WO2016112179A1 - Methods and kits for detecting fungus and bacteria in cannabis - Google Patents

Abstract

A method of detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant includes amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-specific primers as set forth in SEQ ID NOs: 1 and 2 and in the presence of at least one of a fungus-specific primer and a bacteria- specific primer, to thereby form amplicons. The amplicons are compared with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

Description

METHODS AND KITS FOR DETECTING FUNGUS AND BACTERIA IN CANNABIS RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

62/101,762, filed on January 9, 2015. The entire teachings of the above applications are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

[0002] This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:

[0003] a) File name: 47231012001 SEQLISTING.txt; created January 7, 2016, 40 KB in size.

BACKGROUND OF THE INVENTION

[0004] Very few plant-related deaths are reported annually by the Center for Disease Control. However, there are documented cases of, for example, Cannabis related fatalities due to pathogens, such as the mold Aspergillus, which is often found cohabitating in or with a Cannabis plant. The inhalation of Aspergillus spores can lead to fungal infections in the lungs, which is increased in immuno-compromised patients. Patients seeking the immuno- regulatory benefits of Cannabis are often at increased risk for pathogens found on the plant. In addition to the risk of microbial infection, several molds synthesize mycotoxins that are not readily sterilized from plants. Many mycotoxins require the human liver microsome CYP3 A4 for metabolism and clearance. Phytocannabinoids native to the cannabis plant, such as Cannabidiol, potently inhibit CYP3A4 and CYP2C19 further complicating clearance of toxins produced by fungus, in particular Aflatoxin Bl produced by Aspergillus flavius and Aspergillus parasiticus.

[0005] Thus, a need exists for a rapid, cost effective pathogen and sensitive detection methods capable of detection in a background of plant nucleotides, such as Cannabis DNA, and flower contaminants.

SUMMARY OF THE INVENTION [0006] The invention generally relates to methods and kits for detecting the presence of at least one pathogen in a plant. In particular, the invention is generally directed to methods and kits for detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant.

[0007] In an embodiment, the invention is directed to a method of detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant comprising amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-s e ific primers, and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons. The amplicons are compared with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the

Cannabis plant with at least one of the fungus and bacteria.

[0008] In another embodiment, the invention is directed to a method of detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant comprising amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-specifxc primers as set forth in SEQ ID NOs: 1 and 2, and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons. The amplicons are compared with known nucleotide sequences of the

Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

[0009] In still another embodiment, the invention is directed to a kit for detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant that comprises amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-s ec ic primers and in the presence of at least one of a fungus- specific primer and a bacteria-specific primer, to thereby form amplicons; and comparing the amplicons with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

[0010] In a further embodiment, the invention is directed to a kit for detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant that comprises amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-s^ec ic primers as set forth in SEQ ID NOs: 1 and 2, and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons; and comparing the amplicons with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

[0011] The methods and kits described herein provide rapid, accurate and cost efficient procedures to detect the presence of pathogens in plants. In particular, the methods and kits described herein provide advantages to methods currently in use, which include

pasteurization and ELISA assays, to detect and eliminate pathogens, such as at least one of a fungus and a bacteria, in Cannabis plants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 : MicroSeq® fungal detection kit on Cannabis DNA .Lester= 1 1,250 genomic copies and Blue Dream= 25,000 genomic copies and Aspergillus Niger (ASN)= 1 10,000 genomic copies. The line indicated by the arrow labeled "NTC" (No Template Control ) is for the standard curve primers. MicroSeq® primers demonstrate about 100,000 fold more reactivity on Cannabis DNA than ITS 3 primers redesigned to be more mold specific. The MicroSeq® kit is more reactive for plant DNA than mold DNA. 1 10,000 genomic copies of Aspergillus DNA amplified with ITS primers provides nearly equivalent Ct as 1 1,250 genomic copies of Cannabis using MicroSeq® Primers. These same DNAs amplified with ITS primers are 5 logs lower in Ct signal (Curves indicated by solid arrows for Blue Dream and Lester samples).

[0013] FIG. 2A: Next generation sequencing of 10 cultivars. CLC assembly confirms CMK as the most stable genomic cannabinoid pathway gene. Reads mapped to mRNA sequences from NCBI and van Bakel et al. Genome Biol 12, R102 (2011) suggest putative copy number variation in AAE3, THCA Synthase, CBDA Synthase, and Desaturase. CMK remains consistently covered and a good candidate for qPCR assay development.

[0014] FIGs. 2B-2G: CMK qPCR amplicon assembly. Assembly demonstrates four distinct alleles with homology to CanSat3 scaffolds 8634 and 99544. CanSat3 is a diploid assembly thus 2 alleles are expected. The alignments shown in FIGs. 2B-2D are from forward reads (SEQ ID NOS:25-45) and the alignments shown in FIGs. 2E-2G are from Reverse reads (SEQ ID NOS:46-66). 150 base pair reads do not intersect on the 500 bp amplicon.

[0015] FIG. 3 : Linearity of Aspergillus detection. Copies of Aspergillus DNA spiked into Lester Grinspoon Cannabis DNA. 10, 50 and 200 copies of Aspergillus genome equivalents are easily detected in a background of 2500 copies of Cannabis DNA using the Total Yeast and Mold primers. Cannabis only (Lester 1) and No Template Control (NTC) show some background amplification at cycle 35. ITS2 primers were tested in qPCR with differing copies of Candida and Aspergillus Niger (AsN) DNA spiked into Pathogen Free plant DNA (Lesterl). With 2500 copies of plant genomic DNA, very little Yeast or mold signal is obtained (Similar to negative control amplification signal). When adding 10, 50, 100, 200 copies of Candida or AsN, significant delta CTs are observed suggesting primers that specifically amplify yeast and molds while not amplifying plant genomic DNA.

[0016] FIG. 4: Linearity of E. coli detection. E. coli Detection with . coli specific primers shows no background cannabis amplification or NTC signal. Primers are designed for Bacterial detection to avoid Cannabis DNA amplification. Copies (10, 50, 100, 200) of E. coli genomic DNA equivalents are spiked into 2500 copies of plant gDNA (Lesterl). qPCR is performed to demonstrate linear response.

[0017] FIG. 5: Linearity of Salmonella detection. Salmonella Detection with Salmonella specific primers shows no background with Cannabis only DNA or No Template Controls (NTC). Salmonella primers are designed for unique detection of Salmonella DNA in a background of Cannabis DNA. Copies (10, 50, 100, 200) of Salmonella genomics DNA are spiked into 2500 copies of plant gDNA (Lesterl). qPCR is performed to demonstrate linear response.

[0018] FIG. 6A: Multiplexing Salmonella assays with M13 tails. DNA purified from cultivar Lester Grinspoon is amplified in a multiplex TAQMAN protocol with Salmonella DNA spiked into the amplification. Two multiplexed amplifications are performed using the Salmonella primers and its FAM labeled internal probe in conjunction with the SCCG primers and its respective HEX labeled internal probe. The effect of Ml 3 tails and untailed primers is shown to be a single Ct shift in amplification. M13 Tails are added to the primers to enable easy sequencing of PCR products. Salmonella and SCCG primers (CMK plant genomic DNA) are multiplexed with FAM and FLEX fluorophores incorporation into the probe. SCCG primers are also M13 tailed and multiplexed into the assay for simultaneous amplification of plant genomic DNA (CMK gene) and pathogen genomic DNA. Relative amplification can be measured to assess plant to pathogen DNA quantities.

[0019] FIG. 6B: Multiplexing E.coli assays with M13 tails. E.coli assay performance is shown with and without multiplexing and with and without M13 tailing the amplicons. M13 Tails are added to the primers to enable easy sequencing of PCR products. E. coli and SCCG primers (CMK plant genomic DNA) are multiplexed with FAM and HEX fluorophores incorporation into the probe. SCCG primers are also Ml 3 tailed and multiplexed into the assay for simultaneous amplification of plant genomic DNA (CMK gene) and pathogen genomic DNA. Relative amplification can be measured to assess plant to pathogen DNA quantities.

[0020] FIG. 6C: Multiplexing Yeast and Mold assays with M13 tails. Multiplex PCR repeated with ITS 3 primers to detect Yeast and Mold concurrently with Cannabis genomic DNA. Ml 3 Tails are added to the primers to enable easy sequencing of PCR products. ITS primers (Yeast and Mold) and SCCG primers (CMK plant genomic DNA) are multiplexed with FAM and HEX fluorophores incorporation into the probe. SCCG primers are also M13 tailed and multiplexed into the assay for simultaneous amplification of plant genomic DNA (CMK gene) and pathogen genomic DNA. Relative amplification can be measured to assess plant to pathogen DNA quantities.

[0021] FIG. 7: Decontamination with DREAM PCR (U.S. Patent Application No.

14/341,540) AbaSI is used for decontamination during reaction set up. Hydroxy Methyl (HM) CTP amplified targets are spiked into reactions at 300K, 600K, 3M, 6M of HM modified amplicons to 2500 copies Cannabis DNA. At least a four CT shift is seen with the decontamination suggesting about a 16 fold reduction in contamination or over 5 M contaminating molecules destroyed in the highest condition tested. Hydroxy methylated qPCR is demonstrated to enable decontamination techniques. [0022] FIG. 8: qPCR vs BIOLUMIX^1M culture. BIOLUMIX^1M correlation with qPCR Ct measurements in triplicate demonstrate strong correlation between the two methods.

[0023] FIG. 9: Schematic of a magnetic particle separation showing nucleic acid isolation and purification from a complex biological sample. Depicts DNA purification method for capture of plant and pathogen DNA.

[0024] FIG. 10: An exemplary 5' nuclease fluorescent assay that includes 5' universal tails and 5hmCTP in PCR. Depicts TAQMAN qPCR assay design. M13 Tails were added to all assay designs to enable easy sequencing of the qPCR products.

[0025] FIGs. 11 A-l 10: Sequences of IPP (SEQ ID NO: 15) (FIGs. 11 A-l 1C), GPP lsu (SEQ ID NO: 16) (FIGs. 1 lD-1 IE), GPP ssu (SEQ ID NO: 17) (FIGs. 1 lF-11G), CMK partial (SEQ ID NO: 18) (FIG. 11H), CMK (SEQ ID NO: 19) (FIGs. 1 II- 1 IK) and AAE (SEQ ID NO: 20) (FIGs. 1 lL-110).

[0026] FIG. 12: Samples were cultured with 3 different techniques and compared to quantitative PCR (qPCR). BIOLUMIX™ had the lower sensitivity failing to pick up 4/17 samples detected with other culture-based platforms. qPCR identified 2 samples that were not picked by any other method. Positive qPCR samples were sequenced to identify the contributing signal. Outlined, bolded areas indicate samples that fail the 10,000 CFU/g cutoffs that equates to a Cq of 26 on the qPCR assay, (f) is Fail or over 10,000 CFU/g. (p) is Pass or under 10,000 CFU/g.

[0027] FIGs. 13A-13L: qPCR signal from TYM (Total Yeast & Mold) test run concurrently (multiplexed) with a plant internal control marker. This marker targets a conserved region in the cannabis genome and should show up in every assay (FIGs. 13 A, 13E, 131). SimPlates™ count the number of discolored wells as a proxy for CFU/gram (FIGs. 13B, 13F, 13J). Only total aerobic show growth (FIGs. 13B, 13F, 13J). Petrifilm only demonstrate colonies on Total Aerobic platings (FIGs. 13C, 13G, 13K). BIOLUMIX™ demonstrate no signal across all 4 tests (FIGs. 13D, 13H, 13L). FIGs. 13A-13D: Sample KD4.FIGs. 13E-13H: Sample KD8 fails to culture any Total Yeast and mold yet

demonstrates significant TYM qPCR signal. Sample was graduated to ITS based next generation sequencing. FIGs. 13I-13L: Sample Liberty Haze was tested with three culture based methods and compared to qPCR. Sample was graduated to ITS based next generation sequencing. [0028] FIGs. 14A-14G: DNA sequencing of ITS3 amplicons from culture negative samples that are qPCR positive for Total Yeast and Mold tests. Penicillium and Aspergillus are commonly found (Y axis) but at different read counts in each sample (X axis). Read counts are more a reflection of sample normalization for sequencing than inter sample quantitation provided by qPCR.

