Medicinal Genomics announced today that they have received AOAC International certification for their PathoSEEK® Salmonella and STEC E. coli multiplex assay. In combination with their SenSATIVAx® extraction kits, labs can simultaneously detect Salmonella spp. and STEC E. coli with a single qPCR reaction for flower, concentrates and infused chocolates using the Agilent AriaMx and the BioRad CFx-96 instruments.
The certification came after the multiplex assay was validated according to the AOAC Performance Tested Method Program. According to the press release, the PathoSEEK platform now has more cannabis matrices accredited for Aspergillus, Salmonella, and STEC E. coli than any other product out on the market, according to their press release.
The PathoSEEK microbiological testing platform uses a qPCR assay and internal plant DNA controls for reactions. The two-step protocol verifies performance while detecting microbes, which allegedly helps minimize false negative results from human error or failing conditions.
“AOAC’s validation of our Salmonella/STEC E. coli assay across the various cannabis matrices is further proof of our platform’s robustness and versatility,” says Dr. Sherman Hom, director of regulatory affairs at Medicinal Genomics. “We are excited that our PathoSEEK® platform is moving in concert with the FDA’s new blueprint to improve food safety by modernizing the regulatory framework, while leveraging the use of proven molecular tools to accelerate predictive capabilities, enhance prevention, and enhance our ability to swiftly adapt to pathogen outbreaks that could impact consumer safety.”
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.
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.
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.
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.
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?
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?
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.
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 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.
Cannabis laws are changing at a rapid pace across all 50 states and around the world. Currently, Cannabis is legal in 11 states for adults over the age of 21, and legal for medical use in 33 states.
Across the nation, many states have been struggling to enact a viable medical and potential adult use cannabis system since Initiative 59 and the “Legalization of Marijuana for Medical Treatment Initiative of 1998.”
Unfortunately, the program has been continuously impacted by the federal government’s presence, first with the passage of the Barr Amendment by Congress overturning the early legalization progress and continuing to delay the onset of the first medical sale at a dispensary until 2013. The federal government continues to exert influence and control over the program expansion including adding Congressional riders on every proposed update including the latest “Safe Cannabis Sales Act of 2019.”
In Washington DC for example, 18 organizations including the National Cannabis Industry Association (NCIA), the ACLU and Law Enforcement Action Partnership petitioned the US House and Senate Financial Services Subcommittees to remove the rider given that “[the] Current law has interfered with the District’s efforts to regulate marijuana, which has impacted public safety. Without the ability to regulate marijuana sales, the grey market for marijuana flourishes despite the need and want of the District leadership and residents alike to establish a regulatory model.”
States with limited availability of medical cannabis, possession laws or with the ability to legally gift up to one ounce and the constant pressure by the federal government, the grey market has expanded with public safety and the safety of these pop-up businesses put at risk. The current state health and safety laws require a seed-to-sale tracking system and testing at independent labs for all medical cannabis, however the grey market consumers are afforded no such protection. The District of Columbia is unique in the US cannabis landscape as it grapples with the local government trying to provide clarity, safety and equity to a medical and adult use community, but it is hampered by what it can and cannot control through federal influence.
As the United States continues to recover from the effects of the COVID-19 pandemic, control and use of tax revenue will move to center stage in all these communities and the cannabis tax revenue will return to focus.
Cannabis tax revenue has shown a massive disparity between projection and reality. In 2018, California projected upwards of one billion dollars in cannabis tax revenue, but in reality was only able to recover a third of that amount. California in response continues to increase the excise tax and even proposed changes to taxes dependent on the amount of THC, creating new pressure on producers, in-part pushing some back into the grey market.
During the pandemic, Colorado enacted emergency rules to extend cannabis sales online. Allowing customers to pay for cannabis via the web and then pick up their purchases at the store. In a testament to what is considered a “critical businesses” the cannabis industry is given opportunity to expand during the pandemic, but still hampered by severely limited access to standard e-commerce options as credit card merchants still remain concerned that cannabis sales are illegal under US federal law. Alaska, Massachusetts, Michigan, Illinois and Oregon also allowed online sales and curbside pick-up, but remain limited in sales as federal banking and access to credit is limited as the Secure and Fair Enforcement (SAFE) Banking Act remains in limbo.
