# Patent

## Title
Microarray based multiplex pathogen analysis and uses thereof

## Abstract

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

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part under 35 U.S.C. § 120 of U.S. Ser. No. 16/157,404, filed Oct. 11, 2018, now U.S. Pat. No. 11,421,224, which is a continuation under 35 U.S.C. § 120 of U.S. Ser. No. 15/916,062, filed Mar. 8, 2018, now U.S. Pat. No. 11,542,498, which is a continuation-In-part under 35 U.S.C. § 120 of non-provisional application U.S. Ser. No. 15/388,561, filed Dec. 22, 2016, now abandoned, 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.


SEQUENCE LISTING
A sequence listing is electronically submitted in text format in compliance with 37 C.F.R. § 1.821(c) and is incorporated by reference herein. The ASCII text file is named D7550CIP5SEQ, was created on Jun. 30, 2022 and is 29 KB in size.
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 very 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 ITS2 DNA in yeast or mold 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 very 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 >20 bases in length (>10 nm long) a Probe/linker length ratio>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.
Thus, the prior art is deficient in methods of DNA based pathogen analysis that interrogates a multiplicity of samples, uses fewer chemical and labile products, reduces processing steps and provides faster results while maintaining accuracy, specificity and reliability. 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 manufacturing a microarray system. In the method a plastic substrate comprising a plurality of surface moieties on a front surface thereof is contacted with a formulation comprising a solvent mixture, a plurality of oligodeoxythymidine linkers and a plurality of nucleic acid probes. The solvent mixture comprises a mixture of water and of a water-miscible liquid with a boiling point above 100° C. in a water to water-miscible liquid volume ratio from about 10:1 to about 100:1, where the water-miscible liquid has a boiling point above 100° C. In the plurality of oligodeoxythymidine linkers, where there are a greater number of activated surface moieties attached to the front surface of the plastic substrate as compared to the number of oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers, each consists of 20 to 60 thymidine bases, and each comprises an unmodified 3′ terminus and a fluorescent label covalently linked to its 5′ terminus. The plurality of surface moieties and the plurality of oligodeoxythymidine linkers in the formulation are present in a molar ratio of at least 10. The plurality of nucleic acid probes is selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, where each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143 and each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 comprised of a pathogenic bacteria nucleotide sequence or a pathogenic fungi nucleotide sequence are sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence and each pathogenic fungi nucleotide sequence. In sequential steps in the method the unmodified 3′ terminus of each of the plurality of oligodeoxythymidine linkers is crosslinked, photochemically, to one of the plurality of activated surface moieties, whereby the activated surface moieties in the plurality of surface moieties that are not crosslinked create a lattice width spacing between the crosslinked plurality of oligodeoxythymidine linkers. The water in the solvent mixture is evaporated to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the activated surface moieties. A thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes is crosslinked photochemically to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the plastic substrate and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes is crosslinked photochemically to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers attached to the plastic substrate where each of the plurality of nucleic acid probes attached to the oligodeoxythymidine linkers on the plastic substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the plastic substrate. The plastic substrate is washed at least once, thereby manufacturing the microarray system. The present invention is directed to a related method where in the formulation the plurality of nucleic acid probes further comprises a plurality of control probes in combination with the plurality of pathogenic bacteria nucleotide probes or the plurality of pathogenic fungi nucleotide probes or the combination thereof, where each of the plurality of control probes comprises a nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus thereof.
The present invention is directed to a method for manufacturing a 3-dimensional lattice microarray system related to the method described supra in that the plastic substrate is an unmodified polyester substrate comprising a plurality of aromatic ring moieties on a front surface thereof and in that the plurality of nucleic acid probes are selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, the plurality of nucleic acid probes in combination with a plurality of control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128 and 141, wherein each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143, each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 and each of the control nucleotide probes comprised of a pathogenic bacteria nucleotide sequence, a pathogenic fungi nucleotide sequence or a control nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence, each pathogenic fungi nucleotide sequence and each control nucleotide sequence.
The present invention also is directed to a method for manufacturing a microarray system related to the methods described supra in that the substrate is a solid substrate comprising a plurality of surface moieties on a front surface thereof, in that each oligodeoxythymidine linker in the plurality comprises an unmodified or modified 3′ terminus and in that the plurality of nucleic acid probes selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, where the plurality of nucleic acid probes are in combination with a plurality of Cannabis control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128, where each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143, each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 and each of the Cannabis control nucleotide probes comprised of a pathogenic bacteria nucleotide sequence, a pathogenic fungi nucleotide sequence or a Cannabis control nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence, each pathogenic fungi nucleotide sequence and each Cannabis control nucleotide sequence. In sequential steps in the method the unmodified or modified 3′ terminus of each of the plurality of oligodeoxythymidine linkers is linked to one of the plurality of activated surface moieties, whereby the activated surface moieties in the plurality of surface moieties that are not linked create a lattice width spacing between the linked plurality of oligodeoxythymidine linkers. The water in the solvent mixture is evaporated to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers linked to the surface moieties. A thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes is crosslinked photochemically to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the solid substrate and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes is crosslinked photochemically to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers attached to the solid substrate where each of the plurality of nucleic acid probes attached to the oligodeoxythymidine linkers on the solid substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the solid substrate. The solid substrate is washed at least once, thereby manufacturing the microarray system. The present invention is directed to a related method where in the formulation the plurality of nucleic acid probes further comprises negative control probes selected from the group consisting of SEQ ID NOS: 132 and 141 in combination with the plurality of the Cannabis control probes, where each of the negative control probes comprises a nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus thereof.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.