[0029] FIG. 15: One Codex classification of ITS reads P. paxilli is the most frequently found contaminant in Cannabis flowers. P. citrinum is not in the One Codex database at this time. One Codex utilizes a fast k-mer based approach for whole genome shotgun

classification and can be influenced by read trimming and database content. The reads provided to MG-RAST were trimmed and FLASHed (Paired end reads merged when overlapping) prior to classification. K-mer based approaches can significantly differ from longer word size methods and this underscores the importance of confirmatory PCR in microbiome analysis.

[0030] FIGs. 16A-16B: PaxP PCR demonstrates amplification of a 725bp band in sample KD8(FIG. 16 A). PCR products were made into a shotgun library with Nextera and sequenced on an Illumina MiSeq with 2x75bp reads to over 10,000X coverage. Reads were mapped with CLCbio 4 to NCBI Accession No. HM171111.1. Paired reads are displayed as unbroken lines, dashed lines are unpaired reads. Read Coverage over the amplicon is depicted in the upper histogram over the cluster while paired end read distance is measured in the lower histogram over the region. Off target read mapping is limited (FIG. 16B).

Alignment of PCR primers to P. paxilli reference shows a 5 prime mismatch that is a result of the primers being designed to target spliced RNA according to Saikia et al.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.

[0032] The invention is generally directed to methods and kits for detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant. [0033] In an embodiment, the invention is directed to a method of detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant comprising amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of

primers and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons. The amplicons are compared with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

[0034] In another embodiment, the invention is directed to a kit for detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant that comprises amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of

primers and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons; and comparing the amplicons with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

[0035] In an embodiment, the Cawrafos-specific primers employed in the methods or kits are SEQ ID NOs: 1 and 2.

[0036] Fungus-specific primers for use in the methods and kits described herein can include mold-specific primers, such as at least one of SEQ ID NOs: 4 and 5, as set forth in Table 1, infra. Bacteria-specific primers for use in the methods and kits of the invention can include E. co/z^'-specific primers, such as at least one of SEQ ID NOs: 7 and 8, Salmonella- specific primers, such as at least one of SEQ ID NOs: 10 and 11, as set forth in Table 1, infra.

[0037] In an embodiment, the fungus detected is at least one member selected from the group consisting of Sclerotina and Aspergillus. In another embodiment, the fungus is at least one member selected from the group consisting of a yeast and a mold. In a particular embodiment, the fungus is mold. [0038] In an embodiment, the mold is at least one member selected from the group consisting of Penicillium and Mucor. In a particular embodiment, the mold is Penicillium, such as at least one member selected from the group consisting of P. citrinum and P. paxilli.

[0039] In an additional embodiment, the bacteria detected is at least one member selected from the group consisting of E. coli, Coliform, Salmonella and Enterococcus.

[0040] The method described herein can further include the step of determining an amount of the at least one of the fungus and the bacteria present in the Cannabis plant by determining the amount of amplicons of the nucleotide sequence of the Cannabis plant and the amount of amplicons of the nucleotide sequence of at least one of the fungus and the bacteria. In an embodiment, the method can further include the step of assessing the amount of amplicons of the nucleotide sequence of at least one of the fungus and the bacteria to a standard.

[0041] The methods and kits described herein can include amplification in presence of at least one methylated dATP, dTTP, dGTP and dCTP. In an embodiment, the methylated dNTP is at least on member selected from the group consisting of methylated dCTP and methylated dATP. In a particular embodiment, the dCTP is at least one member selected from the group consisting of 5 -methyl cytosine and 5-hydroxymethyl cytosine and the methylated dATP is N6 methyl adenosine.

[0042] In another embodiment, the methods and kits described herein can include at least one of the Cannabis-specific primers and at least one of the fungus-specific primers and the bacterial specific primers that are labeled with at least one tag. In particular embodiments, the tag includes a fluorescent tag. In yet another embodiment, the at least one tag of the Cannabis-specific primers is distinct from at least one of the fungus-specific primers and the bacterial-specific primers. Exemplary tags includes at least one member selected from the group consisting of a HEX tag, a FAM tag, a TAMRA tag, a JOE tag, a R6G tag, a CY3 tag and a CY5 tag.

[0043] The method and kits described herein can further including the step of maintaining the amplicons of the nucleotide sequences of the Cannabis plant and the amplicons of the nucleotide sequence of at least one of the fungus and the bacteria under conditions in which amplicons that include methylated bases are digested by the at least one methyl specific restriction enzyme. Methyl specific restriction enzymes for use in the methods and kits include at least one member selected from the group consisting of MspJl, FspEl, LpnPI, AspBHI, Rial, SgrTI and AbaSI.

[0044] In an embodiment, the one or more nucleotide sequences of the Cannabis plant includes at least one member selected from the group consisting of a CMK locus, an IPP locus, a GPP lsu locus and a GPP ssu locus.

[0045] In a further embodiment, a portion of the Cannabis plant is cultured prior to amplifying the nucleotide sequences of the Cannabis plant. The Cannabis plant can be cultured for about 2, 4, 6, 8, 10, 12 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours.

[0046] In yet another embodiment, the amount of amplicons of the one or more nucleotide sequences of the at least one of the fungus and the bacteria present in the Cannabis plant cultured prior to amplifying is compared to the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus and the bacteria present in a portion of the Cannabis plant that was not cultured prior to amplifying and an increase in the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus and the bacteria present in the Cannabis plant that was cultured compared to the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus and the bacteria present in the Cannabis plant that was not cultured, indicates the presence of at least one of a live fungus and a live bacteria in the Cannabis plant.

[0047] In a further embodiment, the one or more nucleotide sequences of the Cannabis plant and the one or more nucleotide sequences of at least one the fungus and the bacteria are amplified simultaneously in the methods and kits described herein.

[0048] Exemplary amplification conditions for use in the methods and kits described herein can include a polymerase chain (PCR) reaction that includes an initial denaturation at about 95°C followed by about 40 cycles of 95°C at about 15 seconds, about 60°C for 90 seconds.

[0049] The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. As a result, the detection of mold and bacteria on agricultural products has become an important safety consideration. For example, this risk extends itself to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Described herein is a multiplexed PCR based approach to detect pathogenic DNA in a background of host plant {e.g., Cannabis) nucleic acid {e.g., DNA). This method can be applied in a variety of plant genomes {e.g., the Cannabis genome) as well as a variety of pathogenic microbial genomes.

[0050] Quantitative microbial detection methods traditionally require cell culturing or petri-dish plating. As described herein, these techniques can be augmented with rapid quantitative PCR and next generation sequencing based methods. This is due, in part, to the rapid price decline in nucleic acid based methods, but also because some pathogenic microbes are difficult to culture and therefore evade detection with culture-based approaches. Likewise, many culturing techniques rely on culture media selectivity. Culture media designed for specific yeast and mold is not resistant to all bacterial growth or off target fungal growth. The signal produced by culture-based methods requires either morphological ascertainment of colonies or other tools to verify the colonies generated are the target organism of concern. The exclusive use of culture-based methods is complicated by the existence of benign fungicidal endophytic microbes on Cannabis. To complement a culture- based methods lack of specificity, nucleic acid based methods have been developed to offer a DNA based signature with more species specificity. This specificity is more informative than the signal generated from an optical density reading of a culture or the numerical information given from a colony forming unit count (CFU) on an agar lawn.

[0051] However, current mold and bacteria qPCR based detection methods target DNA sequences present in most organisms known as rDNA sequences or Internal Transcribed Spacer regions (ITS). These sequences have highly conserved DNA primer sequences that exist in most species followed by penultimate hyper variable regions unique to each species. As a result ITS sequences have become popular DNA barcodes for species identification and phylogenetics. Since every mold species has some version of an ITS sequence, use of these sequences can afford detection of any mold species present in a mammalian cell culture. However, applying these primers to detecting mold with a plant DNA background has proven to be less effective (FIG. 1). These primers do not exclusively amplify mold and can amplify Cannabis DNA more readily than mold. ITS sequences can vary in copy number in plant species and this variable copy number can obscure the sensitive detection of microbial DNA targeting similar sequences in pathogens. [0052] As described herein, to properly detect pathogen nucleotide (e.g., DNA, RNA), such as fungal (e.g., mold) and bacterial nucleotide in the presence of plant nucleotide, primers were designed to more uniquely differentiate the pathogenic microbial nucleotide from endophytic microbial and host plant nucleotide. In an embodiment, Cannabis DNA is the plant DNA for which primers are designed to differentiate at least one of fungal or bacteria nucleotides from Cannabis DNA.

[0053] Designing qPCR primers that detect yeast and mold contaminating a plant with plant genomic DNA background is an iterative and challenging process. Initial experiments were performed with yeast and mold detection primers from Life Technologies, known as MicroSeq^®. These primes are reported to target conserved ITS sequences adjacent to hypervariable genomic DNA that can be used to phylotype a microbiome. These

hypervariable amplicons can also vary in length depending on the ITS2 region in each microbe. As a result of the hypervariable internal sequence, SYBR green was considered as a qPCR detection method.

[0054] TAQMAN is a more specific method for multiplex qPCR detection, yet requires knowledge of an additional conserved region for hybridization of detection probes. This third conserved region in the ITS regions can be challenging to find and requires extensive human review. These MicroSeq^® primers did not use TAQMAN probes and, thus, produced SYBR signals with pathogen free plant gDNA suggesting cross reactivity for plant ITS2 primer sequences. The MicroSeq^® primers were designed to detect yeast and mold DNA in a mammalian gDNA background as a singleplex assay with no internal control. Plants are more closely related to yeast and mold and, therefore, may require more specific primer designs and internal controls to properly differentiate yeast and mold DNA sequences from plant DNA sequences.

[0055] The singleplex SYBR green detection is also not ideal for multiplexing assays. Design of a plant gDNA internal control would require TAQMAN multiplex methods and more careful design of a probe sequence capable of detecting yeast and molds in plants. An internal control that helped to itemize signal from plant gDNA relative to microbial ITS2 signal would help to normalize the data.

[0056] In addition, the capacity to easily sequence the amplicons should be considered in the primer design by using PCR tails compatible with next generation sequencing. Primers focused on the ITS2 region were therefore modified to avoid cannabis genomic sequences for both primers and probe sequences and configured to contain sequencing tails. Probes were designed to not interact and fluoresce in unique wavelengths.

[0057] Internal control plant genomic DNA primers and probes were also designed to target a critical gene in the cannabinoid synthase pathway. There have been multiple reports of copy number variation in cannabinoid synthase related genes

(http://biorxiv.org/content/early/2015/10/08/028654). The increased polymorphism rate of this highly selected chemotype has resulted in decades of published primer design

optimization in the literature related to THCAS and CBDAS. To address this primer design problem, extensive sequencing was performed to identify SNPs and Indels in the general population so we could design primer pairs that would fall on conserved sequences unique and conserved in cannabis plants. This internal control plant gDNA signal is compared to the ITS2 signal to quantitate Microbial risks.

