Tag Archives: PCR

Hop Latent Viroid (HLVd) & Pathogen Diagnostics: A Comprehensive Overview

By Tassa Saldi, Ph.D.
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Hop latent viroid (HLVd) has gained attention as the molecular cause of “dudding disease” and is causing significant economic losses in the cannabis industry.1,2 Estimates indicate that upwards of 4 billion dollars of market value are lost each year to this pathogen alone.3 The impact of HLVd on cannabis plants necessitates the development and implementation of effective pathogen diagnostics to mitigate its spread and minimize crop damage. With collaborative research efforts, we can gain valuable insights into the characteristics, spread, symptoms and preventive measures associated with HLVd in the cannabis industry.

Viroids: A Brief Overview

Figure 1: Virus vs Viroid

Viroids are unique infectious agents composed solely of genetic material, distinct from viruses. Unlike viruses, viroids lack a protective protein layer and solely rely on the host plant for replication and spread. Their stability and ability to persist in various environments make viroids a formidable threat to plant health.

Hop Latent Viroid: Origin and Global Spread

Hop latent viroid was initially identified in hop plants in 19884 and was found to be largely asymptomatic in this crop. Consequently, HLVd has spread worldwide, mostly unchecked by the hops industry. This pathogen has been identified on most continents and in some fields more than 90% of hops plants are infected.5 Hop latent viroid very likely jumped from hops into cannabis, due to similar genetics. The timing and mechanism of cross-species transmission to cannabis remains unknown, but the prevalence of HLVd suggests this viroid has been circulating within cannabis for an extended period. Data collected at TUMI Genomics indicates that HLVd is present in all states in the United States where cannabis is legal as well internationally including; Canada, the United Kingdom, France, the Netherlands, Thailand, Austria and Switzerland.

Symptoms and Impacts on Cannabis Plants 

Figure 2: HLVd Symptoms

HLVd exhibits a wide range of symptoms, which can vary from severe to subtle, affecting the growth, leaf development, flower quality and overall vitality of cannabis plants. Understanding these symptoms is crucial for timely diagnosis and appropriate disease management strategies.  However, HLVd can also present asymptomatically, especially in vegetative plants. The only way to determine if your plants are infected is by routine molecular testing.

Modes of Transmission

Mechanical Transmission: HLVd primarily spreads mechanically through contact with infected sap during activities like trimming and handling. Additionally, transmission through contaminated water and the potential role of insects, fungal pathogens and seeds in spreading HLVd have also been observed.

Seed Transmission: Although no published studies exist in cannabis describing the frequency of seed transmission, HLVd does transmit through seeds in hop plants at a rate of around 8%.7 Preliminary studies performed by TUMI Genomics in collaboration with EZ-genetics suggest cannabis seed transmission does occur at variable rates depending on strain and level of infection of the parent plants.

Water Transmission: It has also been observed that viroids are in high concentration in the roots8 and can move from the root into runoff water.9 Plants sharing a common water source with infected plants, such as recirculating water systems or flood and drain procedures, are at risk for transmission of the viroid.

Insect and Other Vector Transmission: The jury is still out as to whether or not insects can transmit HLVd. However, multiple viroids are transmitted via insects, so it is likely that HLVd insect transmission occurs. Recent studies also indicate that fungal pathogens, like Fusarium, can transmit viroid infections.6 While pathogenic fungus is a major concern for cannabis growers in its own right, limiting the prevalence and spread of fungal pathogens in your facility could help limit hop latent viroid transmission as well.

Therefore, implementing proper sanitation practices and limiting pest access can help minimize transmission risks.

Preventive Measures

Prevention plays a vital role in safeguarding cannabis crops against HLVd. The STOP program, developed by TUMI Genomics, offers a comprehensive approach that includes maintaining a Sterile environment, Testing mother plants regularly, Organizing the facility to minimize pathogen spread, and Protecting the facility’s borders from introduction of infected plant material, insects and contaminated water. More details on these preventative measures can be found here.

Pathogen Diagnostics

Protecting your plants from hop latent viroid requires accurate identification and removal of infected plants before the infection spreads to other plants. To accomplish this, several critical factors should be considered:

Type of test: HLVd and all viroids can only be detected by a molecular test (a test that detects the presence of DNA/RNA). Among common molecular tests, PCR is generally the most sensitive and accurate method. PCR can provide both a diagnosis and an approximate viroid level, allowing informed management decisions. Other types of molecular tests, such as LAMP and RPA, can formally be as sensitive as PCR, but the classic versions of these assays often suffer from false positive/negative results, reducing accuracy.

Figure 3: HLVd Levels and Distribution

Tissue type: An important consideration for HLVd detection is the plant tissue selected for testing, especially when identifying low-level or early infections when HLVd is not yet systemic. Studies completed by TUMI Genomics and others show root tissue contains the highest levels of HLVd and is the most reliable tissue for detection of viroid infection. While upper root tissue appears to contain the highest levels of viroid, roots from anywhere in the root ball are predictive of infection. Samples taken from the leaves/foliage tend to have lower levels of viroid and may produce false negative results.

Figure 4: Testing Schedule

Testing frequency: Routine pathogen testing is standard practice in general agriculture and is critical to maintain a healthy cannabis crop. Testing of mother plants every 4-6 weeks for economically critical pathogens (such as HLVd) will help ensure a successful run and a high-quality product.

Disinfection Methods

Studies have shown that viroids can remain infectious for longer than 24 hours on most common surfaces11 and 7 weeks in water.10 Making effective disinfection methods essential to limit the spread of HLVd. While common disinfectants like alcohol and hydrogen peroxide are ineffective against viroids, a 10% bleach solution has shown efficacy in destroying HLVd. Proper tool sterilization practices, such as soaking tools in bleach for 60 seconds, are crucial to prevent transmission during plant handling.

Figure 5: Bleach Dilution

Hop latent viroid poses a significant threat to the cannabis industry, leading to substantial economic losses. Timely and accurate pathogen diagnostics, along with stringent preventive measures, are essential for minimizing the impact of HLVd. Regular testing, proper disinfection protocols and adherence to pathogen prevention programs can help ensure the health and vitality of cannabis crops in the face of this global pandemic.


