Microarray-Based Multiplex Fungal Pathogen Analysis
20210317540 · 2021-10-14
Assignee
Inventors
- Melissa May (Tucson, AZ, US)
- Frederick H. Eggers (Sahuarita, AZ, US)
- Kevin M. O'Brien (Sahuarita, AZ)
- Peaches R. Ulrich (Tucson, AZ, US)
- Benjamin A. Katchman (Tucson, AZ, US)
- Shayla Freeman (Tucson, AZ, US)
- Michael E. Hogan (Stony Brook, NY, US)
Cpc classification
G01N21/6428
PHYSICS
G01N33/5308
PHYSICS
C12Q2537/143
CHEMISTRY; METALLURGY
C12Q2537/143
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
International classification
C12Q1/6806
CHEMISTRY; METALLURGY
Abstract
Provided herein is a method of quantitating a fungus in a plant, plant product or agricultural product. Total nucleic acids are isolated from a sample of the plant or plant product, and an asymmetric PCR amplification reaction is performed using fluorescent labeled primer pairs to obtain fluorescent labeled fungal amplicons. These amplicons are hybridized to fungus specific nucleic acid probes that are attached on a microarray support. The microarray is imaged to detect fluorescent signals from the fluorescent labeled fungal amplicons. The fluorescent signal intensity is correlated to the quantity of fungus.
Claims
1. A method for quantitating a fungus on a plant, comprising: a) obtaining a sample from the plant; b) isolating from the sample, total nucleic acids; c) performing on the total nucleic acids an asymmetric PCR amplification reaction using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus to generate at least one fluorescent labeled fungal amplicon; d) hybridizing the fluorescent labeled fungal amplicons to a plurality of nucleic acid probes each having a sequence corresponding to a sequence determinant in the fungus, each of said nucleic acid probes attached at a specific position on a solid microarray support; e) washing the microarray at least once; f) imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled fungal amplicons; and g) calculating an intensity of the fluorescent signal, said intensity correlating with a quantity of the fungus in the sample, thereby quantitating the fungus on the plant.
2. The method of claim 1, further comprising isolating a total DNA after step b, said step c comprising performing the asymmetric PCR amplification reaction on the total DNA.
3. The method of claim 1, wherein the fluorescently labeled primer is in an excess of about 4-fold to about 8-fold over the unlabeled primer in the fluorescent labeled primer pair.
4. The method of claim 1, wherein the fungus is a yeast or a mold.
5. The method of claim 4, wherein the fungus is an Aspergillus species.
6. The method of claim 1, wherein the unlabeled primer is a forward primer comprising the nucleotide sequences of SEQ ID: 13, SEQ ID: 15, SEQ ID: 31, SEQ ID: 33, SEQ ID: 133, or SEQ ID: 135.
7. The method of claim 1, wherein the fluorescently labeled primer is a reverse primer comprising the nucleotide sequences of SEQ ID: 14, SEQ ID: 16, SEQ ID: 32, SEQ ID: 34, or SEQ ID: 134.
8. The method of claim 1, wherein the nucleic acid probes have at least one probe nucleotide sequence selected from the group consisting of SEQ ID NOS: 86-126 and 136-140.
9. The method of claim 1, wherein the plant is a cannabis or a hemp, or a product derived therefrom.
10. The method of claim 9, wherein the product is an oil.
11. A method for quantitating at least one fungus in an agricultural product, comprising: a) obtaining a sample of the agricultural product; b) isolating total nucleic acids from the sample; c) performing on the total nucleic acids an asymmetric PCR amplification reaction using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus to generate at least one fluorescent labeled fungal amplicon; d) hybridizing the fluorescent labeled fungal amplicons to a plurality of nucleic acid probes each having a sequence corresponding to a sequence determinant in the fungus, each of said nucleic acid probes attached at a specific position on a solid microarray support; e) washing the microarray at least once; f) imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled fungal amplicons; and g) calculating an intensity of the fluorescent signal, said intensity correlating with a quantity of the fungus in the sample, thereby quantitating the at least one fungus in the agricultural product.
12. The method of claim 11, further comprising isolating a total DNA after step b, said step c comprising performing the asymmetric PCR amplification reaction on the total DNA.
13. The method of claim 11, wherein the fluorescently labeled primer is in an excess of about 4-fold to about 8-fold over the unlabeled primer in the fluorescent labeled primer pair.
14. The method of claim 11, wherein the fungus is a yeast or a mold.
15. The method of claim 14, wherein the fungus is an Aspergillus species.
16. The method of claim 11, wherein the unlabeled primer is a forward primer comprising the nucleotide sequences of SEQ ID: 13, SEQ ID: 15, SEQ ID: 31, SEQ ID: 33, SEQ ID: 133, or SEQ ID: 135.
17. The method of claim 11, wherein the fluorescently labeled primer is a reverse primer comprising the nucleotide sequences of SEQ ID: 14, SEQ ID: 16, SEQ ID: 32, SEQ ID: 34, or SEQ ID: 134.
18. The method of claim 11, wherein the nucleic acid probes have at least one probe nucleotide sequence selected from the group consisting of SEQ ID NOS: 86-126 and 136-140.
19. The method of claim 11, wherein the agricultural product is obtained from a cannabis, or a hemp.
20. The method of claim 11, wherein the agricultural product is an oil.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] 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:
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DETAILED DESCRIPTION OF THE INVENTION
[0036] 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.
[0037] 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.”
[0038] 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.
[0039] In one embodiment of this invention, there is provided a method for quantitating a fungus on a plant, comprising obtaining a sample from the plant; isolating total nucleic acids from the sample; performing on the total nucleic acids an asymmetric PCR amplification reaction using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus to generate at least one fluorescent labeled fungal amplicon; hybridizing the fluorescent labeled fungal amplicons to a plurality of nucleic acid probes each having a sequence corresponding to a sequence determinant in the fungus, each of said nucleic acid probes attached at a specific position on a solid microarray support; washing the microarray at least once; imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled fungal amplicons; and calculating an intensity of the fluorescent signal, said intensity correlating with a quantity of the fungus in the sample, thereby quantitating the fungus on the plant.
[0040] In this embodiment, the plant is a cannabis or a hemp or a product produced thereof. For example, the product is an oil such as cannabidiol produced from cannabis and hemp.
[0041] In this embodiment, the fungus is any fungus capable of infecting the plants including, but not limited to a yeast, a mold, an Aspergillus species and a Penicillium species.
[0042] In this embodiment, an asymmetric PCR amplification is performed on the total nucleic acids using at least one fluorescent labeled primer pair. Each of the fluorescent labeled primer pairs comprise an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus. In this embodiment, the fluorescently labeled primer in about 4-fold to about 8-fold excess of the unlabeled primer whereby, upon completion of the reaction, the fluorescently labeled amplicon will be primarily single stranded (that is, the reaction is a type of “asymmetric PCR”). In this embodiment, the fluorescent labeled primer pairs have forward (odd numbers) and reverse (even number) sequences shown in SEQ ID: 13-16, 31-34, 133-135 (Table 6). Commercially enzymes and buffers are used in this step. Also, any fluorescent label may be used, including, but not limited to a CY3, a CY5, SYBR Green, a DYLIGHT DY647, a ALEXA FLUOR 647, a DYLIGHT DY547 and a ALEXA FLUOR 550.
[0043] Further in this embodiment, the fluorescent labeled fungal amplicons generated are hybridized to a plurality of nucleic acid probes. The nucleic acid probes have a sequence corresponding to sequence determinants in the fungus and have sequences SEQ ID NOS: 86-126 (Table 4) and 136-140 (Table 9). The nucleic acid probes are attached to a solid microarray support. The solid support is any microarray including but not limited to a 3-dimensional lattice microarray.
[0044] Further in this embodiment, after hybridization, unhybridized amplicons are removed by washing the microarray. Washed microarrays are imaged to detect a fluorescent signal corresponding to the fluorescent labeled fungal amplicons. Further in this embodiment, an intensity for the fluorescent signal is calculated. The calculated intensity is correlated with the number of fungus specific genomes in the sample, thereby quantitating the fungus in the sample. Based on analysis of fungus-free samples, an experimentally determined intensity threshold is established for the hybridization to each probe on the microarray, such that a fluorescent intensity above that threshold signifies the presence of fungus, while fluorescence intensities below the threshold signifies that fungus was not detected. Also, the fluorescence intensity correlates with a quantity of the fungus in the sample.
[0045] Further to this embodiment, the method comprises isolating total DNA after the isolating step and further performing the asymmetric PCR amplification on the total DNA as described above.
[0046] In another embodiment of this invention, there is provided a method for quantitating at least one fungus in an agricultural product, comprising obtaining a sample of the agricultural product; isolating total nucleic acids from the sample; performing on the total nucleic acids an asymmetric PCR amplification reaction using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the at least one fungus to generate at least one fluorescent labeled fungal amplicon; hybridizing the fluorescent labeled fungal amplicons to a plurality of nucleic acid probes each having a sequence corresponding to a sequence determinant in the fungus, each of said nucleic acid probes attached at a specific position on a solid microarray support; washing the microarray at least once; imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled fungal amplicons, and calculating an intensity of the fluorescent signal, the intensity correlating with a quantity of the fungus in the sample, thereby quantitating the at least one fungus in the agricultural product.
[0047] In this embodiment, the plant is a cannabis or a hemp or a product produced thereof. For example, the product is an oil such as cannabidiol produced from cannabis and hemp.
[0048] In this embodiment, the fungus is any fungus capable of infecting the plants including, but not limited to a yeast, a mold, an Aspergillus species and a Penicillium species.
[0049] In this embodiment, an asymmetric PCR amplification is performed on the total nucleic acids using at least one fluorescent labeled primer pair. Each of the fluorescent labeled primer pairs comprise an unlabeled primer, and a fluorescently labeled primer, selective for a target nucleotide sequence in the fungus. In this embodiment, the fluorescently labeled primer in about 4-fold to about 8-fold excess of the unlabeled primer whereby, upon completion of the reaction, the fluorescently labeled amplicon will be primarily single stranded (that is, the reaction is a type of “asymmetric PCR”). In this embodiment, the fluorescent labeled primer pairs have forward (odd numbers) and reverse (even number) sequences shown in SEQ ID: 13-16, 31-34, 133-135 (Table 6). Commercially enzymes and buffers are used in this step. Also, any fluorescent label may be used, including, but not limited to a CY3, a CY5, SYBR Green, a DYLIGHT DY647, a ALEXA FLUOR 647, a DYLIGHT DY547 and a ALEXA FLUOR 550.
[0050] Further in this embodiment, the fluorescent labeled fungal amplicons generated are hybridized to a plurality of nucleic acid probes. The nucleic acid probes have a sequence corresponding to sequence determinants in the fungus and have sequences SEQ ID NOS: 86-126 (Table 4) and 136-140 (Table 9). The nucleic acid probes are attached to a solid microarray support. The solid support is any microarray including but not limited to a 3-dimensional lattice microarray.
