qPCR: A Powerful Tool for the Microbiological Testing of Cannabis
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The suitability of certain methods for microbiological cannabis testing has become a sizable debate in the world of cannabis science.
The issue has been hitting the headlines most recently as state legislators in Massachusetts came under fire for refusing to standardize microbiological testing methods in the state. The criticism followed the decision to approve two cannabis testing laboratories for recreational cannabis testing licenses despite one testing facility using DNA-based quantitative polymerase chain reaction (qPCR) technology, and the other using traditional culture methods (TCM).
Contrasting views have also led to rule changes surrounding the use of the techniques in other states; California’s Bureau of Cannabis Control recently proposed a new suite of testing regulations which favours the use of strain-specific PCR-based methods and does not include a requirement for the more general Total Yeast and Mold Count (TYMC), a quality control test that uses TCM. This move went against the advice of the California Cannabis Industry Association’s Quality Control Committee, who endorse both techniques but felt that the exclusion of a TYMC requirement was an unnecessary risk to public safety.
To learn more about the qPCR testing technique, we spoke with Yvonne Helbert, a Senior Research and Development Scientist at Medicinal Genomics. Medicinal Genomics is a specialist in cannabis genetics research and created the first cannabis-specific validated microbial safety testing platform, known as PathoSEEK, using qPCR methods.
Alex Beadle (AB): What makes qPCR such a powerful analysis technique for the cannabis matrix?
Yvonne Helbert (YH): The cannabis matrix is very complicated. In addition to the many microbes that live on the plant’s surface, there are also endophytes, which are bacteria or fungus that live inside the plant without causing apparent disease to the plant. Since most culture-based techniques rely on viable cells growing in a medium for detection, they do not lyse plant cells open to see the endogenous pathogens like DNA-based methods do.
Aspergillus is a known plant endophyte, and four Aspergillus species (flavus, fumigatus, niger and terreus) are known human pathogens. It is therefore imperative that the flower sample be lysed so that the endophytes are accurately captured.
AB: How widely used or recognized is qPCR as a technique?
YH: qPCR techniques are widely used in gene expression, genotyping, pathogen detection, viral quantification, prenatal genetic testing, blood screening, oncology testing and much more. The qPCR market is considered to be the "gold standard" for accurate, sensitive and fast quantification of nucleic acid sequences. The global qPCR and digital PCR (dPCR) market is expected to reach USD 6.3 billion by 2025, according to a report by Grand View Research, Inc.
AB: In your opinion, what are the main pitfalls of using culture-based plating techniques as a form of microbiological analysis?
YH: Culture-based plating techniques fail to differentiate between pathogenic, beneficial, and benign microbes, making them poor indicators of safety. Also, as stated earlier, culture-based plating techniques fail to detect plant endophytes which can result in false negative results. AB: What advantages does qPCR have over the traditional plating methods? YH: qPCR is faster than plating - results can be obtained in hours, not days. qPCR is also more accurate than plating. qPCR detection assays actively look for specific DNA sequences shared by a family of microbes. If the DNA is present, qPCR will detect it. This active approach means we can design species-specific assays that target only pathogenic species, such as Aspergillus and STEC E.coli.
Culture plating is much more passive. You are essentially placing cells in a growth medium and hoping it’s favorable enough for them to grow so you can count them. The problem with that is only a fraction of microbes culture. And if you have multiple microbes in the same medium, they will compete with one another. We have demonstrated that the makeup of the microbial population after culturing can be radically different than what is actually on the plant.
AB: Is there anything else you'd like to highlight in terms of the work that Medicinal Genomics is doing?
YH: Medicinal Genomics has developed and validated the SenSATIVAx DNA extraction kit and PathoSEEK qPCR detection assays specifically for cannabis microbial testing.
This is important because many other microbial testing solutions for cannabis have been lifted from the food industry and as far as we know have not been validated on cannabis. Cannabis is a very complicated matrix and comes in three major forms: dried flower, cannabis extracts, and concentrates and edibles.
The flower is lipid-rich, making it very sticky. This presents a challenge because lipid-rich matrices cannot be sampled with simple aqueous emersions. The lipids need to be dissolved in water to fully sample such a matrix which requires a strong lysis buffer. While quick and simple aqueous preps are appealing for quick labor-free extraction they fail to fully sample the microbial diversity of the flower. The flower is also full of trichomes, terpenes, and cannabinoids adding another layer of difficulty in evaluating the microbial diversity. Extracts and concentrates can range from alcohol-based oils to tinctures, to whole plant extracts. Edibles can be just about anything.
Our DNA extraction kits were designed with all of these matrices in mind.
Another unique feature of the MGC technology is that the DNA extraction kit extracts the microbial DNA and the cannabis DNA. That cannabis DNA is then used as an internal positive control in all of the PathoSEEK detection assays. This internal positive control signal shows the DNA extraction process, the qPCR assay setup, and the detection run were all successful.