Overarching technologies such as DNA tracking that provide a clear indicator that the cannabis is produced and tested from legal sources, can be proven safe and protects local legal businesses’ products against out of market cannabis would provide such clarity.
As the country moves forward from the COVID-19 health crisis, all legal and safe ways to rapidly restart the economy will be needed, the cannabis economy will be no exception. We should be looking to this emerging market right now to help safely drive revenue and taxes into our states.
In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.
The global food supply chain moves much faster today than it did in the late 19th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21st century pathogen detection solutions.”
Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”
He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”
While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.
Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”
When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”
The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:
Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
Drop sample in solution. Swab should already be in solution
Transfer 1ml of solution into 1.5ml tube
Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
Conduct Loci enhancement PCR of sample for 1 hour
Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
Conduct wash cycle for 15 minutes
Dry and image the slide in imager
The imager will create a TIFF file where software will analyze and deliver results and a report
Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.
Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.
Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.
Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.
Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.
Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.
As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).
When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.
1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample
The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.
Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.
One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.
The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.
The Live Dead Problem
One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).
2. Method Must Be Able to Detect Specific Harmful Species
Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.
Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.
3. The Method Must Work on Multiple Matrices
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, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.
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 from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.
This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 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.
For years we have heard about and sometimes experienced, white powdery mildew when growing cannabis. It is a problem we can see, and we have numerous ways to combat it. But now more and more states are introducing regulatory testing on our harvests and they are looking for harmful substances like Escherichia coli., Aspergillis Fumigatus, Aspergillis terreus, … just to name a few. Mycotoxins, mold and bacteria can render a harvest unusable and even unsellable- and you can’t see these problems with the naked eye. How much would it cost you to have to throw away an entire crop?
You bring in equipment to control the humidity. You treat the soil and create just the right amount of light to grow a superior product. You secure and protect the growing, harvesting, drying and production areas of your facility. You do everything you can to secure a superior yield… but do you?
Many of the organisms that can hurt our harvest are being multiplied, concentrated and introduced to the plants by the very equipment we use to control the growing environment. This happens inherently in HVAC equipment.
Your air conditioning equipment cools the air circulating around your harvest in a process that pulls moisture from the air and creates a perfect breeding ground in the wet cooling coil for growth of many of the organisms that can destroy your yield. As these organisms multiply and concentrate in the HVAC system, they then spew out into the very environment you are trying to protect at concentrated levels far greater than outside air. In effect, you are inoculating the very plants you need to keep safe from these toxins if you want to sell your product.
The cannabis industry is starting to take a page from the healthcare and food safety industries who have discovered the best way to mitigate these dangers is the installation of a proper UVC solution inside their air conditioning equipment.
Why? How does UVC help? What is UVC?
What is Ultraviolet?
Ultraviolet (UV) light is one form of electromagnetic energy produced naturally by the sun. UV is a spectrum of light just below the visible light and it is split into four distinct spectral areas – Vacuum UV or UVV (100 to 200 nm), UVC (200 to 280 nm), UVB (280 to 315 nm) and UVA (315 to 400 nm). UVA & UVB have been used in the industry to help promote growth of cannabis.
What is UVC (Ultraviolet C)?
The entire UV spectrum can kill or inactivate many microorganism species, preventing them from replicating. UVC energy at 253.7 nanometers provides the most germicidal effect. The application of UVC energy to inactivate microorganisms is also known as Germicidal Irradiation or UVGI.
UVC exposure inactivates microbial organisms such as mold, bacteria and viruses by altering the structure and the molecular bonds of their DNA (deoxyribonucleic acid). DNA is a “blue print” these organisms use to develop, function and reproduce. By destroying the organism’s ability to reproduce, it becomes harmless since it cannot colonize. After UVC exposure, the organism dies off leaving no offspring, and the population of the microorganism diminishes rapidly.