[0051] Further in this embodiment, after hybridization, unhybridized amplicons are removed by washing the microarray. Washed microarrays are imaged to detect a fluorescent signal corresponding to the fluorescent labeled fungal amplicons. Further in this embodiment, an intensity for the fluorescent signal is calculated. The calculated intensity is correlated with the number of fungus specific genomes in the sample, thereby quantitating the at least one fungus in the agricultural product. Based on analysis of fungus-free samples, an experimentally determined intensity threshold is established for the hybridization to each probe on the microarray, such that a fluorescent intensity above that threshold signifies the presence of fungus, while fluorescence intensities below the threshold signifies that fungus was not detected. Also, the fluorescence intensity correlates with a quantity of the fungus in the sample.
[0052] Further to this embodiment, the method comprises isolating total DNA after the isolating step and further performing the asymmetric PCR amplification on the total DNA as described above.
[0053] Described herein is a method for detecting a fungus in a plant sample such as for example a cannabis, or a plant product such as for example a cannabidiol. Total nucleic acids or total DNA is isolated, and an asymmetric PCR amplification reaction performed to generate fluorescent labeled fungal amplicons. The fluorescent labeled fungal amplicons are hybridized to nucleic acid probes attached to a microarray. This method allows positive hybridization signals to be validated on each sample tested based on internal “mismatched” and “sequence specific” controls. The method steps may be performed concurrently, performed in a single assay, which is beneficial since it enables streamlined detection of fungus in a single assay. The method may be employed to detect any fungus in the plant or plant product.
[0054] In the embodiments described above, the microarray is made of any suitable material known in the art including but not limited to borosilicate glass, a thermoplastic acrylic resin (e.g., poly(methyl methacrylate-VSUVT) a cycloolefin polymer (e.g. ZEONOR 1060R), a metal including, but not limited to gold and platinum, a plastic including, but not limited to polyethylene terephthalate, polycarbonate, nylon, a ceramic including, but not limited to TiO.sub.2, and Indium tin oxide (ITO) and engineered carbon surfaces including, but not limited to graphene. A combination of these materials may also be used. The solid support has a front surface and a back surface and is activated on the front surface by chemically activatable groups for attachment of the nucleic acid probes. In this embodiment, the chemically activatable groups include but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide. These materials 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.
[0055] The nucleic acid probes are attached either directly to the microarray support, or indirectly attached to the support using bifunctional polymer linkers. 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. In one aspect 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.
[0056] In this embodiment, the bifunctional polymer linker may be unmodified with a fluorescent label. Alternatively, the bifunctional polymer linker has a fluorescent label attached covalently to the top domain, the bottom end, or internally. The second fluorescent label is different from the fluorescent label in the fluorescent labeled primers. Having a 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 fungal 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 fungus 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 fungi. Examples of fluorescent labels include, but are not limited to CY5, DYLIGHT DY647, ALEXA FLUOR 647, CY3, DYLIGHT DY547, or ALEXA FLUOR 550. The fluorescent labels may be attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. In one aspect, 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.
[0057] Further in this embodiment, when the bifunctional polymer linker is also fluorescently labeled a second fluorescent signal image is detected in the imaging step. Superimposing the first fluorescent signal image and second fluorescent signal image allows identification of the fungus by comparing the sequence of the nucleic acid probe at one or more superimposed signal positions on the microarray with a database of signature sequence determinants for a plurality of fungal DNA. This embodiment is particularly beneficial since it allows identification of more than one type of fungus in a single assay.
[0058] QuantX TYM enables quantitating fungus in plants or plant products. The microarray has the capacity to test for multiple fungus and/or multiple plants and/or plant products in parallel. The testing may be performed in triplicate along with a panel of controls as needed, enabling rapid and reliable quantitation of fungus from multiple plant samples.
[0059] 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
[0060] 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.
[0061] 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
[0062] (a) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCR or isothermal amplicon, plasmid or genomic DNA;
[0063] (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.
[0064] (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
[0065] (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).
[0066] 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-US-00001 TABLE 1 Covalent Attachment of Bifunctional Polymeric Linker to an Activated Surfaces Matched Reactive Group on Specific Implementation Activated Surface Bifunctional as Bifunctional polymeric Moiety Linker linker Epoxysilane Primary Amine (1) Amine-modified OligodT (20-60 bases) (2) Chitosan (20-60 subunits) (3) Lysine containing polypeptide (20-60aa) EDC Activated Primary Amine (4) Amine-modified OligodT Carboxylic Acid (20-60 bases) (5) Chitosan (20-60 subunits) (6) Lysine containing polypeptide (20-60aa) N-hydroxy- Primary Amine (7) Amine-modified OligodT succinimide (20-60 bases) (NHS) (8) Chitosan (20-60 subunits) (9) Lysine containing polypeptide (20-60aa)
[0067] 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.
[0068] 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 (
[0069] 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 (
[0070] In the third step, the terminal Thymidine bases in the nucleic acid probes are UV crosslinked to the bifunctional linker within the evaporated surface (
Microarray System with Unmodified Solid Support for Non-Covalent Attachment
[0071] 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
[0072] (1) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCR or isothermal amplicon, plasmid or genomic DNA;
[0073] (2) an unmodified solid support 1;
[0074] (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
[0075] (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);
[0076] 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-US-00002 TABLE 2 Non-Covalent Attachment of Bi-Functional Polymeric Linker to an Inert Surface Representative Matched Adsorptive support Group on Bifunctional Specific Bifunctional surface Linker (R.sub.n) polymeric linker glass Single Stranded Nucleic OligodT (30-60 bases) Acid > 10 bases glass Amine-Polysaccharide Chitosan (30-60 subunits) glass Extended Planar OligodT (30-60 bases)-5′- Hydrophobic Groups, Digoxigenin e.g. Digoxigenin polycarbonate Single Stranded Nucleic Oligo-dT (30-60 bases) Acid > 10 bases polycarbonate Amine-Polysaccharide Chitosan (30-60 subunits) polycarbonate Extended Planar OligodT (30-60 bases)-5′- Hydrophobic Groups, Digoxigenin e.g. Digoxigenin graphene Extended Planar OligodT (30-60 bases)-5′ Hydrophobic Groups, pyrene e.g. pyrene graphene Extended Planar OligodT (30-60 bases)-5′- Hydrophobic Groups, CY-5 dye e.g. CY-5 dye graphene Extended Planar OligodT (30-60 bases)-5′- Hydrophobic Groups, Digoxigenin e.g. Digoxigenin gold Extended Planar OligodT (30-60 bases)-5′ Hydrophobic Groups, pyrene e.g. pyrene gold Extended Planar OligodT (30-60 bases)-5′- Hydrophobic Groups, CY-5 dye e.g. CY-5 dye gold Extended Planar OligodT (30-60 bases)-5′ Hydrophobic Groups, Digoxigenin e.g. Digoxigenin
[0077] 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.
[0078] 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 (
[0079] 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 (
[0080] In the third step, the terminal Thymidine bases in the nucleic acid probes are UV crosslinked to the bifunctional linker within the evaporated surface (
[0081] 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.
[0082] 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.
[0083] DNA nucleic acid probes were formulated as described in Table 3, to be deployed as described above and illustrated in
TABLE-US-00003 TABLE 3 Representative Conditions of use of the Present Invention Unique sequence Oligonucleotide 5′ labelled OligodT Nucleic acid 30-38 bases Long Fluorescent marker 30 probe Type 7 T's at each end bases Long(marker) Nucleic acid 50 mM 0.15 mM probe Concentration Bifunctional Linker OligodT 30 bases long Primary amine at 3′ terminus Bifunctional Linker 1 mM Concentration High Boiling Water:Propanediol, point Solvent 100:1 Surface Epoxysilane on borosilicate glass UV Crosslinking 300 millijoule Dose (mjoule)
TABLE-US-00004 TABLE 4 Nucleic acid probes Linked to the Microarray Surface via the Present Invention SEQ ID NO: 132 Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTT T SEQ ID NO: 129 Imager Calibration TTTTCTATGTATCGATGTTGAGAAATTTTTTT (High) SEQ ID NO: 130 Imager Calibration TTTTCTAGATACTTGTGTAAGTGAATTTTTTT (Low) SEQ ID NO: 131 Imager Calibration TTTTCTAAGTCATGTTGTTGAAGAATTTTTTT (Medium) SEQ ID NO: 126 Cannabis ITS1 DNA TTTTTTAATCTGCGCCAAGGAACAATATTTTT Control 1 TT SEQ ID NO: 127 Cannabis ITS1 DNA TTTTTGCAATCTGCGCCAAGGAACAATATTTT Control 2 TT SEQ ID NO: 128 Cannabis ITS1 DNA TTTATTTCTTGCGCCAAGGAACAATATTTTAT Control 3 TT SEQ ID NO: 86 Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACGCAT Mold (High TTTTTT sensitivity) SEQ ID NO: 87 Total Yeast and TTTTTTTTGAATCATCGARTCTCCTTTTTTT Mold (Low sensitivity) SEQ ID NO: 88 Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACGTTTT Mold (Medium TTT sensitivity) SEQ ID NO: 132 Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTT T SEQ ID NO: 92 Aspergillus TTTCTTTTCGACACCCAACTTTATTTCCTTATT fumigatus 1 T SEQ ID NO: 90 Aspergillus flavus 1 TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTT T SEQ ID NO: 95 Aspergillus niger 1 TTTTTTCGACGTTTTCCAACCATTTCTTTT SEQ ID NO: 100 Botrytis spp. TTTTTTTCATCTCTCGTTACAGGTTCTCGGTT CTTTTTTT SEQ ID NO: 108 Fusarium spp. TTTTTTTTAACACCTCGCRACTGGAGATTTTT TT SEQ ID NO: 89 Alternaria spp TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTT T SEQ ID NO: 123 Rhodoturula spp. TTTTTTCTCGTTCGTAATGCATTAGCACTTTTT T SEQ ID NO: 117 Penicillium paxilli TTTTTTCCCCTCAATCTTTAACCAGGCCTTTTT T SEQ ID NO: 116 Penicillium oxalicum TTTTTTACACCATCAATCTTAACCAGGCCTTT TT SEQ ID NO: 118 Penicillium spp. TTTTTTCAACCCAAATTTTTATCCAGGCCTTTT T SEQ ID NO: 102 Candida spp. TTTTTTTGTTTGGTGTTGAGCRATACGTATTTT Group 1 T SEQ ID NO: 103 Candida spp. TTTTACTGTTTGGTAATGAGTGATACTCTCAT Group 2 TTT SEQ ID NO: 124 Stachybotrys spp TTTCTTCTGCATCGGAGCTCAGCGCGTTTTAT TT SEQ ID NO: 125 Trichoderma spp. TTTTTCCTCCTGCGCAGTAGTTTGCACATCTT TT SEQ ID NO: 105 Cladosporium spp. TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTT T SEQ ID NO: 121 Podosphaera spp. TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTT T SEQ ID NO: 132 Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTT T SEQ ID NO: 37 Total Aerobic TTTTTTTTTCCTACGGGAGGCAGTTTTTTT bacteria (High) SEQ ID NO: 38 Total Aerobic TTTTTTTTCCCTACGGGAGGCATTTTTTTT bacteria (Medium) SEQ ID NO: 39 Total Aerobic TTTATTTTCCCTACGGGAGGCTTTTATTTT bacteria (Low) SEQ ID NO: 47 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGTGTGTRTGTCT negative (High) TTTT SEQ ID NO: 48 Bile-tolerant Gram- TTTTTCTATGCAGCCATGCTGTGTGTRTTTTT negative (Medium) TT SEQ ID NO: 49 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGCGTGTRTTTTT negative (Low) TT 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 TTTTTTCCATCAAAGTTGGTGAAGAATCTTTT TT SEQ ID NO: 132 Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTT T SEQ ID NO: 65 Listeria spp. TTTTCTAAGTACTGTTGTTAGAGAATTTTT SEQ ID NO: 56 Aeromonas spp. TTATTTTCTGTGACGTTACTCGCTTTTATT SEQ ID NO: 78 Staphylococcus TTTATTTTCATATGTGTAAGTAACTGTTTTATT aureus 1 T SEQ ID NO: 49 Campylobacter spp. TTTTTTATGACACTTTTCGGAGCTCTTTTT SEQ ID NO: 72 Pseudomonas TTTATTTTAAGCACTTTAAGTTGGGATTTTATT spp. 3 T 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 TTTTTTTATTGATGCCGATTTGAAGGCCTTTTT T
[0084] 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
Example 2
[0085] Using the 3-dimensional lattice microarray system for DNA analysis
Sample Processing
[0086] Harvesting Pathogens from plant surface comprises the following steps:
[0087] 1) Wash the plant sample or tape pull in 1× phosphate buffered saline (PBS);
[0088] 2) Remove plant material/tape;
[0089] 3) Centrifuge to pellet cells & discard supernatant;
[0090] 4) Resuspend in PathogenDx (PathogenDX, Inc.) Sample Prep Buffer pre-mixed with Sample Digestion Buffer;
[0091] 5) Heat at 55° C. for 45 minutes;
[0092] 6) Vortex to dissipate the pellet;
[0093] 7) Heat at 95° C. for 15 minutes; and
[0094] 8) Vortex and centrifuge briefly before use in PCR.
