Cannabis microbiome sequencing reveals mycotoxic fungi. Kevin McKernan Courtagen Life Science Medicinal Genomics

Transcription

1

2 Cannabis microbiome sequencing reveals mycotoxic fungi Kevin McKernan Courtagen Life Science Medicinal Genomics

3 Mold and Bacterial qpcr Testing qpcr provides very specific microbial risks in under 24 hours Most Culture bases systems are failing too much material and failing to grow important risks. $8/test Confidential 3

4 Cannabis Microbiomes w/ ITS Sequencing PathogINDICAtor

5 PathoSEEK Reveals Microbiomes qpcr PathogINDICAtor Sequencing Microbiome ID OUT Count

6 R Square In PathoSEEK

7 Majority of microbes don t culture

8 Penicillium Citrinum & Paxilli Confirmation Paxilline Confirm the Paxilline and Citrinin Genes are also present Citrinin

9 Diverse Microbiomes Discovered

10 DNA sequence Doesn t = Expression x ESI Product Ion (3.860 min) Frag=85.0V CID@20.0 ( > **) Paxilline_PIS_LongColumn-01.d Design an LC-MS/MS method to detect this in a flower matrix. Uhlig et al Counts vs. Mass-to-Charge (m/z) Orser, Marquez replicate Uhlig et al at DigiPath

11 Microbiological Contamination* * SC Labs, CannMed 2016

12 Positive Feed Back Loop High False Positive microbe tests = More pesticide use Less beneficial microbe use Cyanide inhalation

13 Pesticides* * SC Labs, CannMed

14 Experimental Design 3M & Biomeriuex vs. qpcr & Sequencing Step 1. Capture DNA Before Culture Purified DNA 4. Perform both 16S and 18S ITS qpcr (3 Hours) Step 2. Culture Sample on 3M or Biomerieux Platforms 3M Film (3/5 days to Grow) Biomerieux Cartridge (3 days) Step 3. Extract Positive Cultures in Step 2 5. Sequence both 16S and 18S PCR product (before and after Tempo DNA)

15 Perform both 16S and 18S ITS qpcr Capture DNA After Culture NextGen Sequence

16 Questions 1. Do original microbials match post culture microbials? 2. What % of the CFU/g are actually Yeast and Mold?

17 qpcr, BMX, 3M TAC Cq BMX-TAC 3M Before N/A After TYM Cq BMX-TYM 3M Before N/A 37K/140K Fail After BMX evidence for low levels of bacteria 2. BMX and 3M show high levels of TYM. qpcr demonstrates TYM after growth. 3. We have seen 3M TYM grow Staph. Will it grow Acne?

18 16S qpcr and Sequencing demonstrate bacteria grow very well in TYM cartridges PathoSEEK TAC OUT Counts Before vs. After DNA TYM Biomerieux Culture Before TYM Culture OTU Counts w/ Sequencing Bacillus_coagulans Ralstonia_syzygii_subsp._indonesiensis bacterium_oxscc-14 Tilletiopsis_pallescens After TYM OTU Counts w/ Sequencing

19 Some Are Harmless Acne, Some Not PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture Before TYM Culture OTU Counts w/ Sequencing Plant Background DNA Uncultured bacterium Klebsiella_sp. Raoultella_ornithinolytica Propionibacterium (Acne) Kluyvera_ascorbata Enterobacter_sp._10-17 Kluyvera_sp After TYM Culture OTU Counts w/ Sequencing

20 18S Shows P.citrinum Growth PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture Before TYM Culture OTU Counts w/ 18S Sequencing Penicillium_sp._568_FbG fungal_endophyte Delftia_sp._JC257 Penicillium_citrinum Penicillium_citrinum Penicillium_sp._2433_FbLB fungal_endophyte fungal_endophyte Penicillium_citrinum Penicillium_sp._568_FbG After TYM Culture OTU Counts w/ 18S Sequencing

21 18S and Aspergillus Failure to Grow PathoSEEK TYM OTU Counts Before TYM Culture OTU Counts w/ 18S Sequencing Fusarium Aspergillus_niger funga_endophyte Aspergillus_niger Aspergillus_niger After TYM Culture OTU Counts w/ 18S Sequencing

22 16S primers on 3M petri film vs Biomerieux bacterium_cicc_23843 Bacillus_coagulans uncultured_bacterium Aureimonas_sp._PP-WC-4G-234 uncultured_bacterium uncultured_bacterium Ralstonia_pickettii uncultured_bacterium

23 Discordant Before vs After data with culture Raises a Hiesenberg Uncertainty problem for regulators -The act of measuring microbial load, changes the microbial load. But do the differing culture systems agree with one another?

24 18S qpcr, 3M petri film vs Biomerieux Penicillium_citrinum fungal_endophyte Penicillium_citrinum Penicillium_brevicompactum fungal_endophyte

25 Correlations with Bacteria are weak Correl =0.196 Correl =.992

26 18S qpcr specificity When 18S qpcr & Sequencing is performed how many times do we see 16S sequence hits?

27 Future Directions If we want to reduce Pesticide usage, we must have specific microbial testing Beneficial microbes are known to reduce the need for pesticides. Beneficial microbes should not be included in a CFU/g count.

28 Nevada On The Shoulder of Giants

29 Current Microbial Tests Lack Specificity Culture based CFU/g has no bearing on safety Bacteria Grow on/in Yeast and Mold culture (FP) Staphyloccus (Human introduced) Pseudomonas (ubiquitous, harmless except Aeruginosa) Propionibacteria (Human introduced- Acne) Bacillus Likely beneficial Many forms of visible mold don t grow at all (FN) Culture systems failed to detect some Penicillium species (FN) and some aspergillus species

30 FDA is Moving to Sequence Based Safety Testing Lets get cannabis on the same footing!

31 DNA Sequence Verification of Cannabis Strains Genetic Fingerprints of Cannabis Products in the market place. Kannapedia.net Links to METRC and Blockchain based proof of existence databases.

32 Blockchains Enable Encryption Proof Time Stamps Create Encryption Key of SNP file with SHA e20b9db09c2af093ef926a3ec77efaec1fc2df Spend BTC with Key. Key is placed into a block and the time stamp is distributed to millions of computers and can never be reversed. Customer can demonstrate in Court that a Key in a timestamped Block unlocks their genetic file and therefore the file had to exist at the time of the block

33 Acknowledgements Medicinal Genomics Yvonne Helbert Jessica Spangler Ryan Lynch Wendell Orphe Lei Zhang Jeremy Rualo Adrian Devitt-Lee Christine Sutera Lei Zhang Ted Foss Doug Smith DigiPath Cindy Orser Glen Marquez Proverde Chris Hudalla Matthew Silva SCLabs Josh Wurzer Alec Dixon