Ultraviolet germicidal lamps provide a much more powerful and concentrated effect of ultraviolet energy than can be found naturally. Germicidal UV provides a highly effective method of destroying microorganisms.
To better understand how Steril-Aire UVC works, it is important to understand the recommended design. Directed at a cooling coil and drain pan, UVC energy destroys surface biofilm, a gluey matrix of microorganisms that grows in the presence of moisture. Biofilm is prevalent in HVAC systems and leads to a host of indoor air quality (IAQ) and HVAC operational problems. UVC also destroys airborne viruses and bacteria that circulate through an HVAC system and feed out onto the crop. HVAC cooling coils are the largest reservoir and amplification device for microorganisms in any facility.
For the most effective microbial control, UV germicidal Emitters are installed on the supply side of the system, downstream from the cooling coil and above the drain pan. This location provides more effective biofilm and microbial control than in-duct UVC installations. By irradiating the contaminants at the source – the cooling coils and drain pans – UVC delivers simultaneous cleaning of surface microorganisms as well as destruction of airborne microorganisms and mycotoxins. Steril-Aire patented this installation configuration in 1998.
The recirculating air in HVAC systems create redundancy in exposing microorganisms and mycotoxins to UVC, ensuring multiple passes so the light energy is effective against large quantities of airborne mycotoxins and cleaning the air your plants live by.
Where are these mycotoxins coming from?
Aspergillus favors environments with ample oxygen and moisture. Most pre-harvest strategies to prevent these mycotoxins involve chemical treatment and are therefore not ideal for the cannabis industry.
Despite the lack of cannabis protocols and guidelines for reducing mycotoxin contamination, there are some basic practices that can be utilized from other agricultural groups that will help avoid the production of aflatoxins and ochratoxins.
When guidelines are applied correctly to the cannabis industry, the threat of aflatoxin and ochratoxin contamination can be significantly reduced. The place to start is a clean air environment.
Design to win
The design of indoor grow rooms for cannabis is critical to the control of airborne fungal spores and although most existing greenhouses allow for the ingress of fungal spores, experience has shown that they can be retrofitted with air filters, fans, and UVC systems to make them relatively free of these spores. Proper designs have shown clearly that:
Prevention via air and surface disinfection using germicidal UVC is much better than chemical spot treatment on the surface of plants
High levels of air changes per hour enhance UVC system performance in reducing airborne spores
Cooling coil inner surfaces are a hidden reservoir of spores, a fertile breeding ground and constitute an ecosystem for a wide variety of molds. Continuous UVC surface decontamination of all coils should be the first system to be installed in greenhouses to reduce mildew outbreaks.
UVC can virtually eliminate airborne contaminants
Steril-Aire was the first and is the market leader in using UVC light to eliminate mold and spores to ensure your product will not be ruined or test positive.
Mold and spores grow in your air handler and are present in air entering your HVAC system.
Steril-Aire UVC system installs quickly and easily in your existing system.
The Steril-Aire UVC system destroys up to 99.999% of mold/spores.
Plants are less likely to be affected by mold…with a low cost and no down time solution.
It’s time to protect your harvest before it gets sick. It’s time to be confident your yield will not test positive for the contaminants that will render it unusable. It’s time to win the testing battle. It’s time for a proper UVC solution to be incorporated throughout your facilities.
Genome sequencing has made remarkable strides since the initiation of “The Human Genome Project” in 1990. Still, there are many challenges that must be overcome before this methodology can reach its fullest potential and be useful in serving as a method of Cannabis sativa genetics verification and tracking throughout the cannabis supply chain. Several major milestones that must be realized include end-to-end haploid type (single, unpaired set of chromosomes instead of complete paired set or “diploid”), long read, resolved genome sequences at a reasonable cost within a reasonable timeframe and with confidence in accuracy (Mostovoy et al.). These genomes are typically generated as shorter reads that are then scaffolded (Fig 1.) or matched to reference genomes in order to build a longer continuous read. While shorter sequencing reads indeed lower the cost barrier for producing more genomic data, it has created another issue as a result of this short-read technology.
