Optimized Cannabis Microbial Testing: Combined Use of Extraction Methods and Pathogen Detection Tests Using Quantitative Polymerase Chain Reaction: Page 3 of 4

August 22, 2019
Volume: 
2
Issue: 
4
Figure 6: MIP inhibition on petrifilm.
Figure 6 (click to enlarge): MIP inhibition on petrifilm. Comparative colony growth begins to even out at 96 h; however, 3M specifies counting should occur after 24 h.
Table I: DNA spiked into different MIP samples
Table I (click to enlarge): DNA spiked into different MIP samples
Table II: STEC O111 E. coli DNA spiked at different levels into chocolate
Table II (click to enlarge): STEC O111 E. coli DNA spiked at different levels into chocolate
Table III: Live cultures spiked into a single flower sample
Table III (click to enlarge): Live cultures spiked into a single flower sample
Table IV: List of species tested and corresponding results
Table IV (click to enlarge): List of species tested and corresponding results
Figure 7
Figure 7 (click to enlarge): (a) TAC qPCR dilution curves. Elevated TAC illustrates frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold. (b) qPCR efficiency (E). Across all 12 points an R2 of 0.999 was determined thus illustrating an efficiency approaching 100%.

Marijuana Infused Products Interference and Impact on Results

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils, and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, we spiked in live E. coli cells into various MIPs to measure the qPCR signal. In many of the spiked MIP samples, E. coli failed to grow compared to tryptic soy broth (TSB) controls.

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast the qPCR signal was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

Figure 6 compares plating E. coli with and without MIP on 3M coliform films. (See upper right for Figure 6, click to enlarge.) Growth of E. coli CFU was severely hindered in presence of oil or candy.

Method Accuracy

To show that the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table I, and at different numbers of DNA copies into chocolate (Table II). (See upper right for Tables I and II, click to enlarge.) The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and our PathoSEEK detection assay can successfully detect that range of DNA. Table III demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a  variety of organisms spiked onto cannabis flower samples. (See upper right for Table III, click to enlarge.)

Specificity

More than 60 species in total were used to verify the specificity of the PathoSEEK Microbial Safety Testing Solution, with a minimum of 6 to 10 and a maximum of 45 for each detection assay. The data shows 100% concordance with the current list of species tested. A subset of the data is shown in Table IV. (See upper right for Table IV, click to enlarge.)

Linearity

Linearity shows the relationship between a group of known dilutions and resulting Cq. To test the linearity of our PathoSEEK assays, we conducted 12, two-fold serial dilutions of a known DNA concentration in triplicate. Figures 7a and 7b show the results of the total aerobic count (TAC) assay and the qPCR efficiency (E) of the assays as inferred by the linear coefficient of determination (R2) which is a measure of how well a dependent variable fits a linear model. (See upper right for Figure 7, click to enlarge.)

Limit of Detection

Using PathoSEEK to determine the presence or absence of E. coli, STEC, Salmonella, and Aspergillus spp. a limit of detection (LOD) was determined by performing triplicate analyses of 12, two-fold serial dilutions starting at two copies through 5000 copies. In each case, a LOD of 10 copies were determined.

References: 
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  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.