References

  1. Bektas, A., et al. “Occurrence of Hop Latent Viroid in Cannabis Sativa with Symptoms of Cannabis Stunting Disease in California.” APS Journals, 21 Aug. 2019, doi.org/10.1094/PDIS-03-19-0459-PDN.
  2. Warren, J.G., et al. “Occurrence of Hop Latent Viroid Causing Disease in Cannabis Sativa in California.” APS Journals, 21 Aug. 2019, doi.org/10.1094/PDIS-03-19-0530-PDN.
  3. Cooper, Benjie. “Hop Latent Viroid Causes $4 Billion Cannabis Industry Loss – Candid Chronicle.” Candid Chronicle – Truthful, Straightforward, Blunt Cannabis News, 16 Aug. 2021, candidchronicle.com/hop-latent-viroid-causes-4-billion-cannabis-industry-loss/.
  4. Puchta H, Ramm K, Sänger HL. The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res. 1988 May 25;16(10):4197-216. doi: 10.1093/nar/16.10.4197. PMID: 2454454; PMCID: PMC336624.
  5. Faggioli, Franceso, et al. “Geographical Distribution of Viroids in Europe.” Viroids and Satellites, 31 July 2017, www.sciencedirect.com/science/article/abs/pii/B9780128014981000449#bib47.
  6. Wei S, Bian R, Andika IB, Niu E, Liu Q, Kondo H, Yang L, Zhou H, Pang T, Lian Z, Liu X, Wu Y, Sun L. Symptomatic plant viroid infections in phytopathogenic fungi. Proc Natl Acad Sci U S A. 2019 Jun 25;116(26):13042-13050. doi: 10.1073/pnas.1900762116. Epub 2019 Jun 10. PMID: 31182602; PMCID: PMC6600922.
  7. Singh RP. The discovery and eradication of potato spindle tuber viroid in Canada. Virus disease. 2014 Dec;25(4):415-24. doi: 10.1007/s13337-014-0225-9. Epub 2014 Dec 2. PMID: 25674616; PMCID: PMC4262315.
  8. Jama, Aisha, et al. TUMI Genomics, Fort Collins, CO, 2022, Hop Latent Viroid Levels and Distribution in Cannabis Plant Tissue.
  9. Mackie AE, Coutts BA, Barbetti MJ, Rodoni BC, McKirdy SJ, Jones RAC. Potato spindle tuber viroid: Stability on Common Surfaces and Inactivation With Disinfectants. Plant Dis. 2015 Jun;99(6):770-775. doi: 10.1094/PDIS-09-14-0929-RE. Epub 2015 May 15. PMID: 30699527.
  10. Mackie AE, Coutts BA, Barbetti MJ, Rodoni BC, McKirdy SJ, Jones RAC. Potato spindle tuber viroid: Stability on Common Surfaces and Inactivation With Disinfectants. Plant Dis. 2015 Jun;99(6):770-775. doi: 10.1094/PDIS-09-14-0929-RE. Epub 2015 May 15. PMID: 30699527.
  11. Mackie AE, Coutts BA, Barbetti MJ, Rodoni BC, McKirdy SJ, Jones RAC. Potato spindle tuber viroid: Stability on Common Surfaces and Inactivation With Disinfectants. Plant Dis. 2015 Jun;99(6):770-775. doi: 10.1094/PDIS-09-14-0929-RE. Epub 2015 May 15. PMID: 30699527.

Perfecting Your Packaging for Cannabis Beverages

By Julie Saltzman
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Some consumers participating in the legal cannabis market want to avoid inhalable products. They are concerned about the effects of the smoke or they want their usage to be discreet — without the pungent aroma emanating from burning cannabis flower. For those consumers, edibles are the preferred option and a growing product category.

Within the edibles space, the beverage segment — with limited product options in some states — may offer significant potential for growth. In 2021, cannabis-infused beverages accounted for only 1% of total legal cannabis product sales and about 5% of the edibles segment in the United States, reports market researcher BDSA. But cannabis beverage sales are growing around the U.S.

In California, cannabis drinks grew their market share in the edibles category from 4% in 2018 to 7% in 2021. Nevada saw beverages increase their share of edibles revenues from 7% to 10% in the same time frame. And cannabis beverages’ share of edibles sales in Massachusetts went from less than 1% in 2019 to 8% in 2021.

Pegged at $180 million in revenue last year, the cannabis beverage market is projected to reach nearly $500 million by 2026, predicts BDSA.

Today, gummies and chocolate products dominate the edibles category. Although beverages are currently a small segment of edible sales, they may have some inherent advantages — familiarity, faster-acting products from improved bioavailability, and taste and flavor innovations — over other cannabis products. Since beverages can incorporate many different flavors from fruity, cola and sweet to coffee, tea, sour and bitter, these myriad flavor variations can mask or minimize any off-tastes associated with THC.

Viewed as part of their everyday regimen, consumers drink beverages for hydration, nourishment, refreshment and enjoyment. Cannabis beverages are well-suited for consumers’ lifestyles, while gummies and chocolates may be perceived as sugary treats and special occasion items.

Cruise Beverage B Happy Nitro-Infused CBD Drinks.

Brand owners are beginning to recognize the limited availability of products and growth potential of cannabis-infused beverages and are looking to enter the category. Packaging plays a key role in cannabis beverages, with sustainability, regulatory compliance (e.g., child-resistant), labeling compliance (e.g., warning symbols and text), convenience and branding all contributing to the success of the expanding product category.

Sustainable Packaging

Consumers, especially younger generations, are concerned about the environment and support brands that align with their values. According to the 2020 Sustainable Market Share Index from the NYU Stern Center for Sustainable Business, sustainability-marketed products delivered about 55% of the market growth in consumer packaged goods (CPG) from 2015 to 2019 in the U.S. despite being only 16% of the market. Sustainability-marketed goods grew seven times faster than products not marketed as sustainable and nearly four times faster than the overall CPG market.

As a primary consumer touchpoint, packaging is a good way for cannabis beverage brands to show their commitment to the environment. But finding the most sustainable packaging option for a particular application may not always be as straightforward as it seems. Many considerations are involved — material choice (e.g., plastic, glass, or aluminum), recyclability of the material, the weight of the material, recycled content of the final package, package design (minimalist vs. excessive), transportation costs and other factors like reusability.

To help facilitate the process, Berlin Packaging uses life cycle analysis to determine a product’s environmental impact or carbon footprint over its entire life cycle, including sourcing & raw materials extraction (minerals resource use), manufacturing (energy and water usage), distribution (freight miles, fuel usage) and end-of-life (recovery, recycling, reprocessing).

We have the know-how to improve the sustainability of any packaging material — whether it be lightweighting, use of post-consumer recycled (PCR) content, greater recycling rates and more.

Regulatory Compliance

Because legal cannabis products are regulated by individual states and not at the federal or national level, the regulations for cannabis packaging requirements can vary widely from one state to another. However, there are some common rules that all states follow.

Child-resistant capable cap fits snugly over the top of a can.