Amplification by PCR
[0095] 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.
[0096] 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.
[0097] The product of PCR Reaction #1 (1 μL) 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 (50 μL) was then diluted 1-1 with hybridization buffer (4×SSC+5×Denhardt's solution) and then applied directly to the microarray for hybridization.
TABLE-US-00005 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 PCR Reagent Primary PCR Concentration Secondary PCR Concentration PCR Buffer 1X 1X MgCl.sub.2 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 Polymerase 1.5 Units 1.5 Units Program for PCR Reaction #1 95 ° C., 4 min 98 ° C., 30s 61 ° C., 30s 72 ° C., 60s 72 ° C., 7 min 25X Program for PCR Reaction #2 95 ° C., 4 min 98 ° C., 20s 61 ° C., 20s 72 ° C., 30s 72 ° C., 7 min 25X *FP, Forward Primer; *RP, Reverse Primer
Hybridization
[0098] 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.
[0099] 1) Pipette the amplified DNA+binding buffer onto the microarray
[0100] 2) Incubate for 30 minutes to allow DNA binding to the microarray (typically at room temperature, RT)
[0101] 3) Remove the DNA+binding buffer by pipetting
[0102] 4) Pipette 50 uL of wash buffer onto the microarray (0.4×SSC+0.5×Denhardt's) and incubate 5 min at RT.
[0103] 5) Remove the wash buffer by pipetting
[0104] 6) Repeat steps 4 and 5
[0105] 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
[0106] 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.
[0107]
[0108]
[0109]
Example 3
Position of Pathogen Specific PCR Primers
[0110]
[0111] 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
[0112]
[0113]
TABLE-US-00006 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 TTTGACTACCAGGGTATCTAATCCTG (Bacteria) T SEQ ID NO: 3 Stx1 Locus TTTATAATCTACGGCTTATTGTTGAA (Pathogenic E. coli) CG SEQ ID NO: 4 Stx1 Locus TTTGGTATAGCTACTGTCACCAGACA (Pathogenic E. coli) ATG SEQ ID NO: 5 Stx2 Locus TTTGATGCATCCAGAGCAGTTCTGC (Pathogenic E. coli) G SEQ ID NO: 6 Stx2 Locus TTTGTGAGGTCCACGTCTCCCGGCG (Pathogenic E. coli) TC SEQ ID NO: 7 InvA Locus (Salmonella) TTTATTATCGCCACGTTCGGGCAATT CG SEQ ID NO: 8 InvA Locus (Salmonella) TTTCTTCATCGCACCGTCAAAGGAAC CG SEQ ID NO: 9 tuf Locus (All E. coli) TTTCAGAGTGGGAAGCGAAAATCCT G SEQ ID NO: 10 tuf Locus (All E. coli) TTTACGCCAGTACAGGTAGACTTCTG SEQ ID NO: 11 16S rDNA TTACCTTCGGGCCTCTTGCCATCRG Enterobacteriaceae HV3 ATGTG Locus SEQ ID NO: 12 16S rDNA TTGGAATTCTACCCCCCTCTACRAGA Enterobacteriaceae HV3 CTCAAGC Locus SEQ ID NO: 13 ITS2 Locus TTTACTTTYAACAAYGGATCTCTTGG (All Yeast, Mold/Fungus) SEQ ID NO: 14 ITS2 Locus TTTCTTTTCCTCCGCTTATTGATATG (All Yeast, Mold/Fungus) SEQ ID NO: 15 ITS2 Locus TTTAAAGGCAGCGGCGGCACCGCGT (Aspergillus species) CCG SEQ ID NO: 16 ITS2 Locus TTTTCTTTTCCTCCGCTTATTGATATG (Aspergillus species) SEQ ID NO: 17 ITS1 Locus TTTGCAACAGCAGAACGACCCGTGA (Cannabis/Plant) SEQ ID NO: 18 ITS1 Locus TTTCGATAAACACGCATCTCGATTG (Cannabis/Plant) Second PCR Primers (P2) for the second labeling amplification step SEQ ID NO: 19 16S rDNA HV3 Locus TTTACTGAGACACGGYCCARACTC (All Bacteria) SEQ ID NO: 20 16S rDNA HV3 Locus TTTGTATTACCGCGGCTGCTGGCA (All Bacteria) SEQ ID NO: 21 Stx1 Locus TTTATGTGACAGGATTTGTTAACAGG (Pathogenic E. coli) AC SEQ ID NO: 22 Stx1 Locus TTTCTGTCACCAGACAATGTAACCGC (Pathogenic E. coli) TG SEQ ID NO: 23 Stx2 Locus TTTTGTCACTGTCACAGCAGAAG (Pathogenic E. coli) SEQ ID NO: 24 Stx2 Locus TTTGCGTCATCGTATACACAGGAGC (Pathogenic E. coli) SEQ ID NO: 25 InvA Locus TTTTATCGTTATTACCAAAGGTTCAG (All Salmonella) SEQ ID NO: 26 InvA Locus TTTCCTTTCCAGTACGCTTCGCCGTT (All Salmonella) CG SEQ ID NO: 27 tuf Locus (All E. coli) TTTGTTGTTACCGGTCGTGTAGAAC SEQ ID NO: 28 tuf Locus (All E. coli) TTTCTTCTGAGTCTCTTTGATACCAA CG SEQ ID NO: 29 16S rDNA TTATATTGCACAATGGGCGCAAGCCT Enterobacteriaceae HV3 GATG Locus SEQ ID NO: 30 16S rDNA TTTTGTATTACCGCGGCTGCTGGCA Enterobacteriaceae HV3 Locus SEQ ID NO: 31 ITS2 Locus TTTGCATCGATGAAGARCGYAGC (All Yeast, Mold/Fungus) SEQ ID NO: 32 ITS2 Locus TTTCCTCCGCTTATTGATATGC (All Yeast, Mold/Fungus) SEQ ID NO: 33 ITS2 Locus TTTCCTCGAGCGTATGGGGCTTTGT (Aspergillus species) C SEQ ID NO: 34 ITS2 Locus TITTTCCTCCGCTTATIGATATGC (Aspergillus species) SEQ ID NO: 133 ITS2 Locus TTTGCATCGATGAAGAACGCAGC (All Yeast, Mold/Fungus) SEQ ID NO: 134 IT52 Locus (All Yeast, TTTTCCTCCGCTTATTGATATGC Mold/Fungus) SEQ ID NO: 135 Fungal RSG Primers TTTACTTTCAACAAYGGATCTCTTG (All Fungus) G SEQ ID NO: 35 ITS1 Locus TTTCGTGAACACGTTTTAAACAGCTT (Cannabis/Plant) G SEQ ID NO: 36 ITS1 Locus TTTCCACCGCACGAGCCACGCGAT (Cannabis/Plant)
[0114]
[0115]
[0116]
[0117]
[0118]
[0119] 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
[0120] 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
[0121] 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-US-00007 TABLE 7 Oligonucleotide probe sequence for the 16S Locus SEQ ID NO: 37 Total Aerobic bacteria (High) TTTTTTTTTCCTACGGGAGGCAG TTTTTTT SEQ ID NO: 38 Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATT (Medium) TTTTTT SEQ ID NO: 39 Total Aerobic bacteria (Low) TTTATTTTCCCTACGGGAGGCTTT TATTTT SEQ ID NO: 40 Enterobacteriaceae (Low TTTATTCTATTGACGTTACCCATT sensitivity) TATTTT SEQ ID NO: 41 Enterobacteriaceae (Medium TTTTTTCTATTGACGTTACCCGTT sensitivity) TTTTTT SEQ ID NO: 42 Escherichia coli/Shigella 1 TTTTCTAATACCTTTGCTCATTGA CTCTTT SEQ ID NO: 43 Escherichia coli/Shigella 2 TTTTTTAAGGGAGTAAAGTTAATA TTTTTT SEQ ID NO: 44 Escherichia coli/Shigella 3 TTTTCTCCTTTGCTCATTGACGTT ATTTTT SEQ ID NO: 45 Bacillus spp. Group1 TTTTTCAGTTGAATAAGCTGGCA CTCTTTT SEQ ID NO: 46 Bacillus spp. Group2 TTTTTTCAAGTACCGTTCGAATAG TTTTTT SEQ ID NO: 47 Bile-tolerant Gram-negative TTTTTCTATGCAGTCATGCTGTGT (High) GTRTGTCTTTTT SEQ ID NO: 48 Bile-tolerant Gram-negative TTTTTCTATGCAGCCATGCTGTGT (Medium) GTRTTTTTTT SEQ ID NO: 49 Bile-tolerant Gram-negative TTTTTCTATGCAGTCATGCTGCGT (Low) GTRTTTTTTT SEQ ID NO: 50 Campylobacter spp. TTTTTTATGACACTTTTCGGAGCT CTTTTT SEQ ID NO: 51 Chromobacterium spp. TTTTATTTTCCCGCTGGTTAATAC CCTTTATTTT SEQ ID NO: 52 Citrobacter spp. Group1 TTTTTTCCTTAGCCATTGACGTTA TTTTTT SEQ ID NO: 53 Clostridium spp. TTTTCTGGAMGATAATGACGGTA CAGTTTT SEQ ID NO: 54 Coliform/Enterobacteriaceae TTTTTTCTATTGACGTTACCCGCT TTTTTT SEQ ID NO: 55 Aeromonas TTTTTGCCTAATACGTRTCAACTG salmonicida/hydrophilia CTTTTT SEQ ID NO: 56 Aeromonas spp. TTATTTTCTGTGACGTTACTCGCT TTTATT SEQ ID NO: 57 Alkanindiges spp. TTTTTAGGCTACTGRTACTAATAT CTTTTT SEQ ID NO: 