 
               
               
                 
                   A. flavus 
                 
                 210 
                 65535 
                 116 
                 102 
                 115 
                 128 
               
               
                 
                   A. niger 
                 
                 113 
                 235 
                 24867 
                 108 
                 115 
                 112 
               
               
                 
                   Botrytis 
                 
                 189 
                 205 
                 435 
                 101 
                 126 
                 121 
               
               
                 
                   Penicillium 
                 
                 171 
                 282 
                 121 
                 100 
                 5891 
                 316 
               
               
                 
                   F. solani 
                 
                 112 
                 131 
                 174 
                 16578 
                 113 
                 140 
               
               
                 
                   Mucor 
                 
                 118 
                 127 
                 113 
                 150 
                 113 
                 29886 
               
               
                   
               
            
           
         
       
     
       FIG. 13  displays representative DNA microarray analysis of an embodiment. In this case, one unpurified (raw)  Cannabis  leaf wash sample was used and was compared to data obtained from a commercially-obtained homogenous yeast vitroid culture of live  Candida  to test for affinity and specificity subsequent to the two-step PCR reaction and microarray-based hybridization analysis. Both  Cannabis  leaf wash and cultured fungal analysis have been performed by processing them each for analysis via probes specific for fungi (see Tables 9 and 11). 
     The data of  FIG. 13  were obtained by combining the outcome of analysis of both the leaf wash and yeast vitroid culture samples. The data of  FIG. 13  show that the combination of two step PCR and microarray hybridization analysis, as described in  FIG. 9 , can be used to interrogate the fungal complement of a routine  Cannabis  leaf wash as adequately as can be done with a pure (live) fungal sample. It is expected that fungal analysis via such washing of any plant material could be performed similarly. 
       FIG. 14  shows a graphical representation of the position of PCR primers employed in a variation of an embodiment for low level detection of Bacteria in the Family Enterobacteriaceae including  E. coli . These PCR primers are used to selectively amplify and dye label DNA from targeted organisms for analysis via microarray hybridization. 
       FIGS. 15A-15C  illustrate representative DNA microarray analysis demonstrating assay sensitivity over a range of microbial inputs. In this case, certified reference material consisting of enumerated bacterial colonies of  E. coli  O157:H7 , E. coli  O111 ( FIGS. 15A-15B ) and  Salmonella enterica  ( FIG. 15C ) were spiked as a dilution series onto a hops plant surrogate matrix then processed using the assay version described for  FIG. 14 . Hybridization results from relevant probes from  FIG. 7  are shown. The larger numbers on the x-axis represents the total number of bacterial colony forming units (CFU) that were spiked onto each hops plant sample, whereas the smaller numbers on the x-axis represent the number of CFU&#39;s of the spiked material that were actually inputted into the assay. Only about 1/50 of the original spiked hops sample volume was actually analyzed. The smaller numbers upon the x-axis of  FIGS. 15A-15C  are exactly 1/50 th  that of the total (lower) values. As is seen,  FIGS. 15A-15C  show that the microarray test of an embodiment can detect less than 1 CFU per microarray assay. The nucleic acid targets within the bacterial genomes displayed in  FIGS. 15A-15C  comprise 16s rDNA. There are multiple copies of the 16s rDNA gene in each of these bacterial organisms, which enables detection at &lt;1 CFU levels. Since a colony forming unit approximates a single bacterium in many cases, the data of  FIGS. 15A-15C  demonstrate that the present microarray assay has sensitivity which approaches the ability to detect a single (or a small number) of bacteria per assay. Similar sensitivity is expected for all bacteria and eukaryotic microbes in that it is known that they all present multiple copies of the ribosomal rDNA genes per cell. 
     Tables 14A and 14B show a collection of representative microarray hybridization data obtained from powdered dry food samples with no enrichment and 18-hour enrichment for comparison. The data shows that bacterial microbes were successfully detected on the microarrays of the present invention without the need for enrichment. 
       FIG. 16  and Tables 15-17 describes embodiments for the analysis of fruit, embodiments for the analysis of vegetables and embodiments for the analysis of other plant matter. The above teaching shows, by example, that unprocessed leaf and bud samples in  Cannabis  and hops may be washed in an aqueous water solution, to yield a water-wash containing microbial pathogens which can then be analyzed via the present combination of RSG and microarrays. 
     If fresh leaf, flower, stem or root materials from fruit and vegetables are also washed in a water solution in that same way (when fresh, or after drying and grinding or other types or processing, then the present combination of RSG and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes in those other plant materials. At least two methods of sample collection are possible for fruit and vegetables. One method is the simple rinsing of the fruit, exactly as described for  Cannabis , above. Another method of sample collection from fruits and vegetables is a “tape pull”, wherein a piece of standard forensic tape is applied to the surface of the fruit, then pulled off. Upon pulling, the tape is then soaked in the standard wash buffer described above, to suspend the microbes attached to the tape. Subsequent to the tape-wash step, all other aspects of the processing and analysis (i.e., raw sample genotyping, PCR, then microarray analysis) are exactly as described above. 
     
       
         
           
               
             
               
                 TABLE 14A 
               
             
            
               
                   
               
               
                 Representative microarray data obtained from powdered dry food samples. 
               
            
           
           
               
               
            
               
                   
                 Sample Type 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Whey Protein 
                 Whey Protein 
                 Chewable 
                 Vanilla 
                 Pea 
               
               
                   
                 Shake Vanilla 
                 Shake Chocolate 
                 Berry Tablet 
                 Shake 
                 Protein 
               
            
           
           
               
               
            
               
                   
                 Enrichment time 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 0 
                 18 
                 0 
                 18 
                 0 
                 18 
                 0 
                 18 
                 0 
                 18 
               
               
                   
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
                 hours 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Negative Control Probe 
                 289 
                 318 
                 349 
                 235 
                 327 
                 302 
                 358 
                 325 
                 321 
                 299 
               
               
                 Total Aerobic Bacteria Probes 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 High sensitivity 
                 26129 
                 38896 
                 16629 
                 11901 
                 3686 
                 230 
                 32747 
                 12147 
                 41424 
                 40380 
               
               
                 Medium sensitivity 
                 5428 
                 6364 
                 3308 
                 2794 
                 876 
                 215 
                 7310 
                 2849 
                 15499 
                 8958 
               
               
                 Low sensitivity 
                 2044 
                 3419 
                 1471 
                 990 
                 446 
                 181 
                 2704 
                 1062 
                 4789 
                 3887 
               
               
                 Bile-tolerant Gram-negative Probes 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 High sensitivity 
                 2639 
                 350 
                 1488 
                 584 
                 307 
                 305 
                 1041 
                 472 
                 15451 
                 8653 
               
               
                 Medium sensitivity 
                 1713 
                 328 
                 892 
                 493 
                 322 
                 362 
                 615 
                 380 
                 6867 
                 4997 
               
               
                 Low sensitivity 
                 974 
                 600 
                 749 
                 621 
                 595 
                 688 
                 821 
                 929 
                 2459 
                 1662 
               
               
                 Total Enterobacteriaceae Probes 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 High sensitivity 
                 1131 
                 306 
                 363 
                 310 
                 346 
                 318 
                 273 
                 331 
                 4260 
                 3149 
               
               
                 Medium sensitivity 
                 479 
                 296 
                 320 
                 297 
                 329 
                 339 
                 314 
                 342 
                 1489 
                 990 
               
               
                 Low sensitivity 
                 186 
                 225 
                 203 
                 165 
                 205 
                 181 
                 207 
                 200 
                 216 
                 259 
               
               
                 16S rDNA Species Probes 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Escherichia coli/Shigella  spp. 
                 233 
                 205 
                 255 
                 219 
                 207 
                 255 
                 215 
                 214 
                 242 
                 198 
               
               
                   S. enterica/enterobacter  spp. 
                 203 
                 183 
                 186 
                 281 
                 212 
                 299 
                 197 
                 257 
                 308 
                 303 
               
               
                   Bacillus  spp. 
                 154 
                 172 
                 189 
                 114 
                 307 
                 156 
                 169 
                 153 
                 233 
                 259 
               
               
                   Pseudomonas  spp. 
                 549 
                 201 
                 202 
                 251 
                 148 
                 216 
                 303 
                 276 
                 2066 
                 983 
               