All cannabis products — including beverages — require child-resistant packaging to meet the standards of the Consumer Product Safety Commission. For aluminum cans, Berlin Packaging offers a child-resistant capable mechanism that fits snugly over the top of a can. Available in polypropylene or a bio-based resin, the single-use device can be custom developed to fit the exact specifications of the customer’s cans. In-stock products are available for standard 202 can ends.

Along with child-resistant capable packaging, states also require some type of warning symbol and statement on the label to indicate the product contains cannabis. Depending on the state, the symbol may be a triangular or diamond shaped in a bright or contrasting color to call attention to it on the label. The symbol typically houses a cannabis leaf image or “THC”.

Convenience

Like any packaged drink, cannabis beverages need to check all the boxes for consumer convenience — easy to drink, portability, cupholder friendly and resealable.

Users can easily reseal PET and glass bottles with continuous thread or lug finish closures, but cans present a challenge. Berlin Packaging offers a solution with a resealable can that opens like a traditional stay-on-tab. Here’s how it works. Lifting the pull tab breaks the tamper-evident band and unlocks the slider mechanism. Pulling the slider opens the can and makes the familiar venting sound — even after reopening.

The configuration of the opening creates a smooth laminar flow to improve the drinking experience. Moving the slider back to its original position and pushing down the pull tab, which produces a clicking sound, reseal the closure. The tamper-evident band remains on the can underneath the pull tab.

Branding

Cannabis beverages come in drops, shots, syrups, carbonated, iced tea, lemonade, fruity, water, sports & energy, mocktails, tea, coffee and hot cocoa.

Because cannabis has been associated with medicinal uses, many consumers use cannabis products to manage their wellbeing and health. Thus, some cannabis products have been positioned to relieve stress, promote relaxation and sleep, reduce pain and inflammation, improve mental focus, enhance mood or simply for indulgence and enjoyment.

Product positioning and the experience the brand owner wants to create for the consumer can help inform the brand design, personality, and narrative or storytelling. It’s also important that the brand design and messaging resonate with the product’s target audience.

Studio One Eleven, Berlin Packaging’s in-house innovation division, can help cannabis beverage marketers with their product branding from concept to commercialization. We offer market research and consumer insights, brand strategy and visual branding design, brand name development, structural package design, and more. Our services are available at no additional charge in exchange for a customer’s packaging business.

Cruise Beverage distributes nitrogen-infused CBD drinks with all-natural ingredients in 12-oz aluminum cans under the B Happy brand. The team at Studio One Eleven helped Cruise Beverage and its B Happy brand tell their story of free-spirited enjoyment with updated branding, expressive flavor names (i.e., Loosen Up Lemon, Peaceful Pear, Mellow Mango, and Blissful Blood Orange), and unique packaging graphics.

Uplifting illustrations speak to the brand’s sense of freedom and relaxation, and the hand-drawn style reflects the craftsmanship of the CBD beverage product. A white background with flavorful pops of color says clean and fresh, while tiny bubble imagery communicates the delightful effervescence of the fizzy drinks.

Detecting Microbial Contamination in Cannabis

By Mike Clark
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Increasing cannabis use across the US has come with increased scrutiny of its health effects. Regulators and healthcare providers are not just concerned about the direct effects of inhaling or consuming cannabinoids, however, but also about another health risk: microbial contamination in cannabis products. Like any other crop, cannabis is susceptible to contamination by harmful pathogens at several points throughout the supply chain, from cultivation and harvesting to distribution. Many state regulators have set limits for microbial populations in cannabis products. Consequently, testing labs must adopt efficient screening protocols to help companies remain compliant and keep their customers safe.

Some of the pathogens common to cannabis flower include Aspergillus fungus species such as A. flavus, A. fumigatus, A. niger and A. terreus. Cannabis might also harbor harmful E. coli and Salmonella species, including Shiga toxin-producing E. coli (STEC). Regulations vary by state, but most have set specific thresholds for how many colony forming units (CFUs) of particular species can be present in a sellable product.

The gold standard method for detecting microbes is running cultures.

Growers and testing labs need to develop a streamlined approach to remain viable. Current methods, including running cultures on every sample, can be expensive and time-consuming, but by introducing a PCR-based screening step first, which identifies the presence of microbial DNA – and therefore the potential for contamination – laboratories can reduce the number of cultures they need to run, saving money and time.

The Risk of Aspergillus Contamination

Contamination from Aspergillus species can bring harm to cannabis growers and their customers. The state of Michigan is currently undergoing the largest cannabis recall in its history from Aspergillus contamination.

If contamination grows out of control, the pathogen can damage the cannabis plant itself and lead to financial losses. Aspergillus can also cause serious illness in consumers, especially those that are immunocompromised. If an immunocompromised person inhales Aspergillus, they can develop aspergillosis, a lung condition with a poor prognosis.

A Two-Step Screening Process

The gold standard method for detecting microbes is running cultures. This technique takes weeks to deliver results and can yield inaccurate CFU counts, making it difficult for growers to satisfy regulators and create a safe product in a timely manner. The use of polymerase chain reaction (PCR) can greatly shorten the time to results and increase sensitivity by determining whether the sample has target DNA.

Using PCR can be expensive, particularly to screen for multiple species at the same time, but a qPCR-based Aspergillus detection assay could lead to significant cost savings. Since the average presumptive positive rate for Aspergillus contamination is low (between 5-10%), this assay can be used to negatively screen large volumes of cannabis samples. It serves as an optional tool to further speciate only those samples that screened positive to comply with state regulations.

Conclusion

Overall, screening protocols have become a necessary part of cannabis production, and to reduce costs, testing labs must optimize methods to become as efficient as possible. With tools such as PCR technology and a method that allows for initial mass screening followed by speciation only when necessary, laboratories can release more samples faster with fewer unnecessary analyses and more success for cannabis producers in the marketplace.

bioMérieux Gets AOAC Approval for PCR Detection of STEC and Salmonella in Cannabis

By Cannabis Industry Journal Staff
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bioMérieux, a leader in the in vitro diagnostics space and a supporter of the cannabis testing market, announced last month that they have achieved the first ever AOAC International approval for PCR Multiplex Detection of STEC and Salmonella in cannabis flower for their GENE-UP® PRO STEC/Salmonella Assay. The performance tested method approval for their new assay accomodates simultaneous enrichment and detection of STEC (Shiga Toxigenic Escherichia coli) and Salmonella spp. in cannabis samples.

The method is aimed at increasing efficiency in cannabis testing labs by reducing sample preparation time for microbiological testing. With the single enrichment and real-time multiplex PCR detection, bioMérieux says their new assay can provide reliable detection of STEC and Salmonella in 24 hours using just a single test.