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. 10 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 (Rn) 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 Rn 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 Rn 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 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.
FIGS. 17A-17B show the inkjet printing of probe formulations onto polyethylene terephthlate (PET) substrate before (FIG. 17A) and after (FIG. 17B) UV crosslinking of the probes to bifunctional oligothymidine linkers.
FIGS. 18A-18C illustrate hybridization of CY3 labeled Pseudomonas aeruginosa and Staphylococcus aureus amplicons to a 3-dimensional microarray formed on a PET substrate. FIG. 18A is a CY5 image of the PET microarray showing the positions of the S. aureus, P. aeruginosa and negative control spots. FIG. 18B is a CY3 image of P. aeruginosa hybridization. FIG. 18C is a CY3 image of S. aureus hybridization.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein, “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements, or steps but not the exclusion of any other item, element or step or group of items, elements, or steps unless the context requires otherwise. Similarly, “another” or “other” may mean at least a second or more of the same or different claim element or components thereof.
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® 1060Rn 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 TiO2, 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 a Cy5, a DYLIGHTTM DY647, a ALEXA FLUOR® 647, a Cy3, a DYLIGHTTM DY547, or a 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 a Cy5, DYLIGHTTM DY647, or ALEXA FLUOR® 647, while the amplicons may be labeled with acceptors such as Cy3, DYLIGHTTM 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 TiO2, 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 “Rn”) 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, Cy-5 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 a 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 a Cy5, a DYLIGHTTM DY647, a ALEXA FLUOR® 647, a Cy3, a DYLIGHTTM DY547, or a 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 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 a Cy5, DYLIGHTTM DY647, or ALEXA FLUOR® 647, while the amplicons may be labeled with acceptors such as Cy3, DYLIGHTTM DY547, or ALEXA FLUOR® 550. Preferably, the emitter is Cy5 and the acceptor is Cy3.
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 an 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 “Rn”) 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, Cy-5 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 Cy5, DYLIGHTTM DY647, ALEXA FLUOR® 647, Cy3, DYLIGHTTM 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 1/10th 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 a related embodiment of this invention, there is provided a method for manufacturing a microarray system comprising the steps of contacting a plastic substrate comprising a plurality of surface moieties on a front surface thereof with a formulation comprising a solvent mixture comprising a mixture of water and of a water-miscible liquid with a boiling point above 100° C. in a water to water-miscible liquid volume ratio from about 10:1 to about 100:1; wherein the water-miscible liquid has a boiling point above 100° C.; a plurality of oligodeoxythymidine linkers, wherein there are a greater number of surface moieties on the front surface of the plastic substrate as compared to the number of oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers, each of the plurality of oligodeoxythymidine linkers consisting of 20 to 60 thymidine bases, wherein each oligodeoxythymidine linkers comprises an unmodified 3′ terminus and a fluorescent label covalently linked to its 5′ terminus, and wherein the plurality of surface moieties and the plurality of oligodeoxythymidine linkers in the formulation are present in a molar ratio of at least 10; and a plurality of nucleic acid probes selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, wherein each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143 and each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 comprised of a pathogenic bacteria nucleotide sequence or a pathogenic fungi nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence and each pathogenic fungi nucleotide sequence; performing, in sequence, the steps of crosslinking, photochemically, the unmodified 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of surface moieties, whereby the surface moieties in the plurality of surface moieties that are not crosslinked create a lattice width spacing between the crosslinked plurality of oligodeoxythymidine linkers; evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the surface moieties; crosslinking, photochemically, a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the plastic substrate; and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers crosslinked to the plastic substrate, wherein each of the plurality of nucleic acid probes crosslinked to the oligodeoxythymidine linkers on the plastic substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the plastic substrate; and washing the plastic substrate at least once, thereby manufacturing the microarray system.
Further to this embodiment the plurality of nucleic acid probes comprises a plurality of control probes in combination with the plurality of pathogenic bacteria nucleotide probes or the plurality of pathogenic fungi nucleotide probes or the combination thereof, each of the plurality of control probes comprised of a nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus thereof. In this further embodiment the plurality of control probes is selected from the group consisting of negative control nucleotide probes selected from the group consisting of SEQ ID NOS: 132 and 141, a plurality of Cannabis positive control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128, and a combination thereof.
In both embodiments the plastic substrate is a polyethylene terephthalate, a thermoplastic acrylic resin, a cycloolefin polymer, a polycarbonate, a nylon, or a combination thereof. Also in both embodiments the fluorescent label is a cyanine fluorescent dye or other chemically equivalent fluorescent dye. In addition the molar ratio of the plurality of oligodeoxythymidine linkers to the plurality of nucleic acid probes in the formulation is at least 0.1. Furthermore the water-miscible liquid is selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), and propanediol.
In another related embodiment of this invention, there is provided a method for manufacturing a 3-dimensional lattice microarray system comprising the steps of contacting an unmodified polyester substrate comprising a plurality of aromatic ring moieties on a front surface thereof: a solvent mixture comprising a mixture of water and of a water-miscible liquid with a boiling point above 100° C. in a water to water-miscible liquid volume ratio from about 10:1 to about 100:1; wherein the water-miscible liquid has a boiling point above 100° C.; a plurality of oligodeoxythymidine linkers, wherein there are a greater number of aromatic ring moieties on the front surface of the polyester substrate as compared to the number of oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers, each of the plurality of oligodeoxythymidine linkers consisting of 20 to 60 thymidine bases, wherein each oligodeoxythymidine linkers comprises an unmodified 3′ terminus and a fluorescent label covalently linked to its 5′ terminus, and wherein the plurality of aromatic ring moieties and the plurality of oligodeoxythymidine linkers in the formulation are present in a molar ratio of at least 10; and a plurality of nucleic acid probes selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, the plurality of nucleic acid probes in combination with a plurality of control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128 and 141, wherein each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143, each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 and each of the control nucleotide probes comprised of a pathogenic bacteria nucleotide sequence, a pathogenic fungi nucleotide sequence or a control nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence, each pathogenic fungi nucleotide sequence and each control nucleotide sequence; performing, in sequence, the steps of crosslinking, photochemically, the unmodified 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of aromatic ring moieties, whereby the aromatic ring moieties in the plurality of aromatic ring moieties that are not crosslinked create a lattice width spacing between the crosslinked plurality of oligodeoxythymidine linkers; evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the aromatic ring moieties; and crosslinking, photochemically, a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified polyester substrate; and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified polyester substrate, wherein each of the plurality of nucleic acid probes crosslinked to the oligodeoxythymidine linkers on the unmodified polyester substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the unmodified polyester substrate; and washing the glass support at least once, thereby manufacturing the 3-dimensional lattice microarray system.