               
                 Organism Specific Gene Probes 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 tuf gene( E. coli ) 
                 204 
                 129 
                 180 
                 272 
                 158 
                 190 
                 191 
                 183 
                 186 
                 192 
               
               
                 stx1 gene( E. coli ) 
                 241 
                 178 
                 171 
                 240 
                 289 
                 304 
                 195 
                 245 
                 149 
                 191 
               
               
                 stx2 gene( E. coli ) 
                 145 
                 96 
                 136 
                 125 
                 182 
                 224 
                 130 
                 142 
                 85 
                 127 
               
               
                 invA ( Salmonella  spp.) 
                 215 
                 265 
                 210 
                 284 
                 204 
                 256 
                 239 
                 285 
                 237 
                 229 
               
               
                   
               
            
           
         
       
     
                     TABLE 14B                  Representative microarray data obtained from powdered dry food samples.                         Sample Type                                     Rice   Work-out   Work-out   Vanilla           Protein   Shake FP   Shake BR   Shake                         Enrichment time                                                     0   18   0   18   0   18   0   18           hours   hours   hours   hours   hours   hours   hours   hours                                                         Negative Control Probe   351   351   271   309   299   332   246   362       Total Aerobic Bacteria Probes                                       High sensitivity   471   288   17146   266   19207   261   41160   47198       Medium sensitivity   161   187   3120   229   3309   311   10060   22103       Low sensitivity   186   239   1211   261   1223   264   3673   6750       Bile-tolerant Gram-negative Probes                                       High sensitivity   326   372   375   380   412   363   1418   358       Medium sensitivity   304   362   341   391   308   356   699   394       Low sensitivity   683   942   856   689   698   864   848   665       Total Enterobacteriaceae Probes                                       High sensitivity   277   329   317   327   298   326   290   349       Medium sensitivity   326   272   296   291   297   263   262   307       Low sensitivity   215   207   204   288   213   269   195   247       16S rDNA Species Probes                                         Escherichia coli/Shigella  spp.   228   229   216   267   221   253   220   207         S. enterica/enterobacter  spp.   226   281   238   268   197   254   255   216         Bacillus  spp.   157   166   812   208   915   216   415   168         Pseudomonas  spp.   199   225   247   251   211   259   277   225       Organism Specific Gene Probes                                       tuf gene( E. coli )   150   149   126   206   163   212   215   166       stx1 gene( E. coli )   270   247   211   299   239   307   175   185       stx2 gene( E. coli )   158   178   127   205   138   175   128   100       invA ( Salmonella  spp.)   257   241   249   264   220   258   239   245                    
The data of Tables 15-17 demonstrates that simple washing of the fruit and tape pull sampling of the fruit generate similar microbial data. The blueberry sample is shown to be positive for  Botrytis , as expected, since  Botrytis  is a well-known fungal contaminant on blueberries. The lemon sample is shown to be positive for  Penicillium , as expected, since  Penicillium  is a well-known fungal contaminant for lemons.
 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Representative microarray hybridization data 
               
               
                 obtained from blueberry and lemon washes. 
               
            
           
           
               
               
            
               
                   
                 Sample 
               
            
           
           
               
               
               
            
               
                   
                 Blueberry 
                 Lemon 
               
            
           
           
               
               
            
               
                   
                 Collection Type 
               
               
                   
                 Produce Wash 
               
               
                   
                 Protocol 
               
            
           
           
               
               
               
            
               
                   
                 Wash 1 blueberry in 2 ml 
                 Wash 1 piece moldy 
               
               
                   
                 20 mM Borate, vortex 30 
                 lemon in 2 ml 20 mM 
               
               
                   
                 seconds 
                 Borate, vortex 30 seconds 
               
            
           
           
               
               
            
               
                   
                 Dilution Factor 
               
            
           
           
               
               
               
               
               
            
               
                   
                 NONE 
                 1:20 
                 NONE 
                 1:20 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   A. fumigatus  1 
                 65 
                 61 
                 62 
                 57 
               
               
                   A. fumigatus  2 
                 66 
                 61 
                 58 
                 131 
               
               
                   A. fumigatus  3 
                 69 
                 78 
                 55 
                 127 
               
               
                   A. fumigatus  4 
                 80 
                 198 
                 63 
                 161 
               
               
                   A. fumigatus  5 
                 98 
                 68 
                 59 
                 70 
               
               
                   A. flavus  1 
                 111 
                 65 
                 197 
                 58 
               
               
                   A. flavus  2 
                 64 
                 66 
                 71 
                 49 
               
               
                   A. flavus  3 
                 72 
                 79 
                 54 
                 49 
               
               
                   A. flavus  4 
                 95 
                 71 
                 66 
                 125 
               
               
                   A. flavus  5 
                 59 
                 55 
                 45 
                 47 
               
               
                   A. niger  1 
                 91 
                 75 
                 61 
                 61 
               
               
                   A. niger  2 
                 185 
                 68 
                 61 
                 57 
               
               
                   A. niger  3 
                 93 
                 66 
                 62 
                 61 
               
               
                   A. niger  4 
                 1134 
                 74 
                 75 
                 64 
               
               
                   Botrytis  spp. 1 
                 26671 
                 27605 
                 60 
                 55 
               
               
                   Botrytis  spp. 2 
                 26668 
                 35611 
                 59 
                 57 
               
               
                   Penicillium  spp. 1 
                 63 
                 69 
                 2444 
                 4236 
               
               
                   Penicillium  spp. 2 
                 71 
                 69 
                 4105 
                 7426 
               
               
                   Fusarium  spp. 1 
                 175 
                 69 
                 59 
                 78 
               
               
                   Fusarium  spp. 2 
                 71 
                 73 
                 84 
                 62 
               
               
                   Mucor  spp. 1 
                 71 
                 57 
                 58 
                 61 
               
               
                   Mucor  spp. 2 
                 61 
                 290 
                 66 
                 61 
               
               
                 Total Yeast and Mold 1 
                 20052 
                 21412 
                 8734 
                 7335 
               
               
                 Total Yeast and Mold 2 
                 17626 
                 8454 
                 5509 
                 5030 
               
               
                   
               
            
           
         
       
     
                     TABLE 16                  Representative microarray hybridization data obtained from blueberry washes and tape pulls.                         Sample           Moldy Blueberry           Collection Type           Tape Pull           ID                                                                     1A1   1A1   1A2   1A2   1A3   1A3   1B1   1B1   1B2   1B2   1B3   1B3                                 Collection Point 1   500 ul 20 mM Borate Buffer, vortex 30 seconds   500 ul 20 mM Borate + Triton Buffer, vortex 30 seconds                                 Collection Point 2       Add 15 mg zirconia beads,       Add 15 mg zirconia beads,               vortex, Heat 5 min 95° C.,       vortex, Heat 5 min 95° C.,               Vortex 15 seconds       Vortex 15 seconds                                         Collection Point 3           Heat 5 min           Heat 5 min                   95° C. vortex           95° C. vortex                   15 seconds           15 seconds                                                                 Dilution Factor   NO   1:20   NO   1:20   NO   1:20   NO   1:20   NO   1:20   NO   1:20         A. fumigatus  1   66   388   83   77   97   313   95   68   76   55   75   60         A. fumigatus  2   97   100   82   118   69   56   87   67   185   76   58   52         A. fumigatus  3   77   94   82   1083   87   61   93   84   75   378   73   64         A. fumigatus  4   84   151   94   118   96   80   115   85   85   93   190   88         A. fumigatus  5   63   75   96   71   78   61   98   74   68   98   70   533         A. flavus  1   200   107   113   61   204   58   105   73   62   68   64   65         A. flavus  2   70   104   64   57   133   281   111   78   377   314   57   50         A. flavus  3   83   90   94   150   99   90   96   222   1162   86   80   73         A. flavus  4   76   125   92   146   87   174   241   78   115   69   105   85         A. flavus  5   80   153   77   72   78   439   71   86   280   58   62   57         A. niger  1   409   178   122   72   80   70   76   71   152   117   65   53         A. niger  2   78   292   79   65   715   666   74   70   68   731   70   54         A. niger  3   86   76   87   558   78   60   70   81   96   63   478   58         A. niger  4   164   70   92   108   197   69   130   75   76   148   73   65         Botrytis  spp. 1   41904   26549   28181   29354   25304   25685   57424   33783   57486   49803   33176   32153         Botrytis  spp. 2   36275   25518   29222   27076   26678   27675   49480   32899   52817   34322   29693   32026         Penicillium  spp. 1   80   81   83   64   96   60   79   80   176   60   385   53         Penicillium  spp. 2   90   93   81   80   114   59   98   69   470   65   478   56         Fusarium  spp. 1   77   71   69   62   112   55   61   274   617   81   59   757         Fusarium  spp. 2   91   82   107   74   101   65   91   66   123   63   71   583         Mucor  spp. 1   90   314   73   88   105   61   77   79   741   180   172   74         Mucor  spp. 2   83   69   73   69   91   67   111   102   455   88   70   133       Total Y &amp; M 1   23637   18532   15213   17668   18068   19762   18784   15550   20625   17525   25813   18269       Total Y &amp; M 2   12410   8249   9281   11526   8543   13007   14180   14394   9905   8972   15112   12678                    
The data embodied in  FIG. 16  and Tables 15-17 demonstrate the use of an embodiment, for the recovery and analysis of yeast microbes on the surface of fruit (blueberries and lemons in this case), but an embodiment could be extended to other fruits and vegetables for the analysis of both bacterial and fungal contamination.
 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Representative microarray hybridization data 
               
               
                 obtained from lemon washes and tape pulls. 
               
