Are all Aspergillus qPCR tests the same?

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August 28 2025

Abstract

Safety testing in the cannabis industry has been migrating to more clinically informative microbial testing with the implementation of Aspergillus testing in place of Total Yeast and Mold testing in states like California and 24 other states. While this focus on human pathogens has resulted in a near log scale reduction in Cannabis failure rates, the Aspergillus tests in use in the field still show some discordancies. Next generation sequencing of the PCR products from various kits in use has revealed their target amplicon sequences. These sequences were analyzed with BLAST to construct in-silico inclusion and exclusion potential and provide sequence specificity of the amplicons in use. Some vendors own published data supports these findings suggesting some tests are hitting off target Aspergillus tamarii. Since the prevalence of Aspergillus tamarii in the Cannabis field was relatively unknown, these results could not be fully understood in terms of their potential to inflate Aspergillus failure rates. To address this, we whole genome sequenced 1,249 Cannabis genomes from tissue sourced from Roots, Stems, Leaves and Flowers and discovered Aspergillus tamarii is more prevalent in cannabis than any of the pathogenic Aspergillus species the tests are designed to target.

Introduction

Aspergillus genus has over 400 species but only A.niger, A.terreus, A.flavus and A.fumigatus are deemed a human pathogenic risk. With the recent release of Cannabis lab testing data by MCR labs, Aspergillus failure rates can be charted by lab, across time and from many states. In the state of Michigan, Aspergillus positivity has seasonal increases in the Fall across multiple labs (Figure 1).

In Hawaii, Aspergillus went extinct once one lab closed down leaving a single testing lab in operation (Figure 2).

These data have left many to question the methodological differences between Aspergillus testing and the importance of seasonality adjustments when making such comparisons. Knowing some tests also test positive on off target species like Aspergillus tamarii, we sequenced 1,249 cannabis whole genomes and profiled the microbial content in the sequencing data using KRAKEN/BRAKEN.

Methods

Lab testing data was downloaded from MCR. This data was displayed with a Shiny app and is publicly viewable at Medicinal Genomics Lab Testing Data Portal. Cannabis DNA samples were made into Whole Genome Shotgun libraries using Watchmakers DNA Library Prep Kits. Libraries were sequenced in Illumina HiSeq 4000 and Illumina NovoSeq with 2x150 read lengths. Reads are trimmed with Trimmomatic and mapped with the Illumina DRAGEN mapper to the Jamaican Lion Cannabis genome. Unmapped reads are then classified with KRAKEN/BRACKEN and assembled using Megahit. Assemblies are then scanned with PathoFact2.0 and ResFungi for virulence factors, antibiotic resistance genes, and antifungal resistance genes.

Results

After Illumina sequences are mapped to the Jamaican Lion cannabis genome, unmapped reads are processed with KRAKEN/BRACKEN and reads per million of each Aspergillus species is charted (Figure 3).

Evident from this data is that Aspergillus tamarii (grey) is more prevalent than the 4 regulated Aspergillus species (A.niger, A.terreus, A.fumigatus, A.flavus) and qPCR kits that include A.tamarii will likely produce much higher failure rates. Aspergillus luchensis has also been discussed as potential off target hit for some of these tests but its prevalence is an order of magnitude lower than Aspergillus tamarii.

We then charted the geographic distribution of Aspergillus tamarii hits in the database (n=362) demonstrating they are not constrained to a single geographic location.

We also charted the average RPM for these samples showing the highest RPM in the state of Massachussetts which performs no Aspergillus testing. This may be an unrelated correlation as only stem samples can be shipped across state lines to our Massachussetts lab, leaving many more flower samples being surveyed from the state of Massachusetts.

We further examined these microbiomes for Azole resistance genes. Azoles are a commonly used class of antifungals used to treat Aspergillosis. The CDC lists azole resistant Aspergillus fumigatus as an emerging health threat. COViD-19 and Influenza patients are particularly at risk of this.

These Azole resistance genes also metabolize many of the commonly used fungicides in the cannabis space.

Myclobutanil, Propiconazole, and Tebuconazole have all been listed as fungicides involved in recent cannabis recalls, begging the question if the industries use of these fungicides is selecting for Azole resistant microbiomes.

We then surveyed California lab testing data to see if the migration to more species specific testing (which offers lower microbial failure rates) delivered lower pesticide/fungicide failure rates. This is indeed the case. As California abandoned TYM testing and migrated to more species specific testing, the microbial fail rate dropped below 1% and a concurrent drop in Pesticide/fungicide failure rates is observed.

TYM failure rates in other states are 10 fold higher. This log scale higher cannabis failure rate may be inducing more pesticide and fungicide use. When California had TYM testing in place, SCLabs reported very high Myclobutanil failure rates (39.9%) at the CannMed conference

While many pesticides and fungicides are tested for in cannabis safety testing labs, the thresholds are not always cumulative. While 100ppb of myclobutanil may be the testing threshold for a single fungicide, the use of 3-4 different fungicides under the 100ppb level is allowable. One can use multiple below threshold pesticides to compensate for any stringent thresholds placed on any given pesticide.

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Conclusions

Several methodological differences can account for lab to lab discordances in Aspergillus testing. Some tests don’t enrich likely creating false negatives due to sampling bias. We believe this may be the cause of the Aspergillus ‘extinction’ event in Hawaii as the only platform left in the state advertises no enrichment. Other labs may only enrich for 24 hours vs 48 hours.

In some states the use of antibiotics in the enrichment growth media is recommended as excessive bacterial growth in the broth may suffocate the Aspergillus growth during enrichment or overload the DNA preps and PCR steps. This may be the source of additional false negatives. This data clearly shows that it is important to not assume that the highest failure rate kit is the truth set as off target amplification of A.tamarii is an additional source of false positives.

High off target failure rates can cause the industry to lose confidence in the Aspergillus testing programs and litigate for Aspergillus test removal. This is occurred in the state of Oregon before lab testing data was public. Had this testing data been public, Oregon would have recognized they had the highest Aspergillus failure rate in the country and perhaps more lab calibration was required before booting up an Aspergillus testing program.

The presence of Azole resistance genetics in these cannabis microbiomes begs the question if non-specific, high failure rate TYM testing is inducing more fungicide use and in turn pouring fuel on the fire for Azole resistance in cannabis microbiomes. More targeted species specific testing has been shown in California to reduce Cannabis microbial failure rates concurrently with reductions in pesticide and fungicide failure rates.

Acknowledgements.

Many people contributed to this work from Medicinal Genomics, including Dr. Sherman Hom, Stephen McLaughlin, Liam Kane and Yvonne Helbert.

All of these contributors are employees of Medicinal Genomics who manufacture qPCR testing kits for the Cannabis industry.