58 Bacillus pumilus TTTATTTAAGTGCRAGAGTAACTG CTATTTTATT SEQ ID NO: 59 etuf gene TTTTTTCCATCAAAGTTGGTGAAG AATCTTTTTT SEQ ID NO: 60 Hafnia spp. TTTTTTCTAACCGCAGTGATTGAT CTTTTT SEQ ID NO: 61 invA gene TTTTTTTATTGATGCCGATTTGAA GGCCTTTTTT SEQ ID NO: 62 Klebsiella oxytoca TTTTTTCTAACCTTATTCATTGAT CTTTTT SEQ ID NO: 63 Klebsiella pneumoniae TTTTTTCTAACCTTGGCGATTGAT CTTTTT SEQ ID NO: 64 Legionella spp. TTTATTCTGATAGGTTAAGAGCTG ATCTTTATTT SEQ ID NO: 65 Listeria spp. TTTTCTAAGTACTGTTGTTAGAGA ATTTTT SEQ ID NO: 66 Panteoa agglomerans TTTTTTAACCCTGTCGATTGACGC CTTTTT SEQ ID NO: 67 Panteoa stewartii TTTTTTAACCTCATCAATTGACGC CTTTTT SEQ ID NO: 68 Pseudomonas aeruginosa TTTTTGCAGTAAGTTAATACCTTG TCTTTT SEQ ID NO: 69 Pseudomonas cannabina TTTTTTTACGTATCTGTTTTGACT CTTTTT SEQ ID NO: 70 Pseudomonas spp. 1 TTTTTTGTTACCRACAGAATAAGC ATTTTT SEQ ID NO: 71 Pseudomonas spp. 2 TTTTTTAAGCACTTTAAGTTGGGA TTTTTT SEQ ID NO: 72 Pseudomonas spp. 3 TTTATTTTAAGCACTTTAAGTTGG GATTTTATTT SEQ ID NO: 73 Salmonella bongori TTTTTTTAATAACCTTGTTGATTG TTTTTT SEQ ID NO: 74 Salmonella TTTTTTTGTTGTGGTTAATAACCG enterica/Enterobacter 1 ATTTTT SEQ ID NO: 75 Salmonella TTTTTTTAACCGCAGCAATTGACT enterica/Enterobacter 2 CTTTTT SEQ ID NO: 76 Salmonella TTTTTTCTGTTAATAACCGCAGCT enterica/Enterobacter 3 TTTTTT SEQ ID NO: 77 Serratia spp. TTTATTCTGTGAACTTAATACGTT CATTTTTATT SEQ ID NO: 78 Staphylococcus aureus 1 TTTATTTTCATATGTGTAAGTAAC TGTTTTATTT SEQ ID NO: 79 Staphylococcus aureus 2 TTTTTTCATATGTGTAAGTAACTG TTTTTT SEQ ID NO: 80 Streptomyces spp. TTTTATTTTAAGAAGCGAGAGTGA CTTTTATTTT SEQ ID NO: 81 stx1 gene TTTTTTCTTTCCAGGTACAACAGC TTTTTT SEQ ID NO: 82 stx2 gene TTTTTTGCACTGTCTGAAACTGCC TTTTTT SEQ ID NO: 83 Vibrio spp. TTTTTTGAAGGTGGTTAAGCTAAT TTTTTT SEQ ID NO: 84 Xanthamonas spp. TTTTTTGTTAATACCCGATTGTTC TTTTTT SEQ ID NO: 85 Yersinia pestis TTTTTTTGAGTTTAATACGCTCAA CTTTTT
TABLE-US-00008 TABLE 8 Calibration and Negative Controls SEQ ID NO: Imager TTTTCTATGTATCGATGTTGAGAAAT 129 Calibration TTTTTT (High) SEQ ID NO: Imager TTTTCTAGATACTTGTGTAAGTGAAT 130 Calibration TTTTTT (Low) SEQ ID NO: Imager TTTTCTAAGTCATGTTGTTGAAGAAT 131 Calibration TTTTTT (Medium) SEQ ID NO: Negative TTTTTTCTACTACCTATGCTGATTCA 132 control CTCTTTTT
TABLE-US-00009 TABLE 9 Oligonucleotide probe sequence for the ITS2 Locus SEQ ID NO: 86 Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACG Mold (High CATTTTTTT sensitivity) SEQ ID NO: 87 Total Yeast and TTTTTTTTGAATCATCGARTCTCCTTTTTT Mold (Low T sensitivity) SEQ ID NO: 88 Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACG Mold (Medium TTTTTTT sensitivity) SEQ ID NO: 89 Alternaria spp. TTTTTTCAAAGGTCTAGCATCCATTAAGT TTTTT SEQ ID NO: 90 Aspergillus flavus 1 TTTTTTCGCAAATCAATCTTTTTCCAGTCT TTTT SEQ ID NO: 91 Aspergillus flavus 2 TTTTTTTCTTGCCGAACGCAAATCAATCT TTTTTTTTTTT SEQ ID NO: 92 Aspergillus TTTCTTTTCGACACCCAACTTTATTTCCTT fumigatus 1 ATTT SEQ ID NO: 93 Aspergillus TTTTTTTGCCAGCCGACACCCATTCTTTT fumigatus 2 T SEQ ID NO: 94 Aspergillus TTTTTTGGCGTCTCCAACCTTACCCTTTT nidulans T SEQ ID NO: 95 Aspergillus niger 1 TTTTTTCGACGTTTTCCAACCATTTCTTTT SEQ ID NO: 96 Aspergillus niger 2 TTTTTTTTCGACGTTTTCCAACCATTTCTT TTTT SEQ ID NO: 97 Aspergillus niger 3 TTTTTTTCGCCGACGTTTTCCAATTTTTTT SEQ ID NO: 98 Aspergillus terreus TTTTTCGACGCATTTATTTGCAACCCTTT T SEQ ID NO: 99 Blumeria TTTATTTGCCAAAAMTCCTTAATTGCTCT TTTTT SEQ ID NO: 100 Botrytis spp TTTTTTTCATCTCTCGTTACAGGTTCTCG GTTCTTTTTTT SEQ ID NO: 101 Candida albicans TTTTTTTTTGAAAGACGGTAGTGGTAAGT TTTTT SEQ ID NO: 102 Candida spp. TTTTTTTGTTTGGTGTTGAGCRATACGTA Group 1 TTTTT SEQ ID NO: 103 Candida spp. TTTTACTGTTTGGTAATGAGTGATACTCT Group 2 CATTTT SEQ ID NO: 104 Chaetomium spp. TTTCTTTTGGTTCCGGCCGTTAAACCATT TTTTT SEQ ID NO: 105 Cladosporium spp TTTTTTTTGTGGAAACTATTCGCTAAAGT TTTTT SEQ ID NO: 106 Erysiphe spp. TTTCTTTTTACGATTCTCGCGACAGAGTT TTTTT SEQ ID NO: 107 Fusarium TTTTTTTCTCGTTACTGGTAATCGTCGTT oxysporum TTTTT SEQ ID NO: 108 Fusarium spp TTTTTTTTAACACCTCGCRACTGGAGATT TTTTT SEQ ID NO: 109 Golovinomyces TTTTTTCCGCTTGCCAATCAATCCATCTC TTTTT SEQ ID NO: 110 Histoplasma TTTATTTTTGTCGAGTTCCGGTGCCCTTT capsulatum TATTT SEQ ID NO: 111 Isaria spp. TTTATTTTTCCGCGGCGACCTCTGCTCTT TATTT SEQ ID NO: 112 Monocillium spp. TTTCTTTTGAGCGACGACGGGCCCAATT TTCTTT SEQ ID NO: 113 Mucor spp. TTTTCTCCAWTGAGYACGCCTGTTTCTTT T SEQ ID NO: 114 Myrothecium spp. TTTATTTTCGGTGGCCATGCCGTTAAATT TTATT SEQ ID NO: 115 Oidiodendron spp. TTTTTTTGCGTAGTACATCTCTCGCTCAT TTTTT SEQ ID NO: 116 Penicillium TTTTTTACACCATCAATCTTAACCAGGCC oxalicum TTTTT SEQ ID NO: 117 Penicillium paxilli TTTTTTCCCCTCAATCTTTAACCAGGCCT TTTTT SEQ ID NO: 118 Penicillium spp TTTTTTCAACCCAAATTTTTATCCAGGCC TTTTT SEQ ID NO: 119 Phoma/Epicoccum TTTTTTTGCAGTACATCTCGCGCTTTGAT spp. TTTTT SEQ ID NO: 120 Podosphaera spp TTTTTTGACCTGCCAAAACCCACATACCA TTTTT SEQ ID NO: 121 Podosphaera spp. TTTTTTTTAGTCAYGTATCTCGCGACAGT TTTTT SEQ ID NO: 122 Pythium TTTTATTTAAAGGAGACAACACCAATTTT oligandrum TATTT SEQ ID NO: 123 Rhodoturula spp TTTTTTCTCGTTCGTAATGCATTAGCACT TTTTT SEQ ID NO: 124 Stachybotrys spp TTTCTTCTGCATCGGAGCTCAGCGCGTT TTATTT SEQ ID NO: 125 Trichoderma spp TTTTTCCTCCTGCGCAGTAGTTTGCACAT CTTTT SEQ ID NO: 136 Total Yeast and TTTTTTTTGCATCATAGAAACTTTGTAC Mold Quantitative GCATTT TTTT Control (internal reference standard) SEQ ID NO: 137 Golovinomyces TTTATTTAATCAATCCATCATCTCAAGT spp. CTTTTT SEQ ID NO: 138 Mucor spp. TTTTTTCTCCAWTGAGYACGCCTGTTTC AGTAT CTTTTTT SEQ ID NO: 139 Aspergillus terreus TTTTTTACGCATTTATTTGCAACTTGCCT TTTTT SEQ ID NO: 140 Podosphaera spp. TTTTTCGTCCCCTAAACATAGTGGCTTT TT
[0122] 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-US-00010 TABLE 10 Oligonucleotide probe sequence for the Cannabis ITS1 Locus SEQ ID Cannabis ITS1 TTTTTTAATCTGCGCCAAGGAACAATA NO: 126 DNA Control 1 TTTTTTT SEQ ID Cannabis ITS1 TTTTTGCAATCTGCGCCAAGGAACAAT NO: 127 DNA Control 2 ATTTTTT SEQ ID Cannabis ITS1 TTTATTTCTTGCGCCAAGGAACAATAT NO: 128 DNA Control 3 TTTATTT
TABLE-US-00011 TABLE 11 Representative Microarray Probe Design for the Present Invention: Bacterial Toxins, ITS1 (Cannabis) SEQ ID NO: 81 stx1 gene TTTTTTCTTTCCAGGTACAACAG CTTTTTT SEQ ID NO: 82 stx2 gene TTTTTTGCACTGTCTGAAACTGC CTTTTTT SEQ ID NO: 59 etuf gene TTTTTTCCATCAAAGTTGGTGAA GAATCTTTTTT SEQ ID NO: 61 invA gene TTTTTTTATTGATGCCGATTTGA AGGCCTTTTTT SEQ ID NO: Cannabis ITS1 TTTTTTAATCTGCGCCAAGGAAC 126 DNA Control 1 AATATTTTTTT
[0123]
[0124]
[0125]
[0126] The data of
[0127]
[0128]
[0129] 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.