            
           
           
               
               
            
               
                   
                 Sample 
               
               
                   
                 Moldy Lemon 
               
               
                   
                 Collection Type 
               
               
                   
                 Tape Pull 
               
               
                   
                 ID 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 1A1 
                 1A2 
                 1A3 
                 1B1 
                 1B2 
               
               
                   
                 Lemon 
                 Lemon 
                 Lemon 
                 Lemon 
                 Lemon 
               
               
                   
                   
               
            
           
           
               
               
            
               
                 Collection Point 1 
                 500 ul 20 mM Borate + Triton Buffer, vortex 30 seconds 
               
            
           
           
               
               
               
               
               
            
               
                 Collection Point 2 
                   
                 Add 15 mg 
                   
                 Add 15 mg 
               
               
                   
                   
                 zirconia beads, 
                   
                 zirconia beads, 
               
               
                   
                   
                 vortex, Heat 5 min 
                   
                 vortex, Heat 5 min 
               
               
                   
                   
                 95° C., Vortex 
                   
                 95° C., Vortex 
               
               
                   
                   
                 15 seconds 
                   
                 15 seconds 
               
            
           
           
               
               
               
               
               
               
            
               
                 Collection Point 3 
                   
                   
                 Heat 5 
                   
                   
               
               
                   
                   
                   
                 min 95° C. 
               
               
                   
                   
                   
                 vortex 15 
               
               
                   
                   
                   
                 seconds 
               
            
           
           
               
               
            
               
                 Dilution Factor 
                 NONE 
               
            
           
           
               
               
               
               
               
               
            
               
                   A. fumigatus  1 
                 96 
                 83 
                 75 
                 83 
                 64 
               
               
                   A. fumigatus  2 
                 221 
                 73 
                 71 
                 66 
                 101 
               
               
                   A. fumigatus  3 
                 87 
                 88 
                 85 
                 92 
                 122 
               
               
                   A. fumigatus  4 
                 83 
                 85 
                 91 
                 72 
                 97 
               
               
                   A. fumigatus  5 
                 448 
                 100 
                 84 
                 114 
                 78 
               
               
                   A. flavus  1 
                 85 
                 79 
                 70 
                 66 
                 63 
               
               
                   A. flavus  2 
                 77 
                 82 
                 77 
                 79 
                 63 
               
               
                   A. flavus  3 
                 133 
                 66 
                 86 
                 60 
                 67 
               
               
                   A. flavus  4 
                 96 
                 85 
                 81 
                 98 
                 88 
               
               
                   A. flavus  5 
                 68 
                 62 
                 65 
                 106 
                 59 
               
               
                   A. niger  1 
                 73 
                 88 
                 77 
                 73 
                 73 
               
               
                   A. niger  2 
                 74 
                 84 
                 81 
                 71 
                 103 
               
               
                   A. niger  3 
                 90 
                 86 
                 87 
                 74 
                 78 
               
               
                   A. niger  4 
                 82 
                 93 
                 104 
                 86 
                 161 
               
               
                   Botrytis  spp. 1 
                 82 
                 75 
                 75 
                 77 
                 68 
               
               
                   Botrytis  spp. 2 
                 91 
                 74 
                 83 
                 67 
                 62 
               
               
                   Penicillium  spp. 1 
                 3824 
                 5461 
                 5500 
                 4582 
                 5290 
               
               
                   Penicillium  spp. 2 
                 7586 
                 8380 
                 11177 
                 6528 
                 8167 
               
               
                   Fusarium  spp. 1 
                 101 
                 62 
                 61 
                 70 
                 279 
               
               
                   Fusarium  spp. 2 
                 77 
                 122 
                 78 
                 68 
                 233 
               
               
                   Mucor  spp. 1 
                 74 
                 110 
                 89 
                 76 
                 57 
               
               
                   Mucor  spp. 2 
                 132 
                 1302 
                 90 
                 84 
                 61 
               
               
                 Total Yeast and Mold 1 
                 8448 
                 12511 
                 9249 
                 12844 
                 8593 
               
               
                 Total Yeast and Mold 2 
                 9275 
                 8716 
                 11585 
                 10758 
                 4444 
               
               
                   
               
            
           
         
       
     
     Table 18 shows embodiments for the analysis of environmental water samples/specimens. The above teaching shows by example that unprocessed leaf and bud samples in  Cannabis  and hops may be washed in an aqueous water solution, to yield a water-wash containing microbial pathogens which can then be analyzed via the present combination of Raw Sample Genotyping (RSG) and microarrays. If a water sample containing microbes were obtained from environmental sources (such as well water, or sea water, or soil runoff or the water from a community water supply) and then analyzed directly, or after ordinary water filtration to concentrate the microbial complement onto the surface of the filter, that the present combination of RSG and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Representative microarray data from raw water filtrate. 
               
            
           
           
               
               
            
               
                   
                 Sample ID 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Negative 
               
               
                   
                 2 H 
                 2 H 
                 9 D 
                 9 D 
                 21 
                 21 
                 23 
                 23 
                 25 
                 25 
                 Control 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Imager Calibration High 
                 311 
                 335 
                 322 
                 379 
                 341 
                 348 
                 345 
                 325 
                 354 
                 343 
                 333 
               
               
                 Imager Calibration Med 
                 280 
                 314 
                 268 
                 286 
                 288 
                 231 
                 253 
                 295 
                 267 
                 295 
                 244 
               
               
                 Imager Calibration Low 
                 245 
                 296 
                 302 
                 324 
                 254 
                 268 
                 293 
                 285 
                 271 
                 340 
                 275 
               
               
                   Cannabis  cont. 
                 310 
                 330 
                 313 
                 255 
                 323 
                 368 
                 313 
                 322 
                 274 
                 332 
                 322 
               
               
                   Cannabis  cont. 
                 313 
                 237 
                 298 
                 271 
                 298 
                 288 
                 296 
                 280 
                 249 
                 284 
                 297 
               
               
                   Cannabis  cont. 
                 208 
                 265 
                 276 
                 250 
                 267 
                 327 
                 255 
                 258 
                 253 
                 282 
                 370 
               
               
                 Total Yeast and Mold 
                 284 
                 324 
                 290 
                 307 
                 272 
                 361 
                 296 
                 288 
                 271 
                 321 
                 469 
               
               
                 Total Yeast and Mold 
                 251 
                 259 
                 294 
                 290 
                 309 
                 308 
                 285 
                 281 
                 275 
                 299 
                 293 
               
               
                 Total Yeast and Mold 
                 282 
                 280 
                 294 
                 280 
                 299 
                 284 
                 275 
                 286 
                 299 
                 259 
                 232 
               