PCR technology is one of the most widely utilized testing methods for detecting pathogens in a variety of matrices. bioMérieux claims it is easy to use, scientifically robust and reduces costs, time spent testing and errors.

Maria McIntyre, cannabis strategic operations business manager at bioMérieux, says that AOAC performance tested method approval is setting the bar for cannabis testing laboratories and furthering cannabis science. “AOAC International impacts cannabis science by setting analytical method standards that act as the benchmark for method validation,” says McIntyre. “This simplifies the validations needed by cannabis laboratories and assures the utmost confidence in product safety and human health.”

Leaders in Cannabis Testing – Part 1: A Q&A with Milan Patel, CEO and Co-Founder of PathogenDx

By Aaron Green
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In this “Leaders in Cannabis Testing” series of articles, Green interviews cannabis testing laboratories and technology providers that are bringing unique perspectives to the industry. Particular attention is focused on how these businesses integrate innovative practices and technologies to navigate a rapidly changing landscape of regulatory constraints and B2B demand.

PathogenDx is an Arizona-based provider of microbial testing technologies. Since their inception in 2014, they have broadened their reach to 26 states in the US. In addition to cannabis product testing, PathogenDx also provides technologies for food safety testing, environmental testing and recently started offering human diagnostics testing to support COVID-19 response efforts.

We interviewed Milan Patel, CEO and co-founder of PathogenDx. Milan founded PathogenDx as a spin-off from one of his investments in a clinical diagnostics company testing for genetic markers in transplant organs. Prior to PathogenDx, Milan worked in finance and marketing at Intel and later served as CFO at Acentia (now Maximus Federal).

Aaron Green: What’s the history of PathogenDx?

Milan Patel: PathogenDx was effectively a spin-off of a clinical diagnostics company that my partner Dr. Mike Hogan, the inventor of the technology, had founded when he was a professor at the University of Arizona, but previously at Baylor Medical College back in 2002. I had invested in the company back then and I had realized that his technology had a broad and wide sweeping impact for testing – not just for pathogens in cannabis specifically, but also for pathogens in food, agriculture, water and even human diagnostics. In the last 14 months, this became very personal for every single person on the planet having been impacted by SARS-CoV-2, the viral pathogen causing Covid-19. The genesis of the company was just this, that human health, food and agricultural supply, and the environment has and will continue to be targeted by bacterial, fungal and viral pathogens impacting the safety and health of each human on the planet.

We founded PathogenDx and we pivoted the company from its original human organ transplant genetics market scope into the bigger markets; we felt the original focus was too niche for a technology with this much potential. We licensed the technology, and we repurposed it into primarily cannabis. We felt that achieving commercial success and use in the hands of cannabis testing labs at the state level where cannabis was first regulated was the most logical next step. Ultimately, our goal was and is to move into markets that are approved at the federal regulatory side of the spectrum, and that is where we are now.

Green: What year was that?

Milan Patel, CEO and Co-Founder of PathogenDx
Photo credit: Michael Chansley

Patel: 2014.

Green: So, PathogenDx started in cannabis testing?

Patel: Yes, we started in cannabis testing. We now have over 100 labs that are using the technology. There is a specific need in cannabis when you’re looking at contamination or infection.

In the case of contamination on cannabis, you must look for bacterial and fungal organisms that make it unsafe, such as E. coli, or Salmonella or Aspergillus pathogens. We’re familiar with recent issues like the romaine lettuce foodborne illness outbreaks at Chipotle. In the case of fungal organisms such as Aspergillus, if you smoke or consume contaminated cannabis, it could have a huge impact on your health. Cannabis regulators realized that to ensure public health and safety there was more than just one pathogen – there were half a dozen of these bugs, at a minimum, that could be harmful to you.

The beauty of our technology, using a Microarray is that we can do what is called a multiplex test, which means you’re able to test for all bacterial and fungal pathogens in a single test, as opposed to the old “Adam Smith” model, which tests each pathogen on a one-by-one basis. The traditional approach is costly, time consuming and cumbersome. Cannabis is such a high value crop and producers need to get the answer quickly. Our tests can give a result in six hours on the same day, as opposed to the two or three days that it takes for these other approved methods on the market.

Green: What is your business model? Is there equipment in addition to consumables?

Patel: Our business model is the classic razor blade model. What that means is we sell equipment as well as the consumables – the testing kits themselves.

The PathogenDx technology uses standard, off-the-shelf lab equipment that you can find anywhere. We didn’t want to make the equipment proprietary so that a lab has to buy a specific OEM branded product. They can use almost any equipment that’s available commercially. We wanted to make sure that labs are only paying a fraction of the cost to get our equipment, as opposed to using other vendors. Secondly, the platform is open-ended, meaning it’s highly flexible to work with the volumes that different cannabis labs see daily, from high to low.

One equipment set can process many different types of testing kits. There are kits for regulated testing required by states, as well as required environmental contamination.

Green: Do you provide any in-house or reference lab testing?

Patel: We do. We have a CLIA lab for clinical testing. We did this about a year ago when we started doing COVID testing.

We don’t do any kind of in-house reference testing for cannabis, though we do use specific reference materials or standards from Emerald Scientific, for example, or from NCI. Our platform is all externally third-party reference lab tested whether it’s validated by our external cannabis lab customers or an independent lab. We want our customers to make sure that the actual test works in their own hands, in their own facility by their own people, as opposed to just shrugging our shoulders and saying, “hey, we’ve done it ourselves, believe us.” That’s the difference.

Green: Can you explain the difference between qPCR and endpoint PCR?

Patel: The difference between PathogenDx’s Microarray is it uses endpoint PCR versus qPCR (quantitative real time PCR). Effectively, our test doesn’t need to be enriched. Endpoint PCR delivers a higher level of accuracy, because when it goes to amplify that target DNA, whether it’s E. coli, Salmonella or Aspergillus pieces, it uses all the primer reagent to its endpoint. So, it amplifies every single piece of an E. Coli (for example) in that sample until the primer is fully consumed. In the case of qPCR, it basically reaches a threshold and then the reaction stops. That’s the difference which results in a much greater level of accuracy. This provides almost 10 times greater sensitivity to identify the pathogen in that sample.

The second thing is that we have separated out how the amplified sample hybridizes to the probe. In the case of our assay, we have a microarray with a well in it and we printed the actual probe that has the sequence of E. coli in there, now driving 100% specificity. Whereas in the qPCR, the reaction is not only amplifying, but it’s also basically working with the probe. So, in that way, we have a higher level of efficiency in terms of specificity. You get a definite answer exactly in terms of the organism you’re looking for.