[0130]
[0131] 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.
[0132] 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-US-00012 TABLE 12A Representative microarray data obtained from powdered dry food samples. Sample Type Whey Protein Whey Protein Chewable Shake Shake Berry Vanilla Vanilla Chocolate Tablet Shake Pea Protein Enrichment time (hours) 0 18 0 18 0 18 0 18 0 18 Negative Control 289 318 349 235 327 302 358 325 321 299 Probe Total Aerobic Bacteria Probes High sensitivity 26129 38896 16629 11901 3686 230 32747 12147 41424 40380 Medium sensitivity 5428 6364 3308 2794 876 215 7310 2849 15499 8958 Low sensitivity 2044 3419 1471 990 446 181 2704 1062 4789 3887 Bile-tolerant Gram-negative Probes High sensitivity 2639 350 1488 584 307 305 1041 472 15451 8653 Medium sensitivity 1713 328 892 493 322 362 615 380 6867 4997 Low sensitivity 974 600 749 621 595 688 821 929 2459 1662 Total Enterobacteriaceae Probes High sensitivity 1131 306 363 310 346 318 273 331 4260 3149 Medium sensitivity 479 296 320 297 329 339 314 342 1489 990 Low sensitivity 186 225 203 165 205 181 207 200 216 259 16S rDNA Species Probes Escherichia 233 205 255 219 207 255 215 214 242 198 coli/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 549 201 202 251 148 216 303 276 2066 983 spp. 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 215 265 210 284 204 256 239 285 237 229 spp.)
TABLE-US-00013 TABLE 12B Representative microarray data obtained from powdered dry food samples. Sample Type Work-out Work-out Rice Protein Shake FP Shake BR Vanilla Shake Enrichment time (hours) 0 18 0 18 0 18 0 18 Negative Control 351 351 271 309 299 332 246 362 Probe 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 304 362 341 391 308 356 699 394 sensitivity 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 228 229 216 267 221 253 220 207 coli/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 199 225 247 251 211 259 277 225 spp. 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 257 241 249 264 220 258 239 245 spp.)
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-US-00014 TABLE 13 Representative microarray hybridization data obtained from blueberry and lemon washes. Sample Blueberry Lemon Collection Type Produce Wash Protocol Wash 1 piece moldy Wash 1 blueberry in 2 ml lemon in 2 ml 20 mM 20 mM Borate, vortex 30 Borate, vortex 30 seconds 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
[0133] The data embodied in
TABLE-US-00015 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, vortex 30 seconds 500 ul 20 mM Borate + Triton Buffer, vortex 30 seconds Collection Point 2 Add 15 mg zirconia beads, vortex, Add 15 mg zirconia beads, vortex, Heat 5 min 95° C., Vortex 15 seconds Heat 5 min 95° C., Vortex 15 seconds Collection Point 3 Heat 5 min 95° C. Heat 5 min 95° C. vortex 15 seconds vortex 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. 1 80 81 83 64 96 60 79 80 176 60 385 53 Penicillium spp. 2 90 93 81 80 114 59 98 69 470 65 478 56 Fusarium spp. 1 77 71 69 62 112 55 61 274 617 81 59 757 Fusarium spp. 2 91 82 107 74 101 65 91 66 123 63 71 583 Mucor spp. 1 90 314 73 88 105 61 77 79 741 180 172 74 Mucor spp. 2 83 69 73 69 91 67 111 102 455 88 70 133 Total Y & 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
TABLE-US-00016 TABLE 15 Representative microarray hybridization data obtained from lemon washes and tape pulls. Sample Moldy Lemon Collection Type Tape Pull ID 1A1 Lemon 1A2 Lemon 1A3 Lemon 1B1 Lemon 1B2 Lemon Collection Point 1 500 ul 20 mM Borate + Triton Buffer, vortex 30 seconds Collection Point 2 Add 15 mg Add 15 mg zirconia zirconia beads, beads, vortex, vortex, Heat 5 Heat 5 min 95° C., min 95° C., Vortex Vortex 15 seconds 15 seconds Collection Point 3 Heat 5 min 95° C. vortex 15 seconds Dilution Factor NONE A. fumigatus 1 96 83 75 83 64 A. fumigatus 2 221 73 71 66 101 A. fumigatus 3 87 88 85 92 122 A. fumigatus 4 83 85 91 72 97 A. fumigatus 5 448 100 84 114 78 A. flavus 1 85 79 70 66 63 A. flavus 2 77 82 77 79 63 A. flavus 3 133 66 86 60 67 A. flavus 4 96 85 81 98 88 A. flavus 5 68 62 65 106 59 A. niger 1 73 88 77 73 73 A. niger 2 74 84 81 71 103 A. niger 3 90 86 87 74 78 A. niger 4 82 93 104 86 161 Botrytis spp. 1 82 75 75 77 68 Botrytis spp. 2 91 74 83 67 62 Penicillium spp. 1 3824 5461 5500 4582 5290 Penicillium spp. 2 7586 8380 11177 6528 8167 Fusarium spp. 1 101 62 61 70 279 Fusarium spp. 2 77 122 78 68 233 Mucor spp. 1 74 110 89 76 57 Mucor spp. 2 132 1302 90 84 61 Total Y & M 1 8448 12511 9249 12844 8593 Total Y & M 2 9275 8716 11585 10758 4444
[0134] 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.
[0135] 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-US-00017 TABLE 16 Representative microarray data from raw water filtrate. Negative Sample ID 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 High 40101 42007 47844 47680 45102 44041 43520 41901 46459 46783 135 Total Aerobic bacteria Medium 14487 12314 24189 26158 19712 16210 17943 15474 25524 18507 157 Total Aerobic bacteria Low 4885 5629 7625 6456 5807 4505 5316 6022 6264 6974 159 Negative Control 293 359 303 339 312 329 306 377 307 335 307 Aspergillus fumigatus 285 291 284 268 289 265 271 281 269 248 228 Aspergillus flavus 184 211 201 344 237 179 212 213 163 204 171 Aspergillus niger 226 213 228 273 190 195 245 206 222 209 172 Botrytis spp. 219 285 258 302 275 219 202 288 221 248 214 Alternaria spp. 81 97 76 89 58 76 75 175 117 174 167 Penicillium paxilli 135 162 215 142 127 161 103 115 238 190 200 Penicillium oxalicum 119 107 161 131 135 241 178 158 140 143 194 Penicillium spp. 50 123 179 177 128 138 146 163 148 115 184 Can. alb/trop/dub 261 236 235 230 250 213 276 244 245 237 194 Can. glab/Sach & Kluv spp. 146 165 196 128 160 215 185 217 215 177 225 Podosphaera spp. 111 119 100 122 192 105 95 43 169 27 143 Bile-tolerant Gram-negative 16026 9203 13309 8426 16287 14116 10557 17558 15343 14285 183 High Bile-tolerant Gram-negative 12302 11976 9259 10408 13055 10957 11242 8416 9322 11785 196 Medium Bile-tolerant Gram-negative 5210 7921 3818 3984 7224 6480 4817 6933 5021 5844 240 Low Total Enterobacteriaceae High 193 248 389 357 215 214 198 220 276 208 210 Total Enterobacteriaceae Med 246 214 297 246 244 224 219 245 252 229 207 Total Enterobacteriaceae Low 165 140 158 119 151 180 150 167 182 174 132 Total Coliform 121 148 158 117 129 117 155 157 125 178 152 Escherichia coli specific gene 31821 115 132 155 127 62 86 121 59 90 234 stx1 gene 67 0 2 0 0 23 21 28 0 0 116 stx2 gene 17 36 174 0 61 47 0 51 33 0 85 Salmonella specific gene 181 172 245 172 178 212 157 243 174 156 146 Bacillus spp. 137 135 174 112 164 143 163 182 168 152 149 Pseudomonas spp. 271 74 332 56 366 133 91 114 60 179 555 Escherichia coli/Shigella spp. 103 124 221 124 90 144 130 121 137 143 158 Salmonella 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/Shigella spp. 163 193 220 202 308 280 121 271 341 317 124 Aspergillus fumigatus 713 865 862 830 784 657 827 803 746 812 793 Aspergillus flavus 155 261 198 156 239 171 250 218 210 258 219 Salmonella enterica 136 98 85 43 109 47 23 123 70 100 135 Salmonella enterica 68 53 52 41 60 92 26 28 55 81 116
[0136] 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.
[0137] 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.
[0138] 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.
[0139] While the foregoing written description of an embodiments enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The present disclosure should therefore not be limited by the above described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the present disclosure.
Example 4
[0140] PathogenDx QuantX assay for the detection of fungal contaminants in plants.
Definitions
[0141] Probability of Detection (POD): The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated; POD.sub.R (reference method POD), POD.sub.C (confirmed candidate method POD), POD.sub.CP (candidate method presumptive result POD) and POD.sub.CC (candidate method confirmation result POD).
[0142] Difference of Probabilities of Detection (dPOD): Difference of probabilities of detection is the difference between any two POD values. If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.
[0143] Microarray: A laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are 96-well plates that are printed as a matrix of oligonucleotide probe “Spots” in defined positions, with each spot containing a known DNA sequence.
Materials and Methods
Test Kit—PathogenDx QuantX Assay (Catalog Number.—QF-003 PathogenDx, LLC).
Test Kit Components
[0144] a) QuantX Sample Preparation Kit [0145] 1) Lysis Buffer, 1 bottle (4 mL) [0146] 2) Neutralization Buffer, 1 vial (700 μL) [0147] 3) Sample Prep Buffer, 1 bottle (3.2 mL) [0148] 4) Sample Digestion Buffer, 1 vial (200 μL) [0149] 5) Promega RELIAPREP DNA Clean-up and Concentration System—100 reaction kit
[0150] b) PCR Master Mix [0151] 1) PCR Master Mix, 1 bottle (9 mL) [0152] 2) Primer Set 2: Quant Fungal, 1 vial (250 μL) [0153] 3) Standard, 1 vial (12 μL) [0154] 4) Taq polymerase, 1 vial (75 μL)
[0155] c) Hybridization and Analysis [0156] 1) Buffer 1, 1 bottle (7.5 mL) [0157] 2) Buffer 2, 1 bottle (3.5 mL) [0158] 3) QuantX Bacterial 96 well plate, 96 per 96-well plate [0159] 4) AUGURY software
Key Equipment (Not part of the kit)
[0160] a) SENSOSPOT Fluorescence Microarray Analyzer (Sensospot Milteny Imaging GmbH, Germany)
[0161] b) MiniAmp Thermocycler, PN A37834 (ThermoFisher Scientific)
[0162] c) PCR Plate Spinner Centrifuge, PN C2000 (Light Labs)
Sample Preparation
[0163] a) Cannabis flower (10 g). Mix 10 g of sample with 90 mL of PBS in a Whirl-Pak filter bag.
[0164] b) Perform a wash of the matrix by homogenizing for 10 sec.
