Page 1 of 12 Please refer to http://www.medicinalgenomics.com/product-literature/ for updated protocols and Material Safety Data Sheets (MSDS). Consult MSDS before using any new product. PATHOGINDICATOR is a registered trademark of Medicinal Genomics Corporation, and is for laboratory use only. Table of Contents Introduction... 1 Process Overview... 2 Kit Specifications... 3 Materials Supplied in the Kit... 3 Materials Supplied by the User... 4 Consumables & Hardware... 4 Reagents... 4 Decontamination Protocol (Optional Step)... 5 Real-Time Quantitative PCR (qpcr) Protocol... 8 Glossary and Definitions... 12 LIMITED USE LABEL LICENSE... 12 Introduction PathogINDICAtor utilizes a novel PCR based assay that is contamination-free and provides an internal plant DNA control for every reaction. It is a simple two-step protocol, which is flexible and automation compatible. PathogINDICAtor microbial detection assays use a multiplexing strategy with an internal plant DNA reaction control to ensure accurate detection of microbial species for every reaction. Unlike other techniques, this multiplexing strategy verifies the performance of the assay when detecting pathogens, resulting in the minimization of false negatives due to reaction set-up errors or failing experimental conditions.
Page 2 of 12 Process Overview 1. Decontamination Step, OPTIONAL: Use of restriction enzyme to digest the potential contaminant amplicon DNA from a previous qpcr. For more detail on this method see http://www.ncbi.nlm.nih.gov/pmc/articles/pmc4008621/ 2. Real-time quantitative PCR (qpcr) using a multiplex system of primers to detect potential pathogens within the plant material sample. Below is a simplified depiction of the qpcr assays. The forward and reverse primers have universal primer tails to enable potential Next Generation Sequencing of resulting products. Step%1:% Primers%and%probe%% bind%to%target%dna.%% Forward Primer Probe = Fluorophore = Quencher Reverse Primer Step%2:% PCR%occurs,%primers%are% extended%on%forward%and% reverse%dna%strands.%% Polymerization Probe Degradation Step%3:% Probe%is%degraded%as%a%result% of%polymerizaeon%and% fluorescent%signal%is% generated.%% Step%4:% Target%DNA%is%amplified%and% fluorescent%signal%can%be% measured%and%quanefied%.%% Result +" PCR Amplified DNA Fluorescent Signal
Page 3 of 12 Kit Specifications The qpcr Master Kit contains 125 reactions (Medicinal Genomics # 420002). Each PathogINDICAtor Detection Assay Probe Mix contains 200 reactions worth of reagents. Each PathogINDICAtor Positive Control contains 60 reactions worth of reagents. Materials Supplied in the Kit qpcr Master Kit, store at -15 to -20 o C upon arrival [Medicinal Genomics #420002]. Decontamination Buffer (10x) Decontamination Enzyme (10 Units/µL) qpcr Master Mix (5x) Nuclease Free Water PathogINDICAtor Detection Assays and Positive Controls, ordered separately, store at -15 o C to -20 o C upon arrival. Salmonella and E.coli Multiplex Detection Assay (Medicinal Genomics #420113) o Salmonella and E.coli Positive Control (Medicinal Genomics #420313) Salmonella and Total Pathogenic E.coli (STEC) Detection Assay (Medicinal Genomics #420122) o Salmonella and Total Pathogenic E.coli (STEC) Positive Control (Medicinal Genomics #420322) Yeast & Mold Detection Assay (Medicinal Genomics #420103) o Yeast & Mold Positive Control (Medicinal Genomics #420303) Total Aerobic Count (TAC) Detection Assay (Medicinal Genomics #420106) o Total Aerobic Count (TAC) Positive Control (Medicinal Genomics #420306) Total Enterobacteriaceae and Coliform Detection Assay (Medicinal Genomics #420114) o Total Enterobacteriaceae and Coliform Positive Control (Medicinal Genomics #420314) Aspergillus niger Detection Assay (Medicinal Genomics #420109) o Aspergillus niger Positive Control (Medicinal Genomics #420309) Aspergillus flavus Detection Assay (Medicinal Genomics #420111) o Aspergillus flavus Positive Control (Medicinal Genomics #420311) Aspergillus fumigatus Detection Assay (Medicinal Genomics #420110) o Aspergillus fumigatus Positive Control (Medicinal Genomics #420310) Aspergillus terreus Detection Assay (Medicinal Genomics #420129) o Aspergillus terreus Positive Control (Medicinal Genomics #420329) Single Copy Control Gene (SCCG) Positive Control (Medicinal Genomics #420326)