               
                 Total Aerobic bacteria High 
                 
                   40101 
                 
                 
                   42007 
                 
                 
                   47844 
                 
                 
                   47680 
                 
                 
                   45102 
                 
                 
                   44041 
                 
                 
                   43520 
                 
                 
                   41901 
                 
                 
                   46459 
                 
                 
                   46783 
                 
                 135 
               
               
                 Total Aerobic bacteria Medium 
                 
                   14487 
                 
                 
                   12314 
                 
                 
                   24189 
                 
                 
                   26158 
                 
                 
                   19712 
                 
                 
                   16210 
                 
                 
                   17943 
                 
                 
                   15474 
                 
                 
                   25524 
                 
                 
                   18507 
                 
                 157 
               
               
                 Total Aerobic bacteria Low 
                 
                   4885 
                 
                 
                   5629 
                 
                 
                   7625 
                 
                 
                   6456 
                 
                 
                   5807 
                 
                 
                   4505 
                 
                 
                   5316 
                 
                 
                   6022 
                 
                 
                   6264 
                 
                 
                   6974 
                 
                 159 
               
               
                 Negative Control 
                 293 
                 359 
                 303 
                 339 
                 312 
                 329 
                 306 
                 377 
                 307 
                 335 
                 307 
               
               
                 
                   Aspergillus fumigatus 
                 
                 285 
                 291 
                 284 
                 268 
                 289 
                 265 
                 271 
                 281 
                 269 
                 248 
                 228 
               
               
                 
                   Aspergillus flavus 
                 
                 184 
                 211 
                 201 
                 344 
                 237 
                 179 
                 212 
                 213 
                 163 
                 204 
                 171 
               
               
                 
                   Aspergillus niger 
                 
                 226 
                 213 
                 228 
                 273 
                 190 
                 195 
                 245 
                 206 
                 222 
                 209 
                 172 
               
               
                   Botrytis  spp. 
                 219 
                 285 
                 258 
                 302 
                 275 
                 219 
                 202 
                 288 
                 221 
                 248 
                 214 
               
               
                   Alternaria  spp. 
                 81 
                 97 
                 76 
                 89 
                 58 
                 76 
                 75 
                 175 
                 117 
                 174 
                 167 
               
               
                 
                   Penicillium paxilli 
                 
                 135 
                 162 
                 215 
                 142 
                 127 
                 161 
                 103 
                 115 
                 238 
                 190 
                 200 
               
               
                 
                   Penicillium oxalicum 
                 
                 119 
                 107 
                 161 
                 131 
                 135 
                 241 
                 178 
                 158 
                 140 
                 143 
                 194 
               
               
                   Penicillium  spp. 
                 50 
                 123 
                 179 
                 177 
                 128 
                 138 
                 146 
                 163 
                 148 
                 115 
                 184 
               
               
                   Can. alb /trop/dub 
                 261 
                 236 
                 235 
                 230 
                 250 
                 213 
                 276 
                 244 
                 245 
                 237 
                 194 
               
               
                   Can. glab/Sach  &amp;  Kluv  spp. 
                 146 
                 165 
                 196 
                 128 
                 160 
                 215 
                 185 
                 217 
                 215 
                 177 
                 225 
               
               
                   Podosphaera  spp. 
                 111 
                 119 
                 100 
                 122 
                 192 
                 105 
                 95 
                 43 
                 169 
                 27 
                 143 
               
               
                 Bile-tolerant Gram-negative High 
                 
                   16026 
                 
                 
                   9203 
                 
                 
                   13309 
                 
                 
                   8426 
                 
                 
                   16287 
                 
                 
                   14116 
                 
                 
                   10557 
                 
                 
                   17558 
                 
                 
                   15343 
                 
                 
                   14285 
                 
                 183 
               
               
                 Bile-tolerant Gram-negative Medium 
                 
                   12302 
                 
                 
                   11976 
                 
                 
                   9259 
                 
                 
                   10408 
                 
                 
                   13055 
                 
                 
                   10957 
                 
                 
                   11242 
                 
                 
                   8416 
                 
                 
                   9322 
                 
                 
                   11785 
                 
                 196 
               
               
                 Bile-tolerant Gram-negative Low 
                 
                   5210 
                 
                 
                   7921 
                 
                 
                   3818 
                 
                 
                   3984 
                 
                 
                   7224 
                 
                 
                   6480 
                 
                 
                   4817 
                 
                 
                   6933 
                 
                 
                   5021 
                 
                 
                   5844 
                 
                 240 
               
               
                 Total Enterobacteriaceae High 
                 193 
                 248 
                 389 
                 357 
                 215 
                 214 
                 198 
                 220 
                 276 
                 208 
                 210 
               
               
                 Total Enterobacteriaceae Med 
                 246 
                 214 
                 297 
                 246 
                 244 
                 224 
                 219 
                 245 
                 252 
                 229 
                 207 
               
               
                 Total Enterobacteriaceae Low 
                 165 
                 140 
                 158 
                 119 
                 151 
                 180 
                 150 
                 167 
                 182 
                 174 
                 132 
               
               
                 Total Coliform 
                 121 
                 148 
                 158 
                 117 
                 129 
                 117 
                 155 
                 157 
                 125 
                 178 
                 152 
               
               
                   Escherichia coli  specific gene 
                 31821 
                 115 
                 132 
                 155 
                 127 
                 62 
                 86 
                 121 
                 59 
                 90 
                 234 
               
               
                 stx1 gene 
                 67 
                 0 
                 2 
                 0 
                 0 
                 23 
                 21 
                 28 
                 0 
                 0 
                 116 
               
               
                 stx2 gene 
                 17 
                 36 
                 174 
                 0 
                 61 
                 47 
                 0 
                 51 
                 33 
                 0 
                 85 
               
               
                   Salmonella  specific gene 
                 181 
                 172 
                 245 
                 172 
                 178 
                 212 
                 157 
                 243 
                 174 
                 156 
                 146 
               
               
                   Bacillus  spp. 
                 137 
                 135 
                 174 
                 112 
                 164 
                 143 
                 163 
                 182 
                 168 
                 152 
                 149 
               
               
                   Pseudomonas  spp. 
                 271 
                 74 
                 332 
                 56 
                 366 
                 133 
                 91 
                 114 
                 60 
                 179 
                 555 
               
               
                   Escherichia coli/Shigella  spp. 
                 103 
                 124 
                 221 
                 124 
                 90 
                 144 
                 130 
                 121 
                 137 
                 143 
                 158 
               
               
                   Salmonella enterica/enterobacter  spp. 
                 124 
                 98 
                 131 
                 119 
                 136 
                 88 
                 121 
                 77 
                 128 
                 140 
                 124 
               
               
                   Erysiphe  Group 2 
                 278 
                 221 
                 237 
                 230 
                 245 
                 254 
                 250 
                 220 
                 205 
                 236 
                 233 
               
               
                   Trichoderma  spp. 
                 105 
                 157 
                 204 
                 152 
                 180 
                 154 
                 130 
                 161 
                 201 
                 180 
                 150 
               
               
                 
                   Escherichia coli 
                 
                 429 
                 431 
                 551 
                 576 
                 549 
                 406 
                 407 
                 484 
                 556 
                 551 
                 293 
               
               
                 
                   Aspergillus niger 
                 
                 218 
                 212 
                 216 
                 297 
                 255 
                 312 
                 221 
                 202 
                 238 
                 231 
                 209 
               
               
                   Escherichia coli/Shigella  spp. 
                 163 
                 193 
                 220 
                 202 
                 308 
                 280 
                 121 
                 271 
                 341 
                 317 
                 124 
               
               
                 
                   Aspergillus fumigatus 
                 
                 713 
                 865 
                 862 
                 830 
                 784 
                 657 
                 827 
                 803 
                 746 
                 812 
                 793 
               
               
                 
                   Aspergillus flavus 
                 
                 155 
                 261 
                 198 
                 156 
                 239 
                 171 
                 250 
                 218 
                 210 
                 258 
                 219 
               
               
                 
                   Salmonella enterica 
                 
                 136 
                 98 
                 85 
                 43 
                 109 
                 47 
                 23 
                 123 
                 70 
                 100 
                 135 
               
               
                 
                   Salmonella enterica 
                 
                 68 
                 53 
                 52 
                 41 
                 60 
                 92 
                 26 
                 28 
                 55 
                 81 
                 116 
               
               
                   
               
            
           
         
       
     