[0058] DNA purification from whole cannabis flowers is a metagenomic DNA isolation as many microbes and insects can exist in the sampled flower. This metagenomic DNA purification can produce variable DNA yields based on the genetics of the plant, the degree of trichome formation, cannabinoid and terpene expression, and the extent that the plant matter has been cured or contains high water content. As described herein, this tissue extraction variability can be partially addressed with a quantitative and ratiometric measurement of the total plant DNA present compared to the total pathogenic DNA. This presents a quantitative ratio of host to pathogen DNA that can be used as a proxy for putative colony forming units (CFU). In an embodiment, the methods described herein include a genomic marker in the plant genome that is stable, consistently diploid and/or does not vary substantially in copy number. A stable genomic region is preferred so that a qPCR assay can more accurately estimate the number of plant genome equivalents that are present in the DNA extraction. Examples of unstable regions are described by van Bakel et al, Genome Biol, 72/R102 (2011), which demonstrated that the AAE3 gene in the cannabinoid synthesis pathway is variable in copy number in a THCA positive plant and perhaps played a role in the evolution of higher THCA producing cannabis. Quantitative measurement of a loci under selective pressure and demonstrating variable copy number in the plant genome could lead to variable and inaccurate host to pathogen DNA ratios. A more stable loci unique to the Cannabis plant is desired for methods to detect pathogens, in particular at least one of a fungus and a bacteria in the Cannabis plant. [0059] Several Cannabis genome sequences are now available and one of the most remarkable findings in these genomes is a very high AT content (65%) in addition to a very high polymorphism rate (over 1.3%). This is consistent with the high diversity of

chemotypes and phenotypes reported in the Cannabis literature. Due to the diversity in Cannabis cultivars, selecting a DNA target for amplification must be done with knowledge of the variants in many genomes. This high polymorphism rate and copy number variation can complicate the detection of a control Cannabis amplicon and effect pathogen detection and CFU estimation in any ratio metric analysis. To address this variability provided herein is a multiplexed quantitative amplification {e.g., polymerase chain reaction (PCR)) assay that assesses the Cannabis DNA yield in comparison to the pathogens DNA yield where amplicons have been selected based on many Cannabis genomes. In a particular

embodiment, the multiplexed quantitative amplification {e.g., polymerase chain reaction (PCR)) assay simultaneously assesses the Cannabis DNA yield in comparison to the pathogens DNA yield where amplicons have been selected with the careful review of many Cannabis genomes.

[0060] Comparison of dozens of Cannabis genomes can inform a strategic selection of highly conserved and stable host sequences. An ideal target would be a gene unique to cannabis and cannabinoid production while also demonstrating stable copy number, van Bakel et al. specifically focused on the copy number and transcriptional activity of multiple genes in the cannabinoid synthesis pathway in two Cannabis cultivars (one THCA- Finola hemp and one THCA+ Purple Kush). As described herein, these data were combined with 24M reads from each of 10 additional strains. This sequencing highlighted CMK as an example of a stable {e.g., consistent copy number) candidate Cannabis quantitative amplicon (FIG. 2A).

[0061] As described herein, multiplex amplification of the CMK loci and a pathogen specific loci enabled a DNA purification system highly tolerant to the diversity of the Cannabis cultivars in the field. The CMK gene was selected as a single copy cannabis gene (SCCG) internal control. Cannabis is a diploid plant so any single copy gene will have two copies in flower tissue so SNPs can still play an important role in primer design. As a result, a 500 base pair amplicon was designed, amplified, cloned and sequenced in 10 diploid cultivars to better understand its variation in the Cannabis population. In some embodiments, amplifying both plant and pathogen amplicons in a single well allows for the determination of how much plant DNA is present in the mixture concurrently with how much ITS sequence is present. As described herein, a single copy plant gene like CMK is a valuable internal control that can normalize for plant to plant variation in DNA yield. Any incremental ITS signal can be attributed to non-plant species.

[0062] An embodiment of the method described herein is shown in FIGs. 9-11. In an embodiment, magnetic particle separation is employed for the purification of both plant and microbial DNA from a raw homogenized sample. Magnetic particle separation is a highly economical, efficient and automatable process to isolate DNA from a single sample or a large batch in less than about 30 minutes. DNA is bound to magnetic particles, which are separated from the sample using a magnetic device. The isolated DNA can then be purified and used for downstream analysis. An amplification (PCR) based assay that is contamination free and provides an internal plant DNA control for every reaction. DNA detection is based on a 5' nuclease assay that directly measures the amount of plant and microbe DNA in a given sample. This technique provides robust sensitivity (e.g., detection down to about 1 molecule), specificity (only targeted DNA sequences are detected), and multiplexing capability (multiple fluorescent molecules can be combined in a single tube to provide detection of multiple pathogens in a single reaction). The methods described herein employ a multiplexing strategy with an internal plant DNA reaction control to ensure accurate detection of microbial species for every reaction. Cannabis genomes were decoded to select a suitable plant control target. Unlike other techniques this multiplexing strategy verifies the performance of the assay when detecting pathogens resulting in an elimination of false negatives due to reaction set-up errors or failing experimental conditions. Amplification reactions, such as PCR, provide a consistent and reproducible level of detection over a large dynamic range from about 1 to less than about 1 billion copies of the target DNA with a linear correlation between input amount and target detection.

[0063] In an embodiment, the methods described herein are detecting one or more pathogens present in plant material from one or more plants comprising amplifying one or more highly conserved and stable nucleotide sequences of the one or more plants (e.g., the control) and one or more nucleotide sequences of the one or more pathogens, thereby producing an amount of amplicons of the one or more highly conserved and stable nucleotide sequences of the one or more plants and an amount of amplicons of the one or more nucleotide sequences of the one or more pathogens. The presence of amplicons of the one or more highly conserved and stable nucleotide sequences of the plants and the presence of amplicons of the one or more nucleotide sequences of the pathogens are determined, wherein if amplicons of the one or more highly conserved and stable nucleotide sequences of the one or more plants and amplicons of the one or more nucleotide sequences of the one or more pathogens are present, then one or more pathogens are present in the plant material.

[0064] In a particular embodiment, the one or more highly conserved and stable nucleotide sequences of the one or more plants and the one or more nucleotide sequences of the one or more pathogens are amplified simultaneously. In an embodiment, the method of detecting (e.g., simultaneously detecting) one or more plant specific DNA sequences concurrently with pathogen DNA sequences comprises detecting a ratio of plant to microbial DNA, such as detecting a ratio of plant amplicons to pathogen amplicons, in particular at least one of a fungus and a bacteria. In an embodiment, the lack of signal in qPCR is being used as a proxy for microbial safety. When lack of a signal in qPCR is a proxy for microbial safety, it is important to confirm the PCR reaction properly performed. The plant amplicon, such as SCCG, acts an internal control to verify that the PCR was properly setup, performed to specification and assists to enumerate the plant DNA purification yield. The yield information can be inferred from the CT value, which can assist in enumerating the relative amount of microbial genomes present relative to the plant genomes equivalents.

[0065] In another embodiment, the method can further include determining the amount of the one or more pathogens present in the plant material by determining the amount of amplicons of the one or more highly conserved and stable nucleotide sequences of the plants and/or the amount of amplicons of the one or more nucleotide sequences of the pathogens.

[0066] In an embodiment, the amount of amplicons is an absolute amount. In another embodiment, the amount of amplicons is a relative amount in comparison to a control or standard. The control or standard can be at least one of the amount of amplicons of the one or more nucleotide sequence of the pathogens relative to the amount of amplicons of the one or more highly conserved and stable nucleotide sequences of the plants or the amount of amplicons of the one or more nucleotide sequence of the pathogens relative to a standard value or reference standard established in the art. In yet another embodiment, the amount of amplicons is detected by determining the cycle threshold (Ct) of the amplicons of at least one of the one or more nucleotide sequences of the pathogens and the amplicons of the one or more highly conserved and stable nucleotide sequences of the plants. As used herein, "Ct" refers to the number of amplification cycles, the accumulation of the signal, such as a fluorescent signal associated with amplification of a particular sequence required for the amplification to reach a particular threshold or baseline. Baseline can be the background or noise level of the method.

[0067] In an embodiment, the method can further include comparing the amount of amplicons of the one or more nucleotide sequence of the pathogens to a control or standard, also referred to as a reference standard. In embodiments, the method can include determining the amount of amplicons of at least one of the one or more nucleotide sequence of the pathogens and the amplicons of the one or more highly conserved and stable nucleotide sequences of the plants that exceed a particular level. In a particular embodiment, the amount is measured as colony forming units/gram (CFU)/g).

[0068] As used herein, "amplifying" "amplification" or an "amplification reaction" refers to methods for amplification of a nucleotide sequence including polymerase chain reaction (PCR), multiplexed PCR, next generation sequencing, ligase chain reaction (LCR), rolling circle amplification (RCA), strand displacement amplification (SDA) and multiple displacement amplification (MDA), serial amplification as will be understood by a person of skill in the art. Methods for amplification include primers that anneal to the nucleotide sequence to be amplified, a DNA polymerase and nucleotides.

[0069] Amplification methods, such as PCR, can be solid-phase amplification, polony amplification, colony amplification, emulsion PCR, bead RCA, surface RCA, surface SDA, etc., as will be recognized by one of skill in the art. It will also be recognized that it is advantageous to use an amplification method that results in exponential amplification of free DNA molecules in solution or tethered to a suitable matrix by only one end of the DNA molecule. Methods that rely on bridge PCR, where both PCR primers are attached to a surface (see, e.g., WO/18957 and Adessi et al, Nucleic Acids Research (2000): 28(20): E87) result in only linear amplification, which does not produce sufficient amounts of product to support efficient library construction for subsequent sequencing. Furthermore, the products of bridge PCR technologies are array -bound and are cleaved from the support as intact double stranded DNA molecules to be useful for subsequent sequencing. In addition, it will be recognized that it is often advantageous to use amplification protocols that maximize the fidelity of the amplified products to be used as templates in DNA sequencing procedures. Such protocols use, for example, DNA polymerases with at least one of a strong discrimination against misincorporation of incorrect nucleotides and strong 3' exonuclease activities (also referred to as proofreading or editing activities) to remove misincorporated nucleotides during polymerization.

[0070] In a particular embodiment, the one or more highly conserved and stable nucleotide sequences of the one or more plants and the one or more nucleotide sequences of the one or more pathogens are amplified by a method comprising contacting the sequences with (i) one or more primers that hybridize to one or more highly conserved and stable nucleotide sequences of the one or more plants, (ii) one or more primers that hybridize to one or more nucleotide sequences of the one or more pathogens, (iii) deoxynucleotide

triphosphates (dNTPs) comprising dATP, dTTP, dGTP and dCTP and (iv) a nucleic acid polymerase, thereby producing a combination. The combination is maintained under conditions in which the one or more highly conserved and stable nucleotide sequences of the one or more plants and the one or more nucleotide sequences of the one or more pathogens are amplified. In some embodiments, at least one of the dNTPs is modified (a modified base).

[0071] As described herein, internal plant (e.g., Cannabis) control amplicons are used to troubleshoot amplification failure for use in e.g., detecting one or more pathogens in plant material. With amplification-based methods, it is also important to safeguard against amplification that should not occur such as amplification that might arise from DNA contamination from previous amplicons. To address this, in an embodiment, the method of detecting one or more pathogens present in plant material from one or more plants can further include a decontamination step or method to afford multiplexed PCR without the

requirements of a cleanroom (see, for example, Published International Application No. WO 2014/081511 and U.S. Application No. 14/341,540. In an embodiment, such a method utilizes amplification, such as PCR, with one or more modified bases, such as a methylated nucleotide, including a methylated deoxynucleotide triphosphates (dNTPs).

[0072] Amplicons generated with a methylated nucleotide (e.g., 5hme-dCTP) are susceptible to digestion with one or more methyl specific enzymes (e.g., AbaSI). Prior to, and of after a PCR reaction, DNA samples are incubated with one or more methyl specific enzymes to ensure no amplicons from previous amplification reactions can be contaminating the reaction set up. This is an important feature for safety testing of plants, such as Cannabis, as the majority of other assays required to test for cannabis safety (cannabinoids, pesticides and heavy metals) do not utilize amplified assays and, thus, rarely require clean rooms.

Microbiological methods require the laboratory exponentially amplify the contaminant for measurements. As a result, sterile technique and decontamination methods become important laboratory procedures.