In this embodiment the plurality of control probes is selected from the group consisting of a plurality of Cannabis positive control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128, negative control nucleotide probes selected from the group consisting of SEQ ID NOS: 132 and 141, and a combination thereof. Also, in this embodiment the fluorescent label, the molar ratio of the plurality of oligodeoxythymidine linkers to the plurality of nucleic acid probes and the water-miscible liquid are as described supra.
In another embodiment of this invention, there is provided a method for manufacturing a microarray system comprising the steps of contacting a solid substrate comprising a plurality of surface moieties on a front surface thereof with a formulation comprising a solvent mixture comprising a mixture of water and of a water-miscible liquid with a boiling point above 100° C. in a water to water-miscible liquid volume ratio from about 10:1 to about 100:1; wherein the water-miscible liquid has a boiling point above 100° C. a plurality of oligodeoxythymidine linkers, wherein there are a greater number of surface moieties attached to the front surface of the solid substrate as compared to the number of oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers, each of the plurality of oligodeoxythymidine linkers consisting of 20 to 60 thymidine bases, wherein each oligodeoxythymidine linker comprises an unmodified or modified 3′ terminus and a fluorescent label covalently linked to its 5′ terminus, and wherein the plurality of surface moieties and the plurality of oligodeoxythymidine linkers in the formulation are present in a molar ratio of at least 10; and a plurality of nucleic acid probes selected from the group consisting of a plurality of pathogenic bacteria nucleotide probes selected from the group consisting of SEQ ID NOS: 37-85 and 142-143, a plurality of pathogenic fungi nucleotide probes selected from the group consisting of SEQ ID NOS: 86-125, and a combination thereof, the plurality of nucleic acid probes in combination with a plurality of Cannabis control nucleotide probes selected from the group consisting of SEQ ID NOS: 126-128, wherein each of the pathogenic bacteria nucleic acid probes of SEQ ID NOS: 37-85 and 142-143, each of the pathogenic fungi nucleotide sequences of SEQ ID NOS: 86-125 and each of the Cannabis control nucleotide probes comprised of a pathogenic bacteria nucleotide sequence, a pathogenic fungi nucleotide sequence or a Cannabis control nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each pathogenic bacteria nucleotide sequence, each pathogenic fungi nucleotide sequence and each Cannabis control nucleotide sequence; performing, in sequence, the steps of linking the unmodified or modified 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of surface moieties, whereby the surface moieties in the plurality of surface moieties that are not linked create a lattice width spacing between the linked plurality of oligodeoxythymidine linkers; evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers covalently linked to the surface moieties; crosslinking, photochemically, a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers linked to the solid substrate; and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers linked to the solid substrate, wherein each of the plurality of nucleic acid probes attached to the oligodeoxythymidine linkers on the solid substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the solid substrate; and washing the solid substrate at least once, thereby manufacturing the microarray system.
Further to this embodiment the plurality of nucleic acid probes comprise negative control probes selected from the group consisting of SEQ ID NOS: 132 and 141 in combination with the plurality of the Cannabis control probes, where each of the negative control probes comprised of a nucleotide sequence sandwiched between two to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus thereof.
In one aspect of both embodiments the solid substrate is an unmodified plastic substrate comprising a plurality of surface moieties on the front surface thereof and each of the plurality of oligodeoxythymidine linkers comprises an unmodified 3′ terminus, said step (2) comprising performing, in sequence, the steps of crosslinking, photochemically, the unmodified 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of surface moieties, whereby the surface moieties in the plurality of surface moieties that are not crosslinked create a lattice width spacing between the crosslinked plurality of oligodeoxythymidine linkers; evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the surface moieties; crosslinking, photochemically, a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified plastic substrate; and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified plastic substrate, wherein each of the plurality of nucleic acid probes crosslinked to the oligodeoxythymidine linkers on the unmodified plastic substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the unmodified plastic substrate; and washing the glass support at least once, thereby manufacturing the 3-dimensional lattice microarray system. In this aspect the unmodified plastic substrate is a polyethylene terephthalate, a thermoplastic acrylic resin, a cycloolefin polymer, a polycarbonate, or a nylon, or a combination thereof.
In another aspect of both embodiments the solid substrate is borosilicate glass comprising a front surface and a plurality of activated surface moieties selected from the group consisting of an epoxysilane group, an N-hydroxysuccinimide group, and an activated carboxylic acid ester attached to the front surface and each of the plurality of oligodeoxythymidine linkers is modified with an amino group at its 3′ terminus, said step (2) comprising performing, in sequence, the steps of attaching, by covalent coupling, the amino group at the 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of activated surface moieties, whereby the activated surface moieties in the plurality of activated surface moieties that are not covalently coupled create a lattice width spacing between the covalently coupled plurality of oligodeoxythymidine linkers; evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers covalently coupled to the activated surface moieties; crosslinking, photochemically, a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers attached to the glass support; and/or a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers attached to the glass support,
wherein each of the plurality of nucleic acid probes attached to the oligodeoxythymidine linkers on the glass support are separated by both a vertical space and a lattice width, such that crosslinking the plurality of nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the glass support; and washing the glass support at least once, thereby manufacturing the 3-dimensional lattice microarray.
Also, in both embodiments and all aspects thereof the fluorescent label, the molar ratio of the plurality of oligodeoxythymidine linkers to the plurality of nucleic acid probes and the water-miscible liquid are as described supra.
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 “Rn”), 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, Cy-5 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, DYLIGHTTM DY647, ALEXA FLUOR® 647, Cy3, DYLIGHTTM 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 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 urn 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 fora 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 the present disclosure there is provided an improved 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 (20-60 aa)