There are two main issues with the more affordable short read sequencing methodology, the first being that sequential variants are typically not detected, especially if they involve a ton of repeats/inverted repeats, due to the limitation of the current referenced Cannabis genomes and the mapping process of the short-read sequences. This is especially unfortunate because larger variants can have up to a 13% variance within a diploid multichromosomal genome, such as Cannabis sativa, and this variance is thought to largely contribute to disease in various species, or maybe terpene profile in Cannabis sativa. Not being able to detect these variances with more affordable sequencing methodologies is particularly problematic and reference genomes produced with short read sequences are typically highly fragmented. The second limitation is the inherent errors, gaps and other ambiguities associated with taking tons of short read sequences and combining them all, like a jigsaw puzzle, in order to draft the larger genomic picture. While there is software with algorithms to assist in deciphering raw sequences, there is still much more work to be done on this challenge, considering that cannabis genome sequencing is new genomics territory. Unfortunately, as researchers seek higher and higher levels of data quality, shortcomings of this type of sequencing technology begin to become apparent. This sort of sequencing methodology relies heavily on reference sequences. This isn’t much of an issue with microbial genomes, which tend to be rather short and typically have one chromosome, however, when seeking to analyze much longer genomes with multiple diploid chromosomes and tons of mono and dinucleotide repeats, problems arise (English et al.).
The other category of sequencing is long read sequencing. Long read sequencing is as it sounds, the deciphering of much longer DNA strands. Of course, the technology is limited by the quality of the DNA captured, therefore, special high molecular weight DNA extraction protocols must be deployed in order to obtain the proper DNA quality (Fig. 3). Once this initial limitation is overcome there is the stark cost of long read sequencing technology. PacBio without a doubt makes one of the highest quality long read sequence generating instruments that has ever graced the field of biotechnology, but due to the steep price tag of the machine, progress in this field has been stifled simply because it just isn’t affordable and the read depth for mammalian and plant genomes is currently almost completely prohibitive until read lengths double in length for this instrumentation. In order to produce what is considered to be a “validated genome” both short read and long read sequencing methodologies are combined. Long read sequencing data is used to produce the reference contigs because they are much easier to assemble, then short read sequencing is scaffolded against the reference contigs as a sort of “consensus validation” of the long read contigs.
Despite the shortcoming of utilizing short read sequencing technology for analysis of the cannabis genome, it is still useful especially when combined with other longer read sequencing technologies or optical mapping technologies. Kevin McKernan, chief scientific officer of Medicinal Genomics, has been working feverishly to bridge the information gap between the cannabis genome and other widely studied plant genomes. As a scientist that worked on the Human Genome Project in 2001, McKernan has a demonstrated history of brilliance in the field of genomics. This paved the way for him to coordinate the first crypto funded and blockchain notarized sequencing project (DASH DAO funded) (Fig. 2), which was completed in 60 days, and surprisingly showed that the cannabis genome is over 1 billion bases long which is 30% larger than any cannabis genome submitted prior to his work. By reaching the standard of 500kb N50 set forth by the Human Genome Project, Kevin McKernan was able to see new aspects of the cannabis genome that were not visible due to the fragmented genomic data previously generated. Information such as a possible linkage of THCA synthase and CBDA synthase genes is crucial when seeking to use the cannabis genome for verification and tracking purposes. This is because special linkages can be considered a type of “genetic marker” that may be used to differentiate cannabis cultivars and lineages. There are many types of genetic markers, including SNP (single nucleotide polymorphisms), VNTR (variable number tandem repeats) and even patterns of gene expression. Funding and recording of cannabis genomics must be further developed in order for potential markers to be identified and validated via larger scale genome-wide association studies.