[0073] As used herein, the term "primer" refers to an oligonucleotide that is capable of acting as a point for the initiation of synthesis of a primer extension product that is complementary to the template polynucleotide sequence (e.g., the conserved and stable nucleotide sequence of the one or more plants; the nucleotide sequence of the one or more pathogens). The primer may occur naturally, as in a purified restriction digest, or be produced synthetically. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from about 5 to about 100; from about 5 to about 75; from about 5 to about 50; from about 10 to about 35; from about 18 to about 22 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently

complementary to hybridize with a template for primer elongation to occur, i.e., the primer is sufficiently complementary to the template polynucleotide sequence such that the primer will anneal to the template under conditions that permit primer extension.

[0074] As used herein, the phrase "conditions in which the nucleotide sequence is amplified," or "conditions that permit primer extension," refers to those conditions, such as salt concentration (metallic and non-metallic salts), pH, temperature, and necessary cofactor concentration, among others, under which a given polymerase enzyme catalyzes the extension of an annealed primer. Conditions for the primer extension activity of a wide range of polymerase enzymes are known in the art. Exemplary conditions permitting the extension of a nucleotide primer by Taq polymerase include the following (for any given enzyme, there can and often will be more than one set of such conditions): reactions are conducted in a buffer containing 50 mM KCl, 10 mM Tris (pH 8.3), 4 mM MgC12, (200 mM of one or more dNTPs and/or a chain terminator may be included, depending upon the type of primer extension or sequencing being performed); reactions are performed at 72°C.

[0075] It will be clear to persons skilled in the art that the size of the primer and the stability of hybridization will be dependent to some degree on the ratio of A-T to C-G base pairings, since more hydrogen bonding is available in a C-G pairing. Also, the skilled person will consider the degree of homology between the extension primer to other parts of the amplified sequence and choose the degree of stringency accordingly. Guidance for such routine experimentation can be found in the literature, for example, Molecular Cloning: a laboratory manual by Sambrook, J., Fritsch E. F. and Maniatis, T. (1989).

[0076] Conditions for amplification will vary depending upon the type of sequence being amplified and the type of amplification being used. Exemplary conditions under which an amplification reaction is maintained in order to amplify a nucleotide sequence include one or more amplification cycles which comprises 98°C for 20 seconds, 60°C for 15 seconds, 72°C for 60 seconds; 12°C for 60 seconds, 98°C for 20 seconds, 60°C for 15 seconds, 72°C for 60 seconds, 12 sequencing cycles at 98°C for 20 seconds, 72°C for 3 minutes; an initial 1 minute denaturization at 94°C followed by 30 cycles of 98°C for 10s, 68°C for 15minutes;

performance of a final 72°C 10 minute extension prior to 4°C hold; 12 Cycles of 72°C for 3 minutes, 98°C for 30 seconds, 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minute. In some embodiments, the amplification reaction can comprise a heat kill which is followed by a Phi-29 isothermal incorporation (e.g., 80°C/20 minutes to heat kill the MspJI/AbaSI and then add Phi29 for methylated isothermal amp at 37°C, Bst polymerase isothermal amps. In an embodiment, the sequences are amplified in a polymerase chain (PCR) reaction comprising an initial denaturation at about 95°C followed by about 40 cycles of 95°C at about 15 seconds, about 60°C for 90 seconds.

[0077] As used herein, the term "base" refers to the heterocyclic nitrogenous base of a nucleotide or nucleotide analog (e.g., a purine, a pyrimidine, a 7-deazapurine). A

"nucleoside" refers to a nitrogenous base linked to a sugar molecule. A "nucleotide" (e.g., "deoxyribonuleotide (dNTP)", "ribonucleotide") is a nitrogenous heterocyclic base (or nucleobase), which can be either a double-ringed purine or a single-ringed pyrimidine; a five- carbon pentose sugar (deoxyribose in DNA or ribose in RNA); and a phosphate group.

Suitable bases for use in the methods of the invention include, but are not limited to, adenine (A) (e.g., dATP), cytosine (C) (e.g., dCTP), guanine (G) (e.g., dGTP), thymine (T) (e.g., dTTP), and uracil (U) (e.g., dUTP). These and other suitable bases will permit a nucleotide bearing the base to be enzymatically incorporated into a polynucleotide chain. The base will also be capable of forming a base pair involving hydrogen bonding with a base on another nucleotide or nucleotide analog. The base pair can be either a conventional (standard) Watson-Crick base pair or a non-conventional (non-standard) non-Watson-Crick base pair, for example, a Hoogstein base pair or bidentate base pair. The terms "base" and

"deoxy nucleotide triphosphate (dNTP)" are at times used interchangeably. [0078] A "modified base" comprises one or more moieties that renders the base cleavable (a cleavable base) by one or more restriction enzymes. The terms "modified base" and "modified deoxynucleotide triphiosphate" are at times used interchangeably. As will be appreciated by those of skill in the art a restriction enzyme can specifically recognize and cleave a particular cleavable base (e.g., a single cleavable base), or can recognize and cleave more than one cleavable base. A variety of modified bases are known in the art, such as modified purine bases (e.g., Hypoxanthine, Xanthine, 7-Methylguanine, Inosine, Xanthosine, 7-Methylguanosine) and modified pyrimidine bases (e.g., 5,6-Dihydrouracil, 5- Methylcytosine, 5-Hydroxymethylcytosine, Dihydrouridine, 5-Methylcytidine).

[0079] In embodiments, the modified base is at least one member selected from the group consisting of a methylated, hydroxymethylated and a formylated base. In one embodiment, the modified base is a formylated deoxynucleotide triphosphate (dNTP). In other

embodiments, the modified base is a methylated dNTP. In yet additional embodiments, the modified base is at least one member selected from the group consisting of a methylated dNTP and a hydroxymethylated dNTP. In additional embodiments, the one or more methylated deoxynucleotide triphosphates is at least one member selected from the group consisting of one or more methylated cytosines, one or more hydroxymethylated dNTPs and one or more methylated adenosines. In yet further embodiments, the one or more methylated cytosines is at least one member selected from the group consisting of 5-methyl cytosine and 5 -hydroxy methyl cytosine. In yet other embodiments, the one or more methylated

adenosines is N6 methyl adenosine.

[0080] In other embodiments, the modified base is employed in an amplification reaction. In some embodiments, all or some of a (one or more) particular dNTP are modified (e.g., methylated). In other embodiments, about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc. of a (one or more) particular dNTP are modified. In yet further embodiments, about 25% of a (one or more) particular dNTP are methylated.

[0081] As described herein, the modified base is cleavable by one or more restriction enzymes. As will be appreciated by those of skill in the art a restriction enzyme can specifically (selectively) recognize and cleave a particular cleavable base (e.g., a single cleavable base) to the exclusion of other cleavable bases, or can recognize and cleave more than one cleavable base. In some embodiments, the restriction enzyme digests a nucleotide sequence at the site of the modified base or at a site (loci) that is distant from the modified base {e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, etc. bases away from the modified based {e.g., methylated base)). In other embodiments, the restriction enzyme can cleave a nucleotide sequence comprising a methylated base {e.g., a methyl specific restriction enzyme), a nucleotide sequence comprising a hydroxymethylated base {e.g., a hydroxymethyl specific restriction enzyme), or a nucleotide sequence comprising methylated bases and hydroxymethylated bases.

[0082] In particular embodiments, the restriction enzyme is capable of being deactivated {e.g., denatured). In other embodiments, the restriction enzyme is deactivated upon a change {e.g., increase; decrease) in temperature {e.g., heat labile; cold labile), a change {e.g., increase; decrease) in pH {e.g., pH labile), contact with a reagent {e.g., cofactors which can differentially chelate (EGTA for Ca2+ and EDTA for Mg2+), or a combination thereof. In other embodiments, the deactivation of the restriction enzyme is permanent. In these embodiments, upon deactivation, the restriction enzyme cannot be reactivated {e.g., renatured; brought back to its native (active) form). In embodiments, in which more than one restriction enzyme is used, the first methyl specific restriction enzyme, the second methyl specific restriction enzyme or both are deactivated upon a change in temperature, a change in pH, contact with a reagent (cofactors which can differentially chelated (EGTA for Ca2+ and EDTA for Mg2+).

[0083] As described herein, an amplification reaction comprising amplicons that include one or modified bases is contacted with a restriction enzyme that is capable of being deactivated to produce a combination, and the combination is maintained under conditions in which amplicons comprising the modified base which is recognized and cleavable by the restriction enzyme are digested by the restriction enzyme prior to amplification. As is known in the art, many amplification reactions comprise one or more steps that involve an increase in temperature {e.g., to denature a nucleotide sequence such as double stranded DNA).

[0084] Thus, in some embodiments, the restriction enzyme used in the methods of the invention is deactivated upon a change in temperature. In a particular embodiment, the restriction enzyme is deactivated upon an increase in temperature {e.g., a heat labile restriction enzyme), such as during amplification of a nucleotide sequence in an amplification reaction. Once the amplification reaction which includes a step that involves an increase in temperature occurs, the restriction enzyme is deactivated. Thus, after amplification, amplicons that include the modified base that is recognized and cleavable by the restriction enzyme, will not be digested by the restriction enzyme since it is longer active.

[0085] In some embodiments, the methyl specific restriction enzyme is at least one member selected from the group consisting of MspJl, FspEl, LpnPI, AspBHI, Rial, SgrTI and AbaSI or a combination thereof.

[0086] As described herein, amplification or extension of a primer (e.g., DNA synthesis) can be accomplished using a nucleic acid polymerase which is capable of enzymatically- incorporating both standard (dNTPs) and modified thiol deoxynucleotides (sdNTPs) into a growing nucleic acid strand. As used herein, the phrase a "nucleic acid polymerase" or "nucleic acid polymerase enzyme" refers to an enzyme (e.g., naturally-occurring,

recombinant, synthetic) that catalyzes the template-dependent polymerization of nucleoside triphosphates to form primer extension products that are complementary to one of the nucleic acid strands of the template nucleotide acid sequence. Numerous nucleotide acid

polymerases are known in the art and are commercially available. In particular embodiments, the nucleic acid or nucleotide polymerases are thermostable, i.e., retain function after being subjected to temperatures sufficient to denature annealed strands of complementary nucleic acids.

[0087] Suitable polymerases for the methods of the present invention include any polymerase known in the art to be useful for recognizing and incorporating standard deoxynucleotides. Exemplary polymerases are described in Table 1 of U.S. Patent No.

6,858,393. Many polymerases are known by those of skill in the art to possess a proofreading, or exonucleolytic activity, which can result in digestion of 3' ends that are available for primer extension. In order to avoid this potential problem, it may be desirable to use a polymerase enzyme that lacks this activity, such as exonuclease-deficient polymerases, referred to herein as "exo- polymerases." Such polymerases are well known to those of skill in the art and include, for example, Klenow fragment of E. Coli DNA polymerase I,

Sequenase, exo- Thermus aquaticus (Taq) DNA polymerase and exo- Bacillus

stearothermophilus (Bst) DNA polymerase. In a particular embodiment, incorporation of deoxynucleotides, including modified deoxynucleotides (dNTPs), into a growing nucleotide strand (e.g., DNA) is accomplished using a nucleic acid amplification reaction, such as PCR. Therefore, especially suitable polymerases for the methods of the present invention include those that are stable and function at high temperatures (i.e., thermostable polymerases useful in PCR thermal cycling). Exemplary polymerases include Thermus aquaticus (Taq) DNA polymerase, TaqFS DNA polymerase, thermosequenase, Therminator DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Q5 polymerase (New England Biolabs) and Vent (exo- )DNA polymerase. In another embodiment, incorporation of triphosphates into RNA is accomplished using an RNA polymerase. Examples of RNA polymerases include E.coli RNA polymerase, T7 RNA polymerase and T3 RNA polymerases.