EDC Activated
Primary Amine
(4) Amine-modified OligodT (20-60 bases)


Carboxylic Acid

(5) Chitosan (20-60 subunits)




(6) Lysine containing polypeptide (20-60 aa)


N-hydroxysuccinimide
Primary Amine
(7) Amine-modified OligodT (20-60 bases)


(NHS)

(8) Chitosan (20-60 subunits)




(9) Lysine containing polypeptide (20-60 aa)









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. 10). Generally, use of pure water as the solvent during matrix fabrication is disadvantageous because water is very 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 1/10th 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. 10) 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 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.







TABLE 2







Non-Covalent Attachment of Bi-Functional Polymeric Linker to an Inert Surface









Representative
Matched Adsorptive Group on
Specific Bifunctional 


support surface
Bifunctional Linker (Rn)
polymeric linker





glass
Single Stranded Nucleic 
OligodT (30-60 bases)



Acid >10 bases



glass
Amine-Polysaccharide
Chitosan (30-60 subunits)


glass
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’-



e.g. Digoxigenin
Digoxigenin


polycarbonate
Single Stranded Nucleic 
Oligo-dT (30-60 bases)



Acid >10 bases



polycarbonate
Amine-Polysaccharide
Chitosan (30-60 subunits)


polycarbonate
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’-



e.g. Digoxigenin
Digoxigenin


graphene
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’pyrene



e.g. pyrene



graphene
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’-CY-5 dye



e.g. CY-5 dye



graphene
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’-



e.g. Digoxigenin
Digoxigenin


gold
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’pyrene



e.g. pyrene



gold
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’ CY-5 dye



e.g. CY-5 dye



gold
Extended Planar Hydrophobic Groups
OligodT (30-60 bases)-5’



e.g. Digoxigenin
Digoxigenin









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 very 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 1/10th 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 very 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 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










Unique sequence 
5’ labelled OligodT



Oligonucleotide
Fluorescent 


Nucleic acid 
30-38 bases Long 
marker 30 bases


probe Type
7 T’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, 100:1



Solvent




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 (Low)
TTTTCTAGATACTTGTGTAAGTGAATTTTTTT


 


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
TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT



(High sensitivity)



 


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.
TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTTTT


 


SEQ ID NO: 108
Fusarium spp.
TTTTTTTTAACACCTCGCTTACTGGAGATTTTTTT


 


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-
TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTTT



negative (High)



 


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 & 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
        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 proteinaseK, 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


 


Cannabis ITS1 1° RP*- TTTCGATAAACACGCATCTCGATTG


 


Enterobacteriaceae 16S 1° FP- TTACCTTCGGGCCTCTTGCCATCRGATGTG


 


Enterobacteriaceae 16S 1° RP- TTGGAATTCTACCCCCCTCTACRAGACTCAAGC


PCR primers (P2) for PCR Reaction #2


Cannabis ITS1 2° FP- TTTCGTGAACACGTTTTAAACAGCTTG


 


Cannabis ITS1 2° RP- (Cy3)TTTTCCACCGCACGAGCCACGCGAT


 


Enterobacteriaceae 16S 2° FP- TTATATTGCACAATGGGCGCAAGCCTGATG


 


Enterobacteriaceae 16S 2° RP- (Cy3)TTTTGTATTACCGCGGCTGCTGGCA













Primary PCR
Secondary PCR


PCR Reagent
Concentration
Concentration


PCR Buffer
1X
1X





MgCl2
 2.5 mM
 2.5 mM


 


BSA
0.16 mg/mL
0.16 mg/mL


 


dNTP'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., 30 s
61° C., 30 s
72° C., 60 s
72° C., 7 min










25X











Program for PCR Reaction #2














95° C., 4 min
98° C., 20 s
61° C., 20 s
72° C., 30 s
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'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's) and incubate 5 min at RT.
        5) Remove the wash buffer by pipetting
        6) Repeat steps 4&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
Primers and Probe Sequences
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 14 of any unpurified DNA sample, such as a raw Cannabis leaf wash for example. About 14 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 Locus
TTTCACAYTGGRACTGAGACACG



(Bacteria)



 


SEQ ID NO: 2
16S rDNA HV3 Locus
TTTGACTACCAGGGTATCTAATCCTGT



(Bacteria)



 


SEQ ID NO: 3
Stx1 Locus (Pathogenic E.
TTTATAATCTACGGCTTATTGTTGAACG



coli)



 


SEQ ID NO: 4
Stx1 Locus (Pathogenic E.
TTTGGTATAGCTACTGTCACCAGACAATG



coli)



 


SEQ ID NO: 5
Stx2 Locus (Pathogenic E.
TTTGATGCATCCAGAGCAGTTCTGCG



coli)



 


SEQ ID NO: 6
Stx2 Locus (Pathogenic E.
TTTGTGAGGTCCACGTCTCCCGGCGTC



coli)



 


SEQ ID NO: 7
InvA Locus (Salmonella)
TTTATTATCGCCACGTTCGGGCAATTCG


 


SEQ ID NO: 8
InvA Locus (Salmonella)
TTTCTTCATCGCACCGTCAAAGGAACCG


 


SEQ ID NO: 9
tuf Locus (All E. coli)
TTTCAGAGTGGGAAGCGAAAATCCTG


 


SEQ ID NO: 10
tuf Locus (All E. coli)
TTTACGCCAGTACAGGTAGACTTCTG


 


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 Yeast,
TTTACTTTYAACAAYGGATCTCTTGG



Mold/Fungus)



 


SEQ ID NO: 14
ITS2 Locus (All Yeast,
TTTCTTTTCCTCCGCTTATTGATATG



Mold/Fungus)



 


SEQ ID NO: 15
ITS2 Locus (Aspergillus
TTTAAAGGCAGCGGCGGCACCGCGTCCG



species)



 


SEQ ID NO: 16
ITS2 Locus (Aspergillus
TTTTCTTTTCCTCCGCTTATTGATATG



species)



 


SEQ ID NO: 17
ITS1 Locus (Cannabis/Plant)
TTTGCAACAGCAGAACGACCCGTGA


 


SEQ ID NO: 18
ITS1 Locus (Cannabis/Plant)
TTTCGATAAACACGCATCTCGATTG


 







Second PCR Primers (P2) for the second labeling amplification step









SEQ ID NO: 19
16S rDNA HV3 Locus (All
TTTACTGAGACACGGYCCARACTC



Bacteria)



 


SEQ ID NO: 20
16S rDNA HV3 Locus (All
TTTGTATTACCGCGGCTGCTGGCA



Bacteria)



 


SEQ ID NO: 21
Stx1 Locus (Pathogenic E.
TTTATGTGACAGGATTTGTTAACAGGAC



coli)



 


SEQ ID NO: 22
Stx1 Locus (Pathogenic E.
TTTCTGTCACCAGACAATGTAACCGCTG



coli)



 


SEQ ID NO: 23
Stx2 Locus (Pathogenic E.
TTTTGTCACTGTCACAGCAGAAG



coli)



 


