SenSATIVAx PathoSEEK Manufacturer Validation

SenSATIVAx® and PathoSEEK® Manufacturer Validation Table of Contents Introduction ............................................................................................................................................. 2 Quality Policy Statement ............................................................................................................................... 2 Matrices Included .............................................................................................................................................. 2 Species Included ............................................................................................................................................... 3 Accuracy ................................................................................................................................................... 5 Single DNA Species Spike ..............................................................................................................................................5 Single DNA Species Spiked at Varying Inoculum Levels ...............................................................................6 Single Cultured Organism Spike ..................................................................................................................................9 Precision .................................................................................................................................................12 Specificity................................................................................................................................................19 Multiple DNA Species Spike in .................................................................................................................................. 24 Multiple live organism spike in ................................................................................................................................... 25 Linearity, PCR Efficiency and Limit of Detection ......................................................................26 Linearity and PCR Efficiency ......................................................................................................................................... 26 Limit of Detection ............................................................................................................................................................... 36 Appendix I: Aspergillus niger spore lysis and growth ............................................................62 Appendix II: Cq to CFU Conversion ................................................................................................64 Cq to CFU/g Equations............................................................................................................................................. 67 Appendix III: Additional Data supporting Cq to CFU/g conversions ................................68 Appendix IV: Grim Reefer Free DNA Removal kit ...................................................................72 Conclusion ..............................................................................................................................................75 References ..............................................................................................................................................76 Page 1 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Introduction Medicinal Genomics Corporation (MGC) has pioneered the genomics of cannabis to build stronger scientific foundation for cannabis-based products. Our unmatched expertise in genetic science has helped cultivars, dispensaries and testing laboratories characterize and understand the quality and consistency of cannabis to ensure patients and consumers have access to safe, quality cannabis. Medicinal Genomics Corporation (MGC) completed a manufacturer’s validation on the SenSATIVAx® Flower/Leaf DNA Extraction Kit and the SenSATIVAx® Marijuana Infused Product (MIP)/Extract DNA Extraction Kit in conjunction with the PathoSEEK® qPCR Microbial Detection Platform. The following experiments were conducted in a Biosafety Level 2 Laboratory when necessary. The following document demonstrates the results of these experiments. Quality Policy Statement MGC is committed to exceeding customers’ expectations. This is achieved through the application of world class operations, continuous process improvement, and premier customer service. MGC’s quality system is designed to meet the quality system management requirements set forth directly in ISO 9001 and ISO 13485 standards. MGC conducts all business in accordance to and in compliance with these standards. It is a requirement of the system that all employees adhere to the spirit and intent of the quality policy and the directives of the quality system manual. Matrices Included The samples that were included in this validation are: 1. Plant – Cannabis flower 2. Concentrate – The product of any process used to concentrate particular plant constituent/s- Marijuana infused products (MIP) – Products using any of the above samples mixed into a consumable, see Table 1. Table 1, Matrices evaluated Concentrate/MIP Tested Gummy Chocolate Lozenge Extract Tincture Resin Page 2 Components Sugar and gelatin Sugar and fat Sugar Oil Alcohol Wax and shatter 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Species Included The live organisms listed in Table 2 were used in the validation of the MGC SenSATIVAx® Flower/Leaf DNA Extraction Kit and SenSATIVAx® MIP/Extract Kits and the PathoSEEK® qPCR platform. Certificates for the organisms that were obtained from the American Type Culture Collection (ATCC) can be found on the ATCC website, https://atcc.org, by searching the ATCC# provided in Table 2. All ATCC organisms were reconstituted and incubated according to ATCC product documentation. The yeast and mold species from ATCC were grown in 5 mL Tryptic Soy Broth (TSB) for 5 days at room temperature and checked visually for turbidity. The Pseudomonas aeruginosa and Staphylococcus aureus from ATCC were grown in 5mL of TSB for 24 hours at 37°C. The E.coli obtained from New England Biolabs and Salmonella from ATCC was grown in 20 mL TSB for 24 hours at 37°C. The S. cerevisiae was used as recommended. Table 2, Live Organisms Evaluated: Live Organisms Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus terreus Aspergillus janponicus Candida albicans Candida catenulate Candida glabralta Candida krusei Candida sphaerica Debaryomyces hanseii Rhodotorula mucilaginosa Trichothecium Roseum Yarrowia lipolytica Salmonella Saccharomyces cerevisiae E.coli Pseudomonas aeruginosa Staphylococcus aureus Source ATCC# 16870 ATCC# 16903 ATCC# 15475 ATCC# 16793 ATCC# 16873 ATCC# 10231 ATCC# 10565 ATCC# 15545 ATCC# 28870 ATCC# 8565 ATCC# 10623 ATCC# 4557 ATCC# 90473 ATCC# 18944 ATCC# 700720 The Gold Pitch, Giga Yeast, Inc. DH10B (New England Biolabs, #C3020K) ATCC# 9027 ATCC# 6538 The DNA species in Table 3 were used in the validation of the MGC SenSATIVAx® Flower/Leaf DNA Extraction Kit, SenSATIVAx® MIP/Extract kits and the PathoSEEK® qPCR assays. Page 3 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 3, DNA Organisms Evaluated DNA Organisms Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus terreus E.coli Klebsiella pneumoniae STEC O111 Salmonella enterica Pseudomonas aeruginosa Staphylococcus aureus Coliform/Entero Positive Control Total Yeast and Mold Positive Control Total Aerobic Count Positive Control E.coli/Salmonella Positive Control Page 4 Source ATCC# 9643D-2 ATCC# 1022D-2 ATCC# 1015D-2 ATCC# 20542D-2 ATCC# 8739D-5 ATCC# 700721D-5 ATCC# BAA-2440D-5 ATCC# 700720D-5 ATCC# 9027D-5 ATCC# 6538D-5 MGC# 420314 MGC# 420303 MGC # 420306 MGC# 420313 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Accuracy Single DNA Species Spike The data below shows that the SenSATIVAx® DNA Extraction kits can successfully extract DNA species spiked into various matrices. 2 μL of a 1:10 dilution of the Medicinal Genomics Total Enterobacteriaceae & Coliform Multiplex Control DNA was spiked into flower sample, hemp oil extract sample, and several MIP samples. For each matrix tested, a non-spiked control was included to show that the product being tested was clean and the qPCR signal was from the spiked DNA. This was followed by DNA extraction using either the SenSATIVAx® Flower/Leaf Extraction Kit or the SenSATIVAx® MIP/Extract DNA Extraction Kit. To show the DNA extraction was successful, the PathoSEEK® Total Entero & Coliform Multiplex Detection Assay was tested on all extracted DNA. Plant Table 4, DNA Spiked into Flower Sample: Sample Type qPCR Assay Flower Total Coliform/Entero Flower No DNA spike Total Coliform/Entero Cq 22.38 Not Detected Extract Table 5, DNA Spiked into Extract Sample: Sample Type qPCR Assay Extract Total Coliform/Entero Extract No DNA spike Total Coliform/Entero Cq 21.21 Not Detected MIP Table 6, DNA Spiked into different MIP Samples: MIP qPCR Assay Shatter Total Coliform/Entero Gummy Total Coliform/Entero Chocolate Total Coliform/Entero Gummy No DNA spike Total Coliform/Entero Page 5 866.574.3582 | www.medicinalgenomics.com Cq 21.07 21.21 22.79 Not Detected version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Single DNA Species Spiked at Varying Inoculum Levels The data below shows that the SenSATIVAx® DNA Extraction kits can successfully extract DNA species spiked into various matrices at three different inoculum levels. 100,000 copies, 50,000 copies, and 10,000 copies of Aspergillus flavus, A. fumigatus, A. niger and A. terreus DNA were spiked into flower samples. 100,000 copies, 50,000 copies, and 10,000 copies of STEC O111 E.coli and Salmonella DNA were spiked into Rick Simpson Oil (RSO) and chocolate. For each matrix and species tested, a nonspiked control was included to show that the product being tested was clean and the qPCR signal was from the spiked DNA. This was followed by DNA extraction using either the SenSATIVAx® Flower/Leaf DNA Extraction Kit or the SenSATIVAx® MIP/Extract DNA Extraction Kit. To show the DNA extraction was successful, the PathoSEEK® qPCR Assay listed in tables 7-14 was used. Table 7, Aspergillus flavus DNA spiked into Flower Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed* Flower 100,000 6,670 Aspergillus flavus 19.14 Flower 100,000 6,670 Aspergillus flavus 18.93 Flower 100,000 6,670 Aspergillus flavus 18.24 Flower 50,000 3,335 Aspergillus flavus 19.97 Flower 50,000 3,335 Aspergillus flavus 19.82 Flower 50,000 3,335 Aspergillus flavus 19.62 Flower 10,000 667 Aspergillus flavus 22.52 Flower 10,000 667 Aspergillus flavus 22.58 Flower 10,000 667 Aspergillus flavus 22.46 Flower None None Aspergillus flavus Not Detected Flower None None Aspergillus flavus Not Detected Table 8, Aspergillus fumigatus DNA spiked into Flower Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed* Flower 100,000 6,670 Aspergillus fumigatus 20.49 Flower 100,000 6,670 Aspergillus fumigatus 19.75 Flower 100,000 6,670 Aspergillus fumigatus 20.55 Flower 50,000 3,335 Aspergillus fumigatus 21.42 Flower 50,000 3,335 Aspergillus fumigatus 21.85 Flower 50,000 3,335 Aspergillus fumigatus 21.04 Flower 10,000 667 Aspergillus fumigatus 23.82 Flower 10,000 667 Aspergillus fumigatus 23.49 Flower 10,000 667 Aspergillus fumigatus 23.29 Flower None None Aspergillus fumigatus Not Detected Flower None None Aspergillus fumigatus Not Detected Page 6 866.574.3582 | www.medicinalgenomics.com %RSD 2.51 0.89 0.27 Not Applicable %RSD 2.20 1.89 1.14 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 9, Aspergillus niger DNA spiked into Flower Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed* Flower 100,000 6,670 Aspergillus niger 26.37 Flower 100,000 6,670 Aspergillus niger 26.11 Flower 100,000 6,670 Aspergillus niger 26.28 Flower 50,000 3,335 Aspergillus niger 27.78 Flower 50,000 