[0165] c) Serially dilute the sample to the action level required for analysis (e.g. 1:1,000, 1:10,000, 1:100,000).
TABLE-US-00018 TABLE 17 Sample Buffer Mix volumes Sample Prep Buffer Sample Digestion Buffer Number of Samples (μL) (μL) 1 23.8 1.2 8 238 12 16 428.4 21.6 24 666.4 33.6 32 856.8 43.2 40 1047.2 52.8 48 1285.2 64.8 56 1475.6 74.4 64 1666 84 72 1856.4 93.6 88 2237.2 112.8 96 2427.6 122.4
Analysis
[0166] a) Transfer 1 mL of the PBS suspension into a clean 1.5 mL conical tube, then centrifuge tube at 50×g for 3 minutes to pellet the excess matrix material.
[0167] b) Transfer the supernatant to a clean 1.5 mL tube, being careful to avoid matrix material. Discard the matrix pellet.
[0168] c) Centrifuge samples at 14,000×g for 3 minutes to pellet the cells.
[0169] d) Decant the supernatant and retain the cell pellet. Remove as much supernatant as possible without disturbing the pellet. It may be necessary to remove excess with a pipette.
[0170] e) Add 35 μL of Lysis Buffer to each tube, vortex to dislodge the pellet and quick spin.
[0171] f) Heat Sample tubes at 95+1° C. for 10 minutes.
[0172] g) Remove the tubes from the heat, vortex and briefly centrifuge.
[0173] h) To each tube add 5 μL of Neutralization Buffer and vortex thoroughly to mix.
[0174] i) Sample buffer Mix (make fresh each time) is prepared as shown in Table 17 by adding volumes of Sample Digestion Buffer and Sample Prep Buffer based on the number of samples being prepared.
[0175] j) Add 25 μL of Sample Buffer Mix to each tube, vortex to mix.
[0176] k) Heat sample tubes at 55+1° C. for 45 minutes.
[0177] l) Vortex for 10 seconds and briefly centrifuge samples to bring the fluid to bottom of the tube.
[0178] m) Heat sample tubes at 95+1° C. for 15 minutes.
[0179] n) Perform the Promega ReliaPrep DNA Clean-Up and Concentration System protocol using the following instructions: [0180] 1) Make sure the Column Wash Solution and Buffer B have had Molecular Biology Grade Ethanol (not provided with the kit) added to them following the instructions for the varying kit sizes. [0181] 2) Add 32.5 μL of Membrane Binding Solution to each prepped lysate and vortex for 5 seconds. [0182] 3) Add 97.5 μL of 100% isopropanol, not provided in the kit, to each prepped lysate, vortex to mix. [0183] 4) Load the sample onto the RELIAPREP Minicolumn seated in a collection tube and centrifuge for 30 seconds at 10,000×g. [0184] 5) Remove the column and discard the contents in the collection tube. Reseat the column into the collection tube. [0185] 6) Add 200 μL of Column Wash Solution (CWE) and centrifuge at 10,000×g for 15 seconds. Remove the column and discard the contents in the collection tube. Reseat the column into the collection tube. [0186] 7) Wash with 300 μL of Buffer B (BWB) and centrifuge at 10,000×g for 15 seconds. Repeat wash with 300 uL of Buffer B and centrifuge at 10,000×g again. [0187] 8) Remove column and discard the contents in the collection tube. Reseat the column into the same collection tube and centrifuge at 10,000×g for 1 minute to dry the column. [0188] 9) Transfer the column to a labelled Elution Tube. [0189] 10) Pipet 15 μL of Nuclease-Free water or TE Buffer, not provided, into the center of the RELIAPREP Minicolumn. The color should change from light to dark tan. Centrifuge at 10,000×g for 30 seconds. [0190] 11) For maximum recovery, repeat elution with an additional 15 μL of Nuclease Free Water or TE Buffer for a final volume of 30 μL.
[0191] c) Samples are now ready for PCR. Vortex and briefly centrifuge the tubes before removing 2 μL for PCR.
PCR amplification
[0192] a) Thaw PCR Master Mix and Primer Set.
[0193] b) Thaw the Standard tube on the Sample Prep Area bench top. [0194] 1) The High Standard is the stock tube. [0195] 2) The Low Standard is prepared by removing 5 μL of the vortexed High Standard tube to a new sterile tube. [0196] 3) Add 495 μL of Molecular Biology Grade Water, vortex to mix. [0197] 4) The Low Standard must be made fresh each time. Discard after use.
Table 18 shows calculations for the appropriate volumes needed for the reaction. Labeling PCR Master Mix is made fresh each run.
TABLE-US-00019 TABLE 18 Labeling PCR Master Mix Volumes Taq Total # of Reactions PCR Master Primer Set Polymerase Volume per Primer Mix (μL) Fungal (μL) (μL) (μL) 1 45.5 2 0.5 48 8 455 20 5 480 16 819 36 9 864 24 1183 52 13 1248 32 1638 72 18 1728 40 2002 88 22 2112 48 2366 104 26 2496 56 2730 120 30 2880 64 3185 140 35 3360 72 3549 156 39 3744 80 3913 172 43 4128 88 4277 188 47 4512 96 4641 204 51 4896 [0198] (a) Vortex all reagents except the Taq Polymerase for 15 seconds; centrifuge at 1000×g speed for 3-5 seconds. [0199] (b) Mix the indicated reagent volumes (calculated from Table 18) in a microfuge tube to prepare PCR Master Mix. [0200] (c) Briefly vortex PCR Master Mix and centrifuge at 1000×g for 3-5 seconds. [0201] (d) Store all reagents at −20° C. after use. [0202] (e) Pipette 48 μL of Labeling PCR Master Mix into the bottom of PCR tubes or wells of a PCR plate. [0203] (f) In the Sample Prep area, pipette 2 μL of sample lysate, 2 μL of the High Standard and 2 μL of the Low Standard into the bottom of the corresponding tube or well for a final volume of 50 μL per PCR reaction. Pipette up and down to mix. [0204] (g) Cap tubes, or seal plates with PCR film ensuring every well is completely sealed. [0205] (h) Centrifuge at 1000×g for 3-5 second. [0206] (i) Move to the Hybridization Area/Post PCR Area. Place tubes or plate into the thermal cycler with a pressure pad if necessary, before closing the thermal cycler lid. [0207] (j) Enter the PCR Program into your thermal cycler as shown in Table 19. Confirm all parameters. [0208] (k) Once the PCR is complete, the plate may be stored at 4° C. for up to 1 weeks.
TABLE-US-00020 TABLE 19 Labeling PCR Program Steps Temp. Time Cycles 1 95° C. 4 Minutes 1 2 95° C. 30 seconds 40 3 55° C. 30 seconds 4 72° C. 1 minute 5 72° C. 7 minutes 1 6 15° C. ∞ 1
Hybridization
[0209] a) Perform all steps in the Hybridization/Post PCR Area.
[0210] b) Before starting, thaw Buffer 2 at room temperature. [0211] 1) Place the plate to be used in the Hybridization Chamber. [0212] 2) Ensure the wells to be used have been clearly tracked. [0213] 3) Carefully remove the foil seal from only the wells that will be hybridized.
Use a clean razor blade or other precision blade to carefully cut the seal between the wells to be used and the wells that should remain covered for future use. Gently peel the seal from the wells you are going to use. [0214] 4) Leave the remainder of the wells covered to avoid any contact with moisture.
[0215] c) Prepare the Pre-hybridization Buffer and Hybridization Buffers in sterile tubes for the number of wells that will be hybridized as per Tables 20 and 21. The tables shown below have the volumes required to make one well. Multiply the reagent volumes by the number of wells to be run. Add extra wells to account for pipetting loss. Vortex briefly to mix.
[0216] d) Apply 200 μL of Molecular Biology Grade Water to each well while being careful to avoid contact with the array.
[0217] e) Aspirate and then again, dispense 200 μL of Molecular Biology Grade Water to each well and allow to sit covered in the Hybridization Chamber for 5 minutes before aspirating water from the plate.
[0218] f) Aspirate the water wash and add 200 μL of Pre-hybridization Buffer to each designated well of the PathogenDx plate without touching the pipette tip to the array surface. Close the Hybridization Chamber box lid.
[0219] g) Allow Pre-hybridization Buffer to stay on the arrays for 5 minutes; do not remove the plate from the Hybridization Chamber.
[0220] h) Briefly centrifuge the tubes or plate containing the Labeling PCR product.
[0221] i) Add 18 μL of Hybridization Buffer to each well of the Labeling PCR product for hybridization within the 96-well PCR plate or tubes, pipette up and down to mix. It is important that no cross-contamination occurs during this step. The PCR product and the Hybridization Buffer mix constitute the Hybridization Cocktail.
[0222] j) Aspirate the Pre-hybridization Cocktail from the arrays.
[0223] k) Immediately add 68 μL of the Hybridization Cocktail to each array being careful not to touch the array surface with the pipette tip. Ensure that the sample ID and location are recorded.
[0224] l) Close the Hybridization Chamber lid.
[0225] m) Allow to hybridize for 30 minutes at room temperature in the Hybridization Chamber.
TABLE-US-00021 TABLE 20 Reagent volumes for preparation of Pre-hybridization Buffer Volumes corresponding to the number of wells Pre-hybridization 1 8 16 24 32 40 48 56 64 72 80 88 96 reagents well wells wells wells wells wells wells wells wells wells wells wells wells Molecular biology 137.6 1651 2752 3853 5229 6330 7430 8531 9907 11008 12109 13210 14310 grade water (μL) Buffer 1 (μL) 40.9 490.8 818 1145 1554 1881 2209 2536 2945 3272 3599 3926 4254 Buffer 2 (μL) 21.5 258 430 602 817 989 1161 1333 1548 1720 1892 2064 2236
TABLE-US-00022 TABLE 21 Reagent volumes for preparation of Hybridization Buffer Volumes corresponding to the number of wells Hybridization 1 8 16 24 32 40 48 56 64 72 80 88 96 reagents well wells wells wells wells wells wells wells wells wells wells wells wells Buffer 1 (μL) 11.8 141.6 236 330.4 448.4 542.8 637.2 731.6 849.6 944 1038 1133 1227 Buffer 2 (μL) 6.2 74.4 124 173.6 235.6 285.2 334.8 384.4 446.4 496 545.6 595.2 644.8
TABLE-US-00023 TABLE 22 Reagent volumes for preparation of Wash Buffer Volumes corresponding to the number of wells Wash Buffer 1 8 16 24 32 40 48 56 64 72 80 88 96 reagents well wells wells wells wells wells wells wells wells wells wells wells wells Buffer 1 (μL) 4.5 54 90 126 171 207 243 279 324 360 396 432 468 Molecular biology 0.5955 6.714 11.19 15.666 21.261 25.737 30.213 34.689 40.284 44.76 49.236 53.712 58.188 grade water (μL)
Post hybridization PathogenDx slide processing
[0226] a) Prepare Wash buffer according to the number of wells to be used (Table 22). Washing must be performed according to the protocol to ensure detectable signal and adequate washing to prevent elevated background signals.
[0227] b) Aspirate the Hybridization Cocktail from the slides.
[0228] c) Add 200 μL of Wash Buffer to each array, then aspirate.