SEQ ID NO: 24
Stx2 Locus (Pathogenic E.
TTTGCGTCATCGTATACACAGGAGC



coli)



 


SEQ ID NO: 25
InvA Locus (All Salmonella)
TTTTATCGTTATTACCAAAGGTTCAG


 


SEQ ID NO: 26
InvA Locus (All Salmonella)
TTTCCTTTCCAGTACGCTTCGCCGTTCG


 


SEQ ID NO: 27
tut Locus (All E. coli)
TTTGTTGTTACCGGTCGTGTAGAAC


 


SEQ ID NO: 28
tut Locus (All E. coli)
TTTCTTCTGAGTCTCTTTGATACCAACG


 


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 Yeast,
TTTGCATCGATGAAGARCGYAGC



Mold/Fungus)



 


SEQ ID NO: 32
ITS2 Locus (All Yeast,
TTTCCTCCGCTTATTGATATGC



Mold/Fungus)



 


SEQ ID NO: 33
ITS2 Locus (Aspergillus
TTTCCTCGAGCGTATGGGGCTTTGTC



species)



 


SEQ ID NO: 34
ITS2 Locus (Aspergillus
TITTTCCTCCGCTTATIGATATGC



species)



 


SEQ ID NO: 35
ITS1 Locus (Cannabis/Plant)
TTTCGTGAACACGTTTTAAACAGCTTG


 


SEQ ID NO: 36
ITS1 Locus (Cannabis/Plant)
TTTCCACCGCACGAGCCACGCGAT









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 tufgene 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'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 & 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 & mold. In all cases, the probe sequences are terminated with a string of T'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 (Low)
TTTATTTTCCCTACGGGAGGCTTTTATTTT


 


SEQ ID NO: 40
Enterobacteriaceae (Low
TTTATTCTATTGACGTTACCCATTTATTTT



sensitivity)



 


SEQ ID NO: 41
Enterobacteriaceae
TTTTTTCTATTGACGTTACCCGTTTTTTTT



(Medium sensitivity)



 


SEQ ID NO: 42
Escherichia coli/Shigella 1
TTTTCTAATACCTTTGCTCATTGACTCTTT


 


SEQ ID NO: 43
Escherichia coli/Shigella 2
TTTTTTAAGGGAGTAAAGTTAATATTTTTT


 


SEQ ID NO: 44
Escherichia coli/Shigella 3
TTTTCTCCTTTGCTCATTGACGTTATTTTT


 


SEQ ID NO: 45
Bacillus spp. Group1
TTTTTCAGTTGAATAAGCTGGCACTCTTTT


 


SEQ ID NO: 46
Bacillus spp. Group2
TTTTTTCAAGTACCGTTCGAATAGTTTTTT


 


SEQ ID NO: 47
Bile-tolerant Gram-negative
TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTTT



(High)



 


SEQ ID NO: 48
Bile-tolerant Gram-negative
TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT



(Medium)



 


SEQ ID NO: 49
Bile-tolerant Gram-negative
TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTT



(Low)



 


SEQ ID NO: 50
Campylobacter spp.
TTTTTTATGACACTTTTCGGAGCTCTTTTT


 


SEQ ID NO: 51
Chromobacterium spp.
TTTTATTTTCCCGCTGGTTAATACCCTTTATTTT


 


SEQ ID NO: 52
Citrobacter spp. Group1
TTTTTTCCTTAGCCATTGACGTTATTTTTT


 


SEQ ID NO: 53
Clostridium spp.
TTTTCTGGAMGATAATGACGGTACAGTTTT


 


SEQ ID NO: 54
Coliform/Enterobacteriaceae
TTTTTTCTATTGACGTTACCCGCTTTTTTT


 


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 aeruginosa
TTTTTGCAGTAAGTTAATACCTTGTCTTTT


 


SEQ ID NO: 69
Pseudomonas cannabina
TTTTTTTACGTATCTGTTTTGACTCTTTTT


 


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 1
TTTATTTTCATATGTGTAAGTAACTGTTTTATTT


 


SEQ ID NO: 79
Staphylococcus aureus 2
TTTTTTCATATGTGTAAGTAACTGTTTTTT


 


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
TTTTCTATGTATCGATG




Calibration
TTGAGAAATTTTTTT




(High)



 



SEQ ID NO: 130
Imager
TTTTCTAGATACTTGTG




Calibration
TAAGTGAATTTTTTT




(Low)



 



SEQ ID NO: 131
Imager
TTTTCTAAGTCATGTTG




Calibration
TTGAAGAATTTTTTT




(Medium)



 



SEQ ID NO: 132
Negative
TTTTTTCTACTACCTAT




control
GCTGATTCACTCTTTTT
















TABLE 9





Oligonucleotide probe sequence for the ITS2 Locus

















SEQ ID NO: 86
Total Yeast and
TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT



Mold (High




sensitivity)



 


SEQ ID NO: 87
Total Yeast and
TTTTTTTTGAATCATCGARTCTCCTTTTTTT



Mold (Low




sensitivity)



 


SEQ ID NO: 88
Total Yeast and
TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT



Mold (Medium




sensitivity)



 


SEQ ID NO: 89
Alternaria spp.
TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT


 


SEQ ID NO: 90
Aspergillus flavus 1
TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT


 


SEQ ID NO: 91
Aspergillus flavus 2
TTTTTTTCTTGCCGAACGCAAATCAATCTTTTTTTTTTTT


 


SEQ ID NO: 92
Aspergillus
TTTCTTTTCGACACCCAACTTTATTTCCTTATTT



fumigatus 1



 


SEQ ID NO: 93
Aspergillus
TTTTTTTGCCAGCCGACACCCATTCTTTTT



fumigatus 2



 


SEQ ID NO: 94
Aspergillus nidulans
TTTTTTGGCGTCTCCAACCTTACCCTTTTT


 


SEQ ID NO: 95
Aspergillus niger 1
TTTTTTCGACGTTTTCCAACCATTTCTTTT


 


SEQ ID NO: 96
Aspergillus niger 2
TTTTTTTTCGACGTTTTCCAACCATTTCTTTTTT


 


SEQ ID NO: 97
Aspergillus niger 3
TTTTTTTCGCCGACGTTTTCCAATTTTTTT


 