3,335 Aspergillus niger 27.43 Flower 50,000 3,335 Aspergillus niger 27.79 Flower 10,000 667 Aspergillus niger 31.46 Flower 10,000 667 Aspergillus niger 31.71 Flower 10,000 667 Aspergillus niger 30.81 Flower None None Aspergillus niger Not Detected Flower None None Aspergillus niger Not Detected Table 10, Aspergillus terreus DNA spiked into Flower Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed* Flower 100,000 6,670 Aspergillus terreus 25.05 Flower 100,000 6,670 Aspergillus terreus 25.05 Flower 100,000 6,670 Aspergillus terreus 24.92 Flower 50,000 3,335 Aspergillus terreus 26.41 Flower 50,000 3,335 Aspergillus terreus 26.34 Flower 50,000 3,335 Aspergillus terreus 26.43 Flower 10,000 667 Aspergillus terreus 28.82 Flower 10,000 667 Aspergillus terreus 29.04 Flower 10,000 667 Aspergillus terreus 28.86 Flower None None Aspergillus terreus Not Detected Flower None None Aspergillus terreus Not Detected Table 11, STEC O111 E.coli DNA spiked into RSO Sample: Copies DNA Copies DNA Sample qPCR Assay Spiked Analyzed** RSO 100,000 2,000 STEC E.coli RSO 100,000 2,000 STEC E.coli RSO 50,000 1,000 STEC E.coli RSO 50,000 1,000 STEC E.coli RSO 10,000 200 STEC E.coli RSO 10,000 200 STEC E.coli RSO None None STEC E.coli RSO None None STEC E.coli Page 7 866.574.3582 | www.medicinalgenomics.com Cq %RSD 0.50 0.74 1.48 Not Applicable %RSD 0.30 0.18 0.41 Not Applicable %RSD 26.97 27.04 28.15 28.21 31.77 31.74 Not Detected Not Detected 0.18 0.15 0.07 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 12, Salmonella enterica DNA spiked into RSO Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed** RSO 100,000 2,000 Salmonella 29.00 RSO 100,000 2,000 Salmonella 29.11 RSO 50,000 1,000 Salmonella 30.11 RSO 50,000 1,000 Salmonella 30.67 RSO 10,000 200 Salmonella 33.22 RSO 10,000 200 Salmonella 33.08 RSO None None Salmonella Not Detected RSO None None Salmonella Not Detected Table 13, STEC O111 E.coli DNA spiked into Chocolate Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed** Chocolate 100,000 2,000 STEC E.coli 27.07 Chocolate 100,000 2,000 STEC E.coli 27.44 Chocolate 50,000 1,000 STEC E.coli 28.57 Chocolate 50,000 1,000 STEC E.coli 28.17 Chocolate 10,000 200 STEC E.coli 31.81 Chocolate 10,000 200 STEC E.coli 31.25 Chocolate None None STEC E.coli Not Detected Chocolate None None STEC E.coli Not Detected Table 14, Salmonella enterica DNA spiked into Chocolate Sample: Copies DNA Copies DNA Sample qPCR Assay Cq Spiked Analyzed** Chocolate 100,000 2,000 Salmonella 30.27 Chocolate 100,000 2,000 Salmonella 30.45 Chocolate 50,000 1,000 Salmonella 31.52 Chocolate 50,000 1,000 Salmonella 31.46 Chocolate 10,000 200 Salmonella 35.06 Chocolate 10,000 200 Salmonella 34.98 Chocolate None None Salmonella Not Detected Chocolate None None Salmonella Not Detected %RSD 0.27 1.30 0.30 Not Applicable %RSD 0.96 1.00 1.26 Not Applicable %RSD 0.42 0.13 0.16 Not Applicable *The copies of DNA analyzed for the spiked flower samples were calculated by the following equations. X number of DNA copies in 300 μL * 0.667 (200 μL of the 300 μL is extracted). 1/10th of the extracted DNA is then assayed in the qPCR reaction. For example: 10,000 copies * 0.667 = 6,670 copies; 6,670 copies * 0.1 = 667 copies analyzed Page 8 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation **The copies of DNA analyzed for the MIP samples were calculated by the following equations. X number of DNA copies in 500 μL * 0.6 (300 μL of the 500 μL is prepped with Chloroform). That number * 0.333 (100 μL of the 300 μL is extracted). 1/10th of the extracted DNA is then assayed in the qPCR reaction. For example, 10,000 copies * 0.6 = 6,000 copies; 6,000 copies * 0.333 = 1,998 copies; 1,998 copies * 0.1 = 200 copies analyzed. Single Cultured Organism Spike The data in tables 15-17 show that the SenSATIVAx® DNA Extraction Kits can successfully extract DNA from a single cultured organism spiked into various matrices. E.coli, Salmonella, and S. cerevisiae live cultures were grown to saturation and a 1:100 dilution was spiked into flower sample, hemp oil extract sample, and several MIP samples. Pseudomonas aeruginosa, and Staphylococcus aureus live cultures were grown to saturation. 100μL of the culture was removed and spiked into either 3.45mL TSB with 0.25g of flower present or 1.65mL MGC Solution A with 0.25g of hemp oil extract or chocolate present. For each matrix tested, a non-spiked control was included to show that the product being tested was clean and the qPCR signal was from the spiked live organism. For the live cultures of Aspergillus flavus, A. fumigatus, A. niger and A. terreus experiments, the organisms were grown to saturation. 100μL of the culture was removed and spiked into either 3.45mL TSB with 0.25g of flower present or 1.65mL MGC Solution A with 0.25g of hemp oil extract present. For each matrix tested, a non-spiked control was included to show that the product being tested was clean and the qPCR signal was from the spiked live organism. DNA extraction was performed using either the SenSATIVAx® Flower/Leaf DNA Extraction Kit or the SenSATIVAx® MIP/Extract DNA Extraction Kit. To show the DNA extraction was successful from live organisms, the PathoSEEK® qPCR Assay listed in tables 15-17 was used. Plant: Table 15, Live culture spiked into a single Flower Sample: Sample Organism Spiked qPCR Assay Flower E.coli Salmonella/E.coli Flower Salmonella Salmonella/E.coli Flower S.cerevisiae Total Yeast and Mold Flower E.coli Total Aerobic Count Flower E.coli Total Coliform Flower E.coli Total Entero Flower Pseudomonas aeruginosa Pseudomonas aeruginosa Flower Staphylococcus aureus Staphylococcus aureus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus niger Aspergillus niger Flower Aspergillus terreus Aspergillus terreus Page 9 866.574.3582 | www.medicinalgenomics.com Cq 22.34 23.09 27.07 18.26 21.36 18.54 17.72 24.99 20.64 20.40 28.47 33.03 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower None None None None None None None None None None Total Yeast and Mold Salmonella/E.coli Total Aerobic Count Total Entero/Coliform Pseudomonas aeruginosa Staphylococcus aureus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus terreus Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Extract Table 16, Live culture spiked into Hemp Extract Sample: Sample Organism Spiked qPCR Assay Extract E.coli Salmonella/E.coli Extract Salmonella Salmonella/E.coli Extract S.cerevisiae Total Yeast and Mold Extract E.coli Total Aerobic Count Extract E.coli Total Coliform Extract E.coli Total Entero qPCR Extract Aspergillus terreus Aspergillus terreus Extract Pseudomonas aeruginosa Pseudomonas aeruginosa Extract Staphylococcus aureus Staphylococcus aureus Extract None Total Yeast and Mold Extract None Total Aerobic Count Extract None Salmonella/E.coli Extract None Total Coliform/Entero Extract None Aspergillus terreus Extract None Pseudomonas aeruginosa Extract None Staphylococcus aureus Page 10 866.574.3582 | www.medicinalgenomics.com Cq 24.48 25.78 27.56 20.48 23.85 21.25 36.04 23.34 28.45 Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation MIP Table 17, Live culture spiked into different MIP Samples: Sample Organism Spiked qPCR Assay Wax E.coli Salmonella/E.coli Gummy E.coli Salmonella/E.coli Chocolate E.coli Salmonella/E.coli Lozenge E.coli Salmonella/E.coli Chocolate E.coli Salmonella/E.coli Gummy Aspergillus terreus Aspergillus terreus Chocolate Pseudomonas aeruginosa Pseudomonas aeruginosa Chocolate Staphylococcus aureus Staphylococcus aureus Gummy None Salmonella/E.coli Gummy None Aspergillus terreus Chocolate None Pseudomonas aeruginosa Chocolate None Staphylococcus aureus Page 11 866.574.3582 | www.medicinalgenomics.com Cq (E.coli) 23.41 25.14 22.71 23.08 23.66 35.61 22.51 26.21 Not Detected Not Detected Not Detected Not Detected version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Precision Precision contains two sub-tasks: repeatability and robustness. Repeatability is defined as the precision under the same operating conditions (intra-assay precision). Robustness is defined as the degree of precision of test results obtained by the analysis of the same samples under a variety of typical test conditions, such as different days and analysts. With this in mind, it was necessary for MGC to display the repeatability and robustness of the SenSATIVAx® Flower/Leaf DNA Extraction Kit and the SenSATIVAx® MIP/Extract DNA Extraction Kit and the PathoSEEK qPCR assays. Plant Repeatability and robustness was tested using SenSATIVAx® Flower/Leaf DNA Extraction Kit, PathoSEEK® qPCR Master Kit, and PathoSEEK ®Total Yeast & Mold Detection Assay. Three sets of experiments were done to confirm precision. All samples for this validation were done in conjunction with an outside, approved Cannabis Testing Laboratory in the state of Massachusetts. The sample used was a flower sample with visible microbial contamination. Experiment 1 addressed repeatability, extracted DNA Cannabis Sample 1) was placed into 6 random wells of the PCR plate and tested using the PathoSEEK® Total Yeast and Mold Assay. Experiment 2 addressed sampling robustness; the same plant sample (Cannabis Sample 2) was split into six 0.25g portions which were processed independently, following MGC released protocols. Experiment 3, one sample (Cannabis Sample 3) was split into six, 0.25g portions and these were processed independently following the standard protocols on Day1 and then repeated on Day 2. The analysis provided displays the relative standard deviation (RSD) of each group of experimental data for, quantification cycle (Cq). The data in Table 18-20 below displays the data and results of the three reproducibility experiments. Table 18, Repeatability: Sample & Well qPCR Assay Cannabis Sample 1 Total Yeast & Mold Cannabis Sample 1 Total Yeast & Mold Cannabis Sample 1 Total Yeast & Mold Cannabis Sample 1 Total Yeast & Mold Cannabis Sample 1 Total Yeast & Mold Cannabis Sample 1 Total Yeast & Mold Cq Percent RSD Page 12 Cq 20.3 20.5 20.4 20.5 20.3 20.6 0.6 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 19, Sampling Robustness: Sample qPCR Assay Cannabis Sample 2 Total Yeast & Mold Cannabis Sample 2 Total Yeast & Mold Cannabis Sample 2 Total Yeast & Mold Cannabis Sample 2 Total Yeast & Mold Cannabis Sample 2 Total Yeast & Mold Cannabis Sample 2 Total Yeast & Mold Cq Percent RSD 1.9 Table 20, Day-to-Day Robustness: Sample & Well Day qPCR Assay Cannabis Sample 3_A01 Day 1 Total Yeast & Mold Cannabis Sample 3_A02 Day 1 Total Yeast & Mold Cannabis Sample 3_A03 Day 1 Total Yeast & Mold Cannabis Sample 3_A04 Day 1 Total Yeast & Mold Cannabis Sample 3_A05 Day 1 Total Yeast & Mold Cannabis Sample 3_A06 Day 1 Total Yeast & Mold Cannabis Sample 3_G07 Day 2 Total Yeast & Mold Cannabis Sample 3_G08 Day 2 Total Yeast & Mold Cannabis Sample 3_G09 Day 2 Total Yeast & Mold Cannabis Sample 3_G10 Day 2 Total Yeast & Mold Cannabis Sample 3_G11 Day 2 Total Yeast & Mold Cannabis Sample 3_G12 Day 2 Total Yeast & Mold Day 1 Cq Percent RSD 3.2 Day 2 Cq Percent RSD 2.2 Overall Day 1 & Day 2 Cq Percent RSD 2.8 Mean Values of Day 1 vs. Day 2 Day 1 Cq Mean 24.0 Cq Percent RSD Page 13 Cq 30.0 29.1 29.8 29.7 28.6 29.0 Cq 23.0 23.3 23.9 24.0 25.2 24.4 22.8 23.3 23.9 23.8 24.1 23.1 Day 2 23.5 1.4 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Extract Repeatability and Robustness was tested using SenSATIVAx® MIP/Extract DNA Extraction Kit, PathoSEEK® qPCR Master Kit, PathoSEEK® Total Coliform Detection Assay and Total Yeast and Mold Assay. All samples for this validation were done in conjunction with an outside-approved Cannabis Testing Laboratory in the state of Massachusetts. The first experiment, Table 21, addressed repeatability. Hemp extract (Elixinol LLC) was spiked with 2 μL of a 1:10 dilution of the MGC Entero/Coliform positive control. The DNA extraction was performed using the SenSATIVAx® MIP/Extract DNA Extraction Kit. The extracted DNA was placed into 5 random