[0088] In embodiments, prior to amplifying the one or more highly conserved and stable nucleotide acid sequences of the one or more plants and the one or more nucleotide sequences of the one or more pathogens, the plant material is contacted with at least one methyl specific restriction enzyme, thereby producing a combination, and the combination is maintained under conditions in which amplicons comprising one or more methylated bases are digested by the at least one methyl specific restriction enzyme. In another embodiment, after determining the amount of amplicons of the one or more highly conserved and stable nucleotide sequences of the one or more plants and the amount of amplicons of the one or more nucleotide sequences of the one or more pathogens, the plant material is contacted with at least one methyl specific restriction enzyme, thereby producing a combination, and the combination is maintained under conditions in which amplicons comprising one or more methylated bases are digested by the at least one methyl specific restriction enzyme.

[0089] In additional embodiments, the primers used in the amplification reaction are labeled with one or more tags. In one embodiment, the one or more tags comprise tailed primers that enable subsequent sequencing {e.g., next generation sequencing). In other embodiments, the one or more of the tags comprises a fluorescent tag {e.g., Rosenblum et al, Nucleic Acids Res., 25(22):4500-4504 (1997); Lee et al., Nucleic Acids Res., 25(14):2%\6- 2822 (1997)). In further embodiments, the tag(s) on the one or more primers that hybridize to the one or more highly conserved and stable nucleotide acid sequences of the one or more plants are the same or different from the tag(s) on the one or more primers that hybridize to one or more nucleotide sequences of the one or more pathogens. For example, in an embodiment, a single tag (the same tag) is used for the one or more primers that hybridize to the one or more highly conserved and stable nucleotide sequences of the one or more plants and the one or more primers that hybridize to one or more nucleotide sequences of the one or more pathogens. In another embodiment, the one or more tags used for the one or more primers that hybridize to the one or more highly conserved and stable nucleotide sequences of the one or more plants are different from tags used for the one or more primers that hybridize to one or more nucleotide sequences of the one or more pathogens (thereby allowing for distinguishing amplicons of plant nucleotide sequences from amplicons of pathogens.

Exemplary tags for use in the method include at least one member selected from the group consisting of a HEX tag, a FAM tag, a TAMRA tag, a JOE tag, a R6G tag, a CY3 tag and a CY5 tag.

[0090] As described herein, the methods of detecting one or more pathogens in plant material from one or more plants comprises the use of one or more highly conserved and stable nucleotide sequences of the one or more plants. As used herein, a highly conserved and stable nucleotide sequences of a plant refers to a sequence that is conserved, has a consistent copy number and/or has a consistent ploidy (e.g., consistently diploid, consistently haploid). In certain embodiments, a conserved DNA sequence is one that is conserved in copy number to relative to the diploid nature of the genome. Conserved regions can have S Ps and insertion and deletions but preferably not vary in copy number relative to other diploid genes in the genome. Examples of highly conserved and stable nucleotide sequences of a Cannabis plant include at least one member selected from the group consisting of CMK locus, an IPP locus, a GPP lsu locus and a GPP ssu locus.

[0091] Pathogens that can be detected employed the methods described herein, include at least one member selected from the group consisting of bacteria, fungus and viruses.

Exemplary bacteria include E. coli, Salmonella (S. enteritidis, S. typhimurium),

Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Listeria

monocytogenes, Shigella, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enter ocolitica. Exemplary fungus include yeast and mold. Exemplary particular fungus include Sclerotina, Aspergillis, Mucor, Actinomycetes (e.g., Thermophilic Actinomycetes), Penicillium and Saccharomyces (e.g., Saccharomyces cerevisiae). Exemplary particular viruses include a Sunn hemp mosaic virus and a Tobacco Mosaic virus.

[0092] The methods described herein can be employed to detect one or more pathogens, in particular at least one member selected from the group consisting of a fungus and a bacteria, in a variety of plants. Exemplary plants include medicinal plants (e.g., Cannabis), agricultural plants (e.g., food, tobacco), landscaping plants (e.g., shrubs) and trees. As is apparent to those of skill in the art, a variety of methods can be used to obtain nucleic acids or nucleotide sequences from one or more plants. Such methods include magnetic separation as shown in FIG. 9.

[0093] The methods described herein can further include determining whether one or more pathogens present in the plant material is alive or dead. Thus, the method can further include the step of culturing a portion of the plant material prior to amplifying the one or more highly conserved and stable nucleotide sequences of the one or more plants and the one or more nucleotide sequences of the one or more pathogens. The amount of amplicons of the one or more nucleotide sequences of the pathogens present in the plant material cultured prior to amplifying is compared to the amount of amplicons of the one or more nucleotide sequences of the pathogens present in a portion of the plant material that was not cultured prior to amplifying, wherein an increase in the amount of amplicons of the one or more nucleotide sequences of the pathogens present in the plant material that was cultured compared to the amount of amplicons of the one or more nucleotide sequences of the pathogens present in the plant material that was not cultured, indicates the presence of live pathogens present in the plant material.

[0094] In another embodiment, the method can further include the step of UV treating a plant material to eliminate one or more pathogens (e.g., mold and bacteria) content by treating the plant sample e.g., with 254nm up to 405nm UV light. UV light can kill the pathogen, but it will not eliminate the DNA. This may help sterilize material, but in the case of Aspergillus, it may kill the microbe, but may not eliminate the toxins, such as aflatoxin. Thus, UV light may have limitations and care should be taken with microbes that make UV resistant toxins.

[0095] As will be apparent to those of skill in the art, the portion of the plant material can be cultured under a variety of conditions and over a variety of times. For example, the portion of the plant material can be cultured for about 2, 4, 6, 8, 10, 12 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours.

[0096] Articles such as "a", "an", "the" and the like, may mean one or more than one unless indicated to the contrary or otherwise evident from the context. EXEMPLIFICATION

[0097] EXAMPLE 1

[0098] Multiplexed quantitative PCR for pathogenic microbial detection on Cannabis sativa L.

[0099] Methods

[00100] DNA Purification

[00101] Genomic DNA was isolated by homogenization of 250mg of cannabis flowers in 3.8 ml Tryptic Soy Broth (TSB). Whirl-pak bags (Nasco #B01385WA) were used to filter homogenized TSB from the grinded plant matter. Eluent (285 μΐ) was combined with 15 μΐ of Lysis buffer (10% LiDS) in a 1.5 mL tube. The tube was vortexed for 5 seconds and incubated for 2 minutes. The tube was then spun in a micro centrifuge for 30 seconds. 200ul was removed (being careful not to disturb the pellet) and placed into a 96 well plate. 200ul of Binding buffer (20% PEG, 2.25M NaCl, 1% solids COOH Seramag Speedbeads) was tip mixed into the solution to capture DNA to magnetic particles. Magnetic particles were collected with a magnet plate and rinsed with 400ul of 70% EtOH twice. Beads were eluted in 50 μΐ of RE1 (lOmM Tris, 0.1 mM EDTA). DNA yields were 1-30 ng/μΐ as measured by a Thermo Fischer Qubit. DNA was tested for lack of cannabinoid content by MCR labs (Framingham, MA).

[00102] MicroSeq ^® qPCR

[00103] Aspergillus DNA was obtained from ATCC. Cannabis DNA was purified with the above methods. The qPCR of the Aspergillus DNA and Cannabis DNA were performed with a MicroSeq^® kit from Life Technologies according to manufacturer's instructions with the exception of the DNA purification suggested methods. Multiple melting temperatures and extension temperatures were explored to improve the detection.

[00104] Cultivar Sequencing

[00105] Approximately 200 mg of flower from 10 different cultivars were collected in Holland from various coffee houses. DNA purification as described above was performed and Nextera Libraries were constructed from 50 ng of DNA according to the manufacturer's instruction. These libraries were quantified and sequenced on Illumina HiSeq and MiSeq platforms with paired 100 and 250 base pair reads. Reads (24 million) were mapped to van Bakel (Genome Biol 12, R102 (2011) mRNA transcript sequences to assess exonic copy number of cannabinoid pathway genes. [00106] Amplicon Sequencing

[00107] CMK amplicons were derived from SCCG primers in Table 1 for 10 cultivars. These amplicons were dA tailed and converted into a barcoded amplicon sequencing library (New England Biolabs #E6053S) for Illumina MiSeq sequencing. 2xl50bp reads were utilized to sequence the 500bp CMK Amplicons. Reads were aligned and assembled with CLC Bio Genomic Workstation version 4.

[00108] ITS qPCR screening

[00109] Cannabis cultivars (n=10) were qPCR screened to identify a sample with the lowest mold and bacterial content using the described primers. Since many agricultural fungal pesticides contain Bacillus (Seranade R), care must be taken to understand if PCR or CFU signals are pathogenic, symbiotic or endophytic. This signal was compared to DNA extracted from a cannabis stem biopsy to better understand external pathogen populations versus lower frequency internally vascularized pathogens. Cultivar "Lester Grinspoon" was selected as having the lowest ITS signal compared to stem biopsy.

[00110] Decontamination

[00111] AbaSI decontamination digestion is performed with 5-150 ng DNA. The decontamination reaction is set up using 5 μΐ of eluent DNA, lul NEB4 buffer, 1U AbaSI, brought up to 10 μΐ total with ddH₂0. The 10 μΐ reaction is incubated at 25°C for 1 hour, with a heat kill at 65°C for 20 minutes before initiating PCR. All 10 μΐ of digestion are included in a 25 μΐ PCR.

[00112] Multiplex Quantitative PCR

[00113] Each assay had a Single Copy Cannabis Gene (SCCG) assay with a 5' HEX label and a pathogen target assay with a FAM label. TAQMAN Probes were quenched with Iowa Black quenchers combined with a Zen internal quencher. PCR primers for the SCCG target the CMK gene and consisted of the sequences in the Table. The primers are designed with and without Next Generation sequencing 5' M13 Tails. PCR was performed utilizing 1-30 ng of decontaminated gDNA. Reaction setup included 10 μΐ decontaminated DNA, 12.5 μΐ of 2 X LongAmp Hot Start Master Mix, 0.5 μΐ of 50x probe mix (for each TaqMan Primer and Probe Mix), and lul 20mM 5hydroxy me-dCTP. The 25 μΐ PCR reaction was cycled with an initial five minute denaturation at 95°C and is followed by 40 cycles of 95°C at 15 s, 60°C for 90 s.

[00114] Comparison to culture based techniques [00115] Benchmarking was performed with a BIOLUMIX instrument (Neogen). E.coli and Salmonella tests were performed using the EC and SAL vials while the mold comparison was performed with the BIOLUMIX™ YM vial.

! Total Mold & Yeast

\ Forward j 5' GCATCGATGAAGAACGCAGC (SEQ ID NO: 4)

Reverse I 5' ATTTGAGCTCTTGCCGCTTCA (SEQ ID NO: 5)

j 5' CGCTGAACTTAAGCATATCAATAAGCGG (SEQ j

I Probe 5 1· AM ID NO: 6)

E. Coli

\ Forward ! 5' GCATCGTGACCACCTTGA 3 ' (SEQ ID NO: 7)

\ Reverse j 5' CAGCGTGGTGGC AAAA3 ' (SEQ ID NO: 8)

i 5' CCGGCAAACATAATGCAATC-BHQ (SEQ ID NO: ! j Probe 5 1· AM j. 9) j

\ Salmonella (2)

\ Forward j 5' TGGTTTCGATTCGGAAGC (SEQ ID NO: 10)

\ Reverse i 5' C GATGAGATC TGGTCGC (SEQ ID NO: 1 1)

! 5' CCGTCAGGAGGCATTACGAAAAGA-BHQ (SEQ 1 i Probe \ ID NO: 12)

[00116] Results

[00117] Evaluation of MicroSeq® kits

[00118] MicroSeq® kits from Life Technologies were initially tested on Cannabis DNA

spiked with Aspergillus DNA. FIG. 1 demonstrates that Cannabis DNA screened to have

low ITS 3 signal demonstrates 5 log orders more signal with the MicroSeq® kits from Life

Technologies. This 100,000 fold higher measurement for mold did not respond to serial dilutions of Aspergillus, as observed with Cannabis DNA. This implied a cross reactivity with a high copy number sequence in the plant.