SEQ ID NO: 98
Aspergillus terreus
TTTTTCGACGCATTTATTTGCAACCCTTTT


 


SEQ ID NO: 99
Blumeria
TTTATTTGCCAAAAMTCCTTAATTGCTCTTTTTT


 


SEQ ID NO: 100
Botrytis spp
TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTTTT


 


SEQ ID NO: 101
Candida albicans
TTTTTTTTTGAAAGACGGTAGTGGTAAGTTTTTT


 


SEQ ID NO: 102
Candida spp.
TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT



Group 1



 


SEQ ID NO: 103
Candida spp.
TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT



Group 2



 


SEQ ID NO: 104
Chaetomium spp.
TTTCTTTTGGTTCCGGCCGTTAAACCATTTTTTT


 


SEQ ID NO: 105
Cladosporium spp
TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT


 


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 spp.
TTTCTTTTGAGCGACGACGGGCCCAATTTTCTTT


 


SEQ ID NO: 113
Mucor spp.
TTTTCTCCAWTGAGYACGCCTGTTTCTTTT


 


SEQ ID NO: 114
Myrothecium spp.
TTTATTTTCGGTGGCCATGCCGTTAAATTTTATT


 


SEQ ID NO: 115
Oidiodendron spp.
TTTTTTTGCGTAGTACATCTCTCGCTCATTTTTT


 


SEQ ID NO: 116
Penicillium
TTTTTTACACCATCAATCTTAACCAGGCCTTTTT



oxalicum



 


SEQ ID NO: 117
Penicillium paxilli
TTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT


 


SEQ ID NO: 118
Penicillium spp
TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT


 


SEQ ID NO: 119
Phoma/Epicoccum
TTTTTTTGCAGTACATCTCGCGCTTTGATTTTTT



spp.



 


SEQ ID NO: 120
Podosphaera spp
TTTTTTGACCTGCCAAAACCCACATACCATTTTT


 


SEQ ID NO: 121
Podosphaera spp.
TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT


 


SEQ ID NO: 122
Pythium oligandrum
TTTTATTTAAAGGAGACAACACCAATTTTTATTT


 


SEQ ID NO: 123
Rhodoturula spp
TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT


 


SEQ ID NO: 124
Stachybotrys spp
TTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT


 


SEQ ID NO: 125
Trichoderma spp
TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT









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
TTTTTTAATCTGCGCCA




ITS1 DNA
AGGAACAATATTTTTTT




Control 1



 



SEQ ID NO: 127
Cannabis
TTTTTGCAATCTGCGCC




ITS1 DNA
AAGGAACAATATTTTTT




Control 2



 



SEQ ID NO: 128
Cannabis
TTTATTTCTTGCGCCAA




ITS1 DNA
GGAACAATATTTTATTT




Control 3
















TABLE 11





Representative Microarray Probe


Design for the Present Invention:


Bacterial Toxins, ITS1 (Cannabis)



















SEQ ID NO: 81
stx1 gene
TTTTTTCTTTCCAGGTA





CAACAGCTTTTTT


 



SEQ ID NO: 82
stx2 gene
TTTTTTGCACTGTCTGA





AACTGCCTTTTTT


 



SEQ ID NO: 59
etuf gene
TTTTTTCCATCAAAGTT





GGTGAAGAATCTTTTTT


 



SEQ ID NO: 61
invA gene
TTTTTTTATTGATGCCG





ATTTGAAGGCCTTTTTT


 



SEQ ID NO: 126
Cannabis
TTTTTTAATCTGCGCCA




ITS1 DNA
AGGAACAATATTTTTTT




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 & mold. In all cases, the DNA of the target bacteria or fungi, yeast & 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 & 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 & 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. 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 & 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.
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'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 1/50th 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 <1CFU 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 very 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 12A and 12B 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 13-15 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 12A







Representative microarray data obtained from powdered dry food samples.









Sample Type













Whey Protein
Whey Protein
Chewable
Vanilla 




Shake Vanilla
Shake Chocolate
Berry Tablet
Shake
Pea Protein


















0
18
0 
18
0
18
0
18
0
18


Enrichment time →
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
12459 
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/
233
205
255
219
207
255
215
214
242
198


Shigella spp.












S.enterica/
203
183
186
281
212
299
197
257
308
303


enterobacter spp.












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 12B







Representative microarray data obtained from powdered dry food samples.









Sample Type












Rice
Work-out
Work-out
Vanilla 



Protein
Shake FP
Shake BR
Shake
















0
18
0
18
0
18
0
18


Enrichment time →
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/
228
229
216
267
221
253
220
207


Shigella spp.










S.enterica/
226
281
238
268
197
254
255
216


enterobacter spp.










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 13-15 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 13







Representative microarray hybridization data obtained from blueberry


and lemon washes.









Sample










Blueberry
Lemon









Collection Type



Produce Wash










Wash 1 blueberry in 2 ml
Wash 1 piece moldy



20 mM Borate, vortex 30
lemon in 2 ml 20 mM


Protocol
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 Y & M 1
20052
21412
8734
7335


Total Y & M 2
17626
8454
5509
5030
















TABLE 14







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, 
500 ul 20 mM Borate + Triton Buffer,



vortex 30 seconds
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
NONE
1:20
NONE
1:20
NONE
1:20
NONE 
1:20
NONE 
1:20
NONE
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.
80
81
83
64
96
60
79
80
176
60
385
53


1














Penicillium spp. 
90
93
81
80
114
59
98
69
470
65
478
56


2














Fusarium spp.
77
71
69
62
112
55
61
274
617
81
59
757


1














Fusarium spp.
91
82
107
74
101
65
91
66
123
63
71
583


2














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 & M 1
23637
18532
15213
17668 
18068
19762
18784 
15550 
20625
17525
25813 
18269


Total Y & M 2
12410
8249
9281
11526 
8543
13007
14180 
14394
9905
8972
15112 
12678









The data embodied in FIG. 16 and Tables 13-15 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 15







Representative microarray hybridization data obtained from lemon washes


and tape pulls.









Sample



Moldy Lemon



Collection 



Tape Pull



Type













1A1
1A2
1A3
1B1
1B2


ID
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 95° C.,




vortex, Heat 5 min

Vortex 15 seconds




95° C., Vortex 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 Y & M 1
8448
12511
9249
12844
8593


Total Y & M 2
9275
8716
11585
10758
4444









Table 16 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.
The data embodied in Table 16 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.







