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Cannabis microbiome sequencing reveals mycotoxic fungi
Kevin McKernanCourtagen Life ScienceMedicinal Genomics
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
3Confidential
Cannabis Microbiomes w/ ITS Sequencing
PathogINDICAtor
PathoSEEK Reveals Microbiomes
PathogINDICAtorqPCR
Sequencing
Microbiome IDOUT Count
R Square In PathoSEEK
Majority of microbes don’t culture
Penicillium Citrinum & Paxilli Confirmation
Confirm the Paxilline and CitrininGenes are also present
Paxilline
Citrinin
Diverse Microbiomes Discovered
DNA sequence Doesn’t = Expression
Uhlig et al.4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
+ESI Product Ion (3.860 min) Frag=85.0V [email protected] (436.3 -> **) Paxilline_PIS_LongColumn-01.d
129.8
181.6
418.0
345.7 400.1
308.0 359.6 208.2 143.8 317.7 164.9 185.8 378.0 300.3 230.7 327.9 289.1 435.8 270.4
Counts vs. Mass-to-Charge (m/z)
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
Orser, Marquez replicate Uhlig et al at DigiPath
Design an LC-MS/MS method to detect this in a flower matrix.
Microbiological Contamination*
* SC Labs, CannMed 2016
Positive Feed Back Loop
Cyanide inhalation
High False Positive microbe tests = More pesticide use Less beneficial microbe use
http://www.medicinalgenomics.com/wp-content/uploads/2016/05/wurzer-CannMed2016.pdf
Pesticides*
* SC Labs, CannMed 2016
Experimental Design3M & Biomeriuex vs. qPCR & Sequencing
5. Sequence both 16S and 18S PCR product (before and after Tempo DNA)
Step 1. Capture DNA Before Culture
3M Film(3/5 days to Grow)
Biomerieux Cartridge(3 days)
Step 2. Culture Sample on 3M or Biomerieux Platforms
Step 3. Extract Positive Cultures in Step 2
4. Perform both 16S and 18S ITS qPCR
Purified DNA(3 Hours)
Capture DNA After Culture
Perform both 16S and 18S ITS qPCR
NextGenSequence
Questions
1. Do original microbials match post culture microbials?
2. What % of the CFU/g are actually Yeast and Mold?
1. 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?
TAC Cq BMX-TAC 3M
Before 23.98 100 N/A
After 30.12
TYM Cq BMX-TYM 3M
Before N/A 37K/140K Fail
After 20.08
qPCR, BMX, 3M
Tilletiopsis_pallescens
Bacillus_coagulans
Ralstonia_syzygii_subsp._indonesiensis
bacterium_oxSCC-14
PathoSEEK TAC OUT Counts Before vs. After DNA TYM Biomerieux Culture
16S qPCR and Sequencing demonstrate bacteria grow very well in TYM cartridges
After TYM OTU Counts w/ Sequencing
Befo
re T
YM C
ultu
re O
TU C
ount
s w/
Sequ
enci
ng
PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture
Some Are Harmless Acne, Some Not
Kluyvera_ascorbatahttp://cid.oxfordjournals.org/content/33/7/e69.full
Raoultella_ornithinolyticahttp://jcm.asm.org/content/47/3/868.full
Klebsiella_sp.
Uncultured bacterium
Propionibacterium (Acne)
Enterobacter_sp._10-17
Plant Background DNA
Kluyvera_sp
Befo
re T
YM C
ultu
re O
TU C
ount
s w/
Sequ
enci
ng
After TYM Culture OTU Counts w/ Sequencing
18S Shows P.citrinum GrowthBe
fore
TYM
Cul
ture
OTU
Cou
nts w
/ 18
S Se
quen
cing
After TYM Culture OTU Counts w/ 18S Sequencing
PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture
fungal_endophyte
Delftia_sp._JC257
Penicillium_citrinum
Penicillium_sp._2433_FbLBPenicillium_citrinum
fungal_endophyte
fungal_endophyte
Penicillium_sp._568_FbG
Penicillium_citrinumPenicillium_sp._568_FbG
18S and Aspergillus Failure to Grow
PathoSEEK TYM OTU Counts
funga_endophyte
Aspergillus_niger
Aspergillus_nigerAspergillus_niger
Fusarium
Befo
re T
YM C
ultu
re O
TU C
ount
s w/
18S
Sequ
enci
ng
After TYM Culture OTU Counts w/ 18S Sequencing
16S primers on 3M petri film vs Biomerieux
uncultured_bacterium
Aureimonas_sp._PP-WC-4G-234
uncultured_bacterium
uncultured_bacterium
bacterium_CICC_23843
Bacillus_coagulans
uncultured_bacteriumRalstonia_pickettii
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?
18S qPCR, 3M petri film vs Biomerieux
Penicillium_citrinum
fungal_endophyte
fungal_endophyte
Penicillium_citrinum
Penicillium_brevicompactum
Correl = .992
Correl =0.196
Correlations with Bacteria are weak
18S qPCR specificity
• When 18S qPCR & Sequencing is performed how many times do we see 16S sequence hits?
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.
Nevada On The Shoulder of Giants
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
Lets get cannabis on the same footing!
FDA is Moving to Sequence Based Safety Testing
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.
Blockchains Enable Encryption Proof Time Stamps
21e20b9db09c2af093ef926a3ec77efaec1fc2df
Create Encryption Key of SNP file with SHA-256
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 time-stamped Block unlocks their genetic file and therefore the file had to exist at the time of the block
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
DigiPathCindy OrserGlen Marquez
ProverdeChris HudallaMatthew Silva
SCLabsJosh WurzerAlec Dixon