wells of the PCR plate and tested using the PathoSEEK® Total Coliform Assay. The second experiment, Table 22, addressed sampling Robustness. The same Hemp extract sample was split into six, 0.25 gram portions, and these were processed independently, using the SenSATIVAx® MIP/Extract DNA Extraction Kit. The last experiment, Table, 23, 3 different Hemp extracts were spiked with S. cerevisiae and samples were processed by 3 independent lab users using the SenSATIVAx® MIP/Extract DNA Extraction Kit. The extracted DNA was then tested using the PathoSEEK® Total Yeast and Mold Detection Assay. The analysis provided displays the relative standard deviation (RSD) of each group of experimental data, for quantification cycle (Cq). The data in Tables 21-23 below display the data and results of the three reproducibility experiments. Table 21, Sampling Repeatability: Sample qPCR Assay Extract Sample 2 Total Coliform Extract Sample 2 Total Coliform Extract Sample 2 Total Coliform Extract Sample 2 Total Coliform Extract Sample 2 Total Coliform Cq Percent RSD 0.534 Table 22, Sample Robustness: Sample & Well qPCR Assay Extract Sample 1 Total Coliform Extract Sample 2 Total Coliform Extract Sample 3 Total Coliform Extract Sample 4 Total Coliform Extract Sample 5 Total Coliform Extract Sample 6 Total Coliform Cq Percent RSD Page 14 Cq 21.07 20.93 20.80 20.99 21.07 Cq 20.58 20.47 20.56 20.62 21.05 20.81 1.034 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 23, User-to-User Robustness: Sample User Extract 1 User 1 Extract 1 User 2 Extract 1 User 3 Extract 2 User 1 Extract 2 User 2 Extract 2 User 3 Extract 3 User 1 Extract 3 User 2 Extract 3 User 3 Extract 1 (3 users) Cq Percent RSD 4.94 Extract 2 (3 users) Cq Percent RSD 1.63 Extract 3 (3 users) Cq Percent RSD 2.73 qPCR Assay Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Total Yeast & Mold Cq 23.95 22.14 21.90 24.45 24.59 25.21 32.13 32.18 33.70 MIP Robustness was tested using SenSATIVAx® MIP/Extract DNA Extraction Kit, PathoSEEK® qPCR Master Kit, PathoSEEK® Total Coliform Detection Assay. The first experiment was conducted at an outside-approved Cannabis Testing Laboratory in the state of Massachusetts. Four different MIPs were spiked with 2 μL of a 1:10 dilution of the MGC Total Entero/Coliform positive control. The DNA extraction was performed using the SenSATIVAx® MIP/Extract DNA Extraction Kit. The extracted DNA was tested at the cannabis testing laboratory using the PathoSEEK® Total Coliform Assay. The results can be found in Table 24. In the second experiment, Table 25, the same four MIPs were tested at Medicinal Genomics and spiked with a 1:100 dilution of live E.coli grown to saturation. The DNA extraction was performed using the SenSATIVAx® MIP/Extract DNA Extraction Kit. The extracted DNA was tested using the PathoSEEK® Total Coliform Assay. Table 24, DNA Spiked into MIP at Cannabis Testing Laboratory Sample qPCR Assay Cq Lozenge Total Coliform 23.30 Wax Total Coliform 20.97 Gummy Total Coliform 21.55 Page 15 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Chocolate Total Coliform 22.00 Table 25, live E.coli Spiked into MIPs at Medicinal Genomics Sample qPCR Assay Cq Lozenge Total Coliform 22.07 Wax Total Coliform 21.57 Gummy Total Coliform 23.56 Chocolate Total Coliform 20.31 Discussion The data in Tables 24 and 25 show that both DNA and live E.coli can be extracted from a variety of different matrices. Cannabis Testing Labs looking to adopt qPCR can validate the MGC SenSATIVAx® Flower/Leaf DNA Extraction Kit, SenSATIVAx® MIP/Extract DNA Extraction Kit, PathoSEEK® qPCR Master Kit, and PathoSEEK® qPCR assays using DNA spike-in experiments because this validation report fully demonstrates the cell lysing capabilities within many matrices (Table 1). Matrices outside the scope of this validation may require live organism spikes in addition to DNA spikes. Precision of Aspergillus species-specific and multiplex assays The Aspergillus multiplex assay combines the Aspergillus A. flavus, A. fumigatus, A. niger and A. terreus detection assays together in one reaction. The detection probe for each species is on the FAM channel; therefore, if a positive result were to occur, the only way to determine which species was present would be to run all four individual Aspergillus assays. Live Aspergillus flavus, fumigatus, niger and terreus organisms were grown to saturation. The species-specific assay vs. multiplex assay was tested by adding 100 μL of the species-specific culture into 3.45 mL of TSB with 0.25g of flower present. The multiplex assay was also tested by adding 100 μL of each species culture to 3.15 mL of TSB with 0.25g of flower present. For each matrix tested, a non-spiked control was included to show that the product being tested was clean and the qPCR signal was from the spiked live organism. This was followed by DNA extraction using the SenSATIVAx® Flower/Leaf DNA Extraction Kit. The extracted DNA was tested using the PathoSEEK® Aspergillus flavus, A. fumigatus, A. niger, A. terreus and or Aspergillus multiplex assay in replicates of six for the single species spikes and in triplicate for the multiple species spike. The data presented in Table 26 shows that Aspergillus flavus, A. fumigatus, A. niger and A. terreus can be detected at similar Ct values when either the Aspergillus speciesspecific assay or the Aspergillus multiplex assay is used. The table also presents the repeatability of the extracted DNA when tested using the Aspergillus flavus, A. fumigatus, A. niger, A. terreus and or Aspergillus multiplex assays. Page 16 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 26, Precision of Aspergillus Assays Sample Organism Spiked qPCR Assay Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus flavus Flower Aspergillus flavus Aspergillus Multiplex Flower Aspergillus flavus Aspergillus Multiplex Flower Aspergillus flavus Aspergillus Multiplex Flower Aspergillus flavus Aspergillus Multiplex Flower Aspergillus flavus Aspergillus Multiplex Flower Aspergillus flavus Aspergillus Multiplex Flower A. flavus, fumigatus, niger, terreus Aspergillus flavus Flower A. flavus, fumigatus, niger, terreus Aspergillus flavus A. flavus, fumigatus, niger, terreus Flower Aspergillus flavus Flower None Aspergillus flavus Flower None Aspergillus flavus Flower None Aspergillus flavus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus fumigatus Flower Aspergillus fumigatus Aspergillus Multiplex Flower Aspergillus fumigatus Aspergillus Multiplex Flower Aspergillus fumigatus Aspergillus Multiplex Flower Aspergillus fumigatus Aspergillus Multiplex Flower Aspergillus fumigatus Aspergillus Multiplex Flower Aspergillus fumigatus Aspergillus Multiplex A. flavus, fumigatus, niger, terreus Flower Aspergillus fumigatus Flower A. flavus, fumigatus, niger, terreus Aspergillus fumigatus Flower A. flavus, fumigatus, niger, terreus Aspergillus fumigatus Flower None Aspergillus fumigatus Flower None Aspergillus fumigatus Flower None Aspergillus fumigatus Flower Aspergillus niger Aspergillus niger Flower Aspergillus niger Aspergillus niger Flower Aspergillus niger Aspergillus niger Flower Aspergillus niger Aspergillus niger Page 17 866.574.3582 | www.medicinalgenomics.com Cq 25.20 25.06 24.94 24.88 24.76 25.19 24.57 24.40 24.47 24.68 25.03 24.58 25.43 24.48 25.01 Not Detected Not Detected Not Detected 18.60 18.60 18.72 18.47 18.53 18.90 20.36 21.04 20.32 19.59 18.82 20.39 21.55 21.54 21.49 Not Detected Not Detected Not Detected 26.66 26.56 26.47 26.65 %RSD 0.70 0.90 1.91 Not Applicable 0.82 3.84 0.15 Not Applicable 0.29 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Flower Page 18 Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus None None None Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus None None None A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus A. flavus, fumigatus, niger, terreus Aspergillus niger Aspergillus niger Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus Multiplex Aspergillus Multiplex Aspergillus Multiplex 866.574.3582 | www.medicinalgenomics.com 26.52 26.52 26.97 26.92 26.95 27.15 26.98 27.09 32.06 32.14 32.39 Not Detected Not Detected Not Detected 25.43 25.35 24.88 24.91 24.88 25.07 27.34 25.03 25.21 25.01 25.26 26.30 26.62 26.81 27.12 Not Detected Not Detected Not Detected 24.04 23.68 21.59 0.33 0.53 Not Applicable 0.98 3.65 0.94 Not Applicable 5.73 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Specificity Specificity was tested using the MGC SenSATIVAx® DNA Extraction Kits, PathoSEEK® qPCR Master Kit, and PathoSEEK® Detection Assays. For the presence/absence assays there was a minimum of 6 species tested, both positive and negative results were expected. Whereas the threshold assays were tested with a minimum of 10 different species. Many of the species tested below were determined by using our published Illumina sequencing methods that target organisms found to be present on the cannabis plant1, 2. The data provided in Table 27 displays all the species tested at Medicinal Genomics and the result of each species on each assay. The species within the table are sorted alphabetically. Most species were purchased from American Type Culture Collection (ATCC). The provided catalog numbers from ATCC contain information about the samples. A “D” is indicative of purified DNA while only numbers are provided as freezedried cells. All freeze-dried cells were reconstituted and incubated according to ATCC product documentation. Table 27, List of species tested and corresponding results: Species Acinetobacter baumanii Aeromonas hydrophilia Aeromonas hydrophilia Strain CDC 35960 Alternia alternata Aspergillus aculeatus Aspergillus brasiliensis Aspergillus brasiliensis WLRI 034 (120) Aspergillus carbonarius Aspergillus carbonarius Aspergillus clavatus Aspergillus clavatus Aspergillus deflectus Aspergillus fijiensis Aspergillus flavus Aspergillus flavus Aspergillus flavus Type ATCC # Total Y&M B 19606 Neg (-) B 7965D B 7966D-5 M 6663 M 24147 M 9642 M 16404D-2 M 1025 M MYA-4641 M 1007D-2 M 1007D M 62502 M 20611 M 9643 M 26768 M 16870 Page 19 TAC TC TE E. coli Sal STEC A.fla A.fum A.nig Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) P. aer S. aur BTGN Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) A. terr Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus M 16883 M 11489 M 11497 M 204304 M MYA-1004 Type ATCC # M MYA-4921 M MYA-3631 M 9643D-2 M 16903 M 1022 M 1022D-2 M 1028 M 96918 M 13073 M 26934 M 14110 M MYA-3626 M MYA-3627 M MYA-4609 M 16873 M 38163D-2 M 38163 Aspergillus niger M 15475 Aspergillus niger M 16888 Aspergillus niger M 1015D-2 Aspergillus niger M 9142 Aspergillus niger M 9029 Aspergillus niger M 10535 Aspergillus niger M 13496 Aspergillus niger M 1015 Aspergillus niger M 6275 Aspergillus niger M 66295 Species Aspergillus flavus Aspergillus flavus Aspergillus flavus SN3 Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus 118 Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus janponicus Aspergillus nidulans Aspergillus nidulans Page 20 Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Total Y&M Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) TAC TC TE Neg (-) Neg (-) Neg (-) Neg (-) E. coli Sal STEC Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) A.fla A.fum A.nig Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) 866.574.3582 | www.medicinalgenomics.com Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) A. terr Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) P. aer version 31.05202020 S. aur BTGN SenSATIVAx® and PathoSEEK® Manufacturer Validation Pos (+) Pos (+) Pos (+) Pos (+) Aspergillus niger M 201201 Aspergillus niger M 208815 M 10075 M 1010 Type ATCC # Total Y&M M 42149D-2 Pos (+) M 10124 M 1005 M 16793 M 1012 M 10690 M 16794 M 24839 M 52430 M 12238 M MYA-4897 M MYA-4898 M 26604 M 20542D-2 M 1041 M 11730D-2 M 11730D B 6633D-5 B 6633 M 11542 Y 10231D-5 Y 10231 Y 10565 Y 28870 Y 344449 Y 8565 B 11437FD-5 