With the introduction of legal cannabis available for sale in the U.S., providing safe, high-quality products defines the industry standard. As part of compliance testing, each state has different requirements for detection of microbial species in cannabis products. An ideal test is one that can be performed quickly with small amounts of cannabis product, is specific for the microbes required, can differentiate between live and dead microbes, and can be automated for high sample throughput. Medicinal Genomics developed a novel quantitative polymerase chain reaction (qPCR)-based test, in a 96-well plate format, that relies on fluorescence to detect amplified deoxyribonucleic acid (DNA). Fluorescence detection indicates the presence of microbial contamination on cannabis. This quantitative PCR method has been adapted for multiple matrices such as flower, leaf, concentrates, and an array of non-flower marijuana infused products (MIP). This review introduces the qPCR assay and compares its performance to culture-based methods.
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.
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.)
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 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.
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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).
The print version of this article mistakenly left off Figure 7a and Table IV. Those elements are included in the correct version found here.