     The data embodied in Table 18 were obtained from 5 well-water samples (named 2H, 9D, 21, 23, 25) along with 2 samples of milliQ laboratory water (obtained via reverse osmosis) referred to as “Negative Control”. All samples were subjected to filtration on a sterile 0.4 um filter. Subsequent to filtration, the filters, with any microbial contamination that they may have captured, were then washed with the standard wash solution, exactly as described above for the washing of  Cannabis  and fruit. Subsequent to that washing, the suspended microbes in wash solution were then subjected to exactly the same combination of centrifugation (to yield a microbial pellet) then lysis and PCR of the unprocessed pellet-lysate (exactly as described above for  Cannabis ), followed by PCR and microarray analysis, also as described for  Cannabis.    
     The data seen in Table 18 demonstrate that microbes collected on filtrates of environmental water samples can be analyzed via the same combination of raw sample genotyping, then PCR and microarray analysis used for  Cannabis  and fruit washes. The italicized elements of Table 18 demonstrate that the 5 unprocessed well-water samples all contain aerobic bacteria and bile tolerant gram-negative bacteria. The presence of both classes of bacteria is expected for unprocessed (raw) well water. Thus, the data of Table 18 demonstrate that this embodiment of the present invention can be used for the analysis of environmentally derived water samples. 
     The above teaching shows that unprocessed leaf and bud samples in  Cannabis  and hops may be washed in an aqueous water solution to yield a water-wash containing microbial pathogens which can then be analyzed via the present combination of RSG and microarrays. The above data also show that environmentally-derived well water samples may be analyzed by an embodiment. Further, if a water sample containing microbes were obtained from industrial processing sources (such as the water effluent from the processing of fruit, vegetables, grain, meat) and then analyzed directly, or after ordinary water filtration to concentrate the microbial complement onto the surface of the filter, that the present combination of RSG and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes. 
     Further, if an air sample containing microbes as an aerosol or adsorbed to airborne dust were obtained by air filtration onto an ordinary air-filter (such as used in the filtration of air in an agricultural or food processing plant, or on factory floor, or in a public building or a private home) that such air-filters could then be washed with a water solution, as has been demonstrated for plant matter, to yield a microbe-containing filter eluate, such that the present combination of Raw Sample Genotyping (RSG) and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes. 
     While the foregoing written description of an embodiments enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The present disclosure should therefore not be limited by the above described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the present disclosure. 
     Example 4 
     Method of Using the 3-Dimensional Lattice Microarray System for Quantitative DNA Analysis. 
     Sample Processing 
     Harvesting Microbes from plant surface comprises the following steps: 
     1. Wash the plant sample or tape pull in 1× phosphate buffered saline (PBS); 
     2. Remove plant material/tape; 
     3. Centrifuge to pellet cells and iscard supernatant; 
     3. Resuspend in PathogenDx® Sample Prep Buffer pre-mixed with Sample Digestion Buffer; 
     3. Heat at 55° C. for 45 minutes; 
     3. Vortex to dissipate the pellet; 
     3. Heat at 95° C. for 15 minutes; and 
     3. Vortex and centrifuge briefly to obtain a sample comprising DNA from one or more types of microbes. 
     Addition of a Synthetic DNA to the Processed Sample 
     A known amount (known copy number) of synthetic DNA is added to the sample obtained in the sample processing step described above. The synthetic DNA has a length and sequence structure similar to that of the 16s (bacteria) or ITS2 (eukaryote) DNA sequence being amplified, but with a central region sequence that is distinct, to distinguish it from bacterial and eukaryotic DNA in the sample. The 5′ and 3′ end sequences of the synthetic DNA are designed to be substantially identical to a consensus sequence in the unknown bacterial or unknown eukaryotic DNA being queried, to allow amplification using the same pair of PCR primers used for amplification of the unknown DNA in the sample. Examples of such consensus sequences are shown in SEQ ID NO: 152 and SEQ ID NO: 153 (Table 19). These features allow unbiased amplification of both the synthetic DNA and the unknown microbial pool DNA in the sample. Examples of synthetic DNA sequences are shown in SEQ ID NO: 154 to SEQ ID NO: 157 (Table 19). 
     Amplification by PCR 
     The sample comprising the synthetic DNA sequence was amplified (PCR Reaction #1) using locus specific primer pairs (Tables 6 and 20). The product of PCR Reaction #1 (14) was then subjected to a second PCR reaction (PCR Reaction #2) using a pair of labeling primers (Tables 6 and 20), which additionally amplified and labeled the two targeted regions to generate fluorophore labeled amplicons. The product of the PCR Reaction #2 (504) was then diluted 1-1 with hybridization buffer (4×SSC+5×Denhardt&#39;s solution) and then applied directly to the microarray for hybridization. 
     Hybridization 
     Because the prior art method of microarray manufacture allows DNA to be analyzed via hybridization without the need for pre-treatment of the microarray surface, the use of the microarray is simple, and involves 6 manual or automated 
     1. Pipette the amplified DNA+binding buffer onto the microarray to which are immobilized oligonucleotide probe sequence for the pathogen gene being queried and the synthetic DNA used as internal reference standard (Tables 7-11 and 21). 
     2. Incubate for 30 minutes to allow DNA binding to the microarray (typically at room temperature, RT). 
     3. Remove the DNA+binding buffer by pipetting 
     4. Pipette 50 uL of wash buffer onto the microarray (0.4×SSC+0.5×Denhardt&#39;s) and incubate 5 min at RT. 
     5. Remove the wash buffer by pipetting. 
     6) Repeat steps 4 and 5 
     7) Perform image analysis at 532 nm and 635 nm to detect the probe spot location (532 nm) and PCR product hybridization (635 nm). 
     
       
         
           
               
             
               
                 TABLE 19 
               
               
                   
               
               
                 Concensus sequences and Synthetic DNA sequences 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 Consensus sequence corresponding to the ITS2 domain in 
               
               
                   
                 eukaryotes including yeast, mold fungi 
               
               
                 SEQ ID NO: 152 
                 (TYM Quant Control) 
               
               
                   
               
            
           
           
               
            
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTTYCAACAAYGGATCTCTTG 
               
               
                 GTTCTGGCATCGATGAAGAACGCAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNGTGAATCATCGARTCTTTGAACGCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCATATCAATAAGCGGAGGAAAANNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Consensus sequence corresponding to the rDNA domain in 
               
               
                 SEQ ID NO: 153 
                 prokaryotes 
               
               
                   
               
            
           
           
               
            
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCACAYTGGRACTGAGACACGG 
               
               
                 NNNNNNCTCCTACGGGAGGCAGCAGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAY 
               
               
                 SSAGCMAYGCCGCGTGDRBGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNATTGACGTTACCCGCNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNTGCCAGCAGCCGCGGTAATACNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Synthetic DNA region compatible for use as an internal 
               
               
                 SEQ ID NO: 154 
                 reference standard with the ITS2 domain in eukaryotes 
               
               
                   
               
            
           
           
               
            
               
                 TACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGAACTTTCAACAACGGATCTCTTGGTT 
               
               
                 CTGGCATCGATGAAGAACGCAGCGACTTGCGATAAGTAATGTGAATTGCAGAATTCAGTG 
               
               
                 CATCATAGAAACTATGTACGCAAATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTC 
               
               
                 GAGCGTCATTTCAACCCTCAAGCTTAGCTTGGTATTGAGTCTATGTCAGTAATGGCAGGCT 
               
               
                 CTAAAATCAGTGGCGGCGCCGCTGGGTCCTGAACGTAGTAATATTTCTTGTCACCGTTTC 
               
               
                 TAGGTGTGCTTCTGTCTATACCCAAATTCTTCTATGGTTGACCTCGGATCAGGTAGGGATA 
               
               
                 CCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGCACGCCGTCTAGAAGCACGATC 
               
               
                 AGAGGCTGAATACTA 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Synthetic DNA region compatible for use as an internal 
               
               
                 SEQ ID NO: 155 
                 reference standard with the rDNA domain in prokaryotes 
               
               
                   
               
            
           
           
               
            
               
                 TACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCC 
               
               
                 AGACTCCATCGGGAGCGAGCATGGGGGAATATTGCACAATGGGCGCAAGCCTGATGGAC 
               
               
                 CCTAGCCGCCACTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAGG 
               
               
                 GAATGAAAGTATATACCTTTCGTCATGTACGTTACTCGCAGAAGAAGCACCGGCTAACTCC 
               
               
                 GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCAC 
               
               
                 GCCGTCTAGAAGCACGATCAGAGGCTGAATACTA 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Synthetic DNA region compatible for use as an internal 
               
               
                   
                 reference standard with the rDNA domain in prokaryotes 
               
               
                 SEQ ID NO: 156 
                 including Bile-tolerant Gram-negative bacteria 
               
               
                   