Aspergillus niveo-glacus Aspergillus oryzae* Species Aspergillus oryzae* Aspergillus oryzae* Aspergillus tamarii Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus terreus Aspergillus ustus Aspergillus versicolor Aspergillus versicolor Bacillus subtilis subsp. Spizizenii Bacillus subtilis subsp. Spizizenii Botrytis cinerea Persoon Candida albicans Candida albicans Candida catenulata Candida krusei Candida lusitiniae Candida sphaerica Clostridium sporogenes Page 21 Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) TAC TC TE Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) E. coli Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Sal STEC Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) A.fla A.fum A.nig Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) A. terr Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) P. aer Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) 866.574.3582 | www.medicinalgenomics.com version 31.05202020 S. aur BTGN SenSATIVAx® and PathoSEEK® Manufacturer Validation Clostridium sporogenes Cryptococcus laurentii Cryptococcus neoformans Debaryomyces hanseii B 11437 Y 18803 Y 208821 Y 10623 Type ATCC # B NEB B 15038D-5 Escherichia coli B 8739D-5 Escherichia coli B 8739 B Species E.coli DH10B Compentent Cells Enterobacter aerogenes Neg (-) Pos (+) Pos (+) Pos (+) Total Y&M TAC TC TE Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) BAA2193D-5 Pos (+) B BAA2219D-5 B B TM Escherichia coli Strain 20003039 Escherichia coli Strain 20023211 Escherichia coli Strain 20033014 Escherichia coli Strain 20063008 Escherichia coli Strain 99-3311 Escherichia coli Strain O111 Fusarium proliferatum Fusarium oxysporum B B A.fla A.fum A.nig A. terr Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) BAA2196D-5 Pos (+) Pos (+) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) BAA2215D-5 Pos (+) Pos (+) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) BAA2192D-5 BAA2440D-5 Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) 52628 M 74170 M 28575 B 700721D-5 B 4357 B 4357D-5 B 19115D-5 M 38592 Mucor hiemalis M 28935 Mucor luteus M 28932 M 22365 M 10106D-2 Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) S. aur BTGN Neg (-) 866.574.3582 | www.medicinalgenomics.com Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Pos (+) Pos (+) Pos (+) P. aer Pos (+) Pos (+) M Page 22 Neg (-) Neg (-) Neg (-) 62506 Mucor racemosus Penicillium chrysogenum STEC Neg (-) Neg (-) Pos (+) M Geotrichum Candidum Geotrichum capitatum Klebsiella pneumoniae Lactobacillus acidophilos Lactobacillus acidophilus Listeria monocytogenes Mucor circinelloides Sal Neg (-) Neg (-) Neg (-) 76097 Neg (-) E. coli Neg (-) Neg (-) Neg (-) Neg (-) M Fusarium solani Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Penicillium citrinum Penicillium paxilli Penicillum marneffei Penicillum rubens Penicillium venetum Pos (+) Pos (+) Pos (+) Pos (+) Pos (+) M 36382 M 96516 M 18224 M 11709 M 16025 Type ATCC # B 9027D-5 B 9027 B 35554 B BAA-871D5 M 11709 Y 4557 Y - B 43975D-5 B 700720D-5 B 700720 B 1715D-5 B BAA-731D5 B BAA1579D-5 B BAA1580D-5 Pos (+) Pos (+) Neg (-) Pos (+) B BAA1582D-5 Pos (+) Pos (+) Neg (-) B BAA1578D-5 Pos (+) Pos (+) Neg (-) B 13313 Shigella flexneri B 12022 Shigella sonnei B 9290 Shigella sonnei B 25931 Species Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas syringae pathovar tomato Rhizopus stolonifer Rhodotorula mucilaginosa Saccharomyces cerevisiae Salmonella bongori strain 124.72 Salmonella enterica serova Typhimurium LT2 Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica strain MZ1445 Salmonella enterica subsp. arizonae Salmonella enterica subsp. diarizonae Salmonella enterica subsp. houtenae (IV) Salmonella enterica subsp. salamae (II) Salmonella enterica subsp. indica (VI) Shigella dysenteriae Page 23 Total Y&M Neg (-) Neg (-) Neg (-) TAC TC TE Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) E. coli Neg (-) Neg (-) Neg (-) Neg (-) Sal STEC Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) A.fla A.fum A.nig A. terr Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) P. aer Pos (+) Pos (+) S. aur Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) BTGN Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Pos (+) Neg (-) Pos (+) Neg (-) Pos (+) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Staphylococcus aureus subsp. Aureus Staphylococcus aureus subsp. aureus Trichoderma virens Trichothecium Roseum B 6538D-5 B 6538 M 13213 M 90473 Vibrio cholerae B 39315D-5 Species Type ATCC # M 18944 M 20390 B BAA1511D-5 Yarrowia lipolytica Yarrowia lipolytica Yersinia pestis K25 Neg (-) Pos (+) Neg (-) Neg (-) Pos (+) Neg (-) Neg (-) Pos (+) Neg (-) Pos (+) TAC TC TE Neg (-) Neg (-) Neg (-) Pos (+) Neg (-) Pos (+) Pos (+) Pos (+) Total Y&M Pos (+) E. coli Sal STEC A.fla A.fum A.nig Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Neg (-) Pos (+) Pos (+) Pos (+) Neg (-) Pos (+) A. terr Neg (-) Neg (-) P. aer S. aur Neg (-) Pos (+) Table 27 Legend: B = bacteria, M = Mold, Y = yeast Note: A blank cell means that the species was not tested with that assay A filled well means that the species was tested with the assay and shows results of the test. * Aspergillus orzyae is a subspecies of Aspergillus flavus and therefore, generates a positive Ct value when using the Aspergillus flavus assay and or the Aspergillus multiplex assay.6,7,8 Multiple DNA Species Spike In The data below shows that the SenSATIVAx® MIP/Extract DNA Extraction Kit can successfully extract DNA from hemp extract sample with multiple DNA or live organism species spiked in. For the DNA example 2 μL of a 1:10 dilution of six different positive controls was spiked into the hemp extract sample. For the live organism example, a 1:100 dilution of each organism grown to saturation was spiked into an oil extract sample. This was followed by DNA extraction using the SenSATIVAx® MIP/Extract DNA Extraction Kit. To show the DNA extraction was successful, multiple PathoSEEK® assays were run and are listed below in tables 28 and 29 with resulting Cq values. Table 28, Six Control DNAs spiked into same Hemp Extract Sample Sample DNA Spiked qPCR Assay TYM, TAC, TC, TE, E.coli, Salmonella Extract Total Yeast and Mold TYM, TAC, TC, TE, E.coli, Salmonella Extract Total Aerobic Count TYM, TAC, TC, TE, E.coli, Salmonella Extract Total Coliform TYM, TAC, TC, TE, E.coli, Salmonella Extract Total Entero TYM, TAC, TC, TE, E.coli, Salmonella Extract Salmonella TYM, TAC, TC, TE, E.coli, Salmonella Extract E.coli Page 24 866.574.3582 | www.medicinalgenomics.com BTGN Cq 19.31 20.35 21.03 20.94 21.78 20.9 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Multiple live organism spike in Table 29, 3 live organisms spiked into one Extract Sample Sample Live organisms qPCR Assay E.coli, Salmonella, Aspergillus niger Tincture Total Yeast and Mold E.coli, Salmonella, Aspergillus niger Tincture Total Aerobic Count E.coli, Salmonella, Aspergillus niger Tincture Total Coliform E.coli, Salmonella, Aspergillus niger Tincture Total Entero E.coli, Salmonella, Aspergillus niger Tincture E.coli E.coli, Salmonella, Aspergillus niger Tincture Salmonella E.coli, Salmonella, Aspergillus niger Tincture Aspergillus niger Cq 27.64 25.00 27.57 26.16 28.63 29.15 32.56 Discussion: More than 60 species in total were used to verify the specificity for the PathoSEEK® Microbial Safety Testing Solution, with a minimum of 6 to 10 and a maximum of 45 for each detection assay. The large data set in Table 27 displays 100% concordance with the current list of species tested. The list of species detected by each assay in-silico, comparing the target regions and the genomes within the NCBI database, is substantially larger than that which we can obtain from ATCC. Page 25 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Linearity, PCR Efficiency and Limit of Detection Linearity and PCR Efficiency Linearity experiments conducted describe the relationship between a group of known dilutions and resulting quantification cycle (Cq). All Medicinal Genomics qPCR detection assays are set to a threshold of 102 in the log view, this threshold was selected because it is constantly above any background noise. Medicinal Genomics developed a calculation to convert the quantification cycle (Cq) value from the PathoSEEK® qPCR Assay to colony forming units (CFU), which is reported when using culture-based methods (see Appendix I: Cq to CFU conversion). To ensure consistency from run to run, a fixed threshold was necessary. The 102 threshold was tested on all assays and confirmed by the resulting R2 value. R2 is used to evaluate the performance of a primer set. The following data in Tables 30-34 and Figures 1-4 demonstrate the experiments used to calculate linearity when using the PathoSEEK® qPCR Master Kit and Detection Assays. The qPCR efficiency was calculated by the Bio-Rad CFX96 TouchTM Real-Time PCR Detection System using the linearity experiments conducted above. The genomic copy number was calculated using the sample DNA concentration and the size of the genome for the species in question using the equation: number of copies = X ng*6.0221 x 1023 molecules/mole / (N x 650 g/mole) * 1x 109 ng/g). Using the slope of the regression line, the PCR efficiency can be calculated using following formula: E=100×(10−1/slope−1). For each target, the slope of the regression curve, when using the log10 transformation of the DNA, should be between −3.9 and −2.9 corresponding to PCR efficiencies ranging from 80% to 120%. 5 Table 47 summarizes the qPCR efficiency (E) and R2 value for all detection assays. Page 26 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation PathoSEEK® Total Yeast and Mold Detection Assay The Linearity of the PathoSEEK® Total Yeast and Mold Detection Assay was tested by performing a two-fold serial dilution of Aspergillus fumigatus DNA (Table 3, DNA Organisms Evaluated) starting at 500,000 copies. Table 30 shows the Cq Value and %RSD for each dilution point. Figure 1 shows the qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Table 30, Serial dilution of A. fumigatus in triplicate on Total Yeast & Mold Assay Assay Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Page 27 Copies of A. fumigatus DNA 500,000 500,000 500,000 250,000 250,000 250,000 125,000 125,000 125,000 62,500 62,500 62,500 31,250 31,250 31,250 15,625 15,625 15,625 7,813 7,813 7,813 3,906 3,906 3,906 1,953 1,953 1,953 977 977 977 488 488 488 244 Cq Value (FAM) 12.72 12.83 12.73 13.37 13.67 13.78 14.87 14.71 14.88 15.93 16.04 15.90 17.06 17.04 17.08 18.22 18.19 18.18 19.22 19.28 19.23 20.38 20.34 20.32 21.56 21.59 21.54 22.66 22.74 22.66 24.06 23.92 24.27 25.08 866.574.3582 | www.medicinalgenomics.com %RSD 0.48 1.56 0.64 0.46 0.11 0.11 0.17 0.15 0.12 0.20 0.73 0.07 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold Total Yeast and Mold 244 244 0 0 0 25.08 25.11 Not Detected Not Detected Not Detected Not Applicable Figure 1, Total Yeast and Mold qPCR Dilution Curves and qPCR Efficiency (E) PathoSEEK® Total Aerobic Bacteria Detection Assay The Linearity of the PathoSEEK® Total Aerobic Bacteria Detection Assay was tested by performing a two-fold serial dilution of E. coli DNA (Table 3, DNA Organisms Evaluated) starting at 1,000,000 copies. Table 31 shows the Cq Value and %RSD for each dilution point. Figure 2 shows the qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Table 31, Serial dilution of E.coli in triplicate on Total Aerobic Assay Assay Copies of E.coli DNA Cq Value (FAM) Total Aerobic Bacteria 1,000,000 14.79 Total Aerobic Bacteria 1,000,000 14.95 Total Aerobic Bacteria 1,000,000 14.61 Total Aerobic Bacteria 500,000 15.61 Total Aerobic Bacteria 500,000 15.57 Total Aerobic Bacteria 500,000 15.55 Total Aerobic Bacteria 250,000 16.77 Total Aerobic Bacteria 250,000 16.65 Total Aerobic Bacteria 250,000 16.73 Total Aerobic Bacteria 125,000 17.82 Total Aerobic Bacteria 125,000 17.90 Total Aerobic Bacteria 