In terms of an analogy, let’s take a zip code for example which has the extra four digits at the end of it.  In the case of endpoint PCR, we have nine digits. We have our primer probes which represent the standard five digits of a zip code, and the physical location of the probe itself in the well which serves as the extra four digits of that zip code. The analyte must match both primary and secondary parts of the nine-digit zip code for it to lock in, like a key and a lock. And that’s the way our technology works in a nutshell.

Endpoint PCR is completely different. It drives higher levels of accuracy and specificity while reducing the turnaround time compared to qPCR – down to six hours from sample to result. In qPCR, you must enrich the sample for 24 to 48 hours, depending on bacteria or fungus, and then amplification and PCR analysis can be done in one to three hours. The accuracies and the turnaround times are the major differences between the endpoint PCR and qPCR.

Green: If I understand correctly, it’s a printed microarray in the well plate?

Patel: That’s correct. It’s a 96-well plate, and in each well, you’ve now printed all the probes for all targets in a single well. So, you’re not running more than one well per target, or per organism like you are for qPCR. You’re running just one well for all organisms. With our well plates, you’re consuming fewer wells and our patented foil-cover, you only use the wells you need. The unused wells in the well plate can be used in future tests, saving on costs and labor.

Green: Do you have any other differentiating IP?

The PathogenDx Microarray

Patel: The multiplex is the core IP. The way we process the raw sample, whether it’s flower or non-flower, without the need for enrichment is another part of the core IP. We do triplicate probes in each well for E. Coli, triplicate probes for Salmonella, etc., so there are three probes per targeted organism in each of the wells. We’re triple checking that you’re definitively identifying that bug at the end of the day. This is the cornerstone of our technology.

We were just approved by the State of New York, and the New York Department of Health has 13 different organisms for testing on cannabis. Think about it: one of the most rigorous testing requirements at a state level – maybe even at a federal level – and we just got approved for that. If you had to do 13 organisms separately, whether it’s plate culture or qPCR, it would become super expensive and very difficult. It would break the very backs of every testing lab to do that. That’s where the multiplexing becomes tremendously valuable because what you’re doing is leveraging the ability to do everything as a single test and single reaction.

Green: You mentioned New York. What other geographies are you active in?

Patel: We’re active in 26 different states including the major cannabis players: Florida, Nevada, California, Arizona, Michigan, New York, Oklahoma, Colorado and Washington – and we’re also in Canada. We’re currently working to enter other markets, but it all comes down to navigating the regulatory process and getting approval.

We’re not active currently in other international markets yet. We’re currently going through the AOAC approval process for our technology and I’m happy to say that we’re close to getting that in the next couple of months. Beyond that, I think we’ll scale more internationally.

I am delighted to say that we also got FDA EUA federal level authorization of our technology which drives significant credibility and confidence for the use of the technology. About a year ago, we made a conscious choice to make this technology federally acceptable by going into the COVID testing market. We got the FDA EUA back on April 20, ironically. That vote of confidence by the FDA means that our technology is capable of human testing. That has helped to create some runway in terms of getting federalized with both the FDA and the USDA, and certification by AOAC for our different tests.

Green: Was that COVID-19 EUA for clinical diagnostics or surveillance?

Patel: It was for clinical diagnostics, so it’s an actual human diagnostic test.

Green: Last couple of questions here. Once you find something as a cannabis operator, whether its bacteria or fungus, what can you do?

Patel: There are many services that are tied into our ecosystem. For example, we work with Willow Industries, who does remediation.

There’s been a lot of criticism around DNA based technology. It doesn’t matter if it’s qPCR or endpoint PCR. They say, “well, you’re also including dead organisms, dead DNA.” We do have a component of separating live versus dead DNA with a biomechanical process, using an enzyme that we’ve created, and it’s available commercially. Labs can test for whether a pathogen is living or dead and, in many cases, when they find it, they can partner with remediation companies to help address the issue at the grower level.

Another product we offer is an EnviroX test, which is an environmental test of air and surfaces. These have 50 pathogens in a single well. Think about this: these are all the bad actors that typically grow where soil is – the human pathogens, plant pathogens, powdery mildew, Botrytis, Fusarium – these are very problematic for the thousands of growers out there. The idea is to help them with screening technology before samples are pulled off the canopy and go to a regulated lab. We can help the growers isolate where that contamination is in that facility, then the remediation companies can come in, and help them save their crop and avoid economic losses.

Green: What are you most interested in learning about?

Patel: I would prefer that the cannabis industry not go through the same mistakes other industries have gone through. Cannabis started as a cottage industry. It’s obviously doubled every year, and as it gets scaled, the big corporations come in. Sophistication, standards, maturity all help in legitimacy of a business and image of an industry. At the end of the day, we have an opportunity to learn from other industries to really leapfrog and not have to go through the same mistakes. That’s one of the things that’s important to me. I’m very passionate about it.

One thing that I’ll leave you with is this: we’re dealing with more bugs in cannabis than the food industry. The food industry is only dealing with two to four bugs and look at the number of recalls they are navigating – and this is a multi-billion-dollar industry. Cannabis is still a fraction of that and we’re dealing with more bugs. We want to look ahead and avoid these recalls. How do you avoid some of the challenges around antimicrobial resistance and antibiotic resistance? We don’t want to be going down that road if we can avoid it and that’s sort of a personal mission for myself and the company.

Cannabis itself is so powerful, both medicinally as well as recreationally, and it can be beneficial for both consumers and industry image if we do the right things, and avoid future disasters, like the vaping crisis we went through 18 months ago because of bad GMPs. We must learn from those industries. We’re trying to make it better for the right reasons and that’s what’s important to me.

Green: Okay, great. That concludes the interview. Thank you, Milan.

Patel: Thank you for allowing me to share my thoughts and your time, Aaron.

Bio-Rad Aspergillus PCR Test Gets AOAC Approval

By Cannabis Industry Journal Staff
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According to a press release published earlier this month, the Bio-Rad iQ-Check Aspergilllus Real-Time PCR Detection Kit has received AOAC International approval. The test covers detection for four different Aspergillus species: A. flavus, A. fumigatus, A. niger, and A. terreus.

The detection kit covers those Aspergillus species for testing in cannabis flower and cannabis concentrates, produced with our without solvents. The PCR detection kit was validated through the AOAC Research Institute’s Performance Tested Method Program. They conducted a study that resulted in “no significant difference” between the PCR detection kit and the reference method.

