There are a number of reasons why qPCR results and plating results may disagree when the methods are compared in a real-life scenario
- Homogenization and sampling technique
- Endophytes
- Off-target growth
- Regional differences in microbiomes
- Insufficient Standards
1. Homogenization and sampling technique
In our experience, the largest factors that influence discordant results between qPCR and Plating are homogenization and sampling techniques. These techniques differ from state to state and lab to lab and our kits cannot control for this. Sampling and homogenization techniques can radically alter plate counts. For example, excessive homogenization can cause viability to decay, which will reduce the number of colonies that grow on a plate without altering the amount of total DNA.
Furthermore, filamentous fungi can clump and create sampling problems for both platforms. Depending on the lab’s implementation of qPCR, a smaller volume of the sample makes it into a PCR reaction than appears on a plate. DNA purification or enrichment steps such as growth or centrifugation often helps to concentrate samples to limit this effect but it is most obvious with organisms that clump. We published on this effect in F1000. We have strong CFU/Cq concordance for many ATCC fungi but find the most discordance is described with plating of filamentous fungi.
It is not possible to Proficiency Test (PT) these techniques between labs since shipping THCA flower across state lines is federally illegal. Our PathoSEEK® assays have strong CFU/Cq concordance in controlled scenarios when live organisms are spiked onto cannabis material, which has been demonstrated in our Manufacturers Validation Document. We included a CFU/Cq formula in our validation document as a starting point that labs can use to create their own equations, but our manufacturers validation was not meant to act as a replacement of the labs’ own internal validation. We stand by our CFU/Cq equation as being accurate when the same amount of culture is being plated as is being qPCR’d, but we cannot anticipate standards that did not exist when we wrote the SOP.
2. Endophytes
Plating methods rely on microbial viability in order to detect contamination. This presents a problem because many species of yeast and mold are endophytic and live inside the plant. Culture-based sample preparations do not lyse the plant cells to access endophytic species because doing so would compromise viability. As a result, plating methods do not accurately detect certain fungal species, including the most harmful, Aspergillus.
3. Off-target growth
Our team has observed off-target bacterial growth in commercially available culture-based Total Yeast Mold (TYM) tests, which can potentially lead to false positives. We published two manuscripts that illustrate this point, which we invite you to review.
4. Regional differences in microbiomes
Another challenge facing manufacturers trying to validate a non-species specific test for the cannabis matrix is a result of the interstate commerce laws regarding the transport of cannabis samples. These laws prohibit the manufacturer from accessing cannabis flower from any other given state but its own to extend its validation. Each jurisdiction is likely to have unique plants and microbiomes that could impact the design of a TYM assay. This hurdle makes it impossible for us, or any other manufacturer for that matter, to adequately sample regional plants and microbiomes to optimize the assay.
5. Insufficient Standards
We believe that PT standards designed for culture-based methods are not compatible with qPCR assays, for several reasons. The live samples are frozen, killing many of the organisms while not changing the total amount of DNA that is present. It should also be noted that freezing is not performed on real cannabis flower. Ideally, proficiency testing can be accomplished with shipping cured hemp samples as a proxy for THCA-rich flower under the USDA farm bill. This requires further legal review but, in our opinion, would be the best solution for the cannabis field.
In the absence of sharing cannabis flower, the field has been forced to share cultures as a proficiency testing standard. This is not an ideal workaround either as it can enrich the sample and creates a selection for only sharing organisms that can culture between labs. We’re currently working with NSI to develop qPCR-based standards for the sector to address some of these issues. While working with NSI, we discovered a number of issues with plating standards such as the live-dead issue, and in response, we have developed an enzyme that can eliminate DNA from dead organisms before running qPCR assays.
We understand our TYM assay and the equation we have in our validation document does not yet line up with the most recently released NSI standards. Our equations were calibrated in Massachusetts years before NSI even offered these standards to the field and we published these results in open access peer-reviewed journals that utilized an open and transparent review system. We will continue to evolve and improve our validation documents as the regulatory standards catch up to our own.
The Ongoing Challenge of Designing an Orthogonal Technology
To better understand these challenges, our lab is working with a number of clients to sequence their colonies in an effort to improve our TYM assay. We are currently performing work for a customer that is having discordant results. One of the samples they shipped to us as an example of an organism our assay failed to detect was grown on a plate and shipped to us for sequencing. Whole genome sequencing revealed this organism to be Penicillium brevicompactum. This is an organism our assay detects and has been reported in our peer reviewed publication. We confirmed successful detection with our TYM assay. It’s important to note that we were not able to sample the actual cannabis flower that failed detection in the customers lab, only the colonies that grew on the plate. This is not the same and it is an important distinction.
We still have additional samples from this customer and we will continue to study them. We appreciate this type of industry collaboration to better understand how to optimize the primer design, cell lysis, and sampling for the TYM Assay.
We have had customers report cases to us in which our qPCR assays failed to detect any signal when their total yeast and mold plates were presenting high counts. When we have sequenced these colonies we found they were either bacteria, endofungal bacteria or filamentous fungi.
In an effort to improve our accuracy, we are evaluating a new sampling protocol that will improve sampling filamentous or clumping fungi that might contribute to qPCR false negatives. Our plan is to purify a larger portion of the sample while employing a heat step during the chemical lysis to ensure we are not failing to lyse open cells. We also developed four species specific Aspergillus assays to help reduce discordance with filamentous fungi and these are used quite successfully to ensure TYM plates are not failing to detect these endophytes.