TABLE 16







Representative microarray data from raw water filtrate









Sample ID





























Negative



2H
2H
9D
9D
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 & Mold
284
324
290
307
272
361
296
288
271
321
469


Total Yeast & Mold
251
259
294
290
309
308
285
281
275
299
293


Total Yeast & Mold
282
280
294
280
299
284
275
286
299
259
232


Total Aerobic bacteria 
40101 
42007
47844
47680 
45102
44041
43520
41901
46459
46783
135


High













Total Aerobic bacteria 
14487 
12314
24189
26158
19712
16210
17943 
15474
25524
18507
157


Medium













Total Aerobic bacteria
4885
5629
7625
6456
5807
4505
5316
6022
6264
6974
159


Low













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 & Kluv
146
165
196
128
160
215
185
217
215
177
225


spp.













Podosphaera spp.
111
119
100
122
192
105
95
43
169
27
143


Bile-tolerant Gram-
16026
9203
13309 
8426
16287
14116
10557 
17558
15343
14285
183


negative High













Bile-tolerant Gram-
12302 
11976
9259 
10408
13055
10957
11242
8416
9322 
11785
196


negative Medium













Bile-tolerant Gram-
5210
7921
3818
3984
7224
6480
4817
6933
5021
5844
240


negative Low













Total
193
248
389
357
215
214
198
220
276
208
210


Enterobacteriaceae













High













Total
246
214
297
246
244
224
219
245
252
229
207


Enterobacteriaceae Med













Total
165
140
158
119
151
180
150
167
182
174
132


Enterobacteriaceae Low













Total Coliform
121
148
158
117
129
117
155
157
125
178
152


Escherichia coli 
31821
115
132
155
127
62
86
121
59
90
234


specific gene













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
181
172
245
172
178
212
157
243
174
156
146


gene













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/
103
124
221
124
90
144
130
121
137
143
158


Shigella spp.













Salmonella
124
98
131
119
136
88
121
77
128
140
124


enterica/enterobacter













spp.













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/
163
193
220
202
308
280
121
271
341
317
124


Shigella spp.













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 seen in Table 16 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 16 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 16 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.
Example 4
Fabrication of 3-Dimensional Lattice Microarray Systems on a Plastic Substrate
Probe and bifunctional polymer linker formulations used herein in the manufacture of 3-dimensional microarray are printed on glass substrates are printed onto optical grade polyethylene terephthalate using standard ink jet printing to produce a microarray with the 3-dimensional microarray lattice structure. FIGS. 17A-17B are microarray images before and after UV crosslinking of probes printed onto the optical grade PET substrate (DUPONT TEIJIN FILMS). The images were obtained by imaging the CY5 fluorescent dye label at a wavelength of 647 nm at 18 ms sec exposure time. The CY5 label was introduced as a 5′ modification onto the bifunctional oligothymidine linkers, which is present at 0.1% by mass relative to the unlabeled sequence specific probes comprising each microarray probe spot.
As seen in the raw microarray image (FIGS. 17A-17B), probe spot morphology on the PET substrate is uniform with a spot-to-spot spacing of 250 μm, an average diameter of 121 μm±2.4 μm which is nearly identical (123 μm±4.0 um) to that for printing of the same formulations onto silanized glass subsrate (Schott Nexterion). Thus, the manufacture of microarray probe content onto a plastic film produces a microarray product that is similar in probe morphology to that printed on a glass substrate.
Example 5
Hybridization Performance Analysis of Probe Formulations on PET Polyester Source of gDNA
Genomic DNA from Staphylococcus aureus subsp. aureus strain Seattle 1945 (ATCC 25923D-5) and genomic DNA from Pseudomonas aeruginosa strain Boston 41501 (ATCC 27853D-5) were used with 80 copies per reaction for each.
PCR Primers and Probes Sequences
PCR primers and hybridization probes for P. aeruginosa and S. aureus to produce CY3 5′-labeled amplicons are shown in Table 17.







TABLE 17







Primer and probe sequences









Sequence 5′-3′











Loci PCR primers










P. aeruginosa
FP
TTTGTTGGGAGGAAGGGCAGTAAGT




(SEQ ID NO: 133)



RP
TTTCTCTACCGTACTCTAGCTCAGT




(SEQ ID NO: 134)


 


S. aureus
FP
TTTCAAGTCGAGCGAACGGACGAGA




(SEQ ID NO: 135)



RP
TTTCCTTACCAACTAGCTAATGCAG




(SEQ ID NO: 136)








Labeling PCR



primers










P. aeruginosa
FP
TTTTTGCTGTTTTGACGTTACCAAC




(SEQ ID NO: 137)



RP
/5Cy3/TTTCTACCGTACTCTAGCT




CAGTAG (SEQ ID NO: 138)


 


S. aureus
FP
TTTACGGGTGAGTAACACGTGGATA




(SEQ ID NO: 139)



RP
/5Cy3/TTTTCCATCTATAAGTGAC




AGCAAG (SEQ ID NO: 140)








Hybridization



Probes



Negative 
TTTTTTCTACTACCTATGCTGATTCA


control
CTCTTTTT (SEQ ID NO: 141)


 


P. aeruginosa
TTTTTTTGTGGTTCAGCAAGTTGTTC



TTCT (SEQ ID NO: 142)


 


S. aureus
TTTTTTTAACCTACCTATAAGACCTT



TTTT (SEQ ID NO: 143)









PCR Amplification Programs

Tables 18 and 19 list the PCR conditions for loci enhancement and CY3 labeling to produce the CY3 5′-labeled P. aeruginosa and S. aureus amplicons.







TABLE 18







PCR for loci enhancement for P. aeruginosa


and S. aureus


Loci Enhancement PCR












Steps
Temp.
Time
Cycles

















1
95° C.
4 
minutes
1



2
95° C.
30 
seconds
30



3
55° C.
30 
seconds




4
72° C.
1 
minute




5
72° C.
7 
minutes
1












6
15° C.
∞
1

















TABLE 19







PCR for CY5 labeling


Loci Enhancement PCR












Steps
Temp.
Time
Cycles

















1
95° C.
4 
minutes
1



2
95° C.
20 
seconds
30



3
55° C.
20 
seconds




4
72° C.
30 
seconds




5
72° C.
7 
minutes
1












6
15° C.
∞
1










Hybridization and Washing Protocol

After amplification of the P. aeruginosa and S. aureus loci the Cy3 5′-labeled amplicons are hybridized to the hybridization probes in Table 17 and the microarray is subsequently washed and imaged in the following protocol.

