Protected: qPCR versus Plating Validation

On September 14th, 2018  Analytical Cannabis published an article discussing cannabis microbial testing methods in Massachusetts.

This is an important topic regarding patient health and we are encouraged the media is taking notice of it. Nevertheless, we feel it is important to correct and address some of the quoted statements in the document. The statements are made by Chris Hudalla who is a well respected scientist in the cannabis testing market. We have worked closely with Chris over the years and we have even co-authored a manuscript on this topic with him. We have great respect for his contributions to the field but feel his comments may have been taken out of context and a comprehensive rebuttal is the most transparent approach to such a complicated topic.

The reader should be aware that we have a financial interest in this story as we manufacture one of the technologies being discussed (qPCR).

A quick paraphrase of the article

The article is discussing the validity of using qPCR versus Plating for Cannabis microbial testing in Massachusetts and why the state needs to step in and pick a winner. The article implies that without the state mandating a technology, growers can “shop around” and therefore this creates “unfair” competition.

The two technologies in dispute are Culture based Plating or “CBP” versus Quantitative PCR or qPCR. Medicinal Genomics sells qPCR kits for microbe detection on cannabis. We developed these because we found tremendous holes in the culture-based plating methods that present serious risks to epileptic and cancer patients.

The article explains how one lab is losing business to another newer lab that uses a faster and more accurate technology with internal controls (qPCR). The lab losing business is complaining to the State and the Cannabis Control Commission asking them to mandate the slower way (culture-based plating), despite this technique having no internal controls or peer-reviewed literature supporting its use in cannabis.

False Statement: The FDA and other industries endorse culture-based plating tests

At one point in this article, the concerned scientist (Chris Hudalla) makes the assertion that (only?) culture-based plating has the backing of the cosmetic industry and the FDA. In the following sentence he said “The method they are using is accepted by nobody“. He is referring to qPCR.

These are false statements. They are made concurrent to statements regarding his financial conflicts and loss of revenue.

Let us address the first straw man argument Chris has presented, which is that other industries such as cosmetics, and the FDA endorse one technology.

The problem with this appeal to antiquity (how it has always been done) is that cannabis has been illegal since 1937 and this prohibition predates the existence of the FDA. “How it has always been done in food” is misleading logic as it has never been done before in Cannabis and Cannabis is not food. This is important to underscore as the scientist surfacing these critiques is distorting language and logic to create a political effect in the market as opposed to competing with technological skill in the marketplace.

The food and cosmetic industries use qPCR for many clinically regulated tests. Below, we have provided a list of references to qPCR being used for FDA approved tests. In addition to many qPCR based tests being approved by the FDA, 98% of the clinical tests being run today are non-FDA approved Laboratory Developed Tests (LDTs). These tests make life and death decisions for human health care in the CLIA lab settings and they have remained outside of FDA review to ensure rapid innovation. More importantly, FDA approval is not required for these LDT tests nor is it required for cannabis testing today.

This appeal to authority actually works against the culture-based plating argument as those very authorities have large programs that are funded to get rid of Plating. This field is known as Culture Independent Diagnostic Testing or (CIDT).

Both the CDC and FDA have multiple independent programs to eliminate culture-based plating from the food testing industry due to its failure to accurately assess food-born illness risk. The FDA program is known as the Genome Trakr network and the CDCs program is known as Pulsenet. Both programs use genome sequencing to survey food outbreaks across a network of labs around the country. This is being done because culture-based plating takes too long and fails to detect too many risks.

In general, agencies are technology agnostic but validation centric. It is important to understand what it means to be validated. This is the second slight of hand delivered by the concerned but arguably conflicted scientist.

False Assertion: Food Testing Techniques are Applicable to Cannabis.

Independent of what testing techniques these agencies recommend for food, we cannot pretend cannabis is food. Cannabis is ground up and inhaled, often by immunocompromised patients. Your food is not. Route of administration is critical in establishing tolerable microbial load. The superimposition of the methods that have been validated in the food industry (by laboratories other than Chris Hudalla’s) onto a new matrix like cannabis is one major error and slight of hand designed to mislead the public and the political organizations into sympathy for a Chris’s lab that has published nothing to support its methods.

The second error in the complaint is that this scientist assumes the validation work done for culture-based plating in other industries can be superimposed on his lab who has no published manuscripts to back up their deployment of other peoples methods with Cannabis. This is an outstanding magic trick that works well in short form media but fails under any prolonged scientific scrutiny.