The iQ-Check Aspergillus Real-Time PCR Kit detects Aspergillus flavus, fumigatus, niger, and terreus in cannabis flower and cannabis concentrates.

The kit was evaluated on “robustness, product consistency, stability, inclusivity and exclusivity, and matrix studies,” the press release says. Bio-Rad also received approval and validation on the iQ-Check Free DNA Removal Solution, part of the workflow for testing cannabis flower.

The test kit uses gene amplification and real-time PCR detection. Following enrichment and DNA extraction, the test runs their PCR technology, then runs the CFX Manager IDE software to automatically generate and analyze results.

Bio’Rad has also recently received AOAC approval for other microbial testing methods in cannabis, including their iQ-Check Salmonella II, iQ-Check STEC VirX, and iQ-Check STEC SerO II PCR Detection Kits.

Drug Plastics & Glass Launches Carbon Footprint Tool

By Cannabis Industry Journal Staff
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According to a press release, Drug Plastics & Glass, a packaging company that specializes in cannabis bottles and closures, announced new tools for their customers to calculate their carbon footprint. The company launched six new sustainability calculators with the goal to help their customers get more informed about their carbon footprint.

According to Jeff Johnson, director of marketing and business development for Drug Plastics, they want to show how small, incremental changes can have a lasting impact on a company’s environmental sustainability.“From switching to more eco-friendly resin and eliminating flame treatment, to calculating the savings gained from choosing PET plastic over glass, or eliminating collateral packaging, these calculators show how making simple changes can have a big impact on the environment,” says Johnson.

Here are some of their sustainability calculators they recently launched:

  • PCR PET Resin Sustainability Calculator: Reduce greenhouse gases by making new products from PCR PET removes plastic from the environment by converting PET plastic discarded by the consumer back into resin that can be used again.
  • Flaming Elimination Calculator: Conserve fossil fuels by opting out of the flame treatment process traditionally used to ensure water-based adhesive labels and silk screening would adhere properly to HDPE, LDPE, and PP bottles. Today, this is not always necessary.*
  • Bag Reduction Calculator: Determine the individual savings when you move to single bagging instead of double bagging bottles and closures inside the carton.
  • Concentrate Elimination Calculator: Switch from white pigmented bottles to those made with resin in its natural color state and eliminate CO2
  • Glass to PET Conversion Calculator: PET requires less energy to produce and saves on transportation costs.
  • Glass to HDPE Conversion Calculator: See the sustainable improvements in weight, transportation costs, and durability when you use HDPE instead of glass.

Analytical Instruments You Need to Start a Cannabis Testing Laboratory

By Bob Clifford
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The cannabis industry is growing exponentially, and the use of cannabis for medical purposes is being adopted across the nation. With this boom in cannabis consumers, there has been an increasing need for knowledge about the product.

The role of testing labs has become crucial to the process, which makes owning and operating a lab more lucrative. Scientists testing for potency, heavy metals, pesticides, residual solvents, moisture, terpene profile, microbial and fungal growth, and mycotoxins/aflatoxins are able to make meaningful contributions to the medical industry by making sure products are safe, while simultaneously generating profits and a return on investment.

Here are the key testing instruments you need to conduct these critical analyses. Note that cannabis analytical testing requirements may vary by state, so be sure to check the regulations applicable to the location of your laboratory.

Potency Testing

High-performance liquid chromatograph (HPLC) designed for quantitative determination of cannabinoid content.

The most important component of cannabis testing is the analysis of cannabinoid profiles, also known as potency. Cannabis plants naturally produce cannabinoids that determine the overall effect and strength of the cultivar, which is also referred to as the strain. There are many different cannabinoids that all have distinct medicinal effects. However, most states only require testing and reporting for the dry weight percentages of delta-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). It should be noted that delta-9-tetrahydrocannabinolic acid (Δ9-THCA) can be converted to THC through oxidation with heat or light.

For potency testing, traditional high-performance liquid chromatography (HPLC) is recommended and has become the gold standard for analyzing cannabinoid profiles. Look for a turnkey HPLC analyzer that delivers a comprehensive package that integrates instrument hardware, software, consumables and proven HPLC methods.

Heavy Metal Testing

ICP-MS instrument for detecting heavy metals in cannabis.

Different types of metals can be found in soils and fertilizers, and as cannabis plants grow, they tend to draw in these metals from the soil. Heavy metals are a group of metals considered to be toxic, and the most common include lead, cadmium, arsenic and mercury. Most labs are required to test and confirm that samples are under the allowable toxic concentration limits for these four hazardous metals.

Heavy metal testing is performed by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS uses the different masses of each element to determine which elements are present within a sample and at what concentrations. Make sure to include accompanying software that provides assistant functions to simplify analysis by developing analytical methods and automatically diagnosing spectral interference. This will provide easy operation and analytical results with exceptionally high reliability.

To reduce running costs, look for a supporting hardware system that reduces the consumption of argon gas and electricity. For example, use a plasma ignition sequence that is optimized for lower-purity argon gas (i.e., 99.9% argon as opposed to more expensive 99.9999%).

Pesticide Testing

The detection of pesticides in cannabis can be a challenge. There are many pesticides that are used in commercial cannabis grow operations to kill the pests that thrive on the plants and in greenhouses. These chemicals are toxic to humans, so confirming their absence from cannabis products is crucial. The number of pesticides that must be tested for varies from state to state, with Colorado requiring only 13 pesticides, whereas Oregon and California require 59 and 66 respectively. Canada has taken it a step further and must test for 96 pesticides, while AOAC International is developing methods for testing for 104 pesticides. The list of pesticides will continue to evolve as the industry evolves.

Testing for pesticides is one of the more problematic analyses, possibly resulting in the need for two different instruments depending on the state’s requirements. For a majority of pesticides, liquid chromatography mass spectrometry (LCMS) is acceptable and operates much like HPLC but utilizes a different detector and sample preparation.

With excellent sensitivity and ultra-low detection limits, LC-MS/MS is an ideal technique for the analysis of pesticides.

Pesticides that do not ionize well in an LCMS source require the use of a gas chromatography mass spectrometry (GCMS) instrument. The principles of HPLC still apply – you inject a sample, separate it on a column and detect with a detector. However, in this case, a gas (typically helium) is used to carry the sample.

Look for a LC-MS/MS system or HPLC system with a triple quadrupole mass spectrometer that provides ultra-low detection limits, high sensitivity and efficient throughput. Advanced systems can analyze more than 200 pesticides in 12 minutes.

For GCMS analysis, consider an instrument that utilizes a triple quadrupole mass spectrometer to help maximize the capabilities of your laboratory. Select an instrument that is designed with enhanced functionality, analysis software, databases and a sample introduction system. Also include a headspace autosampler, which can also be used for terpene profiles and residual solvent testing.