Page 4 of 12 Materials Supplied by the User Consumables & Hardware Agilent AriaMx Real-Time PCR System G8830A Option 010 FAM, ROX, and HEX (Contact Agilent) Agilent HP 650 Notebook PC option 650 (Contact Agilent) 96 well optical qpcr plates (Agilent AriaMx 96 well plates, Agilent # 401490, 401491, or 401494 or Fisher Scientific 96-Well Armadillo PCR Plate, Fisher # AB2396) Adhesive optical seal for qpcr plates (Agilent adhesive plate seals, Agilent # 401492 or USA Scientific TempPlate RT Optical Film # 2978-2100) Multi-channel pipette P50 or P20 (optional) Single channel pipette P10, P20 and P200 Filtered pipette tips for P10, P20, P50, and P200 Crushed ice or cold racks (96 well PCR Cryogenic Rack, VWR #89004-570 and 1.5µL Tube Benchtop Cryogenic Racks, VWR #89004-558 or similar) Freezer, -20 o C Table top mini plate centrifuge (Fisher Scientific #14-100-143 or similar) Table top mini tube centrifuge (VWR Mini Centrifuge #10067-588 or 6-place personal microcentrifuge for 1.5/2.0 ml tubes # 2631-0006, or similar) Table top Vortex Genie (Scientific Industries #SI-0236 or Similar) Reagents 10% bleach
Page 5 of 12 Decontamination Protocol OPTIONAL STEP Note: If not performing Decontamination step, please skip to page 9. 1. Begin with a 10% bleach wipe down of the workspace, including the bench top and all equipment being used (except the Agilent AriaMX Real-Time System). 2. Remove decontamination reagents and positive controls for assays being tested from the -20 o C freezer. 2.1. Decon Buffer, Water and positive controls Allow tubes to defrost at room temperature. Once defrosted, place tubes on ice. 2.2. Decon Enzyme Place directly on ice. Note: The Decon Enzyme will only be used if performing this decontamination step. 2.3. Before preparing the reaction, invert or vortex the tubes and spin-down the reagents. 2.3.1. Decon Buffer and Positive Controls Vortex quickly followed by a pulse spin down in a microcentrifuge. 2.3.2. Decon Enzyme Invert the tube 5 times, followed by a pulse spin-down in a microcentrifuge. 2.4. Return all reagents to the ice. 3. Prepare a 1Unit/µL stock of Decon Enzyme. 3.1. Label a 1.5ml tube 1:10 Decon Enzyme. 3.2. Label a 1.5ml tube 1:10 Decon Buffer. 3.3. Make a 1:10 dilution of Decon Buffer with H 2 O by adding 1ul of Decon Buffer into 9ul H 2 0. 3.4. Make a 1:10 dilution of 10 Unit/µL Decon Enzyme with diluted Decon Buffer prepared in previous step. 3.5. For every 1µL of 10 Unit/µL Decon Enzyme dilute with 9µL of diluted Decon Buffer. Note: It is best to add the largest volume reagent first, in this case diluted Decon Buffer 3.6. Once combined, slowly tip mix or invert the tube 5 times. 3.7. Pulse spin-down in a microcentrifuge. 3.8. Return all reagents to the ice. 4. Make the decontamination master mix using the table below, labeling a fresh 1.5ml tube as Decon MM Reagent 1 Reaction 24 Reactions (plus 2 48 Reactions (plus 4 excess rxn) excess rxn) Decon Buffer (non diluted) 0.75µL 19.5µL 39µL Diluted Decon Enzyme (1Unit/µL) 0.75µL 19.5µL 39µL H 2 O 1.0µL 26µL 52µL Total 2.5µL 65µL 130µL Note: The number of reactions prepared must include an assay specific positive control and negative control for all assays to be tested. (i.e. If testing Yeast & Mold, E.coli, and Salmonella there will need to be 3 assay specific Positive Controls and 3 assay specific Negative Controls)
Page 6 of 12 Below is an example plate setup. This will vary depending on which assays are being run. 4.1. Prepare Positive Controls. 4.1.1. Make a 1:10 dilution of the positive control for each assay being run in separate tubes. 4.1.1.1. Label a clean 1.5mL tube for each positive control being prepared. 4.1.1.2. Dilute 1µl of each Positive Control with 9µl of H 2 O (found in the kit). Note: It is best to add the largest volume reagent first, in this case the H 2 O. 4.1.2. For the negative control H 2 O provided in the kit will be used. 4.2. Place the extraction plate on the magnet. This is only necessary if magnetic beads were carried over during the final elution in the DNA extraction process. It s important that magnetic beads are not transferred into qpcr reactions. 4.3. Use a new 96 well optical qpcr plate and label the plate Decontamination Plate_[date]. Add 2.5ul of prepared Decon MM to each corresponding sample well, positive control well, and negative control well in the qpcr plate. Note: It may be helpful to label each of the corresponding columns or rows on plate to accurately dispense the correct samples. 4.4. Carefully remove the seal from the Extraction Plate and transfer 5uL of each sample into the corresponding well in the qpcr plate and slowly tip mix a few times. Keep the extraction plate on the magnet when aspirating the 5uL. 4.5. Add 5uL of each diluted Positive Control to the corresponding well. Then add 5uL of water to each corresponding Negative Control well. Tip mix after each addition. Note: ALWAYS use a fresh tip for every liquid transfer into the qpcr plate. 4.6. Seal the plate with the adhesive seal, making sure to completely seal the plate wells using a pen or flat object to slide back and forth along the seal. 4.7. Spin down for at least 1 minute in the plate microcentrifuge. 4.8. Place the sealed plate onto the Agilent AriaMX qpcr instrument, positioning the A1 well in the top left corner.