[0229] d) Add 200 μL of Wash Buffer a second time to each array, close the Hybridization Chamber lid and allow buffer to remain on the slides for 10 minutes.
[0230] e) Aspirate the Wash Buffer.
[0231] f) Perform a final wash by dispensing and aspirating 200 μL of Wash Buffer, aspirate immediately.
[0232] g) Following the last aspiration step, remove the slides from the Hybridization Chamber.
[0233] h) Dry the plate using the plate centrifuge for 1 minute. [0234] 1) Place the plate face down with the open wells against paper towels to absorb liquid during centrifugation. [0235] 2) After 1 minute, remove the plate and inspect for any remaining moisture. If moisture is present, repeat the centrifugation step until completely dry.
[0236] i) Prior to scanning, clean the back of the glass microarray with lens paper or Kim wipe (never use paper towels which leave an excess of fibers and interferes with scanning). [0237] 1) If the back of the slide still shows dust and/or streaks, lightly spray the back of the plate with 70% ethanol and wipe dry.
[0238] j) PathogenDx plates should be placed back into a moisture barrier bag with desiccant until scanning may be performed in order to protect the arrays from light. Plates should be scanned within two weeks of hybridization.
Scanning conditions and Data Acquisition
[0239] a) Access the Sensovation scanner desktop, select the application “Array Reader”.
[0240] b) Open the tray, select “Open Tray”.
[0241] c) Place the microarray into the tray oriented with the plate face up and aligned with A1 in the top left corner.
[0242] d) Close the tray, select “Close Tray”.
[0243] e) Select “Scan”.
[0244] f) From the dropdown menu for “Rack Layout” select the Full Slide (96 wells) PDx.
[0245] g) From the dropdown menu for assay layout, select “PathogenDx Assay 002”.
[0246] h) Click on the three dots icon to the right of “Scan Position”.
[0247] i) To scan a full plate, double click the asterisk at the top left of the plate image.
[0248] j) To scan a partial plate, click the desired wells or click on the column number.
[0249] k) Select the Blue Arrow to begin the scanning process.
[0250] l) While the plate is being scanned, select “Result overview” to review the images of the wells.
[0251] m) When the plate is finished scanning and the screen displays the digital image of a plate with all green wells, select the Red X to exit the scanning process.
[0252] n) Open the tray, select “Open Tray”.
[0253] o) Remove the microarray and store inside the moisture barrier bag with the desiccant packets.
[0254] p) Close the tray, select “Close Tray”.
[0255] q) Exit the Array Reader application, select “Exit”.
[0256] r) On the Sensovation Scanner desktop, select the folder “Scan Results”.
[0257] s) Locate the folder associated with your plate and rename the folder with the plate barcode number y scanning the barcode located either on the outside of the barrier bag or on the plate itself. [0258] 1) If a full plate was scanned, rename the scan file to reflect the plate barcode. For example, rename “ScanJob-191108130334_1” to “7024001001”. [0259] 2) If a partial plate was scanned, add the wells scanned to the end of the barcode. For example, if the first two columns were scanned rename“ScanJob-191108130334_1” to “7024001001.well001-well016”.
[0260] t) Submit the whole barcode labeled folder to Portal.
[0261] u) Refer to the Portal instructions for Analysis.
Interpretation and Test Results Report
[0262] a) Data is analyzed automatically by the software.
[0263] b) Table 23 was used to determine the final interpretation.
Confirmation
[0264] For samples that fail an action limit, confirm by streaking the test aliquot onto Dichloran Rose Bengal Chloramphenicol (DRBC) agar. DRBC plates should be incubated for 5-7 days at 25±1° C. Growth on the plate is confirmation that the sample is positive at that action limit level.
TABLE-US-00024 TABLE 23 Interpretation of Results TOTAL YEAST and MOLD Action Limit Evaluated Result (CFU/g) Interpretation 1:1,000 <1,000 Pass >1,000 Fail 1:10,000 <10,000 Pass >10,000 Fail 1:100,000 <100,000 Pass >100,000 Fail
Example 5
AOAC Validation Study
Study Overview
[0265] This validation study was conducted under the AOAC Research Institute Performance Tested Method (PTM) ERV program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces (6). The QuantX method was compared to plating on DRBC for the detection of total viable yeast and mold in cannabis flower at specific dilution thresholds. Inclusivity and exclusivity was also performed. The matrix study was performed by an independent laboratory, SV Laboratories (Kalamazoo, Mich.). The inclusivity and exclusivity analysis was performed by Q Laboratories (Cincinnati, Ohio).
Inclusivity/Exclusivity
[0266] Inclusivity Methodology. Inclusivity and exclusivity strains were evaluated to meet the requirements of the AOAC ERV PTM study protocol. For the ERV study, 50 strains of yeast and mold, and 30 exclusivity strains were evaluated. We are currently in the process of evaluating the remaining exclusive strains. Target strains were cultured in potato dextrose broth or on potato dextrose agar until appropriate growth was observed. After incubation, cultures were diluted in PBS to levels of 100-1000 CFU/mL. Exclusivity strains were cultured onto non-selective agar under optimal conditions for growth and tested undiluted.
[0267] A 1.0 mL aliquot from the diluted target or undiluted non-target culture were randomized, blind coded and analyzed by the QuantX method.
Results
[0268] Of the additional inclusivity strains tested, all were correctly detected. All exclusivity cultures were non-detected. Tables 24 and 25 presents a summary of the results.
TABLE-US-00025 TABLE 24 Results for Inclusivity of the QuantX Method No. Organism QuantX Result 1 Kluyveromyces lactis Pass 2 Saccharomyces kudriavzevii Pass 3 Zygosaccharomyces bailii Pass 4 Kloeckera species Pass 5 Candida albicans Pass 6 Candida lusitaniae Pass 7 Candida tropicalis Pass 8 Dekkera bruxellensis Pass 9 Aureobasidium pullulans Pass 10 Rhodotorula mucilaginosa Pass 11 Cryptococcus neoformans Pass 12 Debaromyces hansenii Pass 13 Purpureocillium lilacinum Pass 14 Yarrowia lipolytica Pass 15 Wickerhamomyces anomala Pass 16 Stemphylium species Pass 17 Penicillium venetum Pass 18 Paecilomyces marquandii Pass 19 Scopulariopsis acremonium Pass 20 Mucor hiemalis Pass 21 Mucor circinelloides Pass 22 Talaromyces pinophilus Pass 23 Aspergillus fumigatus Pass 24 Talaromyces flavus Pass 25 Rhizopus stolonifera Pass 26 Cladosporium halotolerans Pass 27 Rhizopus oryzae Pass 28 Cladosporium herbarum Pass 29 Aspergillus aculeatus Pass 30 Penicillium chrysogenum Pass 31 Chaetomium globosum Pass 32 Arthrinium aureum Pass 33 Aspergillus brasilliensis Pass 34 Aspergillus caesiellus Pass 35 Curvularia lunata Pass 36 Cryptococcus laurentii Pass 37 Aspergillus terreus Pass 38 Byssochlamys fulva Pass 39 Penicillium rubens Pass 40 Geotrichum candidum Pass 41 Aspergillus flavus Pass 42 Fusarium solani Pass 43 Botrytis cinerea Pass 44 Aspergillus niger Pass 45 Aspergillus oryzae Pass 46 Fusarium proliferatum Pass 47 Fusarium oxysporum Pass 48 Paecilomyces variotii Pass 49 Geotrichum silvicola Pass 50 Alternaria alternata Pass
TABLE-US-00026 TABLE 25 Results for Exclusivity of the QuantX Method No. Organism QuantX Result 1 Acinetobacter baumanii Pass 2 Aeromonas hydrophila Pass 3 Burkholderia cepacia Pass 4 Citrobacter braakii Pass 5 Citrobacter farmeri Pass 6 Edwardsiella tarda Pass 7 Enterobacter cloacae Pass 8 Escherichia coli Pass 9 Hafnia alvei Pass 10 Listeria monocytogenes Pass 11 Pantoea agglomerans Pass 12 Proteus mirabilis Pass 13 Pseudomonas aeruginosa Pass 14 Pseudomonas gessardii Pass 15 Rahnella aquatilis Pass 16 Stenotrophomonas maltophilia Pass
Matrix Studies—Methodology
[0269] Cannabis test portions were prepared from Steadfast Analytical Laboratory's inventory of retained samples from its Michigan-licensed grower, patient, and caregiver customers. The samples were screened for yeast and mold prior to the study, using a rapid automated enumeration method in order to prepare matrix batches at the target contamination levels of <1000, ˜1000, ˜10000, and ˜100000 CFU/g.
[0270] Using sterilized aluminum containers, individual samples that produced results within a specified contamination level were combined to produce four batches (control, low, medium and high). Batches were manually mixed in an aseptic manner until homogeneous.
[0271] For each contamination level, five replicates were quantified by spread plating aliquots of the samples onto DRBC agar. Plating results indicated that yeast and mold levels for the control, low, medium, and high batches prepared for analysis were 350, 890, 13000, and 100000 CFU/g, respectively.
[0272] Five replicate test portions at the control and high levels, and 20 replicate test portions at the low and medium levels, were tested. A fractional positive data set (25-75% of test portions positive) was required for at least one of the intermediate levels at a minimum of one test threshold. Individual 10 g test portions from each contamination level were prepared in sterile filter Whirl-Pak bags. Test portions were assigned identification tags following Michigan's Marijuana Regulatory Agency (MRA) seed-to-sale system for distribution and tracking, including blind coding the contamination level of the test portions. The individual samples were also assigned random sample numbers for reporting results to the AOAC Research Institute. A technician at the independent laboratory not involved in the coding process performed the analyses.
[0273] Each test portion was combined with 90 mL PBS. Test portions were homogenized by hand and further 1:100, 1:1000 and 1:10,000 dilutions prepared using PBS as the diluent. From the final 1:1000 and 1:10000 dilutions, 1 mL aliquots were analyzed by the QuantX method.
[0274] For confirmation, 10 μL aliquots of the dilutions evaluated were streaked to DRBC agar. Plates were incubated at 25±1° C. for 5-7 days after which they were examined for yeast or mold growth.
Results
[0275] As per criteria outlined in Appendix J of the Official Methods of Analysis Manual and specified in the study protocol, fractional positive results were obtained for one of the dilution levels evaluated. Fractional positive data sets were obtained for the low level at the >1000 CFU/g test threshold. At this threshold, all control-level test portions produced negative results and all high-level test portions produced positive results.
[0276] Of the 100 data points encompassing all levels and test thresholds, there were seven instances of disagreement between presumptive and confirmed results: three low-level test portions at the >1000 CFU/g threshold were presumptive positive/confirmed negative, one medium-level test portion at the >1000 CFU/g threshold was presumptive positive/confirmed negative, one medium-level test portions at the >1000 CFU/g threshold were presumptive negative confirmed positive, and two high-level test portion at the >10000 CFU/g threshold was presumptive negative/confirmed positive.
[0277] The probability of detection (POD) was calculated for the candidate presumptive results, POD.sub.CP and the candidate confirmed results, POD.sub.CC, as well as the difference in the presumptive and confirmed results, dPOD.sub.CP. The POD analysis between the QuantX assay presumptive and confirmed results indicated that there was not a statistically significant difference. A summary of POD analyses are presented in Table 26.
