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SenSATIVAx and PathogINDICAtor Microbial Detection in Medicinal Cannabis
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Introduction: 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 and pharmaceutical products has become an important safety consideration. This risk extends itself to medical Cannabis and is of particular concern with inhaled, vaporized, and concentrated Cannabis products. Medicinal Genomics (MGC) has developed a novel PCR based assay for the detection of pathogenic microbes in Cannabis materials. This process consists of a proprietary system for DNA extraction called SenSATIVAxTM and a novel PCR assay called PathogINDICAtorTM. Method: SenSATIVAxTM uses a proprietary technique based on magnetic particle separation for the purification of both plant and microbial DNA from a raw homogenized sample. This approach allows for a highly economical, efficient, and automatable process where DNA can be isolated from a single sample or a large batch in under 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 down stream analysis.
Figure 1: Magnetic particle separation showing nucleic acid isolation and purification from a complex biological sample.
PathogINDICAtorTM utilizes a novel 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 (detection down to 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).
Contamination from amplified products is a major concern when working with microbiological based techniques. Both culture and PCR amplify the pathogen in order to detect it, and usually require sterile technique or clean rooms. PathogINDICAtorTM is able to provide a contamination free technique through a proprietary process (continued on page 2) Figure 3: DREAM PCR generates heavily methylated
DNA that cannot survive AbaSI digestion.
= Fluorophore = Quencher
Forward Primer
Reverse Primer
Probe
Polymerization
Probe Degradation
Result
PCR Amplified DNA Fluorescent Signal
+"
Step%1:%Primers%and%probe%%bind%to%target%DNA.%%
Step%2:%PCR%occurs,%primers%are%extended%on%forward%and%reverse%DNA%strands.%%
Step%3:%Probe%is%degraded%as%a%result%of%polymerizaEon%and%fluorescent%signal%is%generated.%%
Step%4:%Target%DNA%is%amplified%and%fluorescent%signal%can%be%measured%and%quanEfied%.%%
Figure 2: Process of a typical 5' nuclease fluorescent assay. PathogINDICAtorTM includes 5’ universal tails and 5hmCTP in PCR.
SenSATIVAx and PathogINDICAtor Microbial Detection in Medicinal Cannabis
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known as DREAM PCR1, 2, which uses methylation specific restriction enzymes to prevent the carryover of amplified products from one reaction to the next. Results: PathogINDICAtorTM microbial detection assays use a multiplexing strategy with an internal plant DNA reaction control to ensure accurate detection of microbial species for every reaction. 12 Cannabis genomes were decoded to select the best plant control target (Figure 4).
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 (Figure 5).
PCR gives a consistent and reproducible level of detection over a large dynamic range from 1 to > 1 billion copies of the target DNA with a linear correlation between input amount and target detection (Figure 6).
Discussion: Traditional microbial detection and quantitative methods require cell culturing or petri dish plating. These techniques can be dramatically improved upon with nucleic acid based techniques such as qPCR. This is in part due 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. Culture based techniques can also be counterfeited with heat killing of the microorganisms prior to testing. While this reduces the viability of the microbes and CFU counts, it does not remove the mycotoxins or DNA. Aflatoxin B1 requires clearance by a human liver enzyme potently inhibited by cannabinoids (CYP3A4)3-‐5. In contrast to culture-‐based techniques, nucleic acid based tools such as SenSATIVAxTM and PathogINDICAtorTM have excellent species specificity, increased multiplexing capabilities based on the use of multiple fluorescent dye labeled molecules, faster result times, and an easily automated protocol. 1. McKernan, K.J., Spangler, J., Helbert, Y., Zhang, L. & Tadigotla, V. DREAMing of a patent-‐free human genome for clinical sequencing. Nat Biotechnol 31, 884-‐887
(2013). 2. McKernan, K.J. et al. Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment. PLoS One 9, e96492 (2014). 3. Langouet, S. et al. Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer
research 55, 5574-‐5579 (1995). 4. Langouet, S. et al. Metabolism of aflatoxin B1 by human hepatocytes in primary culture. Advances in experimental medicine and biology 387, 439-‐442 (1996). 5. Yamaori, S., Ebisawa, J., Okushima, Y., Yamamoto, I. & Watanabe, K. Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic
hydroxyl groups in the resorcinol moiety. Life sciences 88, 730-‐736 (2011).
Figure 4: Sample containing plant and microbe DNA. (Plant DNA = Blue, Microbial DNA = Pink). X-‐axis is cycles (every 1.5 minutes). X intercept is the Ct and is used to calculate the exponential phase of growth. Y-‐axis is log 10 scale.
Figure 6: 10x Serial dilution of sample with plant and pathogen DNA present. (Plant DNA = Blue. Microbial DNA detection = Pink)
Figure 5: Sample with no pathogen DNA present. (Plant DNA = Blue, Microbial DNA detection = Pink)