               
            
           
           
               
            
               
                 TACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCC 
               
               
                 AGACTCCTGCAGGAGACGGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGTAT 
               
               
                 CCCTGACGCAGATATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAG 
               
               
                 GTCGTAAAACTAATACACTTGCTGTTTGAACTTACCCAGAGAAGAAGCACCGGCTAACTCC 
               
               
                 GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCAC 
               
               
                 GCCGTCTAGAAGCACGATCAGAGGCTGAATACTA 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Synthetic DNA region compatible for use as an internal 
               
               
                   
                 reference standard with the rDNA domain prokaryotes 
               
               
                 SEQ ID NO: 157 
                 including the  Enterobacteriaceae  Family 
               
               
                   
               
            
           
           
               
            
               
                 TACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCC 
               
               
                 AGACTCCTAGCGGAGCGAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGACG 
               
               
                 CCAGTCCGCTGGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAG 
               
               
                 GAGGTAAATGTAATACTCTTGCTACTTGAGCTTACCCCGAGAAGAAGCACCGGCTAACTC 
               
               
                 CGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCA 
               
               
                 CGCCGTCTAGAAGCACGATCAGAGGCTGAATACTA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Primers used for Locus PCR and Labeling PCR Amplification Reactions 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO. 
                 Primer target 
                 Primer sequence 
               
               
                   
               
               
                 SEQ ID NO: 133 
                 Universal primers for 
                 TTCAGCCTCTGTACGTGCTTCATG 
               
               
                   
                 preamplification of Fungal 
                   
               
               
                   
                 and Bacterial control 
                   
               
               
                   
                 fragments 
                   
               
               
                   
               
               
                 SEQ ID NO: 134 
                 Universal primers for 
                 TTCAGCCTCTGATCGTGCTTCTAG 
               
               
                   
                 preamplification of Fungal 
                   
               
               
                   
                 and Bacterial control 
                   
               
               
                   
                 fragments 
                   
               
               
                   
               
               
                 SEQ ID NO: 135 
                 Fungal RSG Primers (All 
                 TTTACTTTCAACAAYGGATCTCTTGG 
               
               
                   
                 Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 136 
                 Fungal RSG Primers (All 
                 CTTTTCCTCCGCTTATTGATATG 
               
               
                   
                 Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 137 
                 Bacterial RSG Primers (All 
                 TTTCACACTGGRACTGAGACACG 
               
               
                   
                 Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 138 
                 Bacterial RSG Primers (All 
                 TTTTGTATTACCGCGGCTGCTGGC 
               
               
                   
                 Bacteria) 
                   
               
               
                   
               
               
                 SEQ ID NO: 139 
                 Fungal Labeling 
                 TTTGCATCGATGAAGAACGCAGC 
               
               
                   
                 Primers(All Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 140 
                 Fungal Labeling 
                 TTTTCCTCCGCTTATTGATATGC 
               
               
                   
                 Primers(All Fungus) 
                   
               
               
                   
               
               
                 SEQ ID NO: 141 
                 Bacterial Labeling Primers 
                 TTTCACACTGGRACTGAGACACGG 
               
               
                   
               
               
                 SEQ ID NO: 30 
                 Bacterial Labeling Primers 
                 TTTTGTATTACCGCGGCTGCTGGCA 
               
               
                   
               
            
           
         
       
     
                     TABLE 21               Oligonucleotide probe sequences                                            SEQ ID NO: 132   Negative control   TTTTTTCTACTACCTATGCTGATTCACTCT               TTTT               SEQ ID NO: 37   Total Aerobic bacteria   TTTTTTTTTCCTACGGGAGGCAGTTTTTTT           (High)                   SEQ ID NO: 38   Total Aerobic bacteria   TTTTTTTTCCCTACGGGAGGCATTTTTTTT           (Medium)                   SEQ ID NO: 39   Total Aerobic bacteria   TTTATTTTCCCTACGGGAGGCTTTTATTTT           (Low)                   SEQ ID NO: 47   Bile-tolerant Gram-   TTTTTCTATGCAGTCATGCTGTGTGTRTGT           negative (High)   CTTTTT               SEQ ID NO: 48   Bile-tolerant Gram-   TTTTTCTATGCAGCCATGCTGTGTGTRTTT           negative (Medium)   TTTT               SEQ ID NO: 49   Bile-tolerant Gram-   TTTTTCTATGCAGTCATGCTGCGTGTRTTT           negative (Low)   TTTT               SEQ ID NO: 54   Coliform/   TTTTTTCTATTGACGTTACCCGCTTTTTTT             Enterobacteriaceae                     SEQ ID NO: 41     Enterobacteriaceae     TTTTTTCTATTGACGTTACCCGTTTTTTTT           (Medium sensitivity)                   SEQ ID NO: 40     Enterobacteriaceae     TTTATTCTATTGACGTTACCCATTTATTTT           (Low sensitivity)                   SEQ ID NO: 86   Total Yeast and Mold   TTTTTTTTGAATCATCGARTCTTTGAACGC           (High sensitivity)   ATTTTTTT               SEQ ID NO: 88   Total Yeast and Mold   TTTTTTTTGAATCATCGARTCTTTGAACGT           (Medium sensitivity)   TTTTTT               SEQ ID NO: 87   Total Yeast and Mold   TTTTTTTTGAATCATCGARTCTCCTTTTTTT           (Low sensitivity)                   SEQ ID NO: 142   Total Aerobic Bacteria   TTTTTTTTTCCATCGGGAGCGAGTTTTTTT           Quantitative Control               (internal reference               standard)                   SEQ ID NO: 143   Bile-tolerant Gram-   TTTTTCTATGTATTCCTGATGTAGATRTGT           negative Quantitative   CTTTTT           Control (internal               reference standard)                   SEQ ID NO: 144   Enterobacteriaceae/   TTTTTTCTCTTGAGCTTACCCCGTTTTTTT           Coliform Quantitative               Control (internal               reference standard)                   SEQ ID NO: 145   Total Yeast and Mold   TTTTTTTTGCATCATAGAAACTTTGTACGC           Quantitative Control   ATTTTTTT           (internal reference               standard)                   SEQ ID NO: 103     Candida  spp. Group 2   TTTTACTGTTTGGTAATGAGTGATACTCTC               ATTTT               SEQ ID NO: 146     Golovinomyces  spp.   TTTATTTAATCAATCCATCATCTCAAGTCT               TTTT               SEQ ID NO: 108     Fusarium  spp   TTTTTTTTAACACCTCGCRACTGGAGATTT               TTTT               SEQ ID NO: 107     Fusarium oxysporum     TTTTTTTCTCGTTACTGGTAATCGTCGTTT               TTTT               SEQ ID NO: 42     Escherichia coli /   TTTTCTAATACCTTTGCTCATTGACTCTTT             Shigella  1                   SEQ ID NO: 74     Salmonella     TTTTTTTGTTGTGGTTAATAACCGATTTTT             enterica / Enterobacter  1                   SEQ ID NO: 72     Pseudomonas  spp. 3   TTTATTTTAAGCACTTTAAGTTGGGATTTT               ATTT               SEQ ID NO: 78     Staphylococcus aureus     TTTATTTTCATATGTGTAAGTAACTGTTTTA           1   TTT               SEQ ID NO: 147     Listeria  spp.   TTTATTTTGATAAGAGTAACTGCTTGCTTT               ATTT               SEQ ID NO: 45     Bacillus  spp. Group1   TTTTTCAGTTGAATAAGCTGGCACTCTTTT               SEQ ID NO: 46     Bacillus  spp. Group2   TTTTTTCAAGTACCGTTCGAATAGTTTTTT               SEQ ID NO: 90     Aspergillus flavus  1   TTTTTTCGCAAATCAATCTTTTTCCAGTCT               TTTT               SEQ ID NO: 92     Aspergillus fumigatus  1   TTTCTTTTCGACACCCAACTTTATTTCCTT               ATTT               SEQ ID NO: 95     Aspergillus niger  1   TTTTTTCGACGTTTTCCAACCATTTCTTTT               SEQ ID NO: 100     Botrytis  spp   TTTTTTTCATCTCTCGTTACAGGTTCTCGG               TTCTTTTTTT               SEQ ID NO: 116     Penicillium oxalicum     TTTTTTACACCATCAATCTTAACCAGGCCT               TTTT               SEQ ID NO: 117     Penicillium paxilli     TTTTTTCCCCTCAATCTTTAACCAGGCCTT               TTTT               SEQ ID NO: 118     Penicillium  spp   TTTTTTCAACCCAAATTTTTATCCAGGCCT               TTTT               SEQ ID NO: 89     Alternaria  spp   TTTTTTCAAAGGTCTAGCATCCATTAAGTT               TTTT               SEQ ID NO: 101     Candida albicans     TTTTTTTTTGAAAGACGGTAGTGGTAAGTT               TTTT               SEQ ID NO: 105     Cladosporium  spp   TTTTTTTTGTGGAAACTATTCGCTAAAGTT               TTTT               SEQ ID NO: 99     Blumeria     TTTATTTGCCAAAAMTCCTTAATTGCTCTT               TTTT               SEQ ID NO: 148     Mucor  spp.   TTTTTTCTCCAVVTGAGYACGCCTGTTTCA               GTATCTTTTTT               SEQ ID NO: 120     Podosphaera  spp   TTTTTTGACCTGCCAAAACCCACATACCAT               TTTT               SEQ ID NO: 149     Saccharomyces  spp.   TTTATCTTAGGCGAACAATGTTCTTAAATC               TTTT               SEQ ID NO: 150     Aspergillus terreus     TTTTTTACGCATTTATTTGCAACTTGCCTT               TTTT               SEQ ID NO: 151     Podosphaera  spp.   TTTTTCGTCCCCTAAACATAGTGGCTTTTT                    
Image Analysis
 