125,000 17.94 Total Aerobic Bacteria 62,500 19.12 Total Aerobic Bacteria 62,500 19.06 Total Aerobic Bacteria 62,500 19.01 Page 28 866.574.3582 | www.medicinalgenomics.com %RSD 1.15 0.20 0.37 0.34 0.29 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria Total Aerobic Bacteria 31,250 31,250 31,250 15,625 15,625 15,625 7,813 7,813 7,813 3,906 3,906 3,906 1,953 1,953 1,953 977 977 977 488 488 488 0 0 0 20.13 20.14 20.12 21.07 21.14 21.11 22.25 22.13 22.23 23.39 23.34 23.32 24.40 24.38 24.36 25.57 25.46 25.47 26.82 26.64 26.67 35.02 34.10 34.85 0.05 0.17 0.29 0.15 0.08 0.24 0.36 1.41 Figure 2, Total Aerobic Bacteria qPCR Dilution Curves and qPCR Efficiency (E) PathoSEEK® Total Enterobacteriaceae Detection Assay The Linearity of the PathoSEEK® Total Enterobacteriaceae Detection Assay was tested by performing a two fold serial dilution of Salmonella DNA (Table 3, DNA Organisms Evaluated) starting at 1,000,000 copies. Table 32 shows the Cq Value and %RSD for Page 29 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation each dilution point. Figure 3 shows the qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Table 32, Serial dilution of Salmonella in triplicate on Total Enterobacteriaceae Assay Assay Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Total Enterobacteriaceae Page 30 Copies of Salmonella DNA 1,000,000 1,000,000 1,000,000 500,000 500,000 500,000 250,000 250,000 250,000 125,000 125,000 125,000 62,500 62,500 62,500 31,250 31,250 31,250 15,625 15,625 15,625 7,813 7,813 7,813 3,906 3,906 3,906 1,953 1,953 1,953 977 977 977 488 488 488 0 0 Cq Value (FAM) 16.19 15.73 15.83 16.79 16.69 16.75 18.03 18.00 17.66 18.84 18.93 18.80 20.03 19.85 19.90 20.97 21.13 21.02 22.14 22.21 22.12 23.46 23.12 23.18 24.29 24.29 24.19 25.50 25.46 25.28 26.79 26.55 26.58 28.08 28.15 27.55 Not Detected Not Detected 866.574.3582 | www.medicinalgenomics.com %RSD 1.52 0.30 1.15 0.35 0.47 0.39 0.21 0.78 0.24 0.46 0.49 1.17 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Total Enterobacteriaceae 0 Not Detected Figure 3, Total Enterobacteriaceae qPCR Dilution Curves and qPCR Efficiency (E) PathoSEEK® Total Coliform Detection Assay The Linearity of the PathoSEEK® Total Coliform Detection Assay was tested by performing a two fold serial dilution of Klebsiella pneumoniae DNA (Table 3, DNA Organisms Evaluated) starting at 1,000,000 copies. Table 34 shows the Cq Value and %RSD for each dilution point. Figure 4 shows the qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Table 33, Serial dilution of Klebsiella pneumoniae in triplicate on Total Coliform Assay Copies of Klebsiella Assay Cq Value (FAM) %RSD pneumoniae DNA Total Coliform 1,000,000 18.22 Total Coliform 1,000,000 18.39 0.48 Total Coliform 1,000,000 18.27 Total Coliform 500,000 19.16 Total Coliform 500,000 19.26 0.47 Total Coliform 500,000 19.34 Total Coliform 250,000 20.33 Total Coliform 250,000 20.31 0.26 Total Coliform 250,000 20.41 Total Coliform 125,000 21.53 Total Coliform 125,000 21.55 0.14 Total Coliform 125,000 21.49 Total Coliform 62,500 22.67 Total Coliform 62,500 22.66 0.04 Total Coliform 62,500 22.65 Total Coliform 31,250 23.94 0.07 Page 31 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform Total Coliform 31,250 31,250 15,625 15,625 15,625 7,813 7,813 7,813 3,906 3,906 3,906 1,953 1,953 1,953 977 977 977 488 488 488 0 0 0 23.91 23.91 25.06 25.16 24.93 26.09 26.19 26.17 27.24 27.19 27.28 28.41 28.46 28.58 29.56 29.51 29.87 31.04 30.81 31.04 Not Detected Not Detected Not Detected 0.46 0.20 0.17 0.31 0.66 0.43 Not Applicable Figure 4, Total Coliform qPCR Dilution Curves and qPCR Efficiency (E) PathoSEEK® BTGN + Coliform Detection Assay The Bile Tolerant Gram Negative (BTGN) assay detects Total Enterobacteriaceae and Pseudomonas. While Pseudomonas is considered to be a BTGN is neither an Enterobacteriaceae nor a Colifrom. The BTGN + Coliform multiplex assay utilizes the Page 32 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation FAM channel to detect Total Enterobacteriaceae and Pseudomonas while the ROX channel detects Total Coliform. The Linearity of the PathoSEEK® BTGN + Coliform Detection Assay was tested 2 different ways. The first was by performing a two fold serial dilution of Klebsiella pneumoniae DNA, which is both an Enterobacteriaceae and a Colifrom, (Table 3, DNA Organisms Evaluated) starting at 1,000,000 copies. Table 34 shows the Cq Values and %RSD’s for each dilution point for each target. Figure 5 shows the BTGN qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Figure 6 shows the Total Coliform qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). The second way the BTGN + Coliform Detection Assay was tested was by performing a two fold serial dilution of Pseudomonas aeruginosa (Table 3, DNA Organisms Evaluated), which along with Total Enterobacteriaceae is detected in the FAM channel, starting at 1,000,000 copies. Table 35 shows the Cq Value and %RSD for each dilution point. Figure 7 shows the qPCR curves generated by the dilution series and the DNA genome copies vs. Cq value, resulting R2 value and efficiency (E). Table 34, Serial dilution of Klebsiella pneumoniae in triplicate on BTGN + Coliform Assay Assay BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN Page 33 Copies of Klebsiella pneumoniae DNA 1,000,000 1,000,000 1,000,000 500,000 500,000 500,000 250,000 250,000 250,000 125,000 125,000 125,000 62,500 62,500 62,500 31,250 31,250 31,250 15,625 15,625 15,625 Cq Value (FAM) 15.58 15.71 15.56 16.49 16.53 16.52 17.54 17.49 17.42 18.37 18.28 18.26 19.47 19.52 19.42 20.48 20.59 20.53 21.43 21.44 21.47 Assay %RSD 0.51 0.12 0.35 0.31 0.25 0.28 0.10 Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform 866.574.3582 | www.medicinalgenomics.com Cq Value (ROX) 18.52 18.55 18.50 19.72 19.66 19.72 20.69 20.75 20.72 21.72 21.65 21.64 22.95 22.84 22.90 24.05 24.02 23.92 25.00 25.07 24.97 %RSD 0.14 0.16 0.14 0.21 0.23 0.27 0.20 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN 7,813 7,813 7,813 3,906 3,906 3,906 1,953 1,953 1,953 977 977 977 488 488 488 BTGN 0 BTGN 0 BTGN 0 22.63 22.54 22.51 23.84 23.77 23.90 24.88 24.99 25.00 26.07 26.01 26.06 27.13 27.34 27.11 Not Detected Not Detected Not Detected 0.30 0.27 0.26 0.11 0.48 Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Coliform Not Applicable Coliform Coliform 26.14 26.14 26.15 27.39 27.49 27.29 28.75 28.71 28.68 29.69 29.67 29.65 30.92 31.06 30.99 Not Detected Not Detected Not Detected 0.03 0.36 0.13 0.06 0.22 Not Applicable Figure 5, BTGN qPCR Dilution Curves and qPCR Efficiency (E) Page 34 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 6, Coliform qPCR Dilution Curves and qPCR Efficiency (E) Table 35, Serial dilution of Pseudomonas aeruginosa in triplicate on BTGN + Coliform Assay Copies of Pseudomonas Assay Cq Value (FAM) %RSD aeruginosa DNA BTGN 1,000,000 16.78 BTGN 1,000,000 16.70 0.43 BTGN 1,000,000 16.63 BTGN 500,000 17.71 BTGN 500,000 17.79 0.22 BTGN 500,000 17.74 BTGN 250,000 18.74 BTGN 250,000 18.60 0.39 BTGN 250,000 18.62 BTGN 125,000 19.63 BTGN 125,000 19.59 0.18 BTGN 125,000 19.56 BTGN 62,500 20.85 BTGN 62,500 20.72 0.59 BTGN 62,500 20.96 BTGN 31,250 21.64 BTGN 31,250 21.80 0.37 BTGN 31,250 21.70 BTGN 15,625 22.77 BTGN 15,625 22.76 0.08 BTGN 15,625 22.74 BTGN 7,813 23.75 BTGN 7,813 23.63 0.49 BTGN 7,813 23.52 BTGN 3,906 24.65 BTGN 3,906 24.55 0.27 BTGN 3,906 24.52 BTGN 1,953 25.37 0.20 Page 35 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN BTGN 1,953 1,953 977 977 977 488 488 488 0 0 0 25.38 25.28 26.15 26.06 26.01 27.10 26.87 26.74 Not Detected Not Detected Not Detected 0.28 0.67 Not Applicable Figure 7, BTGN (Pseudomonas) qPCR Dilution Curves and qPCR Efficiency (E) Limit of Detection The limit of detection (LOD) is used to describe the smallest concentration of a species that can be reliably measured by the Medicinal Genomics (MGC) PathoSEEK® Microbial Safety Testing Solution. This is the point where the qPCR signal crosses the set threshold before a Cq of 40. The genomic copy number was calculated using the sample DNA concentration and the size of the genome for the species in question using the equation: number of copies = X ng*6.0221 x 1023 molecules/mole / (N x 650 g/mole) * 1x 109 ng/g). The following data demonstrates the experiments used to calculate limit of detection when using the PathoSEEK® qPCR Master Kit and Detection Assays for the presence/absence assays. The DNA species found in Table 3, DNA Organisms Evaluated, were used for the data below. Page 36 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 36, LOD STEC E.coli Presence/Absence Assay Assay Copies of STEC O111 DNA Cq Value (FAM) STEC E.coli Specific 5,000 24.16 STEC E.coli Specific 5,000 24.15 STEC E.coli Specific 5,000 24.21 STEC E.coli Specific 2,500 25.11 STEC E.coli Specific 2,500 25.01 STEC E.coli Specific 2,500 25.01 STEC E.coli Specific 1,250 25.95 STEC E.coli Specific 1,250 26.07 STEC E.coli Specific 1,250 25.98 STEC E.coli Specific 625 27.02 STEC E.coli Specific 625 26.92 STEC E.coli Specific 625 26.99 STEC E.coli Specific 313 28.20 STEC E.coli Specific 313 28.08 STEC E.coli Specific 313 28.09 STEC E.coli Specific 156 29.03 STEC E.coli Specific 156 29.09 STEC E.coli Specific 156 29.03 STEC E.coli Specific 78 30.10 STEC E.coli Specific 78 29.95 STEC E.coli Specific 78 30.03 STEC E.coli Specific 39 31.21 STEC E.coli Specific 39 31.34 STEC E.coli Specific 39 31.00 STEC E.coli Specific 20 31.64 STEC E.coli Specific 20 31.52 STEC E.coli Specific 20 31.60 STEC E.coli Specific 10 33.78 STEC E.coli Specific 10 33.08 STEC E.coli Specific 10 32.77 STEC E.coli Specific 5 34.14 STEC E.coli Specific 5 34.54 STEC E.coli Specific 5 33.93 STEC E.coli Specific 2 36.15 STEC E.coli Specific 2 35.54 STEC E.coli Specific 2 36.58 STEC E.coli Specific 0 Not Detected STEC E.coli Specific 0 Not Detected STEC E.coli Specific 0 Not Detected Page 37 866.574.3582 | www.medicinalgenomics.com %RSD 0.13 0.23 0.24 0.19 0.24 0.12 0.25 0.55 0.19 1.56 0.91 1.45 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 8, STEC E.coli qPCR Dilution Curves and qPCR Efficiency (E) Table 37, LOD E.coli Presence/Absence Assay Assay Copies of STEC O111 DNA E.coli Specific 5,000 E.coli Specific 5,000 E.coli Specific 5,000 E.coli Specific 2,500 E.coli Specific 2,500 E.coli Specific 2,500 E.coli Specific 1,250 E.coli Specific 1,250 E.coli Specific 1,250 E.coli Specific 625 E.coli Specific 625 E.coli Specific 625 E.coli Specific 313 E.coli Specific 313 E.coli Specific 313 E.coli Specific 156 E.coli Specific 156 E.coli Specific 156 E.coli Specific 78 E.coli Specific 78 E.coli Specific 78 E.coli Specific 39 E.coli Specific 39 E.coli Specific 39 E.coli Specific 20 E.coli Specific 20 E.coli Specific 20 Page 38 Cq Value (FAM) 26.23 26.00 26.01 26.68 26.63 26.72 27.69 27.66 27.81 28.55 28.73 28.63 29.68 29.99 29.93 30.93 30.81 30.94 31.87 31.74 31.72 32.67 32.38 32.87 33.70 33.78 34.08 866.574.3582 | www.medicinalgenomics.com %RSD 0.50 0.17 0.29 0.31 0.55 0.23 0.26 0.75 0.59 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific E.coli Specific 10 10 10 5 5 5 2 2 2 0 0 0 34.24 34.83 35.03 36.37 35.15 36.24 36.15 36.50 37.74 Not Detected Not Detected Not Detected 1.18 1.87 2.27 Not Applicable Figure 9, E.coli qPCR Dilution Curves and qPCR Efficiency (E) Table 38, LOD Salmonella V2 Presence/Absence Assay Assay Copies of Salmonella DNA Cq Value (FAM) Salmonella Specific 5,000 25.20 Salmonella Specific 5,000 25.13 Salmonella Specific 5,000 25.11 Salmonella Specific 2,500 26.15 Salmonella Specific 2,500 26.06 Salmonella Specific 2,500 26.15 Salmonella Specific 1,250 27.07 Salmonella Specific 1,250 27.07 Salmonella Specific 1,250 27.09 Salmonella Specific 625 28.17 Salmonella Specific 625 28.20 Page 39 866.574.3582 | www.medicinalgenomics.com %RSD 0.20 0.20 0.03 0.07 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Salmonella Specific Page 40 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 28.16 29.33 29.29 29.25 30.38 30.32 