How Do You Know You’re Right? qPCR vs. Plating



Cannabis tests to detect microbial contamination is intricate. It might not be rocket science, but it is lifetime science, which implies it’s a moving target, or at the very least, it should really be, as we acquire a lot more and more information and facts about how the environment we stay in will work. We are lucky to be equipped to carry out that evaluation in ever escalating element. For instance, the science of genomics1 was born over 80 yrs ago, and just twenty a long time back, genetics was however a black box. We’ve designed tremendous progress considering that people early days, but we continue to have a extensive way to go, to be sure.

Significantly of that development is because of to our ability to develop additional precise applications, a technological ladder, if you will, that raises our consciousness, skills, and knowledge to new degrees. When a new procedure or technology seems, we assess it towards recognized practice to generate a new paradigm and make the vital adjustments. But men and women have to be ready to alter. In the cannabis sector, immediate improve is a continuous, initially mainly because that is the mother nature of a nascent sector, and 2nd simply because in the absence of some common and unimpeachable normal, it’s tough to know who’s appropriate. In particular when the old, trusted reference system (i.e. plating, which is fundamentally developing microorganisms on the surface area of a nutritional medium) is deeply flawed in its application to cannabis testing vs. molecular solutions (i.e., quantitative polymerase chain response, or qPCR for shorter).

Dr. Sherman Hom, Director of Regulatory Affairs at Medicinal Genomics

Plating devices have been employed faithfully for close to 130 decades in the foodstuff field, and has executed reasonably perfectly.2 But cannabis is not foods and simply cannot be analyzed as if it ended up. In truth, plating techniques have a host of big disadvantages that only clearly show up when they’re utilised to detect cannabis pathogens. They are, in no individual get:

  1. A single plating system just cannot enumerate a team of microorganisms and/or detect distinct bacterial and fungal pathogens. This is further more complex by the point that better than 98% of the microbes in the entire world do not kind colonies.3 And there is no A single Universal bacterial or fungal SELECTIVE agar plate that will let the development of all micro organism or all fungal strains. For example, the 5 genus species of fungal strains implicated in powderly mildew DO NOT plate at all.
  2. Cannabinoids, which can characterize 10-30% of a cannabis flower’s weight, have been demonstrated to have antibacterial activity.4 Antibiotics inhibit the expansion of microbes and in some conditions destroy it completely. Salmonella species & shiga toxin creating coli (STEC) germs, in individual, are really delicate to antibiotics, which qualified prospects to both a false detrimental end result or lower total counts on plates vs. qPCR techniques.
  3. Plating procedures simply cannot detect bacterial and fungal endophytes that reside a aspect or all of their lifetime cycle inside of a hashish plant.5,6 Examples of endophytes are the Aspergillus pathogens (A. flavus, A. fumigatus, A. niger, and A. terreus). Procedures to break open up the plant cells to obtain these endophytes to prepare them for plating strategies also lyse these microbial cells, thereby killing endophytic cells in the course of action. Which is why these endophytes will in no way type colonies, which prospects to possibly wrong damaging benefits or lessen complete counts on plates vs. qPCR techniques.
  4. Selective plating media for molds, such as Dichloran Rose-Bengal Chloramphenicol (DRBC) truly lowers mold growth—especially Aspergillus—by as a great deal as 5-fold.This provides bogus destructive results for this perilous human pathogen. In other text, despite the fact that the DRBC medium is normally utilised to cut down microbes it will come at the charge of lacking 5-fold extra yeast and molds than Potato Dextrose Agar (PDA) + Chloramphenicol or molecular approaches. These observations had been derived from research results of the AOAC emergency reaction validation.7
  5. Ultimately, we’ve not long ago determined four bacterial species, which are human pathogens associated with cannabis that do not develop at the plating method incubation temperature commonly applied.8 They are Aeromonas hydrophila, Pantoea agglomerans, Yersinia enterocolitica, and Rahnella aquatilis. This lowers overall counts on plates qPCR strategies.

So why is plating still so well known? Far better however, why is it even now the advisable strategy for a lot of state regulators? Beats me. But I can hazard a couple of guesses.

A yeast and mould plate exam

Initial, investigation on cannabis has been limited for the improved aspect of the very last 70 a long time, and it’s impossible to assemble a entire body of scientific awareness by retaining anyone in the dim. 10 yrs back, as a single of the first governing administration-employed scientists to analyze hashish, I was tapped to start off the first hashish screening lab at the New Jersey Dept. of Overall health and we had to create it from ground zero. Nobody knew anything about cannabis then.

2nd, due to the fact of a shortage of hashish-skilled professionals, associates of numerous regulatory bodies arrive from the food items industry—where they’ve utilized plating just about completely. So, when it arrives time to draft cannabis microbial tests laws, plating is the default technique. Following all, it worked for them just before and they are at ease with recommending it for their state’s hashish rules.

At last, there is a selected amount of money of discomfort in not remaining suitable. Heading into this entirely new area—remember, the authorized hashish sector didn’t even exist 10 several years ago—we human beings like to have a small certainty to slide back on. The difficulties is, falling again on what we did just before stifles terribly desired development. This is a circumstance in which, if you’re relaxed with your outdated methods and you’re absolutely sure of your effects, you are almost certainly improper.

So let’s acknowledge the reality that we’re all in this uncharted territory alongside one another. We really do not nonetheless know every thing about hashish we require to know, but we do know some items, and we currently have some very good resources, dependent on true science, that transpire to function truly very well. Let’s use them to help light our way.

References

  1. J. Weissenbach. The increase of genomics. Comptes Rendu Biologies, 339 (7-8), 231-239 (2016).
  2. R. Koch. 1882. Die Aetiologie der Tuberculose.  Berliner Klinische Wochenschrift, 19, 221-230 (1882). 
  3. W. Wade. Unculturable bacteria—the uncharacterized organisms that cause oral bacterial infections. Journal of the Royal Society of Drugs, 95(2), 91-93 (2002).
  4. J.A. Karas, L.J.M. Wong, O.K.A. Paulin, A. C. Mazeh, M.H. Hussein, J. Li, and T. Vekov. Antibiotics, 9(7), 406 (2020).
  5. M. Taghinasab and S. Jabaji, Hashish microbiome and the part of endophytes in modulating the generation of secondary metabolites: an overview. Microorganisms 2020, 8, 355, 1-16 (2020).
  6. P. Kusari, S. Kusari, M. Spiteller and O. Kayser, Endophytic fungi harbored in Hashish sativa L.: variety and probable as biocontrol agents towards host plant-precise phytopathogens. Fungal Diversity 60, 137–151 (2013).
  7. K. McKernan, Y. Helbert, L. Kane, N. Houde, L. Zhang, S. McLaughlin, Full genome sequencing of colonies derived from cannabis bouquets & the effect of media choice on benchmarking complete yeast & mildew detection tools,  https://f1000investigation.com/articles or blog posts/10-624 (2021).
  8. K. McKernan, Y. Helbert, L. Kane, L. Zhang, N. Houde, A. Bennett, J. Silva, H. Ebling, and S. McLaughlin, Pathogenic Enterobacteriaceae have to have several lifestyle temperatures for detection in Hashish sativa L. OSF Preprints, https://osf.io/j3msk/, (2022)