Optimized Cannabis Microbial Testing: Combined Use of Extraction Methods and Pathogen Detection Tests Using Quantitative Polymerase Chain Reaction: Page 2 of 4

August 22, 2019
Volume: 
2
Issue: 
4
Figure 3
Figure 3 (click to enlarge): Assay workflow. DNA decontamination means use of a restriction enzyme to digest the potential contaminant amplicon DNA from a previous qPCR.
Figure 4
Figure 4 (click to enlarge): Genomic profiles of before and after culturing. Comparison of classified read percentages for bacterial 16S DNA on samples 2 and 14, before and after culturing on 3M and BMX media. The results represent all species observed down to 1% of classified reads. Large shifts in species prevalence are seen after growth on the two culture-based platforms.
Figure 5
Figure 5 (click to enlarge): Aspergillus niger plated on Sabdex agar (top picture) or 3M petrifilms (bottom picture). Enlarged colonies are the result of 100–1000 conidia clumped into a heterogeneous macro-colony.

Using the Agilent AriaMX Real-Time PCR system, Medicinal Genomics PathoSEEK series of assays detect pathogenic organisms, and has demonstrated robustness, accuracy, and precision for microbial screening in various cannabis flower and cannabinoid product matrices.

To this end, DNA must first be extracted from cannabis plant and microbial cells. To simplify this process, Medicinal Genomics developed SenSATIVAx—a magnetic bead-based DNA extraction kit.

In brief, cannabis flower, leaf, or marijuana-infused product (MIP) is homogenized and, if necessary, allowed to culture in a growth medium, to generate more potential pathogens present on the cannabis product. This culture medium is then subjected to a DNA extraction followed by an optional decontamination step, which rids the sample of any previously amplified DNA.

The extracted and decontaminated DNA sample is then used as a template for the PathoSEEK assay where detection of many of these pathogens is done in multiplex: two to four microbes are targeted in a single PCR reaction. The presence of cannabis DNA and microbial contamination is based on the sample amplification curve achieving an assay-specific fluorescent value within a predetermined number of PCR cycles. Additionally, positive and negative controls show evidence and absence of amplification, respectively. Figure 3 illustrates the assay workflow. (See upper right for Figure 3, click to enlarge.)

Results and Discussion

An Empirical Comparison of PathoSEEK and Culture-Based Methods

Fifteen medicinal cannabis samples were analyzed using PathoSEEK and two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). NGS determines the precise order of nucleotides in a total DNA strand, and MA analyzes genomes collected in environmental samples. These tools respectfully sequence entire genomes of species’, and profile the diversity of genes in a given sample. Figure 4 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms. (See upper right for Figure 4, click to enlarge.)

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

Issues with Aspergillus spp.

Aspergillus spp. grow poorly in culture-based media. One of the common issues is that Aspergillus spp.  clumps over generational growth, so a homogenous spread of the cells in the media is nearly impossible. When Aspergillus niger is plated, clumped conidia cells form heterogeneous macro-colonies which creates significant issues with quantitation (Figure 5). (See upper right for Figure 5, click to enlarge.) What looks like one large colony is hundreds of colonies that are not countable. Additionally, the clumping nature of Aspergillus spp. spores in media makes it difficult to accurately pipette samples. DNA extraction processes performed before running qPCR assays have the benefit of lysis and DNA purification steps that “de-clump” and break open the cells or spores, providing better sampling techniques and therefore more accurate and reliable results.

References: 
  1. C. De Bekker, G.J.  van Veluw, A. Vinck, L.A Wiebenga, and H.A. Wosten, Applied and Environmental Microbiology 77(4),1263–7 (2011). PubMed PMID: 21169437. PubMed Central PMCID: 3067247.
  2. K.J. McKernan, J. Spangler, and L. Zhang et. al., F1000 Research. 4(1422) https://doi.org/10.12688/f1000research.7507.2
  3. Medicinal Genomics, “PathogINDICAtor qPCR microbial detection Assay on the AriaMX Real-Time PCR System optional decontamination step,” Document EUD-00021 1.4.  (2017). (Medicinal Genomics Corporation).
  4. K.J. McKernan, et. al., PLoS One 9(5)e, 96492 (2014).
  5. K.J. McKernan, J. Spangler, and Y. Helbert et. al.,“Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests,” F1000Res. 5(2471) https://doi.org/10.12688/f1000research.7507.2.
  6. S.D. Leppanen and H. Ebling, “Optimized Cannabis Microbial Testing: Combined Use of Medicinal Genomics Extraction Methods with the AriaMx qPCR Instrument,” Application Note 5994-0430EN, Agilent Technologies (2018).

Scott Leppanen is the Senior Field Applications Scientist and Anthony Macherone is the Senior Scientist for Agilent Technologies, Inc.  Heather Ebling is the Senior Applications and Support Manager for Medicinal Genomics. Direct correspondence to: [email protected] com and [email protected].

How to Cite This Article

S.D. Leppanen, H. Ebling, and A. Macherone, Cannabis Science and Technology 2(4), 69-76 (2019).

Editor's Note:

The print version of this article mistakenly left off Figure 7a and Table IV. Those elements are included in the correct version found here.