30.32 31.67 31.33 31.57 32.32 33.02 32.36 33.65 33.75 33.84 34.76 34.28 35.36 35.15 35.66 35.52 36.59 35.97 37.94 Not Detected Not Detected Not Detected 866.574.3582 | www.medicinalgenomics.com 0.15 0.12 0.57 1.20 0.28 1.56 0.73 2.74 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 10, Salmonella V2 Assay qPCR Dilution Curves and qPCR Efficiency Table 39, LOD STEC E.coli/Salmonella V2 Multiplex Presence/Absence Assay Copies of STEC Cq Value Cq Value Assay and Salmonella STEC E.coli %RSD Salmonella DNA (FAM) (ROX) STEC/Salmonella 5,000 26.61 26.45 STEC/Salmonella 5,000 26.07 1.41 25.80 STEC/Salmonella 5,000 26.78 25.62 STEC/Salmonella 2,500 27.22 26.58 STEC/Salmonella 2,500 27.46 0.44 26.52 STEC/Salmonella 2,500 27.30 26.41 STEC/Salmonella 1,250 28.52 27.63 STEC/Salmonella 1,250 28.41 0.46 27.71 STEC/Salmonella 1,250 28.27 27.65 STEC/Salmonella 625 29.73 28.88 STEC/Salmonella 625 29.57 0.62 28.80 STEC/Salmonella 625 29.36 29.03 STEC/Salmonella 313 31.04 30.06 STEC/Salmonella 313 30.19 1.69 29.48 STEC/Salmonella 313 30.11 29.65 STEC/Salmonella 156 31.53 30.75 STEC/Salmonella 156 31.64 0.48 30.61 STEC/Salmonella 156 31.83 31.26 STEC/Salmonella 78 32.91 31.51 STEC/Salmonella 78 32.62 0.71 32.08 STEC/Salmonella 78 32.45 32.71 STEC/Salmonella 39 33.49 33.32 STEC/Salmonella 39 34.19 1.07 33.43 STEC/Salmonella 39 34.00 33.38 STEC/Salmonella 20 35.01 34.08 0.28 STEC/Salmonella 20 34.90 35.44 Page 41 866.574.3582 | www.medicinalgenomics.com %RSD 1.68 0.33 0.16 0.40 1.00 1.10 1.87 0.17 2.02 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella STEC/Salmonella 20 10 10 10 5 5 5 2 2 2 0 0 0 35.09 35.37 35.65 35.77 37.52 36.93 37.89 Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected 0.58 1.30 Not Applicable Not Applicable 34.49 34.21 34.76 34.70 35.51 36.21 36.64 38.23 ND ND Not Detected Not Detected Not Detected 0.87 1.58 Not Applicable Not Applicable Figure 11, STEC E.coli and Salmonella V2 Multiplex qPCR Dilution Curves Figure 12, STEC E.coli and Salmonella Multiplex V2 qPCR Efficiency Page 42 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 40, LOD E.coli/Salmonella V2 Multiplex Presence/Absence Assay Assay E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella E.coli/Salmonella Page 43 Copies of E.coli and Salmonella DNA 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 Cq Value E.coli (FAM) 25.78 25.76 25.73 26.63 26.70 26.75 27.57 27.67 27.62 28.66 28.78 28.93 29.63 29.64 29.78 30.72 30.65 30.76 31.65 31.78 32.04 32.85 32.88 33.12 33.68 33.44 34.19 34.58 34.38 34.58 35.46 36.92 36.06 38.04 37.02 36.02 ND ND ND %RSD 0.10 0.22 0.18 0.47 0.28 0.18 0.62 0.44 1.14 0.32 2.03 2.72 Not Applicable 866.574.3582 | www.medicinalgenomics.com Cq Value Salmonella (ROX) 25.22 24.54 25.52 26.03 25.70 25.81 27.03 27.23 27.06 28.23 28.18 28.08 29.03 29.14 28.93 30.16 30.30 30.35 31.06 31.63 31.57 33.09 32.62 32.55 33.47 33.73 35.01 34.17 35.09 34.10 36.44 35.15 37.27 36.04 35.84 39.07 Not Detected Not Detected Not Detected %RSD 2.02 0.66 0.40 0.27 0.35 0.32 0.98 0.90 2.42 1.60 2.94 4.90 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 13, E.coli and Salmonella V2 Multiplex qPCR Dilution Curves Figure 14, E.coli and Salmonella V2 Multiplex qPCR Efficiency Table 41, LOD A. flavus Presence/Absence Assay Assay Copies of Aspergillus flavus DNA A. flavus specific 5,000 A. flavus specific 5,000 A. flavus specific 5,000 A. flavus specific 2,500 A. flavus specific 2,500 A. flavus specific 2,500 A. flavus specific 1,250 A. flavus specific 1,250 A. flavus specific 1,250 A. flavus specific 625 A. flavus specific 625 A. flavus specific 625 A. flavus specific 313 A. flavus specific 313 A. flavus specific 313 Page 44 Cq Value (FAM) 19.28 19.15 19.20 20.21 20.09 20.26 21.01 21.09 21.26 22.33 22.39 22.25 23.49 23.45 23.44 866.574.3582 | www.medicinalgenomics.com %RSD 0.34 0.43 0.60 0.31 0.11 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific A. flavus specific 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 24.55 24.50 24.45 25.60 25.61 25.58 26.56 26.60 26.60 27.67 27.68 27.78 28.58 28.65 28.25 29.77 30.15 30.14 31.12 31.10 31.17 Not Detected Not Detected Not Detected 0.20 0.06 0.09 0.22 0.75 0.72 0.12 Not Applicable Figure 15, Aspergillus flavus qPCR Dilution Curves and qPCR Efficiency Table 42, LOD A. flavus Presence/Absence Assay Multiplex Assay Copies of Aspergillus flavus DNA Cq Value (FAM) A. flavus Multiplex 5,000 19.01 A. flavus Multiplex 5,000 19.14 A. flavus Multiplex 5,000 19.23 A. flavus Multiplex 2,500 20.06 Page 45 866.574.3582 | www.medicinalgenomics.com %RSD 0.58 0.73 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex A. flavus Multiplex Page 46 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 20.12 20.34 21.05 20.98 21.13 22.19 22.10 22.29 22.95 23.10 23.11 24.17 24.03 23.91 25.24 25.26 25.24 26.40 26.20 26.46 27.33 27.35 27.40 28.22 28.26 28.25 29.39 29.39 29.60 30.57 31.62 31.10 Not Detected Not Detected Not Detected 866.574.3582 | www.medicinalgenomics.com 0.36 0.43 0.39 0.54 0.05 0.52 0.13 0.07 0.41 1.69 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 16, Aspergillus flavus Multiplex qPCR Dilution Curves and qPCR Efficiency Table 43, LOD A. fumigatus Presence/Absence Assay Copies of Aspergillus Assay Cq Value (FAM) fumigatus DNA A. fumigatus specific 5,000 21.30 A. fumigatus specific 5,000 21.17 A. fumigatus specific 5,000 21.33 A. fumigatus specific 2,500 22.36 A. fumigatus specific 2,500 22.19 A. fumigatus specific 2,500 22.17 A. fumigatus specific 1,250 23.30 A. fumigatus specific 1,250 23.20 A. fumigatus specific 1,250 23.45 A. fumigatus specific 625 24.19 A. fumigatus specific 625 24.19 A. fumigatus specific 625 24.18 A. fumigatus specific 313 25.27 A. fumigatus specific 313 25.14 A. fumigatus specific 313 25.57 A. fumigatus specific 156 26.32 A. fumigatus specific 156 26.43 A. fumigatus specific 156 26.17 A. fumigatus specific 78 27.24 A. fumigatus specific 78 27.29 A. fumigatus specific 78 27.26 A. fumigatus specific 39 28.17 A. fumigatus specific 39 28.26 A. fumigatus specific 39 28.16 A. fumigatus specific 20 29.22 A. fumigatus specific 20 29.17 A. fumigatus specific 20 29.12 A. fumigatus specific 10 30.28 Page 47 866.574.3582 | www.medicinalgenomics.com %RSD 0.40 0.47 0.54 0.02 0.87 0.50 0.09 0.20 0.17 0.43 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific A. fumigatus specific 10 10 5 5 5 2 2 2 0 0 0 30.34 30.09 31.41 31.19 31.37 32.14 32.15 32.04 Not Detected Not Detected Not Detected 0.37 0.19 Not Applicable Figure 17, Aspergillus fumigatus qPCR Dilution Curves and qPCR Efficiency Table 44, LOD A. fumigatus Presence/Absence Multiplex Assay Copies of Aspergillus Assay Cq Value (FAM) fumigatus DNA A. fumigatus Multiplex 5,000 21.05 A. fumigatus Multiplex 5,000 21.77 A. fumigatus Multiplex 5,000 20.93 A. fumigatus Multiplex 2,500 22.01 A. fumigatus Multiplex 2,500 22.16 A. fumigatus Multiplex 2,500 22.96 A. fumigatus Multiplex 1,250 23.02 A. fumigatus Multiplex 1,250 23.16 A. fumigatus Multiplex 1,250 23.49 A. fumigatus Multiplex 625 24.14 A. fumigatus Multiplex 625 23.93 A. fumigatus Multiplex 625 24.00 A. fumigatus Multiplex 313 25.16 A. fumigatus Multiplex 313 25.08 A. fumigatus Multiplex 313 24.89 A. fumigatus Multiplex 156 25.75 A. fumigatus Multiplex 156 25.93 Page 48 866.574.3582 | www.medicinalgenomics.com %RSD 2.14 2.28 1.04 0.45 0.55 0.35 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex A. fumigatus Multiplex 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 25.84 27.08 26.99 26.80 27.84 27.98 27.90 28.83 29.03 28.86 30.00 30.08 29.72 30.73 30.98 31.01 31.84 31.90 31.99 Not Detected Not Detected Not Detected 0.53 0.25 0.37 0.63 0.50 0.24 Not Applicable Figure 18, Aspergillus fumigatus Multiplex qPCR Dilution Curves and qPCR Efficiency Table 45, LOD A. niger Presence/Absence Assay Copies of Aspergillus niger Assay DNA Page 49 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com %RSD version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific A. niger specific Page 50 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 26.04 26.10 26.01 27.11 27.06 27.08 28.17 28.16 28.19 29.26 29.35 29.35 30.36 30.35 30.35 31.47 31.43 31.38 32.47 32.64 32.49 33.65 34.10 33.55 34.62 34.29 34.60 35.92 37.25 36.29 36.00 36.48 36.21 37.90 37.98 37.80 Not Detected Not Detected Not Detected 866.574.3582 | www.medicinalgenomics.com 0.18 0.09 0.05 0.18 0.02 0.14 0.29 0.87 0.54 1.88 0.66 0.24 Not Applicable version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 19, Aspergillus niger qPCR Dilution Curves and qPCR Efficiency Table 46, LOD A. niger Presence/Absence Multiplex Assay Copies of Aspergillus niger Assay Cq Value (FAM) DNA A. niger Multiplex 5,000 25.61 A. niger Multiplex 5,000 25.38 A. niger Multiplex 5,000 25.60 A. niger Multiplex 2,500 26.91 A. niger Multiplex 2,500 26.88 A. niger Multiplex 2,500 27.08 A. niger Multiplex 1,250 28.02 A. niger Multiplex 1,250 28.03 A. niger Multiplex 1,250 27.70 A. niger Multiplex 625 28.35 A. niger Multiplex 625 28.27 A. niger Multiplex 625 28.37 A. niger Multiplex 313 29.25 A. niger Multiplex 313 29.44 A. niger Multiplex 313 29.34 A. niger Multiplex 156 30.75 A. niger Multiplex 156 31.14 A. niger Multiplex 156 31.17 A. niger Multiplex 78 31.48 A. niger Multiplex 78 31.68 A. niger Multiplex 78 31.47 A. niger Multiplex 39 32.48 Page 51 866.574.3582 | www.medicinalgenomics.com %RSD 0.51 0.40 0.67 0.19 0.32 0.76 0.38 0.18 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex A. niger Multiplex 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 32.59 32.50 34.33 33.41 34.13 34.70 Not Detected 35.57 37.80 35.59 Not Detected Not Detected Not Detected 37.32 Not Detected Not Detected Not Detected 1.42 1.75 4.26 Not Applicable Not Applicable Figure 20, Aspergillus niger Multiplex qPCR Dilution Curves and qPCR Efficiency Table 47, LOD A. terreus Presence/Absence Assay Copies of Aspergillus Assay terreus DNA A. terreus specific 5,000 A. terreus specific 5,000 A. terreus specific 5,000 A. terreus specific 2,500 A. terreus specific 2,500 A. terreus specific 2,500 A. terreus specific 1,250 A. terreus specific 1,250 Page 52 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 25.46 25.58 25.57 26.93 26.64 26.79 27.92 27.66 %RSD 0.26 0.54 0.48 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific A. terreus specific 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 27.85 29.06 28.91 28.82 29.83 29.81 30.06 31.26 31.12 31.30 31.87 32.05 31.91 33.27 33.72 33.48 34.75 34.32 34.35 35.10 35.17 36.13 36.07 37.48 38.27 37.04 36.80 36.86 Not Detected Not Detected Not Detected 0.42 0.46 0.30 0.30 0.67 0.70 1.62 2.99 0.34 Not Applicable Figures 21, Aspergillus terreus qPCR Dilution Curves and qPCR Efficiency Page 53 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 48, LOD A. niger Presence/Absence Multiplex Assay Assay A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex Page 54 Copies of Aspergillus terreus DNA 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 25.51 25.67 25.92 26.67 26.47 26.59 27.62 27.49 27.85 28.72 28.73 28.60 30.06 29.93 30.09 31.03 30.93 30.99 32.27 32.14 32.09 34.43 33.75 33.92 34.36 34.11 34.37 35.44 36.02 %RSD 0.80 0.38 0.66 0.25 0.28 0.16 0.29 1.04 0.43 1.19 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex A. terreus Multiplex 10 5 5 5 2 2 2 0 0 0 35.20 ND 38.25 ND 36.85 ND ND Not Detected Not Detected Not Detected Not Applicable Not Applicable Not Applicable Figures 22, Aspergillus terreus Multiplex qPCR Dilution Curves and qPCR Efficiency Table 49, LOD Pseudomonas aeruginosa Presence/Absence Assay Assay P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific Page 55 Copies of Pseudomonas aeruginosa DNA 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 24.10 24.07 24.12 24.85 24.81 24.81 25.88 25.84 25.85 26.96 27.06 26.98 28.06 27.94 27.97 28.86 %RSD 0.09 0.10 0.09 0.20 0.21 0.25 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific P. aeruginosa specific 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 