[00119] Sequencing 10 Strains

[00120] After mapping 24M reads to van Bakel's cannabinoid mRNA sequences read depth across 10 strains was utilized as a proxy for copy number changes in the genomes. As expected AAE3 recapitulated the amplification event described by van Bakel et al. for Purple Kush.

[00121] Internal SCCG control

[00122] Sequencing of CMK amplicons revealed two common deletions apparent in the population of CMK alleles but very few SNPs (FIGs. 2A and 2B-2G). One of these gDNA deletions appeared at splice site in the CMK mRNA transcript and may influence expression and activity of the full length CMK gene.

[00123] FIG. 2A is a depiction of whole genome shotgun sequencing across multiple cannabis cultivars. Coverage of various genes in the cannabinoid synthase pathway is depicted on the X axis with depth of coverage reported on the Y axis. The CMK region was chosen based on its low cultivar to cultivar coverage variance. This low coverage variance is a signal for limited copy number variation. This is referred to herein as "Single Copy Cannabis Gene" or "SCCG." Smaller forms of variation like SNPs and Indels may require further sequencing.

[00124] FIGs. 2B-2G depict an iterative amplicon design and sequencing was performed to select for both primer and probe sequences in the CMK gene. Several insertions, deletions and polymorphisms in the CMK target are observed that are designed around to afford detection across the genetic diversity in the plant.

[00125] Pathogenic ITS and Bacteria specific assays

[00126] Multiplexed qPCR demonstrated significant sensitivity above no template controls (NTC) and cannabis background DNA. Ten pathogenic genomic copies of E.coli, S.

typhimurium and S. cerevisiae were readily distinguished in all 3 assays from zero copies. A linear response in pathogenic copy number was demonstrated in a background of Cannabis DNA (FIGs. 3, 4, 5). Simultaneous amplification of SCCG assisted in normalizing differential DNA extraction procedures or plant to plant variable extraction. Some lots of TSB showed occasional TSB only qPCR amplification very late in the amplification (35 Ct) with ITS primers. This is likely due to residual DNA in the yeast extract of TSB and the randomness of its appearance is likely related to sampling bias associated with single copy digital PCR at those levels.

[00127] Conversion to Multiplex assays

[00128] The MicroSeq^® kit employs a universal SYBER green qPCR protocol that cannot be multiplexed. To reduce costs and the number of pipetting steps the SCCG amplicon was multiplexed into every test for the best internal control performance. Converting the assays from singleplex assays into multiplex assays required a simple exchange of a FAM dye for a FLEX dye on the internal taqman probe. The SCCG internal control was thus multiplexed into each assay providing Cannabis DNA estimates concurrently with pathogenic DNA estimates in every test. Failure to obtain SCCG amplification indicated a plant gDNA isolation failure and a potential under-sampling of microbial DNA.

[00129] Forward compatibility with DNA Sequencing for phylotyping

[00130] To enable Sanger and next generation sequencing of any PCR product in the test, universal tailed primer sites (M13 tails) were added to each assays primers and screened to ensure minimal impact on performance. Ml 3 tailed primers demonstrated marginal differences from un-tailed primers (FIG. 7). These tails enabled convenient Sanger or next generation sequencing of the amplicon for future phylotyping of the mold and potentially the Cannabis DNA.

[00131] Correlation to growth based methods

[00132] Correlation with culture-based methods (BIOLUMIX™) was greater than about 98.0 % and the time to generate a result was compressed from days to hours (FIG. 8). There was one sample (n=5) that tested positive for salmonella on the BIOLUMIX™ that showed no evidence via qPCR. This sample was further evaluated with an ELISA based test strip to confirm the negative result. False positives in culture-based systems can arise as many other microbes can grow in a Salmonella culture system. The media does not provide the selectivity of DNA sequence specific to the organism of concern.

[00133] Discussion

[00134] PCR based methods present many attractive features for microbial detection. First, the detection can be obtained in a few hours as opposed to the multiple days required for culturing. In addition, the volume of reagent required is easily performed in 96 and 384 well formats while emulsion and bridge PCR have delivered billions of parallel discrete reactions. Culture-based counting methods have not demonstrated this level of scalability and are limited to microbes that can be cultured while simultaneously lacking specificity in what forms a counted colony.

[00135] Both procedures required sterile technique. PCR based methods can build in additional measures to mitigate laboratory contamination. Emulating assay decontamination in culture-based methods may require the use of multiple different antibiotics and fungicides but is unlikely to be as specific as a DNA signature.

[00136] One benefit of a PCR based method is the ability to count DNA from live and dead microorganisms. This added sensitivity is important to limit false negatives from cannabis samples that have been through a sterilization process to reduce the microbial load. Many sterilization methods such as heat or irradiation will disrupt microbial cell membranes and reduce viability and CFU count. The reduction in microbial viability while beneficial can present cannabis that has mycotoxins and other mold synthesized contaminants that evade CFU based detection.

[00137] Since microbes synthesize a diverse library of mycotoxins and ergo alkaloids a complex collection of ELISA assays and HPLC techniques are required to test this diversity in crude plant extracts. PCR offers a universal assay to detect the enzymatic genes responsible for microbial small molecule synthesis. This can significantly reduce

instrumentation costs and workflow complexity.

[00138] Additionally, PCR methods have been developed to help distinguish between DNA derived from viable or non viable micro-organism. The simplest solution is to amplify a sample with and without a brief culturing. For example, a sample incubated in culture for 4-6 hours prior to performing PCR and comparing this result to amplification performed on a sample directly obtained from the plant. This +/- incubation PCR may provide information regarding a difference between live versus dead DNA. Although this pre-incubation does not require the full 24-48 hour incubation time of culture-based detection, it is limited by what organisms can be cultured while delivering organism specific signal in a shorter time frame.

[00139] Viability PCR or vPCR can also be utilized to differentiate between DNA that is protected with an intact cell membrane from DNA that is exposed or behind a nonviable cell membrane. This method utilizes an ultraviolet (UV) cross-linking dye (PMA) that cannot traverse a cell membrane unless the membrane is broken. It can be performed in about 15-30 minutes, but uses hazardous intercalating dyes and additional ultraviolet light equipment required to cross link the DNA to intercalating dyes. Some wavelengths of UV light have also been utilized in hospitals to decontaminate medical equipment. As a result, the exact level of exposure required to cross link the "dead" DNA may in fact also kill some percentage of the live microbes and expose more live DNA. Thus, care should be taken to ensure these methods are quantitative, cost effective and use appropriate UV exposures.

[00140] A challenge present in both culture-based methods and PCR is related to sampling large plant volumes in small reactions. In the case of Cannabis, sampling over 100 mg of plant material is preferred to ensure an adequate and homogenized sampling of the plant has been considered in the test. For example, in the described method 3.8 ml of TSB was used to hydrate a 250 mg sampling of a cannabis sample. It was difficult to get all 3.8 ml of TSB into a BIOLUMIX™ vial or a PCR reaction. An advantage of DNA based assays includes concentration of the DNA purification step to a volume about 5 to about 10 times compared to culture-based methods. However, for highly stringent single CFU presence/absence testing with E. Coli, all 3.8 ml of sample was not put into a biolumix or a qPCR reaction. In both cases, a pre-growth can be used, if needed, to compensate for the subsampling required to make these methods perform.

[00141] In summary, as described herein, PCR based techniques can be adapted to work with microbial detection on Cannabis given the proper focus on DNA purification and primer design. The method demonstrated high correlation with culture-based methods and has the advantage of rapid, scalable and cost effective means for safety testing in Cannabis. PCR combined with sequencing can provide new feedback mechanisms to identify any infection of unknown origin, including mold and Cannabis phylotypes. Since DNA can legally cross most state and international borders, DNA based testing in Cannabis can bring rapid safety testing to international markets.

[00142] EXAMPLE 2

[00143] Introduction

[00144] Many states in the U.S. are drafting regulations for microbial detection on cannabis in absence of any comprehensive survey of Cannabis microbiomes. A few of these regulations are inducing growers to "heat kill" or pasteurize Cannabis flowers to lower microbial content. However, drying techniques often create false negatives in culture based safety tests used to monitor colony-forming units (CFU). Even though pasteurization may be effective at sterilizing some of the microbial content, it does not eliminate various pathogenic toxins or spores. Aspergillus spores and mycotoxins are known to resist pasteurization (Fujikawa, H. et a/., Applied and Environmental Microbiology 62, 3745-3749 (1996); Kabak, B. et a/., Journal of food protection 72, 2006-2016 (2009)). Similar thermal resistance has been reported for E.coli produced Shiga Toxin (Rasooly, R. et a/., International Journal of Food Microbiology 136, 290-294 (2010)). While pasteurization may reduce CFU employed in petri-dish or plating based safety tests, it does not reduce the microbial toxins, spores or DNA encoding these toxins.

[00145] Mycotoxin monitoring in Cannabis preparations is important since aflatoxin produced by Aspergillus species is a carcinogen. The clearance of aflatoxin requires the human liver enzyme CYP3 A4 and this liver enzyme is potently inhibited by cannabinoids (Langouet, S. et a/., Adv Exp Med Biol 387, 439-442 (1996); Yamaori, S. et a/., Life Sci 88, 730-736 (2011)). Modern day cannabis flowers can produce up to about 25% (w/v) cannabinoids presenting potent inhibition of CYP3A4 and CYP2C19. Health compromised patients exposed to aflatoxin and clearance-inhibiting cannabinoids raise new questions in regards to the current safety tolerances to aflatoxin. Similarly, Fusarium species are known to produce fungal toxins and has proven to be difficult to selectively culture with tailored media (Bragulat, M R. et al, Journal of Food Protection 67, 207-211 (2004); Castella, G. et al, Mycopathologia 137, 173-178 (1997); Desjardins, A.E. et al., International Journal of Food Microbiology 119, 47-50 (2007)). This is a common limitation of culture-based systems since carbon sources are not exclusive to certain microbes and only about 1% of microbial species are believed susceptible to be culture (Stewart, E.J., Journal of Bacteriology 194, 4151-4160 (2012)).

[00146] While these risks have been well studied in the food markets, the presence of the microbial populations present on cannabis flowers has never been surveyed with next generation sequencing techniques (Kusari P, K.S. et al., Fungal Diversity. 60, 137-151 (2013); McPartland, Phytopathology 72:797 (1983); McPartland, Plant Dis 75, 226-227 (1991); McPartland, J. Int Hemp Assoc 1, 41-44 (1994); McPartland, Mycologia 86, 870- 878 (1995); McPartland, J Int Hemp Assoc 3, 19-23 (1996)). As described herein, yeast and mold species are determined in dispensary-derived Cannabis samples by quantitative PCR and sequencing, and demonstrate the presence of several mycotoxin producing fungal strains that are not detected by widely used culture-based assays. [00147] Methods

[00148] Culture based methods

[00149] Tryptic Soy Broth (TSB) (3.55 ml) was used to wet 250 mg of homogenized flower in a whirlpack bag. TSB was aspirated from the reverse side of the 100 μιη mesh filter and placed into a BIOLUMIX™ growth vial and spread onto a 3M Petri Film™ and a SimPlate™ according to the respective manufacturer's recommendations. BIOLUMIX™ vials were grown and monitored for 48 hours while Petri-films™ and SimPlates™ were grown for 5 days. Petri-films™ and SimPlates™ were colony counted manually by three independent observers. Samples were tested on Total Coliform, Total Entero, Total Aerobic, and Total Yeast and Mold. Only Total Yeast and Mold discrepancies were graduated to sequencing.

[00150] DNA Purification

[00151] Plant DNA was extracted with SenSATIVAx™ (Medicinal Genomics, #420001), as described herein. DNA is eluted with 50 μΐ ddH₂0.