Discussion
[0278] In the matrix study, the QuantX.sup.−Fungal assay successfully detected the target analyte from cannabis flower samples. The QuantX method demonstrated a high level of specificity in detecting the 50 inclusive organisms and no detection of the 30 exclusive organisms (Table 8 and 9). The POD statistical analysis in Table 10, indicated that the candidate method performance was identical to the reference method at low levels (320 CFU/g) but at the 890 CFU/g was statistically different than the reference method (95% CI −0.05, 0.35) with the candidate method detecting more positive samples. The two methods performed identical at the 13,000 CFU/g, both detecting 90% of the samples at the >1000 threshold and 0% at the >10,000 threshold. While it should be noted that the samples used in this study were held longer for analysis and may have resulted in the lower detection at the high level, the results of the QuantX and DRBC plating method align closely.
[0279] Thus, data from this study supports the product claim that the QuantX assay can detect total yeast and mold from cannabis flower at specific action thresholds used by state regulatory agencies. Data from the inclusivity and exclusivity analysis indicates the method is highly specific and can detect a wide range of target organisms and discriminate them from background organisms and near neighbors. The results obtained by the POD analysis of the method comparison study demonstrated that the candidate methods performance was not statistically different than that of the culture confirmation method.
TABLE-US-00027 TABLE 26 QuantX TYM presumptive and confirmed results fortesting of dried cannabis flower. Comparison between QuantX assay and plating (MH/PU). Test Level Threshold QuantX TYM Presumptive Quant TYM Confirmed Matrix Strain (CFU/g).sup.a (CFU/g).sup.b N.sup.c x.sup.d POD.sub.CP.sup.e 95% CI x POD.sub.CC.sup.f 95% CI dPOD.sub.CP.sup.g 95% CI.sup.h Dried Naturally 320 >1000 5 0 0 0.00, 0 0 0.00, 0.00 −0.47, Cannabis Contaminated 0.43 0.43 0.47 Flower >10000 5 0 0 0.00, 0 0 0.00, 0.00 −0.47, 0.43 0.43 0.47 890 >1000 20 9 0.45 0.26, 6 0.30 0.14, 0.15 −0.05, 0.66 0.52 0.35 >10000 20 0 0.00 0.00, 0 0.00 0.00, 0.00 −0.13, 0.16 0.16 0.13 13000 >1000 20 18 0.90 0.70, 18 0.90 0.70, 0.00 −0.19, 0.97 0.97 0.19 >10000 20 0 0.00 0.00, 0 0.00 0.00, −0.05 −0.13, 0.16 0.16 0.13 100000 >1000 5 5 1 0.57, 5 1 0.57, 0.00 −0.47, 1.00 1.00 0.47 >10000 5 0 1 0.00, 2 0.40 0.12, −0.40 −1.00, 0.43 0.77 0.21 .sup.aFrom aerobic viable yeast and mold plate count (DRBC). .sup.bBased on dilution and volume of sample tested. A positive result indicates contamination above the test threshold level. .sup.cN = Number of test portions. .sup.dx = Number of positive test portions. .sup.ePOD.sub.CP = Candidate method presumptive positive outcomes divided by the total number of trials. .sup.fPOD.sub.CC = Candidate method confirmed positive outcomes divided by the total number of trials. .sup.gdPOD.sub.CP = Difference between the candidate method presumptive result and candidate method confirmed result POD values. .sup.h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.
Example 6
[0280] Detection of Fungus in a plant sample
[0281] The method described below shows the developed trendline used for mathematical modeling modifications to the Augury Software (Augury Technology, NY).
Materials & Methods
Extraction of Fungal Nucleic Acids
[0282] 1 mL aliquots of A. nidulans (10{circumflex over ( )}5-10{circumflex over ( )}2) is transferred into a clean 1.5 mL tube and centrifuged (14,000×g for 3 minutes). The resulting supernatant from this step is decanted and the cell pellet retained. Lysis buffer (35 μl) is added to each tube, vortexed and heated at 95° C. for 10 min. The samples are removed from the heat source and centrifuged (2000×g for 5 seconds). To each tube, 5 μl of neutralization buffer is added and vortexed thoroughly to mix. Sample Buffer Mix (Table 17) is prepared and 25 μl added to each tube and vortexed to mix. The sample tubes are heated at 55° C. for 45 min to allow complete sample digestion. The samples are removed from the heat source and vortexed for 10 s. The sample tubes are then heated at 95° C. for 15 min.
Sample cleanup using RELIAPREP Kit
[0283] To each prepped lysate was added 32.5 μl of membrane binding solution and vortexed for 5 s. Isopropanol (97.54 of 100%) was added and vortexed for another 5 s. The sample was then loaded onto a RELIAPREP mini column seated in a collection tube, and centrifuged (10,000×g, 30 s). The contents in the collection tube were discarded, the column reseated into the collection tube and bound sample washed with 200 μL of Column Wash Solution (centrifuge at 10,000×g, 15 s). The contents were discarded, and the bound sample washed with 300 μL of Buffer B (centrifuge at 10,000×g, 15 s), repeating the wash one more with 300 μL of Buffer B. The contents were discarded, and the column centrifuged for 1 min to dry the column. The column was then transferred to a labelled Elution Tube, 154 of Nuclease-Free water or TE Buffer added and centrifuged for 30 s. Elution was repeated with an additional 154 of Nuclease Free Water or TE Buffer to maximize recovery.
Labeling PCR amplification
[0284] Reagents (PCR Master Mix, Primer Set, and High Standard) were thawed. The Low Standard was prepared by mixing 5 μl of the vortexed High Standard tube with 495 μl of Molecular Biology Grade Water and vortexed to mix. Table 18 was used as reference to calculate the appropriate reagent volumes needed based on the number of samples. All reagents (except Taq polymerase) were vortexed for 15 s and centrifuged (1000×g for 5 s). The indicated reagent volumes were mixed in a microfuge tube to prepare the Labeling PCR Master Mix. The PCR master mix was briefly vortexed and centrifuged (1000×g for 5 s). Amplification conditions were as shown in Table 19. The following primers were used—Forward primer SEQ ID NO:133, final concentration 50 nM) and Reverse primer (SEQ ID NO:134, 5′Cy3 labeled, final concentration 200 nM).
Hybridize PCR Amplified Product to Microarray
[0285] The Pre-hybridization Buffer and Hybridization Buffers were prepared in sterile tubes for the number of wells that will be hybridized (Tables 27 and 28) and vortexed to mix. The plate was placed in the Hybridization Chamber and the foil seal carefully removes from the wells to be hybridized. Molecular Biology Grade water (200 μL) was applied to each well, aspirated and another 200 μL of Molecular Biology Grade water added to each well. The plate was incubated in the Hybridization Chamber for 5 min and the water aspirated. Pre-hybridization Buffer (200 μL) was added to each designated well and allowed to sit covered in the Hybridization Chamber for 5 min. Hybridization Buffer (18 μL) was added to each well for hybridization within the 96-well PCR plate and pipetted up and down to mix. The Pre-hybridization Cocktail was aspirated from the array and the Hybridization Cocktail (68 μL) added immediately to each array. The plate was allowed to hybridize for 30 min at room temperature in the Hybridization Chamber. Wash Buffer was prepared (Table 29) and vortexed briefly to mix prior to adding (200 μl) to each array followed by aspirating immediately. Another 200 μL of Wash Buffer was added and incubated for 10 min. A final wash was performed by dispensing 200 μL of Wash Buffer and aspirating immediately. The plate was dried using a plate centrifuge for 5 min.
TABLE-US-00028 TABLE 27 Pre-hybridization buffer volumes Pre-hybridization reagents Volumes corresponding to one well Molecular Biology Grade water 137.6 μL Buffer 1 40.9 μL Buffer 2 21.5 μL
TABLE-US-00029 TABLE 28 Hybridization buffer volumes Hybridization reagents Volumes corresponding to one well Buffer 1 40.9 μL Buffer 2 21.5 μL
TABLE-US-00030 TABLE 29 Wash buffer volumes Wash buffer reagents Volumes corresponding to one well Buffer 1 5 μL Molecular Biology Grade water 555 μL
Results
[0286] A. nidulans cells prepared at 10.sup.5 down to 10.sup.2 dilutions were run to establish a trendline for Augury software calculations. The high, medium, and low Total Yeast and Mold RFU values correspond to the CFU values in the cell curve data.
Discussion
[0287] As the cannabis industry enters an era of acceptance at a national level, the methods developed by PathogenDx as disclosed in this invention are of direct relevance to cannabis testing at the national level. The suite of advanced testing and reporting technologies raises cannabis testing closer to the level of efficacy and standardization required of labs in other mainstream industries.
[0288] The one-step PCR for its QuantX fungal assay method described in this invention employs sample preparation step using RELIAPREP (Promega Corporation, WI). RELIAPREP shortens the assay process by consolidating the two-step PCR into a single PCR step, enabling results to be delivered in 4.5 hours instead of 6 hours, and helps concentrate the sample for improved sensitivity. Overall, the new methodology for preparing and analyzing cannabis improves assay reliability by reducing PCR inhibition and minimizing all types of dim signal.
[0289] Implementation of the expanded 96-well microarray format introduces to the cannabis industry a best practice commonly used in clinical labs. Instrumentation, reagents, and consumables are naturally fitted to a 96-well plate format for a higher level of efficiency, throughput, leading to economical scaling compared to prior 12-well formats. The methods described in this invention are supported by other improvements including, the industry-first foil-sealed wells that enable lab technicians to uncover only the wells needed to test samples received on that day or shift, thereby realizing significant cost savings from reduced waste of unused wells and test media. Moreover, the expanded microarray is made with higher quality glass that provides improved performance for both specificity and imaging accuracy.
[0290] To provide another level of granularity in test results reporting, PathogenDx is migrating from Dropbox to a custom PathogenDx Reporting Portal for cannabis compliance reporting. PathogenDx's intuitive, user-friendly portal drives customer ease and efficiency by reducing the number of steps necessary to obtain lab results and COAs. This also improves data visibility with multi-user access to real-time results tracking and prior history reports.
[0291] While the foregoing written description of an embodiments enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The present disclosure should therefore not be limited by the above-described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the present disclosure.
The following references are cited herein. [0292] 1. Emerald Scientific Cannabis Testing Regulations by State: Increase Your Knowledge. Emerald scientific.com/blog/cannabis-testing-regulations-by-state-increase-your-knowledge/(2018). [0293] 2. Verweij, et al. JAMA 284:2875 (2000). [0294] 3. Thompson, et al. Clinical Microbiology and Infection. 23(4):269-70 (2017). [0295] 4. Kern, R. and Green, J. R. Cannabis Science and Technology, November/December 2:(6) (2019). Cannabissciencetech.com/view/its-not-too-late-post-harvest-solutions-microbial-contamination-issues. [0296] 5. Colorado Department of Revenue Enforcement Division-Marijuana. MED 2019 Annual Update (2019). Drive.google.com/file/d/1rCWw9AquV9Pr1STMbv8dySrU6L2wJHfl/view. [0297] 6. Official Methods of Analysis 21st Ed., Appendix J: AOAC INTERNATIONAL, Rockville, Md., (2019). Eoma.aoac.org/app_j.pdf.