Page 7 of 12 4.9. Start the Decontamination Incubation. 4.9.1. Select User-Defined in the New Experiment page under Experiment Types setup. Select UDG (DNA)segment. 4.9.2. In the Thermal Profile page, add (+) 2 more UDG (DNA) segments and create and run the following profile: 1 hour at 25 o C, 20 minutes at 65 o C, 1 hour at 25 o C 4.9.3. Close the lid and click Start Run. You will see the following warning message, Click No and Yes to use Quick Setup. 4.9.4. Once the run has reached the final 25 o C incubation you can stop the run and place the plate on ice.
Page 8 of 12 Real-Time Quantitative PCR (qpcr) Protocol 1. If you did not perform the decontamination step, Begin with a 10% bleach wipe down of the workspace, including the bench top and all equipment being used (except the Agilent AriaMX Instrument). 2. Remove qpcr Reagents and Assay Probe Mix tubes from the -20 o C freezer. 2.1. qpcr Master Mix, water and Assay Probe Mix tubes Place qpcr Master Mix immediately on ice and allow water and assay probe mix tubes to defrost at room temperature. Once defrosted, immediately place tubes on ice. 2.2. If Decon was not performed, also remove positive control tubes. 2.2.1. Positive controls Allow tubes to defrost at room temperature. Once defrosted, place tubes on ice. 3. Before preparing the reaction, invert or vortex and spin-down the reagents. 3.1. Assay Probe Mix tubes, positive controls and water Vortex quickly followed by a pulse spin-down in a microcentrifuge. 3.2. qpcr Master Mix Invert the tube 5 times (do not vortex), followed by a pulse spin-down in a microcentrifuge. 3.3. Return all reagents to the ice. Note: Do not vortex the qpcr Master Mix at any point during the protocol. 4. Make a separate master mix in a 1.5mL tube for each different assay type being run (all probe mixes contain the internal plant control, SCCG probe mix, in addition to the probe for the microbial target(s)), labeling them [Assay Name] MM. Always prepare enough master mix for 1 or 2 reactions more than you are processing to account for pipetting and dead volumes. The set up process and master mix formulations are different depending on if you performed the decontamination step. Make sure to use the correct table below. 4.1. How to prepare Master Mix WITH Decontamination Step Incorporated Note: It is best to add the largest volume reagent first, in this case H 2 O. Reagents 1 Reaction 24 reactions (plus 1 excess rxn) 48 reactions (plus 2 excess rxn) qpcr Master Mix 3.75µL 93.75µL 187.5µL Assay Probe Mix (Assay Specific) 1µL 25µL 50µL H2O 6.5µL 162.5µL 325µL Total 11.25µL 281.25µL 562.5µL 4.1.1. Once combined gently tip mix or invert the tube 5 times to combine the Assay master mix. It s very important not to add bubbles when mixing qpcr MM. 4.1.2. Pulse spin-down tube in microcentrifuge. 4.1.3. Place MM tubes on ice until used. 4.1.4. Retrieve the decontamination plate from the Bio-Rad qpcr instrument and spin it down for 30 seconds in the plate microcentrifuge. 4.1.5. Remove the seal from the Decontamination Plate carefully (pay attention not to spill any liquid). Add 11.25µL of probe specific MM to each of the corresponding detection assay wells in the spun-down decontamination plate. 4.1.6. Tip mix wells by pipetting up and down 2-3 times. Be careful not to introduce bubbles. Use a fresh tip for every transfer.