[00152] Primers used for PCR and sequencing

[00153] PCR was performed using 5ul of DNA (3ng^l) 12.5 μΐ 2X LongAmp (NEB) with 1.25 μΐ of each 10uM MGC-ITS3 and MGC-ITS3 primer (MGC-ITS3;

TACACGACGTTGTAAAACGACGCATCGATGAAGAACGCAGC) (SEQ ID NO: 21) and (MGC-ITS3R; AGGATAACAATTTCACACAGGATTTGAGCTCTTGCCGCTTCA) (SEQ ID NO: 22) with 10 μΐ ddH₂0 for a 25 μΐ total reaction. An initial 95°C 5 minute denaturization was performed followed by 40 cycles of 95°C for 15 sec and 65° C for 90 sec. Samples were purified with 75 μΐ SenSATIVAx™, washed twice with 100 μΐ 70% EtOH and bench dried for 5 minutes at room temperature. Samples were eluted in 25 μΐ ddH₂0.

[00154] Tailed PCR Cloning and Sequencing

[00155] DNA libraries were constructed with 250 ng DNA using NEBNext Quick™ ligation module (NEB # E6056S). End Repair used 3 μΐ of Enzyme Mix, 6.5 μΐ of Reagent Mix, 55.5ul of DNA + ddH₂0. Reaction was incubated at 30°C for 20 minutes. After End Repair™, Ligation was performed directly with 15 μΐ of Blunt End TA Mix™, 2.5 μΐ of Ilumina Adaptor™ (10 μΜ) and 1 μΐ of Ligation enhancer (about 20% PEG 6000). After 15- minute ligation at 25°C, 3 μΐ of USER™ enzyme was added to digest the hairpin adaptors and prepare for PCR. The USER™ enzyme was tip-mixed and incubated at 37°C for 20 minutes. After USER™ digestion, 86.5 μΐ of SenSATIVAx™ was added and mixed. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 150 μΐ of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25 μΐ of lOmM Tris-HCl.

[00156] Library PGR

[00157] 25 μΐ 2X Q5 Polymerase was added to 23 μΐ of DNA with 1 μΐ of i7 index primer (25 μΜ) and 1 μΐ Universal primer (25 μιη). After an initial 95°C for 10 sees, the library was amplified for 15 cycles of 95°C 10 sec, 65°C 90 sec. Samples were purified by mixing 75 μΐ of SenSATIVAx™ into the PCR reaction. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 150 μΐ of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25 μΐ of lOmM Tris-HCl. Samples were prepared for sequencing on the MiSeq V2™ chemistry according to the manufactures instructions. Reads 2x250 bp (bp refers to base pair) were selected to obtain maximal ITS sequence information.

[00158] PaxP Verification PCR

[00159] Primers described by Shirazi-zand et al. were utilized to amplify a segment of the 725bp PaxP gene. LongAmp (25 μΐ) (NEB), 4 μΐ of 10 μΜ Primer, Ι μΐ DNA (14 ng/μΐ) and 20 μΐ dd¾0 were combined to make a 50 μΐ PCR reaction. Cycling conditions were modified to accommodate a different polymerase. 95°C for 30 sec followed by 28 cycles of 95°C 15 sec, 55°C for 30 sec, 65°C for 2.5 minutes. Samples were purified with 50 μΐ of SenSATIVAx™ as described above. Purified PCR product (1 μΐ) was sized on Agilent™ HS 2000 chip. Nextera libraries and sequencing were performed according to instructions from Illumina™ using 2 x 75bp sequencing on a version 2 MiSeq.

[00160] Citrinin Verification PCR

[00161] Citrinin Forward GATTTTCCAAAATGCCGTCT (SEQ ID NO: 23) and Citrinin Reverse GCTCAAGCATTAATCTAGCTA (SEQ ID NO: 24) primers were used with identical PCR conditions as described above with the exception of 35 cycles of PCR.

Samples were purified with 50 μΐ of SenSATIVAx™ as described above. Purified PCR product (1 μg) was sized on Agilent™ HS 2000 chip. Nextera libraries and sequencing were performed according to instructions from Illumina™ using 2x75bp sequencing on a version 2 MiSeq. Reads were mapped to Genbank accession number LKUP01000000. Mappings were confirmed using BLAST to NCBI to ensure the strongest hits were to P.Citrinum.

[00162] Analysis [00163] Reads were demultiplex and trimmed with Casava 1.8.2 and trim galore. FLASH 19 was used to merge the reads using max overlap 150. The reads were aligned to microbial references using MG-RAST (Glass, E.M. et al., Cold Spring Harbor protocols 2010, pdb prot5368 (2010)). Alignments and classifications were confirmed with a second software tool from One Codex and critical pathways identified for further evaluation with PCR of toxin producing genes. Reads are deposited in NCBI under SRA accession: SRP065410. Nextera (2 x 75bp) sequencing of the PaxP gene was mapped to accession number

HM171111.1 with CLCbio Workstation V4 at 98% identity over about 80% of the read. One Codex analysis was put into Public mode under the following public URLs:

[00164] Australian Bastard:

https://app.onecodex.com/analysis/public/201e7fl642e04a3c

https://app.onecodex.com/analysis/public/58fle03cl0434bfa

[00165] KD4:

https://app.onecodex.com/analysis/public/2e86e262817246c4

https://app.onecodex.com/analysis/public/labd5b60446140a0

[00166] KD6:

https://app.onecodex.com/analysis/public/a92d3dff5485499d

https://app.onecodex.com/analysis/public/8d72e2514e564ecd

[00167] KD8:

https://app.onecodex.com/analysis/public/8d72e2514e564ecd

https://app.onecodex.com/analysis/public/d6e2e0bcfba3469f

[00168] Liberty Haze:

https://app.onecodex.com/analysis/public/7bcd650fa5544f2c

https://app.onecodex.com/analysis/public/7f0feb6cb0a94d56

[00169] Girls Scout Cookie:

https://app.onecodex.com/analysis/public/a71blce8331c461d

https://app.onecodex.com/analysis/public/8d6fl0c7ee684f93

[00170] Jakes Grape:

https://app.onecodex.com/analysis/public/bc8af5edl9e5407a

https://app.onecodex.com/analysis/public/99d7a4a2f7af486b [00171] RECON:

https://app.onecodex.com/analysis/public/8a22al6cc2e24731

https://app.onecodex.com/analysis/public/0af6ae26a01f48d5

[00172] GreenCrack:

https://app.onecodex.com/analysis/public/6114843d2eb3425e

https://app.onecodex.com/analysis/public/3eee642786c54a88

[00173] LA Confidential:

https://app.onecodex.com/analysis/public/01e8aefb0d4f4f62

https://app.onecodex.com/analysis/public/b74c2988fcd84e38

[00174] NYC Diesel:

https://app.onecodex.com/analysis/public/441cfad759f64dcc

https://app.onecodex.com/analysis/public/d97b39cae96c4a44

[00175] Results

[00176] A Total Yeast and Mold qPCR assay as described above, was used to screen for fungal DNA in a background of host Cannabis DNA. The TYM qPCR assay targets the 18S rDNA ITS (Internal Transcribed Spacer) region using modified primers described previously (Borneman, J. et a/., Applied and Environmental Microbiology 66, 4356-4360 (2000); White, T.J. et a/., A Guide to Methods and Applications, 315-322 (1990)). Fungal DNA amplified using these primers may also be subjected to next generation sequencing to identify the contributing yeast and mold species. ITS sequencing has been widely used to identify and enumerate fungal species present in a given sample (Mason, M. et a/., Journal of Prevention & Intervention in the Community 37, 21-34 (2009)).

[00177] DNA from Cannabis samples obtained from two different geographic regions (Amsterdam and Massachusetts) several years was purified. The majority of samples purified and screened with ITS qPCR were negative for amplification signal implying reagents clean of fungal contamination. Six of the 17 dispensary-derived Cannabis samples tested positive for yeast and mold in the TYM qPCR assay. These results were compared with the results derived from three commercially available culture based detection systems for each of the 17 samples (3M Petrifilm™ 3M Microbiology, St. Paul, MN, USA,

SimPlates™ Biocontrol Systems, Bellevue,WA, USA, BioLumix™ Neogen, Lansing MI, USA). Of the 6 qPCR positive samples, two tested negative in all 3 culture-based assays and four tested negative in 1 or 2 of the culture-based assays (FIG. 12). None of the qPCR negative samples tested positive in any of the culture based assays. Each of the 6 discordant samples was subjected to ITS sequencing to precisely identify the collection of microbes present. Four additional samples from a different geographic origin (Amsterdam) were also subjected to ITS sequencing, for a total of 10 Cannabis samples.

[00178] Each discordant sample presented with an array of microbial species, as shown in FIGs. 14A-14G. No sample presented with a single dominant species, and each sample displayed multiple species of interest. Of particular concern were the identified DNA sequences from toxin producing species: Aspergillus versicolor (Abd Alia, E.A. et al., Die Nahrung 40, 310-313 (1996); Aly, S.A. et al, The Journal of Dairy Research 74, 74-78 (2007); Engelhart, S. et al, Applied and Environmental Microbiology 68, 3886-3890 (2002); Kocic-Tanackov, S. et al, Journal of Food Science 77, M278-284 (2012); Song, F. et al, Applied Microbiology and Biotechnology 98, 3753-3758 (2014)), Aspergillus terreus (El- Say ed Abdalla, A. et al, Mycotoxin Research 14, 83-91 (1998)), PenicUlium citrinum (Ames, D.D. et al, Poultry Science 55, 1294-1301 (1976); Mazumder, P.M., et al, Ancient Science of Life 21, 191-197 (2002); Park, S.Y. et al, FEMS Microbiology Ecology 65, 229- 237 (2008)), PenicUlium paxilli (Itoh, Y., et al, Current Genetics 25, 508-513 (1994); Shibayama, M. et al, Current Genetics 42, 59-65 (2002).

[00179] The ITS sequence alignments were analyzed using the whole genome shotgun based microbiome classification software known as One Codex (Minot, S.S. et al, bioRxiv (2015)). Nine of the ten samples sequenced showed the presence of P. paxilli (FIG. 15). To verify the accuracy of this ITS phylotyping, a gene involved in the Paxilline toxin

biosynthesis pathway of P. paxilli was amplified with PaxPssl and PaxPss2 primers described by Saikia e/ a/. (Saikia, S. et al, The Journal of Biological Chemistry 282, 16829- 16837 (2007)). The resulting 725bp amplicon (expected size) was sequenced to confirm the presence of the P. paxilli biosynthesis gene in the cannabis sample KD8 (FIG. 16A-16B). This was successfully repeated with primers designed to target genes in the citrinin pathway of P. citrinum. While there are some discrepancies between the two software platforms, the analysis employed merged paired reads with MG-RAST and correlate better with PCR results. While One Codex predicted and confirmed KD8 as having the highest Paxilli content, the One Codex platform is optimized for whole genome shotgun data and may not be able to differentiate the 18S sequence differences (391/412 aligned bases) between these two species with a K-mer based approach.

[00180] With the confirmed presence of P. paxilli, the presence of the toxin, paxilline, was determined in the samples. Development of monoclonal antibodies to paxilline has recently been described (Maragos, CM. et al, Toxins 7, 3903-3915 (2015)), but commercial ELISA assays with sensitivity under 50ppb do not appear to be available at this time. A >50ppb multiplexed ELISA assay is available from Randox Food Diagnostics (Crumlin, UK).

Detection with LC-MS/MS has also been described (Vishwanath, V. et al, Analytical and Bioanalytical Chemistry 395, 1355-1372 (2009); Uhlig, S. et al., Rapid Communications in Mass Spectrometry : RCM 28, 1621-1634 (2014)), however, and experiments are underway to determine whether Paxilline can be identified in the background of cannabinoids and terpenes present in Cannabis samples.