As anyone versed in cannabis testing can assure you, if you change the matrix, you must revalidate your technology. One cannot just superimpose the testing techniques from tomatoes onto Cannabis and assume the method’s function. One must revalidate their methods using a given technology on the new matrix. There are no publications from Chris Hudalla validating his methods on cannabis yet there are two manuscripts published by our group comparing qPCR to various plating methods.

Both of these peer-reviewed studies point to massive clinical liabilities present with culture-based plating technologies. Chris Hudalla is even a collaborating author on one of these papers, and the paper made it very clear that culture-based plating methods are failing to detect real clinical risk while also suffering from very high false positive rates. Chris is correct to highlight that there are discordant results between qPCR and CBP but is incorrect to assume CBP is the gold standard in Cannabis.

There are two reasons these Plating methods fail in cannabis.

  1. Plating methods can’t detect endophytes. It is now well published that many of the harmful cannabis pathogens (fusarium, aspergillus etc) are endophytes. These live inside the plant as shown in this publication (hemp_endophytes). Culture based techniques need to gently soak the sample in broth so as not to kill any microbes. It is impossible to survey the endophytes inside of cannabis without lysing open cells and culture-based plating cannot detect lysed cells. As a result, culture-based platings’ achillies heal is that it cannot pick up endophytes without killing the epiphytes. qPCR can see both.
  2. Most organisms do not culture and fewer culture in 48 hours. Many fungi need at least 120 hours for spores to culture from dried produce. This has been published on 5 different medias. Any culturing of a dried produce for only 48 hours is security theatre and thus the viability it claims to be capable of estimating is grossly flawed.

While Chris Hudalla is an author on a manuscript highlighting these shortcomings, he has not published anything from his lab suggesting he has addressed these shortcomings of his platform. His public statements seem to differ from the work he put his name on in the scientific record.

Correct Statement: Plating methods grow Off-Target Organisms

We have demonstrated this in several publications. Most recently, we sent Chris Hudalla a saturated bacterial culture of an antibiotic-resistant Pseudomonas aeruginosa from ATCC. This is a Bile-Tolerant Gram-Negative (BTGN) bacteria, which is a required target in the Massachusetts regulations. The USP has listed Pseudomonas aeruginosa as required for any valid BTGN test. However, both of the lab’s culture-based plating technologies (Biomerieux and Hardy plates) failed to detect it in their BTGN tests. As predicted from our peer-reviewed manuscript, their Total Yeast and Mold test detected the Pseudomonas aeruginosa when it should not have detected a bacteria. We published in a peer reviewed journal that over 60% of the DNA in their yeast and mold tests are bacteria. These Pseudomonas aeruginosa failed results were repeated and confirmed at MCR laboratory, which is known for using the same culture-based plating methods (Biomerieux) as Chris Hudalla’s lab.

If these platforms cannot detect the proper organisms from a saturated culture without any cannabis present, you cannot rely on the results they give you with matrix or cannabis present. These methods lack proper controls and spike in experiments. Likewise, there is not any published validation data for their microbial methods. For public safety it is imperative labs have transparent methods and preferably peer-reviewed. Chris Hudalla has neither of these.

The qPCR methods he accuses of cutting corners has more peer-reviewed publications and transparency than the methods used in his laboratory.

Transparency is critical for patient safety

While we do our best to publish data regarding our methods, any laboratory using our reagents is expected to revalidate the use of our reagents in their laboratory setting. This would include proper inclusion and exclusion analysis with live ATCC controls so the public knows what you can detect and what you cannot detect. These same studies are required in the presence of cannabis matrix to understand how this oily matrix (that often contains 30% antimicrobial cannabinoids and terpenoids) alters the growth of various pathogenic threats. This is a pillar of ISO 17025 required for Massachusetts testing compliance. It is very clear to us that Chris has not performed these spike-in controls in his laboratory or he would have known his BTGN test was invalid. This has been sold as a defective test in Massachusetts for nearly 4 years now. This can negatively impact Cystic Fibrosis patients known to use cannabinoids to manage inflammation.

There are employee lawsuits in Massachusetts for exposure to Powdery mildew and Botrytis (Yeast and Molds). These do not culture in Chris Hudalla’s methods (yet we have qPCR tools to detect them) and hence growers using his service didn’t believe the employees exposure. Perhaps if his methods and validation data were public, we could have avoided these blind spots and employee/employer conflicts. This is why each lab must perform their own validations and not rely on the manufacturer’s validation work. Even Chris’ manufacturer has no public data to support the use of their products in cannabis. To the contrary, there are peer-reviewed papers warning of the platforms failure to perform with certain spices (tempo_interferance).