     Image Analysis was performed at two wavelengths (532 nm and 635 nm) on a raster-based confocal scanner: GenePix 4000B Microarray Scanner, with the following imaging conditions: 33% Laser power, 400PMT setting at 532 nm/33% Laser Power, 700PMT setting at 635 nm.  FIG. 3  shows an example of the structure and hybridization performance of the microarray. 
       FIG. 3A  reveals imaging of the representative microarray, described above, after hybridization and washing, as visualized at 635 nm. The 635 nm image is derived from signals from the (red) CY5 fluor attached to the 5′ terminus of the bifunctional polymer linker OligodT which had been introduced during microarray fabrication as a positional marker in each microarray spot (see  FIG. 1  and Table 3). The data in  FIG. 3A  confirm that the CY5-labelled OligodT has been permanently linked to the microarray surface, via the combined activity of the bi-functional linker and subsequent UV-crosslinking, as described in  FIG. 1 . 
       FIG. 3B  reveals imaging of the representative microarray described above after hybridization and washing as visualized at 532 nm. The 532 nm image is derived from signals from the (green) CY3 fluor attached to the 5′ terminus of PCR amplified DNA obtained during PCR Reaction #2. It is clear from  FIG. 3B  that only a small subset of the 48 discrete probes bind to the CY3-labelled PCR product, thus confirming that the present method of linking nucleic acid probes to form a microarray ( FIG. 1 ) yields a microarray product capable of sequence specific binding to a (cognate) solution state target. The data in  FIG. 3B  reveal the underlying 3-fold repeat of the data (i.e., the array is the same set of 48 probes printed three times as 3 distinct sub-arrays to form the final 48×3=144 element microarray. The observation that the same set of 48 probes can be printed 3-times, as three repeated sub-domains show that the present invention generates microarray product that is reproducible. 
       FIG. 3C  reveals imaging of the representative microarray, described above, after hybridization and washing, as visualized with both the 532 nm and 635 nm images superimposed. The superimposed images display the utility of parallel attachment of a CY5-labelled OligodT positional marker relative to the sequence specific binding of the CY3-labelled PCR product. 
     Example 5 
     Quantitation of Absolute DNA Copy Number 
     Quantitation of absolute DNA copy number for a microbe of interest in a sample as disclosed in the present invention is achieved by introducing into the sample, a known copy number of a synthetic DNA before the first PCR amplification step. The synthetic DNA has a length and sequence structure similar to that of the 16s (bacteria) or ITS2 (eukaryote) DNA amplicons generated in the first PCR ( FIGS. 17A-17B and 18 ), but with a central region sequence that is distinct, to distinguish it from bacterial and eukaryotic DNA in the sample. The 5′ and 3′ end sequences of the synthetic DNA are designed to be substantially identical to a consensus sequence in the unknown bacterial or unknown eukaryotic DNA being queried, to allow amplification using the same pair of PCR primers used for amplification of the unknown DNA in the sample. These features allow the synthetic DNA to be amplified by the same pair of PCR primers used to amplify the hypervariable region of the unknown microbes which are being queried. As a result, the synthetic DNA is indistinguishable from the microbial pool DNA in the sample in terms of primer/template hybridization during the first and second PCR amplification reactions. This ensures unbiased amplification of the synthetic (internal reference standard) and microbial (unknown) DNA, such that signal intensity for amplicons from the queried DNA and synthetic DNA are proportional to the original copy number of each DNA respectively (discussed below). Examples of such consensus sequences are shown in SEQ ID NO: 152 and SEQ ID NO: 153 (Table 19). Examples of synthetic DNA sequences are shown in SEQ ID NO: 154 to SEQ ID NO: 157 (Table 19). 
     PCR, as deployed in the present microarray-based analysis is generally performed as a type of end-point PCR. Although in its simplest approximation, the polymerase chain reaction (PCR) has been described as a type of chain reaction because in the beginning of the PCR reaction process, when the DNA target strands are extremely dilute, each cycle of PCR will, in general lead to a geometric increase in product DNA, nearly exactly a 2-fold increase in amplified DNA product for each new PCR thermal cycle. If such a 2-fold increase were to occur indefinitely, the concentration of amplified DNA product should theoretically increase exponentially to infinity. In practice however, such a limitless DNA product increase does not occur, and one observes the well-known “S-shaped” sigmoidal PCR response curve wherein one or more components of the PCR reactant mixture or, one or more components of the PCR product mixture leads to a saturation in amplicon production. It is generally believed that such “leveled-off” S-shaped PCR curves occurs due to consumption of the PCR primers in the reaction mixture. Therefore, in the present invention, the concentration of PCR primers in both of the PCR reactions (first, locus PCR and second, labeling PCR, see  FIGS. 17A, 17B and 18 ) is intentionally kept at a large molar excess, so that primer depletion cannot contribute to saturation of PCR amplification. However, even with an excess of PCR reactants, it was observed that amplicon generated from both PCRs continue to “level-off” to produce a complex “end-point” PCR wherein the number of amplicons will no longer increase despite increasing concentration of the reactants. This saturation in response occurs due to enzymatic end-product inhibition. In the present invention, such inhibition is enabled to generate a substantial amount of PCR product, so as to ensure that microarray hybridization remains highly selective and sensitive to low microbial density in the sample. 
     The key to the present invention is therefore based upon deployment of the above-mentioned synthetic DNA internal reference standards so that even as the PCR reaction saturates by means of end-product inhibition (or any other mechanism of PCR leveling) the relative abundance of any DNA derived from an unknown amount of its microbial source, can be directly compared to that of the internal reference standard, so that the abundance of the unknown DNA, relative to that of the known synthetic DNA (internal reference standard) becomes insensitive to the extent of PCR reaction. This effect was exploited in this invention with the understanding that as the PCR reaction approaches saturation (associated with end-product inhibition), the amounts of various amplified species begin to interact as discussed below: 
     i) Unknown DNA Copies=Standard DNA Copies 
     When the number of copies of the synthetic DNA (standard species) is equal to the number of copies of an microbial DNA (unknown species) in the original sample, the ratio of microbial DNA to synthetic DNA will be equal to 1 from the beginning of the PCR reaction (where amplification is exponential) to the end of the PCR reaction (where amplification begins to saturate via end-product inhibition).
 