These technologies, when combined, often reduce the number of scaffolds while increasing the percent of resolved genome by filling in gaps within the drafted genome. Nanopore sequencing is an especially interesting and innovative sequencing technology that is useful in many ways. One of the most powerful uses of this technology is its ability to upgrade the quality of draft and pushed genomes by resolving poorly organized genomes and genomic structure for a fraction of the time and cost of other long read sequencing platforms (Jian et al.), making it an excellent candidate for solving cost and time constraints. Nanopore’s portability and convenience makes it a real-time solution to solving genetics-based problems and questions. A notable use of this technology is recorded during an epidemiological outbreak in Africa, its proof of concept in pathogen detection in space, and its ability to detect base modifications during sequencing process. Even still there are more uses to this exciting technology and it has the potential to elevate cannabis genomics and the field of genomics entirely, while remaining portable and expeditious. A shortcoming of the Nanopore sequencing platform is its low sequencing coverage, which makes this platform inefficient for applications like haplotype phasing and single nucleotide variant detection due to the number of variants to be detected being smaller than the published variant-detection error rates of algorithms using MinION data. Single nucleotide variants can be considered to be genetic markers, especially markers for disease, so this is what inhibits Nanopore from resolving our cannabis genome sequencing problems, as of today.
There are genetic markers to discover, molecular biology protocols to optimize, and industry wide potential for exciting collaborationMany algorithmic problems seem to occur due to input data quality. Typical input data quality suffers as the reads get longer and the sequencing depth gets shorter, resulting in not enough data being generated by the sequencing to provide confidence in the genome assembly. To mitigate this, scientists may decide to fractionate a genome, sequence it, or they may clone a difficult to sequence region with highly repetitive regions in order to produce reads with greater depth and thus resolve the region. They can then perform single molecule sequencing to resolve genome structure then determine and confirm the place of the cloned region. Thus, it seems that the best solution to the limitation of algorithms is to be aware of sequencing platform limitations and compensate for these limitations by using more than one sequencing platform to obtain enough pertinent data to confidently produce authentic, “validated” genome assemblies (Huddleston et al.). With input data being critical in producing accurate sequencing data, standardization of DNA isolation protocols, extraction reagents and any enzymes utilized may be deemed necessary.
To conclude, the field of cannabis genomics is teeming with opportunities. There are genetic markers to discover, molecular biology protocols to optimize, and industry wide potential for exciting collaboration. More states will need to take into account the lack of federal government research grant availability and begin to think of creative ways to get cannabis science funds to continue the development of this industry. Specifically speaking, developing a feasible method for genetic tracking of cannabis plants will require improvements within the availability of sequencing technology, improvements in deploying the resources to these projects in order for them to be completed expeditiously, and standardization/validation of methods and SOPs used in order to increase confidence in the accuracy of the data generated.
A special thank you to all of my cannabis industry mentors that have molded and elevated my understanding of current needs and applied technologies within the cannabis industry, without you there would be no career within this industry for me. You are immensely appreciated.
Bickhart, D. M., Rosen, B. D., Koren, S., Sayre, B. L., Hastie, A. R., Chan, S., . . . Smith, T. P. (2017). Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome. Nature Genetics,49(4), 643-650. doi:10.1038/ng.3802
English, A. C., Salerno, W. J., Hampton, O. A., Gonzaga-Jauregui, C., Ambreth, S., Ritter, D. I., . . . Gibbs, R. A. (2015). Assessing structural variation in a personal genome—towards a human reference diploid genome. BMC Genomics,16(1). doi:10.1186/s12864-015-1479-3
Huddleston, J., Ranade, S., Malig, M., Antonacci, F., Chaisson, M., Hon, L., . . . Eichler, E. E. (2014). Reconstructing complex regions of genomes using long-read sequencing technology. Genome Research,24(4), 688-696. doi:10.1101/gr.168450.113
Jain, M., Olsen, H. E., Paten, B., & Akeson, M. (2016). The Oxford Nanopore MinION: Delivery of nanopore sequencing to the genomics community. Genome Biology,17(1). doi:10.1186/s13059-016-1103-0
Mostovoy, Y., Levy-Sakin, M., Lam, J., Lam, E. T., Hastie, A. R., Marks, P., . . . Kwok, P. (2016). A hybrid approach for de novo human genome sequence assembly and phasing. Nature Methods,13(7), 587-590. doi:10.1038/nmeth.3865
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.
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:
The double stranded DNA in the cannabis sample is denatured by heat. This refers to splitting the double strand into single strands.
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.
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.
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 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.
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