This also underscores real and immediate risks when you insist on having qPCR match the results of a century-old process. This is harming patients and employees and is not supported by the USP <1223> stance on CFU/g equivalence. The reason the Chris Hudalla is losing customers is because his customers cannot afford this laissez faire approach to the clinical blind spots presented with culture-based plating. It has left his customers exposed to a lawsuit and harmed several employees.

A final critique voiced by this scientist in other forums is that the Massachusetts regulations reference the AHP which uses the metric of CFU/g and that qPCR doesn’t report in CFU/g. The platform Chris uses (Biomerieux) doesn’t form colonies either. It measures a fluorescent change based on a pH shift.


qPCR also uses a fluorescent signal but the signal is based on far more specific DNA signatures than a simple pH shift. Many acid producing bacteria can falsely alter a pH-based readout. This near-religious attachment to CFU is not reflected in other regulatory agencies that allow other technologies to provide equivalent metrics. This insistence on a CFU/g metric exposes patients to harmful microorganisms that do not form colonies. This has been peer-reviewed and published. Many of the Biomerieux shortcomings are revealed in the document we submitted to the USP.

In order to design TYM qPCR assays to perfectly mimic the flaws of culture-based plating we have to intentionally remove the microbes we target with qPCR that do not form colonies and make independent assays for these fungi (more cost). This exposes the State of Massachusetts to the lawsuits they are currently seeing with employees being exposed to high level of unculturable but sickening microbes.

There is no transparency on this scientists microbial methods despite this flawed and irresponsible attack on the far more transparent technologies that are replacing him.

There is one topic we agree with Chris Hudalla on. The regulations as written overly speak in terms of CFU/g and this should change to reflect the USP <1223> language regarding other equivalent or validated technologies. The EP has similar guidance on this. This would enable technologies that can detect the harmful microorganisms that do not form colonies but are creating lawsuits in Massachusetts. This same complaint has been raised in other states struggling to implement the AHP monographs vague definitions. The state of Washington Cannabis Laboratory Association has petitioned for changes to these regulations due to the lack of specificity of CFU/g and the explicit statement in the monograph that these values do not represent pass-fail criteria but are reflective of average microbial loads found on trichome rich plants. The state of Alaska, California abandoned these requirements and move to species specific presence or absence regulations.

Combine the AHPs lack of trust in CFU/g being an adequate pass fail criteria and that of the USP <1223> describing why CFU/g cannot be the only form of microbial enumeration and Chris Hudalla’s comments become even more suspect given his financial conflicts.

In summary, often the tool of last resort for a failing technology is to beg the state to mandate its flaws. Likewise, often labs vested in expensive and antiquated capital equipment will resist the entrance of a faster and cheaper alternatives as they enable new entrants to the market to compete with less equipment amortization and lower overhead. Massachusetts being the hub of the genomics industry, has seen this before and is more likely to let the market competition sort out the winners and losers. We recommend labs that do not have public documentation on the microbes they can and cannot detect in a cannabis matrix be avoided. Lack of method transparency is a sign of the lab not having confidence in what their tests actually measure.

There are labs in Massachusetts that have received ISO17025 accreditation with our qPCR methods.

There are labs in Pennsylvania (Steep Hill Labs) that have received ISO17025 accreditation with our qPCR methods.

We have over 100 labs in the US and Canada that use these qPCR methods.

Our Validation documentation is public on our website here.

We have submitted much our work to the USP expert review panel on this topic.

Peer Reviewed manuscripts for our qPCR being used on Cannabis.

Authors other publications in microbiology

qPCR Methods Used in Other Industries

Food Methods

  1. Diagnostic Real-Time PCR for Detection of Salmonella in Food:
  2. Rapid detection of Salmonella in foods using real-time PCR.
  3. A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products. :
  4. Rapid, Specific, and Sensitive Detection of Spoilage Molds in
    Orange Juice Using a Real-Time Taqman PCR Assay:
  5. foodProof Yeast & Mold Quantification in Dairy Products:

AOAC Validated qPCR Methods

  1. Bio-Rad’s iQ-Check Salmonella II Real-Time PCR Kit Earns Approval as an AOAC Official Method of Analysis
  2. AOAC Certifies BAX® System Real-Time PCR Assays for Non-O157:H7 STEC in 25g Flour and Ground Beef:

Clinical Microbiology

  1. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing:
  2. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything:
  3. Real-time PCR as a diagnostic tool for bacterial diseases:
  4. Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora:
  5. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set:

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