 C   n   /C   o   =S   n   /S   o  where,
 
C n =the number of microbial DNA copies of each type (n) present in the original sample mixture added to the first of two tandem PCR reactions used to prepare amplicons for microarray analysis,
 
C o =the number of known synthetic DNA copies (internal reference standard) added to the first of two PCR reactions used to prepare amplicons for microarray analysis,
 
S n =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the nth microbial species, followed by image analysis,
 
S o =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the synthetic DNA species, followed by image analysis,
 
     ii) Unknown DNA Copies&gt;Standard DNA Copies 
     When the microbial DNA (unknown species) were in large excess over the known synthetic DNA (standard species), the ratio of microbial DNA to synthetic DNA will be &gt;1. In this situation, at the beginning of the PCR reaction, the relative abundance of amplicons (PCR product) will be a truthful representation of the original input strand ratio (unknown:standard). As the reaction proceeds towards saturation however, the more abundant species (unknown species in this case) will approach end-product saturation first, either due to consumption of one or more reactants or due to enzyme inhibition by PCR reaction products—pyrophosphate and/or amplicons. As a result, amplification of the relatively less abundant standard species will be inhibited. In this situation, abundance of amplified DNA product species will retain the correct qualitative relationship of “unknown species&gt;standard species”, whereas the ratio of DNA products will no longer be linearly related to the unknown:standard ratio in the original input sample.
 
 C   n   /C   o   ≠S   n   /S   o  
 
     Such a non-linear relationship between input and response is well known in chemical and physical analysis such as the relationship between grain density versus photon exposure in chemical film or in a charge-coupled device (CCD) where, the relative brightness of photographic inputs are maintained in relative rank order in the resulting image, yet a detailed understanding of the non-linear response is required to predict relative abundance of the original inputs (analogous to DNA copy number in the original sample) from the response data (analogous to PCR product signal after amplification and microarray hybridization). 
     iii) Unknown DNA Copies&lt;Standard DNA Copies 
     When the microbial DNA (unknown species) were less than the known synthetic DNA (standard species), the ratio of microbial DNA to synthetic DNA will be &lt;1. In this situation, at the beginning of the PCR reaction, the relative abundance of amplicons (PCR product) will be a truthful representation of the original input strand ratio (unknown:standard). As the reaction proceeds towards saturation however, the more abundant species (standard species in this case) will approach end-product saturation first, either due to consumption of one or more reactants or due to enzyme inhibition by PCR reaction products—pyrophosphate and/or amplicons. As a result, amplification of the relatively less abundant unknown species will be inhibited. In this situation, abundance of amplified DNA product species will retain the correct qualitative relationship of “unknown species&gt;standard species”, whereas the ratio of DNA products, will no longer be linearly related to the unknown:standard ratio in the original input sample;
 
 C   n   /C   o   ≠S   n   /S   o  
 
     Such a non-linear relationship between input and response is well known in chemical and physical analysis such as the relationship between grain density versus photon exposure in chemical film or in a charge-coupled device (CCD) where, the relative brightness of photographic inputs are maintained in relative rank order in the resulting image, yet a detailed understanding of the non-linear response is required to predict relative abundance of the original inputs (analogous to DNA copy number in the original sample) from the response data (analogous to PCR product signal after amplification and microarray hybridization). 
     The non-linear relationships described above are similar to that observed in chemical and physical analysis, such as the relationship between grain density versus photon exposure in chemical films or charge-coupled devices (CCD) where, the relative brightness of photographic inputs are maintained in relative rank order in the resulting image, yet a detailed understanding of the non-linear response is required to predict relative abundance of the original inputs (analogous to DNA copy number in the original sample) from the response data (analogous to PCR product signal after amplification and microarray hybridization). In the present invention, it is determined that the observed competition between unknown microbial DNA and standard DNA display a useful and generally unexpected “X-shaped” relationship, of the kind displayed in  FIG. 19A . 
     Analysis of Microbial DNA (rDNA or ITS2) Copy Number by PCR-Microarray. 
     “Crossover” titration data of the kind shown in  FIGS. 19A and 19B  were determined to be intrinsic to the PCR-Microarray analysis described in this application. As target microbial DNA copy number is increased, with synthetic DNA copy number being held constant, the products of the microbial PCR reaction, and hence signals due to binding of those products to cognate probes on the microarray surface increase with increasing microbial DNA copy number, whereas the signals obtained from the (fixed) matched internal reference standard decrease in concert ( FIGS. 19A-19B ). The two curves cross-over at or near copy number equivalency (arrow,  FIG. 19A-19B ) where the number of microbial DNA copies become equal to the number of internal reference standard DNA copies. 
     Over the range of PCR conditions consistent with the present invention (i.e. conditions of PCR signals saturating due to competition between the unknown microbial DNA and an added DNA standard) the relation between the DNA input and output can be approximated as,
 
 C   n   /C   o   =P ( S   n   /S   o ) x  where,  Equation #1
 
     C n =the number of microbial DNA copies of each type (n) present in the original sample mixture added to the first of two tandem PCR reactions used to prepare amplicons for microarray analysis, 
     C o =the number of known synthetic DNA copies (internal reference standard) added to the first of two PCR reactions used to prepare amplicons for microarray analysis, 
     S n =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the nth microbial species, followed by image analysis, 
     S o =relative fluorescence units (RFU) signal data obtained after PCR amplification, and microarray hybridization of the synthetic DNA species, followed by image analysis, 
     X=a complex exponential factor which defines the functional relationship between the Experimental Microarray Data Ratio (S n /S o ) to the underlying ratio of microbial DNA copies vs synthetic DNA standard copies present in the original sample (C n /C o ). 
     P=A constant which relates the Experimental Microarray Data ratio (S n /S o ) to the concentration of amplified PCR product which binds to the microarray. In general, for the examples presented here ( FIGS. 19A and 19B ), P=1. 
     In general X can be a linear function or exponential or related functional form or a constant which is itself a function of PCR parameters and conditions of microarray analysis and imaging. Based on representative data shown in the present invention, X is approximated as a constant with a value near to 2. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Experimental Data S n /S o  as a function of Experimental Copy Number Data 
               
               
                 C n /C o  from the Representative PCR-Microarray Validation Studies in FIG. 19A 
               
            
           
           
               
               
            
               
                 C n /C o  Measured DNA 
                   
               
            
           
           
               
               
               
            
               
                 Copy Number Ratio in 
                   
                   
               
               
                 the Original Sample to be 
                 (S n /S o ) 2  Adjusted 
               
               
                 analyzed by PCR-Microarray 
                 Experimental Microarray 
                 S n /S o  Measured Experimental 
               
               
                 C o  = 3000 copies 
                 Data Ratio using X = 2 
                 Microarray Data Ratio 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.01 
                 0.01 
                 0.1 
                 5,000 RFU/45,000 RFU) 
               
               
                 0.1 
                 0.25 
                 0.5 
                 (20,000 RFU/40,000 RFU) 
               
               
                 1 
                 1 
                 1 
                 (32,000 RFU/32,000 RFU) 
               
               
                 10 
                 5.3 
                 2.3 
                 (40,000 RFU/15,000 RFU) 
               
               
                   
               
            
           
         
       
     
     In general, the function (X) relates original DNA Copy Number Ratio (C o /C o ) to the Experimental Microarray Data Ratio (S n /S o ) generated from the microarray hybridization RFU signal data. Function (X) can have different functional forms depending on details of the PCR reaction and microarray hybridization. However, it was determined that, if (1) All microbial DNA PCR reactions use the same pair of PCR primers as the internal reference standard; and (2) All hybridization probes applied to the microarray surface have the same affinity for their cognate DNA sequence produced by the PCR reactions; and (3) All PCR products are labeled with the same fluorescent dye for optical detection; then, X will approach a constant, which in the data presented in  FIG. 19A  assumes a value close to 2. Under these conditions Equation #1 may be simplified as
 
 C   n   =C   o ( S   n   /S   n ) 2   Equation #2
 
where
 
     C n =The number of microbial DNA copies present in the original sample. 
     C o =Is adjusted by adding a known number of synthetic DNA standard copies to the original sample. 
     X=2 as estimated from experimental data, as in  FIG. 19A   
     P=1 as determined by experiment, as in  FIG. 19A   
     For  FIG. 19A , the synthetic DNA reference standard copy number (C o ) was intentionally held at 3,000 but may be set at any other value including but not limited to 100, 500, 3,000 and 5,000 depending on the range of unknown microbial copies which might be encountered. 
     In a specific implementation of the present invention for microbial testing in food or  Cannabis  or other plant matter or for water testing, State or Federal Regulations might require a specific minimum allowable value for microbial contamination. Based on that regulated value and knowledge of the number of rDNA or ITS-2 DNA copies per microbial genome, the adjustable standard copy number value C o  added to the original sample before the PCR and Microarray hybridization steps can be seen as having value as a way to “dial” regulatory standards directly into the PCR-Microarray assay.