Residual Solvent Testing

Residual solvents are chemicals left over from the process of extracting cannabinoids and terpenes from the cannabis plant. Common solvents for such extractions include ethanol, butane, propane and hexane. These solvents are evaporated to prepare high-concentration oils and waxes. However, it is sometimes necessary to use large quantities of solvent in order to increase extraction efficiency and to achieve higher levels of purity. Since these solvents are not safe for human consumption, most states require labs to verify that all traces of the substances have been removed.

Testing for residual solvents requires gas chromatography (GC). For this process, a small amount of extract is put into a vial and heated to mimic the natural evaporation process. The amount of solvent that is evaporated from the sample and into the air is referred to as the “headspace.” The headspace is then extracted with a syringe and placed in the injection port of the GC. This technique is called full-evaporated technique (FET) and utilizes the headspace autosampler for the GC.

Look for a GCMS instrument with a headspace autosampler, which can also be used for pesticide and terpene analysis.

Terpene Profile Testing

Terpenes are produced in the trichomes of the cannabis leaves, where THC is created, and are common constituents of the plant’s distinctive flavor and aroma. Terpenes also act as essential medicinal hydrocarbon building blocks, influencing the overall homeopathic and therapeutic effect of the product. The characterization of terpenes and their synergistic effect with cannabinoids are key for identifying the correct cannabis treatment plan for patients with pain, anxiety, epilepsy, depression, cancer and other illnesses. This test is not required by most states, but it is recommended.

The instrumentation that is used for analyzing terpene profiles is a GCMS with headspace autosampler with an appropriate spectral library. Since residual solvent testing is an analysis required by most states, all of the instrumentation required for terpene profiling will already be in your lab.

As with residual solvent testing, look for a GCMS instrument with a headspace autosampler (see above). 

Microbe, Fungus and Mycotoxin Testing

Most states mandate that cannabis testing labs analyze samples for any fungal or microbial growth resulting from production or handling, as well as for mycotoxins, which are toxins produced by fungi. With the potential to become lethal, continuous exposure to mycotoxins can lead to a buildup of progressively worse allergic reactions.

LCMS should be used to qualify and identify strains of mycotoxins. However, determining the amount of microorganisms present is another challenge. That testing can be done using enzyme linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), with each having their advantages and disadvantages.

For mycotoxin analysis, select a high-sensitivity LC-MS/MS instrument. In addition to standard LC, using an MS/MS selective detector enables labs to obtain limits of detection up to 1000 times greater than conventional LC-UV instruments.

For qPCR and its associated needs, look for a real-time PCR amplification system that combines thermal cyclers with optical reaction modules for singleplex and multiplex detection of fluorophores. These real-time PCR detection systems range from economical two-target detection to sophisticated five-target or more detection systems. The real-time detection platform should offer reliable gradient-enabled thermal cyclers for rapid assay optimization. Accompanying software built to work with the system simplifies plate setup, data collection, data analysis and data visualization of real-time PCR results.

Moisture Content and Water Activity Testing

Moisture content testing is required in some states. Moisture can be extremely detrimental to the quality of stored cannabis products. Dried cannabis typically has a moisture content of 5% to 12%. A moisture content above 12% in dried cannabis is prone to fungal growth (mold). As medical users may be immune deficient and vulnerable to the effects of mold, constant monitoring of moisture is needed. Below a 5% moisture content, the cannabis will turn to a dust-like texture.

The best way to analyze the moisture content of any product is using the thermogravimetric method with a moisture balance instrument. This process involves placing the sample of cannabis into the sample chamber and taking an initial reading. Then the moisture balance instrument heats up until all the moisture has been evaporated out of the sample. A final reading is then taken to determine the percent weight of moisture that was contained in the original sample.

A moisture balance can provide accurate determination of moisture content in cannabis.

Look for a moisture balance that offers intuitive operation and quick, accurate determination of moisture content. The pan should be spacious enough to allow large samples to be spread thinly. The halogen heater and reflector plate should combine to enable precise, uniform heating. Advanced features can include preset, modifiable measurement modes like automated ending, timed ending, rapid drying, slow drying and step drying.

Another method for preventing mold is monitoring water activity (aW). Very simply, moisture content is the total amount of water available, while water activity is the “free water” that could produce mold. Water activityranges from 0 to 1. Pure water would have an aW of 1.0. ASTM methods D8196-18 and D8297-18 are methods for monitoring water activity in dry cannabis flower. The aW range recommended for storage is 0.55 to 0.65. Some states recommend moisture content to be monitored, other states monitor water activity, and some states such as California recommend monitoring both.

Final Thoughts

As you can see, cannabis growers benefit tremendously from cannabis testing. Whether meeting state requirements or certifying a product, laboratory testing reduces growers’ risk and ensures delivery of a quality product. As medicinal and recreational cannabis markets continue to grow, analytical testing will ensure that consumers are receiving accurately

labeled products that are free from contamination. That’s why it is important to invest in the future of your cannabis testing lab by selecting the right analytical equipment at the start of your venture.

Swetha Kaul, PhD

Colorado vs. California: Two Different Approaches to Mold Testing in Cannabis

By Swetha Kaul, PhD
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Swetha Kaul, PhD

Across the country, there is a patchwork of regulatory requirements that vary from state to state. Regulations focus on limiting microbial impurities (such as mold) present in cannabis in order for consumers to receive a safe product. When cultivators in Colorado and Nevada submit their cannabis product to laboratories for testing, they are striving to meet total yeast and mold count (TYMC) requirements.In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

TYMC refers to the number of colony forming units present per gram (CFU/g) of cannabis material tested. CFU is a method of quantifying and reporting the amount of live yeast or mold present in the cannabis material being tested. This number is determined by plating the sample, which involves spreading the sample evenly in a container like a petri dish, followed by an incubation period, which provides the ideal conditions for yeast and mold to grow and multiply. If the yeast and mold cells are efficiently distributed on a plate, it is assumed that each live cell will give rise to a single colony. Each colony produces a visible spot on the plate and this represents a single CFU. Counting the numbers of CFU gives an accurate estimate on the number of viable cells in the sample.

The plate count methodology for TYMC is standardized and widely accepted in a variety of industries including the food, cosmetic and pharmaceutical industries. The FDA has published guidelines that specify limits on total yeast and mold counts ranging from 10 to 100,000 CFU/g. In cannabis testing, a TYMC count of 10,000 is commonly used. TYMC is also approved by the AOAC for testing a variety of products, such as food and cosmetics, for yeast and mold. It is a fairly easy technique to perform requiring minimal training, and the overall cost tends to be relatively low. It can be utilized to differentiate between dead and live cells, since only viable living cells produce colonies.