    
    
        1. Dispense 200 μL of water to each well, aspirate, dispense a second time, and allow to sit for 5 minutes.
        2. Prepare pre-hybridization and hybridization buffers.
        3. aspirate water and dispense 200 μL of pre-hybridization to each well, allow to sit for 5 10 minutes.
        4. Combine the CY3 labeled amplicons with 18 μL of hybridization buffer to make the hybridization cocktail.
        5. Aspirate the pre-hybridization buffer and dispense 68 μL of hybridization cocktail to each wee, allow to sit for 30 minutes.
        6. Prepare wash buffer.
        7. Aspirate hybridization cocktail and dispense 200 μL of wash buffer to each well.
        8. Aspirate wash buffer and dispense wash buffer a second time, allow to sit for 10 minutes.
        9. Aspirate wash buffer, dispense, and aspirate for the final wash.
        10. Immediately dry the plate in a plate centrifuge by spinning for 1-2 minutes.
        11. Scan the plates for imaging.

Results

    
    


FIGS. 18A-18C show the hybridization results. FIG. 18B shows that amplified P. aeruginosa DNA hybridizes specifically only to the 12 replicate probes specific to PCR amplified P. aeruginosa DNA, as assessed by imaging (Sensovation Sensospot Imager) of the CY3 dye linked to each amplified DNA molecule. Similarly, FIG. 18C shows that amplified S. aureus DNA hybridizes specifically only to the 12 replicate probes specific to PCR amplified S. aureus DNA, as assessed by imaging (Sensovtion Sensospot Imager) of the CY3 dye linked to each amplified DNA molecule. Additionally, it is seen that amplicon binding is not detected in regions of the PET substrate which do not include Probe DNA, thus demonstrating that the present PET optical plastic substrate displays negligible non-specific binding to the amplified DNA product of the standard PCR reaction protocol. Thus, the method of probe attachment onto an unmodified PET polyester microarray substrate generates hybridization performance, i.e., sensitivity and specificity, that is comparable to that obtained on a glass microarray substrate. The ability to directly UV crosslink oligodeoxythymidines that are unmodified at the 3′ terminus to the aromatic ring moieties on the monomers comprising the PET polyester by passes the need for an organic coating, such as, but not limited to, epoxysilane, that is required for a glass substrate.

## Claims

1. A method for manufacturing a microarray system comprising the steps of:
(1) contacting an unmodified polyester terephthalate substrate comprising a plurality of carbonyl moieties on a front surface thereof with a formulation comprising:
(i) a solvent mixture comprising a mixture of water and of a water-miscible liquid with a boiling point above 100° C. in a water to water-miscible liquid volume ratio from about 10:1 to about 100:1; wherein the water-miscible liquid has a boiling point above 100° C.;
(ii) a plurality of oligodeoxythymidine linkers, wherein the plurality of carbonyl moieties on the front surface of the unmodified polyester terephthalate substrate is greater in number as compared to the number of oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers, each of said plurality of oligodeoxythymidine linkers consisting of 20 to 60 thymidine bases, wherein each oligodeoxythymidine linkers comprises an unmodified 3′ terminus and a fluorescent label covalently linked to its 5′ terminus, and wherein the plurality of surface moieties and the plurality of oligodeoxythymidine linkers in the formulation are present in a molar ratio of at least 10; and
(iii) a plurality of pathogenic bacteria nucleic acid probes comprising nucleotide sequences of both SEQ ID NO: 142 and SEQ ID NO: 143 and comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 42-85 and a combination thereof, wherein each of the plurality of pathogenic bacteria nucleic acid probes of SEQ ID NOS: 142-143 and SEQ ID NOS: 42-85 are comprised of a nucleotide sequence sandwiched between one to seven consecutive thymidine nucleotides attached to both the 3′ terminus and to the 5′ terminus of each nucleotide sequence in the plurality of pathogenic bacteria nucleic acid probes;

(2) performing, in sequence, the steps of:
(a) crosslinking, photochemically, the unmodified 3′ terminus of each of the plurality of oligodeoxythymidine linkers to one of the plurality of carbonyl moieties, whereby the carbonyl moieties in the plurality of carbonyl moieties that are not crosslinked create a lattice width spacing between the crosslinked plurality of oligodeoxythymidine linkers;
(b) evaporating the water in the solvent mixture to progressively concentrate the plurality of pathogenic bacteria nucleic acid probes in the solvent mixture with the plurality of oligodeoxythymidine linkers crosslinked to the carbonyl moieties;
(c) crosslinking, photochemically,
(i) a thymidine nucleotide at the 3′ terminus or the 5′ terminus of each of the plurality of pathogenic bacteria nucleic acid probes to one or more of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified polyester terephthalate substrate; and/or
(ii) a thymidine nucleotide at the 3′ terminus and the 5′ terminus of each of the plurality of pathogenic bacteria nucleic acid probes to two adjacent oligodeoxythymidine linkers of the plurality of oligodeoxythymidine linkers crosslinked to the unmodified polyester terephthalate substrate, wherein each of the plurality of pathogenic bacteria nucleic acid probes crosslinked to the oligodeoxythymidine linkers on the unmodified polyester terephthalate substrate are separated by both a vertical space and a lattice width, such that crosslinking the plurality of pathogenic bacteria nucleic acid probes to the plurality of oligodeoxythymidine linkers forms a 3-dimensional lattice on the unmodified polyester terephthalate substrate; and

(d) washing the unmodified polyester terephthalate substrate at least once, thereby manufacturing the microarray system.

2. The method of claim 1, wherein the fluorescent label is a cyanine fluorescent dye.

3. The method of claim 1, wherein the molar ratio of the plurality of oligodeoxythymidine linkers to the plurality of nucleic acid probes in the formulation is at least 0.1.

4. The method of claim 1, wherein the water-miscible liquid is selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), and propanediol.