Page 9 of 12 4.2. How to prepare Master Mix WITHOUT Decontamination Step Incorporated Note: It is best to add the largest volume reagent first, in this case H 2 O. Reagents 1 Reaction 24 reactions (plus 1 excess rxn) 48 reactions (plus 2 excess rxn) qpcr Master Mix 3.75µL 93.75µL 187.5µL Assay Probe Mix (Assay Specific) Decon Buffer 1µL 25µL 50µL 0.8µL 20µl 40µl H 2 O 8.2 205µL 410µL Total 13.75µL 343.75µL 687.5µL 4.2.1. Once combined gently tip mix or invert the tube 5 times to combine the Assay master mix. 4.2.2. Pulse spin-down tube in microcentrifuge. 4.2.3. Place MM tubes on ice until used. 4.2.4. For the positive control, make a 1:10 dilution of each assay being run. 4.2.4.1. 1µL of Positive Control dilute with 9µL of H 2 O (found in the kit) 4.2.4.2. For the negative control, use H 2 O (found in the kit). Note: It is best to add the largest volume reagent first, in this case the H 2 O then the 1 µl of positive control, pipette mix well to ensure control DNA is in solution 4.2.5. Below is an example plate setup. This will vary depending on which assays are being tested. 4.2.6. Place the Extraction Plate on the magnet. 4.2.7. Use a new 96 well optical qpcr plate and label the plate qpcr Plate_[date]. 4.2.8. Carefully remove the seal from the Extraction Plate and transfer 5µL of each sample into the corresponding well on the qpcr plate. Keep the extraction plate on the magnet when aspirating the 5µL. 4.2.8.1. Add 5µL of the diluted Positive Controls to their corresponding wells. Then add 5µL of water to the corresponding Negative Control wells. Note: ALWAYS use a fresh tip for every liquid transfer into the qpcr plate 4.2.9. Add 13.75µL of specific Assay Probe MM to each corresponding sample well, positive control well, and negative control well in the qpcr plate. Gently tip mix a few times after each addition of qpcr master mix. Be careful to not introduce bubbles during this mix. Note: It may be helpful to label each of the corresponding column wells to accurately dispense the correct samples
Page 10 of 12 5. Seal the plate with the adhesive seal, making sure to completely seal the plate wells using a pen or flat object to slide back and forth along the seal. 5.1. Spin-down for at least 1 minute in the plate microcentrifuge. Note: Check for bubbles at the bottom of the wells (minimal bubbles on the surface of the liquid is acceptable). If bubbles remain in the bottom of the wells, spin-down for another minute. 5.2. Place the sealed plate onto the Agilent AriaMX instrument, positioning the A1 well in the top left corner. 6. Create a New Experiment on the Agilent qpcr instrument. 6.1. Select Quantitative PCR under Experiment Types. Under Setup>Plate Setup, select FAM,HEX and ROX channel collection. ROX is only necessary if running multiplexed assays, E. coli/salmonella or Coliform/Entero.
Page 11 of 12 6.2 Change the well Types to reflect your plate set up. Add Target names to include pathogen name for FAM or ROX and SCCG (single copy control gene) for HEX. 6.3 Under Setup>Thermal Profile, create the following PCR thermal profile. Hot start at 95 o C for 5minutes, followed by 40 cycles of 95 o C for 15 seconds and 65 o C for 90 seconds. 6.2. Close the lid and click Start Run. 6.3. Save the experiment with the [User] and [date] 6.4. When run is complete, immediately dispose of the plate. Do not open the plate seal after the run to avoid contamination in the lab.
Page 12 of 12 Glossary and Definitions Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms. Polymerase Chain Reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. The Negative Controls are the reactions where no Cq is expected. It helps to ensure that all Assay specific reactions are clean of contaminates. The assay specific Positive Controls are the reactions where a Cq is expected. It helps ensure that all Assay specific reactions are working correctly. The Assay specific Positive Control is targeting the pathogen using the FAM flourophore. The Internal Control is added to every sample reaction where a Cq is expected. It ensures the effectiveness and efficiency of each reaction. The internal control is targeting a Single Copy Control Gene or SCCG, using the HEX flourophore. DISCLAIMER This test was developed and its performance characteristics determined by Medicinal Genomics Company, for laboratory use. Any deviations from this protocol are not supported by MGC. LIMITED USE LABEL LICENSE This product is covered by at least one or more claims of US patents applications, which are exclusively licensed to Medicinal Genomics Corporation. This product is sold strictly for the use of the buyer, and the buyer is not authorized to transfer this product [or any materials made using this product] to any third party. 2017 Medicinal Genomics Corporation. All rights reserved. * All Trademarks are property of their respective owners.