Petri dish containing the fungus Aspergillus flavus
Petri dish containing the fungus Aspergillus flavus.
Photo courtesy of USDA ARS & Peggy Greb.

There is a 24 to 48-hour incubation period associated with TYMC and this impedes speed of testing. Depending on the microbial levels in a sample, additional dilution of a cannabis sample being tested may be required in order to count the cells accurately. TYMC is not species-specific, allowing this method to cover a broad range of yeast and molds, including those that are not considered harmful. Studies conducted on cannabis products have identified several harmful species of yeast and mold, including Cryptococcus, Mucor, Aspergillus, Penicillium and Botrytis Cinerea. Non-pathogenic molds have also been shown to be a source of allergic hypersensitivity reactions. The ability of TYMC to detect only viable living cells from such a broad range of yeast and mold species may be considered an advantage in the newly emerging cannabis industry.

After California voted to legalize recreational marijuana, state regulatory agencies began exploring different cannabis testing methods to implement in order to ensure clean cannabis for the large influx of consumers.

Unlike Colorado, California is considering a different route and the recently released emergency regulations require testing for specific species of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus). While Aspergillus can also be cultured and plated, it is difficult to differentiate morphological characteristics of each species on a plate and the risk of misidentification is high. Therefore, positive identification would require the use of DNA-based methods such as polymerase chain reaction testing, also known as PCR. PCR is a molecular biology technique that can detect species-specific strains of mold that are considered harmful through the amplification and analysis of DNA sequences present in cannabis. The standard PCR testing method can be divided into four steps:

  1. The double stranded DNA in the cannabis sample is denatured by heat. This refers to splitting the double strand into single strands.
  2. Primers, which are short single-stranded DNA sequences, are added to align with the corresponding section of the DNA. These primers can be directly or indirectly labeled with fluorescence.
  3. DNA polymerase is introduced to extend the sequence, which results in two copies of the original double stranded DNA. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA.
  4. Once the double stranded DNA is created, the intensity of the resulting fluorescence signal can uncover the presence of specific species of harmful Aspergillus mold, such as fumigatus.

These steps can be repeated several times to amplify a very small amount of DNA in a sample. The primers will only bind to the corresponding sequence of DNA that matches that primer and this allows PCR to be very specific.

PCR testing is used in a wide variety of applications
PCR testing is used in a wide variety of applications
Photo courtesy of USDA ARS & Peggy Greb.

PCR is a very sensitive and selective method with many applications. However, the instrumentation utilized can be very expensive, which would increase the overall cost of a compliance test. The high sensitivity of the method for the target DNA means that there are possibilities for a false positive. This has implications in the cannabis industry where samples that test positive for yeast and mold may need to go through a remediation process to kill the microbial impurities. These remediated samples may still fail a PCR-based microbial test due to the presence of the DNA. Another issue with the high selectivity of this method is that other species of potentially harmful yeast and mold would not even be detected. PCR is a technique that requires skill and training to perform and this, in turn, adds to the high overall cost of the test.

Both TYMC and PCR have associated advantages and disadvantages and it is important to take into account the cost, speed, selectivity, and sensitivity of each method. The differences between the two methodologies would lead to a large disparity in testing standards amongst labs in different states. In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

Swetha Kaul, PhD

An Insider’s View: How Labs Conduct Cannabis Mold Testing

By Swetha Kaul, PhD
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Swetha Kaul, PhD

As both recreational and medical cannabis legalization continues to progress across the country, each state is tasked with developing regulatory requirements to ensure that customers and patients receive clean cannabis for consumption. This requires cannabis to undergo laboratory testing that analyzes the presence of microbial impurities including yeast and mold.

Some states, such as Colorado, Nevada, Maine, Illinois and Massachusetts use total yeast and mold count testing (TYMC) and set a maximum yeast and mold count threshold that cultivators must fall below. Other states, such as California, require the detection of species-specific strains of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus), which requires analyzing the DNA of a cannabis sample through polymerase chain reaction testing, also known as PCR.

Differences in state regulations can lead to different microbiological techniques implemented for testing.Before diving in further, it is important to understand the scientific approach. Laboratory testing requirements for cannabis can be separated into two categories: analytical chemistry methods and microbiological methods.

Analytical chemistry is the science of qualitatively and quantitatively determining the chemical components of a substance, and usually consists of some kind of separation followed by detection. Analytical methods are used to uncover the potency of cannabis, analyze the terpene profile and to detect the presence of pesticides, chemical residues, residuals solvents, heavy metals and mycotoxins. Analytical testing methods are performed first before proceeding to microbiological methods.

Petri dish containing the fungus Aspergillus flavus
Petri dish containing the fungus Aspergillus flavus. It produces carcinogenic aflatoxins, which can contaminate certain foods and cause aspergillosis, an invasive fungal disease.
Photo courtesy of USDA ARS & Peggy Greb.

Microbiological methods dive deeper into cannabis at a cellular level to uncover microbial impurities such as yeast, mold and bacteria. The techniques utilized in microbiological methods are very different from traditional analytical chemistry methods in both the way they are performed and target of the analysis. Differences in state regulations can lead to different microbiological techniques implemented for testing. There are a variety of cell and molecular biology techniques that can be used for detecting microbial impurities, but most can be separated into two categories:

  1. Methods to determine total microbial cell numbers, which typically utilizes cell culture, which involves growing cells in favorable conditions and plating, spreading the sample evenly in a container like a petri dish. The total yeast and mold count (TYMC) test follows this method.
  2. Molecular methods intended to detect specific species of mold, such as harmful aspergillus mold strains, which typically involves testing for the presence of unique DNA sequences such as Polymerase Chain Reaction (PCR).


Among states that have legalized some form of cannabis use and put forth regulations, there appears to be a broad consensus that the laboratories should test for potency (cannabinoids concentration), pesticides (or chemical residues) and residual solvents at a minimum. On the other hand, microbial testing requirements, particularly for mold, appear to vary greatly from state to state. Oregon requires random testing for mold and mildew without any details on test type. In Colorado, Nevada, Maine, Illinois and Massachusetts, regulations explicitly state the use of TYMC for the detection of mold. In California, the recently released emergency regulations require testing for specific species of
Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus), which are difficult to differentiate on a plate and would require a DNA-based approach. Since there are differences in costs associated and data produced by these methods, this issue will impact product costs for cultivators, which will affect cannabis prices for consumers.