28.72 28.78 29.51 29.76 29.39 30.33 30.41 30.64 31.68 31.44 31.48 32.79 32.83 32.90 33.51 33.42 33.89 34.42 34.30 34.61 Not Detected Not Detected Not Detected 0.64 0.54 0.41 0.18 0.75 0.46 Not Applicable Figures 23, Pseudomonas aeruginosa qPCR Dilution Curves and qPCR Efficiency Table 50, LOD Pseudomonas aeruginosa Presence/Absence Multiplex Assay Assay P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex Page 56 Copies of Pseudomonas aeruginosa DNA 5,000 5,000 5,000 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 24.32 24.10 24.16 %RSD 0.45 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex P. aeruginosa multiplex 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 25.10 25.13 25.10 26.15 26.13 26.21 27.25 27.16 27.23 28.14 28.16 28.17 28.76 29.10 28.98 29.62 29.76 29.75 30.84 30.75 30.47 31.97 31.75 31.95 33.01 32.79 32.86 33.44 32.89 33.66 36.48 35.57 34.73 Not Detected Not Detected Not Detected 0.07 0.17 0.16 0.06 0.60 0.27 0.64 0.38 0.34 1.19 2.45 Not Applicable Figures 24, P. aeruginosa Multiplex qPCR Dilution Curves and qPCR Efficiency Page 57 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 51, LOD Staphylococcus aureus Presence/Absence Assay Assay Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Page 58 Copies of Staphyloccus aureus DNA 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 313 313 313 156 156 156 78 78 78 39 39 39 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 26.31 26.33 26.15 27.33 26.53 26.62 27.81 27.64 27.72 28.94 28.86 28.74 29.77 30.08 29.85 31.33 31.12 31.00 32.11 32.13 32.18 33.02 32.88 33.10 %RSD 0.38 1.64 0.30 0.35 0.54 0.53 0.12 0.34 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific Staph aureus specific 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 34.19 34.17 34.18 35.16 35.17 35.13 35.68 37.13 35.37 37.87 36.52 37.40 Not Detected Not Detected Not Detected 0.03 0.06 2.60 1.84 Not Applicable Figures 25, Staphylococcus aureus qPCR Dilution Curves and qPCR Efficiency Table 52, LOD Staphylococcus aureus Multiplex Presence/Absence Assay Assay Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Page 59 Copies of Staphyloccus aureus DNA 5,000 5,000 5,000 2,500 2,500 2,500 1,250 1,250 1,250 625 625 625 Cq Value (FAM) 866.574.3582 | www.medicinalgenomics.com 26.08 26.29 26.03 26.54 26.47 26.62 28.64 28.29 28.05 28.67 28.68 28.59 %RSD 0.54 0.28 1.03 0.18 version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex Staph aureus multiplex 313 313 313 156 156 156 78 78 78 39 39 39 20 20 20 10 10 10 5 5 5 2 2 2 0 0 0 30.10 29.84 29.78 31.05 30.53 30.50 31.32 31.86 31.87 32.81 32.56 32.86 36.67 33.63 33.67 37.84 35.05 35.09 38.60 35.98 34.96 Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected 0.57 1.00 1.00 0.48 5.03 4.44 5.13 Not Applicable Not Applicable Figures 26, Staph aureus Multiplex qPCR Dilution Curves and qPCR Efficiency Table 53, Summary of qPCR Assay Efficiency Page 60 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Assay Total Yeast and Mold (FAM) Total Aerobic Bacteria (FAM) Total Enterobacteriaceae (FAM) Total Coliform (FAM) BTGN + Coliform (FAM) with Klebsiella pneumoniae BTGN + Coliform (FAM) with Pseudomonas aeruginosa BTGN + Coliform (ROX) with Klebsiella pneumoniae STEC E.coli specific (FAM) E.coli specific (FAM) Salmonella specific (FAM) STEC E.coli/Salmonella multiplex STEC E.coli Target (FAM) STEC E.coli/Salmonella multiplex Salmonella Target (ROX) E.coli/Salmonella multiplex E.coli Target (FAM) E.coli/Salmonella multiplex Salmonella Target (ROX) A. flavus specific (FAM) A. flavus multiplex (FAM) A. fumigatus specific (FAM) A. fumigatus multiplex (FAM) A. niger specific (FAM) A. niger multiplex (FAM) A. terreus specific (FAM) A. terreus multiplex (FAM) Pseudomonas aeruginosa specific (FAM) Pseudomonas aeruginosa multiplex (FAM) Staphylococcus aureus specific (FAM) Staphylococcus aureus multiplex (FAM) Efficiency (E) 84.4% 88.8% 88.8% 82.3% 92.2% 109.3% 85.1% 94.8% 100.4% 103.4% 96.1% 93.1% 92.5% 92.1% 90.1% 92.6% 101.0% 105.6% 92.5% 92.5% 88.7% 84.6% 106.9% 103.9% 96.7% 93.6% R2 0.999 0.999 0.998 0.999 0.998 0.997 0.999 0.990 0.992 0.980 0.993 0.995 0.993 0.986 0.998 0.996 0.999 0.995 0.997 0.982 0.986 0.985 0.997 0.987 0.991 0.970 Discussion: The LOD for the above presence/absence assays were all detected to less then or equal to 10 copies of the genome. The MGC presence/absence assays target specific species that are potentially harmful to humans. In an effort to ensure that the smallest quantities of an organism can be detected, MGC requires 16-24 hour enrichment for E.coli, STEC E.coli and Salmonella testing. For the slower growing Aspergillus species, MGC recommends a minimum 24-hour enrichment. Page 61 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Appendix I: Aspergillus niger spore lysis and growth Aspergillus niger spores were harvested in 1% Tween-80. Thorough vortexing and filtration through a 5um spin column provided individual, de-clumped spores (Figure 27). Once spores were de-clumped they are easy to count. It is important to demonstrate that the MGC lysis buffer can in fact lyse the Aspergillus spores. This was tested by adding MGC lysis buffer to filtered spores vs. not adding MGC lysis buffer to filtered spores. The MGC SenSATIVAx® DNA Extraction Kits protocol was followed and the resulting eluant was tested using the PathoSEEK® qPCR Master Kit and PathoSEEK® Aspergillus Multiplex and Aspergillus niger Detection Assays. That results in Table 54 show that the MGC lysis buffer does lyse Aspergillus niger spores and that if lysis buffer is not added the Aspergillus niger spores remain intact and fail to generate a qPCR result. Figure 27, Aspergillus niger spores unfiltered vs. filtered Table 54, Lysis of Aspergillus niger spores with and without MGC Lysis Buffer Sample A. niger filtered spores A. niger filtered spores A. niger filtered spores A. niger filtered spores MGC Lysis Buffer Added No No Yes Yes Target Assay Aspergillus Multiplex A. niger Aspergillus Multiplex A. niger Cq Value (FAM) Not Detected Not Detected 25.86 26.52 The microbial regulation specific for Aspergillus flavus, fumigatus, niger, and terreus state that none be detected in 1g of sample. In an effort to ensure that the smallest quantities of an organism can be detected, MGC recommends a minimum 24-hour enrichment at 37°C in Tryptic Soy Broth (TSB) for the detection of species specific Aspergillus. An experiment was designed to test the growth of Aspergillus niger spores in TSB vs. Potato Dextrose Broth (PDB) at 37°C. 3.54mL of TSB + 10uL of Aspergillus niger spores (20 spores/uL). The samples was mixed well by vortexing. 285uL was aliquoted into 5 1.5mL tubes and incubated at 37°C. The tubes were labeled 16hrs, 18hrs, 20hrs, 22hrs, and 24hrs. Each tube was then lysed after the specified incubation Page 62 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation time and prepped using the MGC SenSATIVAx® DNA Extraction Kit. The same experimental setup was done using Potato Dextrose Broth. The MGC minimum incubation time of 24 hours at 37°C is adequate for the growth of Aspergillus niger spores both in TSB and PDB, the results can be found in Table 55. Table 55, Growth of Aspergillus niger spores Growth Media TSB TSB PDB PDB Page 63 Assay Tested Aspergillus Multiplex Aspergillus niger Aspergillus Multiplex Aspergillus niger Hours Incubated at 37°C & Cq Values (FAM) 16 18 20 22 24 35.82 30.52 26.88 27.05 26.82 31.75 29.72 27.75 27.84 27.44 30.13 33.98 28.01 27.03 27.55 31.08 31.71 28.31 27.90 27.23 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Appendix II: Cq to CFU Conversion State regulations for microbial thresholds currently use nomenclature from culturebased methods to set acceptable thresholds. With this in mind, it was necessary for Medicinal Genomics to convert the quantification cycle (Cq) value from the PathoSEEK® qPCR Assay to colony forming units (CFU), which is reported when using culture-based methods. This appendix demonstrates the initial development of the equations used to calculate Cq to CFU when using the SenSATIVAx® DNA Extraction Kits and the PathoSEEK® qPCR Detection Assays. Methods: S. cerevisiae culture was used to determine the Cq to CFU conversion equation for the Total Yeast and Mold assay. DH10B E. coli culture was used to determine the Cq to CFU conversion equation for the Total Aerobic Count, Total Coliform, and Total Enterobacteriaceae assays. The analysis and testing was completed using a serial dilution of 1:10 for each species for each of the four detection assays. Due to the limited dynamic range of Petri dishes and culture-based methods, additional intermediate dilutions are required to measure the colony forming units (CFU); 3 intermediate points at 1:5000 from the 1:10 dilution, 1:500 from the 1:100, and 1:50 from the 1:1000. The quantification cycle (Cq) data for each sample was plotted against the CFU from 3M® PetriFilm™ plating. For MIP/extract, initial studies showed gummy behaved differently compared to other MIP samples. Therefore, a separate validation study was performed for gummy only. The summary of the data for the verification is shown on Figures 28-30 for Plant, Extract and Gummy respectively. Page 64 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 28, Cq vs. Log CFU, Plant: Figure 29, Cq vs. LogCFU, Hemp Oil Extract: Page 65 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Figure 30, Cq vs. LogCFU, Gummy: Page 66 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Cq to CFU/g Equations: From the data in the above tables, a line of best fit was created. The coefficient of determination, or R2, for the line of best fit for all detection assays is above 92%. The derived equations can be found below or within the protocols for MGC PathoSEEK® Data Analysis for Agilent Real-Time PCR System or MGC PathoSEEK Data Analysis for BIO-RAD CFX96 Touch Real-Time PCR Detection System. Please reference most up to date SOPs at https://www.medicinalgenomics.com/product-literature/ for most up to date conversion equations. Table 56, Cq to CFU/g Equations for Flower, MIP/Extract and Gummy Matrix Plant Microbial Test Cq to CFU/g Conversion Equation Total Yeast and Mold CFU/g = 10[(36.671 – Cq Value)/3.1194] Plant Total Aerobic Count CFU/g = 10[(35.111 – Cq Value)/2.8883] Plant Total Coliform CFU/g = 10[(40.073 – Cq Value)/3.3417] Plant Total Enterobacteriaceae CFU/g = 10[(41.218 – Cq Value)/4.3708] MIP/Extract Total Yeast and Mold CFU/g = 10[(54.972 – Cq Value)/5.8485] MIP/Extract Total Aerobic Count CFU/g = 10[(38.076 – Cq Value)/3.2249] MIP/Extract Total Coliform CFU/g = 10[(41.935 – Cq Value)/3.6274] MIP/Extract Total Enterobacteriaceae CFU/g = 10[(38.407 – Cq Value)/3.3041] Gummy Total Yeast and Mold CFU/g = 10[(52.989 – Cq Value)/4.9718] Gummy Total Aerobic Count CFU/g = 10[(37.235 – Cq Value)/2.356] Gummy Total Coliform CFU/g = 10[(52.888 – Cq Value)/5.9643] Gummy Total Enterobacteriaceae CFU/g = 10[(44.81 – Cq Value)/4.9665] Discussion: Based on these findings, the Cq to CFU equations developed by Medicinal Genomics Company (MGC) and provided to its partner laboratories, meet all internal specifications and are approved for laboratory use. Any deviations from this protocol are not supported by MGC. The results may vary based on laboratory conditions. For example, altitude and humidity are known to affect the growth of bacterial and fungal species. All thresholds and equations were determined based on the results from the BIO-RAD CFX96 Touch™ Real-Time PCR Detection System and verified on Agilent AriaMX Real Time PCR System. When using a different qPCR machine ramp rates and temperature thresholds can alter the values and thus alter the equation. Page 67 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Appendix III: Additional Data supporting Cq to CFU/g conversions Colony forming units (CFU) are reported when using the 3M™ Rapid Yeast and Mold PetriFilm™ product. The Cq to CFU equation was tested and compared to plating using multiple yeast and mold species listed below. The following species in Table 58 were tested on both 3M™ Rapid Yeast and Mold PetriFilm™ and PathoSEEK Yeast and Mold Detection Assay. The data in this appendix included the decontamination step that precedes the qPCR set-up step of the PathoSEEK workflow. Table 58, Species Evaluated in Cq to CFU comparison experiments: Yeast & Mold Species Aspergillus flavus Aspergillus janponicus Candida albicans Candida catenulate Candida glabralta Candida krusei Candida sphaerica Debaryomyces hanseii Rhodotorula mucilaginosa Trichothecium Roseum Yarrowia lipolytica ATCC # 16870 16873 10231 10565 15545 28870 8565 10623 4557 90473 18944 Organisms were reconstituted and incubated according to the ATCC product documentation. Cultures of ATCC strains were then grown in 5mL Tryptic Soy Broth (TSB) for 5 days at room temperature and checked visually for turbidity. TSB was chosen because it is a general-purpose liquid enrichment medium that has minimum interference with the MGC SenSATIVAx DNA Extraction Kits. The broth recommended by 3M is Butterfields phosphate buffered dilution water. The phosphates in this liquid medium interfere with the magnetic particles in the MGC SenSATIVAx DNA Extraction Kits. 3M states, “Do not use diluents containing citrate, bisulfite or thiosulfate with the 3M PetriFilmTM Coliform Count Plates; they can inhibit growth.” TSB does not contain any of the above ingredients. The undiluted culture, 1:10, 1:100, and 1:1000 dilutions were plated in triplicate on 3M™ PetriFilmTM Rapid Yeast and Mold Count Plates, incubated at room temperature, and counted after 3–5 days. An aliquot of the undiluted culture was extracted using the MGC SenSATIVAx DNA Extraction Kits. The DNA was then tested using PathoSEEK Yeast and Mold detection assay. The Cq value obtained from the qPCR results was plugged into the Cq to CFU/g equation and compared to the CFU obtained from the 3M™ PetriFilm™. The summary of the validation samples is shown on Table 59 below. A graphical representation of Table 59 is shown in Figure 31. Table 59, Validation Data: Page 68 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation PathoSEEK® Cq Figure 31: Cq vs. CFU Concordance Discussion: The concordance was measured between CFU derived from the PathoSEEK qPCR Cq and 3M™ PetriFilm™ CFU pictured in Figure 31. The Aspergillus species CFU counts are approximately three orders of magnitude lower than expected based on Cq estimates that were developed and optimized by plating cultured cells of other species. Excluding the two Aspergillus species and Candida glabralta, the correlation between CFU/g and Cq is greater than or equal to 0.71. The correlation between CFU per gram of plant material and Cq is 0.99 for all fungal species except Candida glabralta.1 Aspergillus has been proven to grow poorly in culture media, and is therefore severely under-reported by current culture-based platforms. This phenomenon has been published in two peer reviewed papers3,4. The differential growth of other toxigenic fungi, depending on the companion species present, further influences the results. Bacterial pathogens are not uncommon, and beneficial bacteria are also capable of influencing the growth or inhibition of other flora. This document and the associated peer reviewed papers1,2 demonstrate that the verification samples processed using Page 69 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation PathoSEEK Total Yeast and Mold Detection Assay meet the standards set forth in the PathoSEEK Cq to CFU Equations document. MIP/Extract Validation Samples: The following species in Table 60 were tested on both 3M™ Rapid Yeast and Mold, Aerobic Count, Enterobacteriaceae Count and Coliform Count Petrifilm™ and PathoSEEK Total Yeast and Mold Detection Assay. Table 60, Species Evaluated: Species S. cerevisiae E.coli Manufacturer The Gold Pitch, Giga Yeast, Inc. DH10B (New England Biolabs, #C3020K) E.coli cultures were grown in 20 mL Tryptic Soy Broth for 24 hours at 37°C and checked visually for turbidity. S.cerevisiae was not grown further in TSB. A 1:100 dilution of the species listed above was spiked into a hemp extract (Elixinol LLC) for testing the four detection assays. Due to the limited dynamic range of Petri films and culture-based methods, an additional intermediate dilution was required to measure the colony forming unit (CFU); a 1:500 dilution was made from the 1:100 dilution and was used for 3M™ PetriFilm™ plating. An aliquot of 1:100 dilutions was extracted using the MGC SenSATIVAx MIP/Extract DNA Extraction Kit. The DNA was then tested using PathoSEEK Yeast and Mold, Total Aerobic Count, Total Coliform and Total Entero detection assays. The Cq value obtained from the 1:100 dilution qPCR results was plugged into the equations found in Table 54 and compared to the CFU obtained from the 3M™ PetriFilm™. The summary of the validation samples is shown below on Table 61. Page 70 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Table 61, Cq vs CFU, Extract: PathoSEEK® Cq The concordance was measured between CFU derived from the PathoSEEK qPCR Cq and 3M™ PetriFilm™ CFU pictured in Figure 32. Figure 32: Extract Cq vs. CFU Concordance Page 71 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Appendix IV: Grim Reefer Free DNA Removal kit The Grim Reefer Free DNA Removal Kit eliminates free DNA from samples prior to qPCR analysis. The Grim Reefer enzyme is used to ensure that DNA from dead organisms does not inflate quantitative results from PathoSEEK® Microbial Safety Testing Assays. The Grim Reefer Process The Grim Reefer buffer and enzyme are added to the sample and incubated at 37C for 10minutes to degrade any dead or free DNA. After incubation, MGC lysis buffer is added to the sample and mixed well. The lysis buffer inactivates the Grim Reefer Enzyme while also lysing the viable cells. After the lysis step, Grim Reefer Positive Control is added, which contains a known amount of free DNA, to ensure that the enzyme has been deactivated. If the Grim Reefer Positive Control appears in the qPCR results, you know the Grim Reefer Enzyme was properly deactivated. Testing the method A hemp sample was inoculated with cultured E. coli in TSB. The resulting TSB was split into 12 tubes. The 12 tubes were split into two sets of 6 tubes. One set of 6 tubes was treated with Grim Reefer and the other 6 tubes were not treated with Grim Reefer. For each set of tubes, salmonella DNA was spiked in at 6 different levels (n=1). Each of the 12 samples was aliquoted into 3 separate wells for lysis, purification, primer/probe addition and thermo cycling (n=3). 1 = 402,000 copies 2 = 804,000 copies 3 = 2,010,000 copies 4 = 4,020,000 copies 5 = 6.030,000 copies 6 = 8,040,000 copies Page 72 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Results for E. coli detection Results for Salmonella detection Results for Grim Reefer Positive Control Page 73 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Results Internal Hemp/Cannabis Control Conclusions There is no statistically significant difference between the E. coli results of the Grim Reefer Method (average Cq = 29.60) and the Original Method (average Cq = 29.9) P-value = 0.0618 > 0.05. The result is consistent with the hypothesis that the Grim Reefer method does not interfere with DNA from live cells. The Salmonella results show that Grim Reefer method can consistently (n=3) deactivate 4.02 million copies of free DNA or less. The results also show that the Grim Reefer method is exclusive of free DNA as long as free DNA ≤ 4.02 million copies. A 10 fold dilution is represented by a 3.3 Ct shift. For the 6.03 million copies there was an approximate 12.93 Ct shift which is about a 7,500 fold reduction. The Grim Reefer positive control results show that there is no statistically significant difference between the Grim Reefer method (average Cq = 27.18) and the original method (average Cq = 27.42) P-value = 0.161 > 0.05. The result is consistent with the hypothesis that the Grim Reefer enzyme is no longer digesting free DNA after incubation at 37C for 10 minutes. The internal hemp/cannabis control results show that there is statistically significant less hemp DNA detected with the Grim Reefer method (average Cq = 28.80) than the Original Method (average Cq = 24.70) P-value = 2.45 x 10-25. This result is consistent with the hypothesis that only hemp DNA inside viable cells is detected with the Grim Reefer method. Page 74 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation Conclusion: Medicinal Genomics SenSATIVAx® Plant/Microbial DNA Extraction Kit, SenSATIVAx® MIP/Extract DNA Extraction Kit in conjunction with the PathoSEEK® Microbial Safety Testing Solution underwent an extensive validation using both live microorganisms and DNA from the same microorganisms. MGC demonstrated that the SenSATIVAx® Plant/Microbial DNA Purification Kit and the SenSATIVAx® MIP/Extract DNA Extraction Kit were able to extract the DNA both accurately and precisely. The specificity of the PathoSEEK® Microbial Safety Testing Solution was tested on over 140 microorganisms with 100% concordance. The linearity of the PathoSEEK® Microbial Safety Testing Solution was assessed for each assay twelve serial dilutions and the resulting R2 values show linearity. The limit of detection for the presence/absence PathoSEEK® Microbial Safety Testing Solution were all detected to less then or equal to 10 copies of the genome. This is a sufficient LOD for methods that employ enrichment. Enrichment incubation should entail at least 4 doublings for 10 copies. LOD copy number = 2^N ; where N is the cell doubling in a given broth/temperature). This validation document demonstrates that the Medicinal Genomics SenSATIVAx® Plant/Microbial DNA Extraction Kit, SenSATIVAx® MIP/Extract DNA Extraction Kit in conjunction with the PathoSEEK® Microbial Safety Testing Solution meet the validation pillars of accuracy, precision, specificity, linearity and limit of detection. Page 75 866.574.3582 | www.medicinalgenomics.com version 31.05202020 SenSATIVAx® and PathoSEEK® Manufacturer Validation References: 1. 2. 3. 4. 5. 6. 7. 8. Page 76 McKernan K, Spangler J, Helbert Y, Lynch RC, Devitt-Lee A, Zhang L, et al. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests. F1000Research. 2016;5:2471. PubMed PMID: 27853518. Pubmed Central PMCID: 5089129. McKernan K, Spangler J, Zhang L, Tadigotla V, Helbert Y, Foss T, et al. Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers. F1000Research. 2015;4:1422. PubMed PMID: 27303623. Pubmed Central PMCID: 4897766. de Bekker C, vanVeluw GJ, Vinck A, Wiebenga LA, Wösten HA. Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures. Appl Environ Microbiol. 2011 Feb;77(4):1263-7. doi: 10.1128/AEM.02134-10. Epub 2010 Dec 17. vanVlew GJ, Teertstra WR, de Bekker C, et al. Heterogeneity in liquid shaken cultures of Aspergillus niger inoculated with melanised conidia or conidia of pigmentation mutants. Stud Mycol. 2013 Mar 15; 74(1): 47–57. Published online 2012 Sep 14. doi: 10.3114/sim0008 S.Broeders, I Huber, L. Grohmann, I. Taverniers et al. Guidelines for validation of qualitative real-time PCR methods. Trends in Food Science & Technology. Volume 37, Issue 2, June 2014. Geiser DM, Dorner JW, Horn BW, Taylor JW. The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae. Fungal Genetics and Biology. Volume 31, Issue 3, December 2000. Akiyama K, Takizawa H, Suzuki M, Miyachi S, Ichinohe M, Yanagihara Y. Allergic Bronchopulmonary Aspergillosis due to Aspergillus oryzae. Chest. Volume 91, Issue 2, February 1987. Stenson S, Brookner A, Rosenthal S. Nilateral Endogenous Necrotizing Scleritis due to Aspergillus oryzae. Annals of Ophthalmology. 1982 Jan;14(1):67-72. 866.574.3582 | www.medicinalgenomics.com version 31.05202020

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