[00181] Discussion

[00182] Several potentially harmful fungal species were detected in dispensary-derived Cannabis samples by qPCR and subsequent sequencing in this study. Three different culture- based assays failed to detect all of the positive samples and one, BioLumix™, detected only one out of 7 positive samples. It has been suggested that Penicillium microbes can be cultured on CYA media, but some may require colder temperatures (21°-24°C) and 7 day growth times (Houbraken, J. et al, Studies in Mycology 70, 53-138 (2011)). Of the

Penicillium, only P. citrinum has been previously reported to culture with 3M Petri-Film (3M http://multimedia.3m.com/mws/media/8985920/3m-petrifilm-rapid-yeast-mold-count- plate.pdf). In addition, several studies have demonstrated plant phytochemicals and terpenoids, such as eugenol, can inhibit the growth of fungi (Zare, M., et al., Iranian

Biomedical Journal 12, 229-236 (2008)). It is possible the different water activity of the culture assay compared to the natural terpene rich flower environment is contributing to the false negative test results.

[00183] Quantitative PCR is agnostic to water activity and can be performed in hours instead of days. The specificity and sensitivity provides important information on samples that present risks invisible to culture based systems. The draw back to qPCR is the indifference of the method to living or non-living DNA. While techniques exist to perform live-dead qPCR, the live status of the microbes is unrelated to toxin potentially produced while the microbes were alive. ELISA assays exists to screen for some toxins (Labs, R. [Online] Available at: http://www.romerlabs.com/en/knowledge/mycotoxins/, Accessed on Oct, 2015). Current State-recommended ELISA's do not detect Citrinin or Paxilline, the toxins produced by P. citrinum and P. paxilli, respectively. The predominance of these Penicillium species in a majority of the samples tested is interesting. Several Penicillium species are known to be endophytes on various plant species, including P. citrinum (Kusari P, K.S. et al., Fungal Diversity. 60, 137-151 (2013);, and this raises the question of whether they are also Cannabis endophytes.

[00184] Paxilline is a tremorgenic and ataxic potassium channel blocker and has been shown to attenuate the anti-seizure properties of cannabidiol in certain mouse models (Shirazi-zand, Z. et al., Epilepsy & Behavior : E&B 28, 1-7 (2013); Sabater-Vilar, M. et al., Journal of Food Protection 66, 2123-2129 (2003); Sanchez-Pastor, E. et al., European Journal of Pharmacology 729, 100-106 (2014)). Paxilline is reported to have tremorgenic effects at nanomolar concentrations and is responsible for Ryegrass-staggers disease (Imlach, W.L. et al., The Journal of Pharmacology and Experimental Therapeutics 327, 657-664 (2008)). Cannabidiol is often used at micromolar concentrations for seizure reduction implying sub- percentage contamination of Paxilline could still be a concern.

[00185] Citrinin is a mycotoxin that disrupts Ca2+ efflux in the mitochondrial

permeability transition pore (mPTP) (Chagas, G.M., et al, JAT 12, 123-129 (1992); Chagas, G.M., et al, Cell Biochemistry and Function 10, 209-216 (1992); Chagas, G.M. et al, Cell Biochemistry and Function 13, 53-59 (1995); Chagas, G.M., et al, Journal of Applied Toxicology: JAT 15, 91-95 (1995); Da Lozzo, E.J. et al, Journal of Biochemical and

Molecular Toxicology 12, 291-297 (1998); Jeswal, P., Cytobios 86, 29-33 (1996); Ribeiro, S.M. et al, Cell Biochemistry and Function 16, 15-20 (1998); Ribeiro, S.M. et al, Cell Biochemistry and Function 15, 203-209 (1997)). Ryan et al. demonstrated that cannabidiol affects this pathway suggesting a potential concern for CBD-mycotoxin interaction (Ryan, D. et al, The Journal of Neuroscience 29, 2053-2063 (2009)). Considering the hydrophobicity of Paxilline and the recent interest in the use of cannabidiol derived from cannabis flower oils for drug resistant Epilepsy, more precise molecular screening of fungal toxins may be warranted (Devinsky, O. et al, Neurotherapeutics 12, 910 (2015); Devinsky, O. et al, Neurotherapeutics 12, 689-691 (2015); Friedman, D. et al, The New England Journal of Medicine 373, 1048-1058 (2015); Rosenberg, E.C. et al, Neurotherapeutics 12, 747-768 (2015); Cilio, M R. et al, Epilepsia 55, 787-790 (2014); Devinsky, O. et al, Epilepsia 55, 791-802 (2014)). [00186] While ELISA assays are easy point of use tests that can be used to detect fungal toxins, they can suffer from lack of sensitivity and cross reactivity. ITS amplification and sequencing provides testing that can complement the lack of specificity in ELISA assays. Appropriate primer design can survey a broad spectrum of microbial genomes while affording rapid iteration of design. Quantitative PCR has also demonstrated single molecule sensitivity and linear dynamic range greater than about 5 orders of magnitude offering a very robust approach for detection of microbial risks. This may be important for the detection of nanomolar potency mycotoxins. Further studies are required to validate better detection methods for these toxins and verify whether Paxilline or Citrinin are present on cannabis at concentrations that present a clinical risk.

[00187] Conclusions

[00188] These results demonstrate that culture based techniques superimposed from the food industry should be re-evaluated based on the known microbiome of actual Cannabis flowers in circulation at dispensaries. Several mycotoxin producing molds were detected that can potentially interfere with the medical use of cannabidiol. These microbes failed to grow on traditional culture based platforms but were rapidly detected with molecular based techniques. Further studies are required to quantitate the presence and concentration of mycotoxin production.

[00189] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00190] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of detecting the presence of at least one of a fungus and a bacteria in a Cannabis plant, comprising the steps of:

(a) amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cawrafos-specific primers as set forth in SEQ ID NOs: 1 and 2, and in the presence of at least one of a fungus-specific primer and a bacteria-specific primer, to thereby form amplicons; and

(b) comparing the amplicons with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the

Cannabis plant with at least one of the fungus and bacteria.

2. The method of Claim 1, wherein the fungus is detected.

3. The method of Claim 1, wherein the fungus is at least one member selected from the group consisting of Sclerotina and Aspergillus.

4. The method of Claim 1, wherein the fungus is at least one member selected from the group consisting of a yeast and a mold.

5. The method of Claim 1, wherein the fungus is mold.

6. The method of Claim 5, wherein the mold is at least one member selected from the group consisting of Penicillium and Mucor.

7. The method of Claim 6, wherein the mold is Penicillium.

8. The method of Claim 7, wherein the Penicillium is at least one member selected from the group consisting of P. citrinum and P. paxilli.

9. The method of Claim 1, wherein the bacteria is detected.

10. The method of Claim 9, wherein the bacteria is at least one member selected from the group consisting of E. Coli, Coliform, Salmonella and Enterococcus.

11. The method of Claim 1, further including the step of determining an amount of the at least one of the fungus and the bacteria present in the Cannabis plant by determining the amount of amplicons of the nucleotide sequence of the Cannabis plant and the amount of amplicons of the nucleotide sequence of at least one of the fungus and the bacteria.

12. The method of Claim 11, further including the step of assessing the amount of

amplicons of the nucleotide sequence of at least one of the fungus and the bacteria to a standard.

13. The method of Claim 1, wherein the amplification step includes at least one

methylated dATP, dTTP, dGTP and dCTP.

14. The method of Claim 13, wherein the at least one methylated dNTP is at least on member selected from the group consisting of methylated dCTP and methylated dATP.

15. The method of Claim 14, wherein the dCTP is at least one member selected from the group consisting of 5-methyl cytosine and 5-hydroxymethyl cytosine.

16. The method of Claim 15, wherein the one or more methylated dATP is N6 methyl adenosine.

17. The method of Claim 1, wherein the at least one of the Cannabis-specific primers and at least one of the fungus-specific primers and the bacterial specific primers are labeled with at least one tag.

18. The method of Claim 17, wherein the at least one tag includes a fluorescent tag.

19. The method of Claim 18, wherein the at least one tag of the Cannabis-specific

primers are distinct from at least one of the fungus-specific primers and the bacterial- specific primers.

20. The method of Claim 17, wherein the at least one tag includes at least one member selected from the group consisting of a HEX tag, a FAM tag, a TAMRA tag, a JOE tag, a R6G tag, a CY3 tag and a CY5 tag.

21. The method of Claim 1, further including the step of maintaining the amplicons of the nucleotide sequences of the Cannabis plant and the amplicons of the nucleotide sequence of at least one of the fungus and the bacteria under conditions in which amplicons that include methylated bases are digested by the at least one methyl specific restriction enzyme.

22. The method of Claim 21, wherein the methyl specific restriction enzyme is at least one member selected from the group consisting of MspJl, FspEl, LpnPI, AspBHI, Rial, SgrTI and AbaSI.

23. The method of Claim 1, wherein the one or more nucleotide sequences of the

Cannabis plant includes at least one member selected from the group consisting of a CMK locus, an IPP locus, a GPP lsu locus and a GPP ssu locus.

24. The method of Claim 1, wherein a portion of the Cannabis plant is cultured prior to amplifying the nucleotide sequences of the Cannabis plant.

25. The method of Claim 24, wherein the amount of amplicons of the one or more

nucleotide sequences of the at least one of the fungus and the bacteria present in the Cannabis plant cultured prior to amplifying is compared to the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus and the bacteria present in a portion of the Cannabis plant that was not cultured prior to amplifying and an increase in the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus, and the bacteria present in the Cannabis plant that was cultured compared to the amount of amplicons of the one or more nucleotide sequences of at least one of the fungus and the bacteria present in the Cannabis plant that was not cultured, indicates the presence of at least one of a live fungus and a live bacteria in the Cannabis plant.

26. The method of Claim 25, wherein the portion of the Cannabis plant is cultured for about 2, 4, 6, 8, 10, 12 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours.

27. The method of Claim 1, wherein the one or more nucleotide sequences of the

Cannabis plant and the one or more nucleotide sequences of at least one the fungus and the bacteria are amplified simultaneously.

28. The method of Claim 1, wherein the fungus-specific primer is a mold-specific primer.

29. The method of Claim 28, wherein the mold-specific primer is at least one of SEQ ID NOs: 4 and 5.

30. The method of Claim 1, wherein the bacteria-specific primer is an E. coli - specific primer.

31. The method of Claim 30, wherein the E. coli - specific primer is at least one of SEQ ID NOs: 7 and 8.

32. The method of Claim 1, wherein the bacteria-specific primer is a Salmonella-specific primer.

33. The method of Claim 32, wherein the Salmonella-specific primer is at least one of SEQ ID NOs: 10 and 11.

34. The method of any one of the preceding claims, wherein the amplification occurs in a polymerase chain (PCR) reaction that includes an initial denaturation at about 95°C followed by about 40 cycles of 95°C at about 15 seconds, about 60°C for 90 seconds.

35. A kit for detecting the presence of at least one of a fungus and a bacteria in a

Cannabis plant that comprises amplifying one or more nucleotide sequences from DNA separated from the Cannabis plant in the presence of Cannabis-specif c primers as set forth in SEQ ID NOs: 1 and 2 and in the presence of at least one of a fungus- specific primer and a bacteria-specific primer, to thereby form amplicons; and comparing the amplicons with known nucleotide sequences of the Cannabis plant and at least one known nucleotide sequence of at least one of the fungus and the bacteria, whereby matching of the amplicons to the known nucleotide sequences of the

Cannabis plant and, separately, of the known nucleotide of at least one of the fungus and the bacteria indicates contamination of the Cannabis plant with at least one of the fungus and bacteria.

36. The kit of Claim 35, wherein the fungus-specific primer is a mold-specific primer.

37. The kit of Claim 36, wherein the mold-specific primer is SEQ ID NOs: 4 and 5.

38. The kit of Claim 35, wherein the bacteria-specific primer is an E. co/z^'-specific primer.

39. The kit of Claim 38, wherein the E. co/z^'-specific primer is at least one of SEQ ID

NOs: 7 and 8.

40. The kit of Claim 35, wherein the bacteria-specific primer is a Salmonella-specific primer.

41. The kit of Claim 40, wherein the Salmonella-specific primer is at least one of SEQ ID NOs: 10 and 11.